WHO FOOD

ADDITIVES
SERIES: 54
J
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Safety evaluation of certain
food additives
Prepared by the
Sixty-third meeting of the Joint FAO/WHO
Expert Committee on Food Additives (JECFA)
54
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International Programme on Chemical Safety
World Health Organization, Geneva
IPCS
This volume contains monographs prepared at the sixty-third meeting of the
Joint FAO/WHO Expert Committee on Food Additives (JECFA), which met in Geneva,
Switzerland, from 8 to 17 June 2004.
The toxicological monographs in this volume summarize the safety data on a
number of food additives, including benzoyl peroxide, -cyclodextrin, hexose oxidase
from Chondrus crispus expressed in Hansenula polymorpha, lutein from Tagetes
erecta L., peroxyacid antimicrobial solutions containing 1-hydroxyethylidene-1,1-
diphosphonic acid, steviol glycosides, D-tagatose, xylanases from Bacillus subtilis
expressed in B. subtilis and zeaxanthin, and a natural constituent, glycyrrhizinic acid.
Monographs on eight groups of related flavouring agents evaluated by the Procedure
for the Safety Evaluation of Flavouring Agents are also included.
This volume and others in the WHO Food Additives Series contain information
that is useful to those who produce and use food additives and veterinary drugs and
those involved with controlling contaminants in food, government and food regulatory
officers, industrial testing laboratories, toxicological laboratories, and universities.
45404 FOOD ADDITIVES 54 COV 9/2/06 12:42 pm Page 1
IPCS International Programme on Chemical
Safety
WHO FOOD
ADDITIVES
SERIES: 54
Safety evaluation of certain
food additives
Prepared by the
Sixty-third meeting of the Joint FAO/WHO
Expert Committee on Food Additives (JECFA)
World Health Organization, Geneva, 2006 6
WHO Library Cataloguing-in-Publication Data
Safety evaluation of certain food additives / prepared by the
sixty-third meeting of the Joint FAO/WHO Expert Committee
on Food Additives (JEFCA).
(WHO food additives series ; 54)
1. Food additives toxicity 2. Flavoring agents
toxicity 3. Food contamination 4. Risk assessment
I. Joint FAO/WHO Expert Committee on Food Additives.
Meeting (63rd : 2004, Geneva, Switzerland II. Series.
ISBN 92 4 166054 6 (NLM classifcation: WA 712)
ISSN 0300-0923
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World Health Organization concerning the legal status of any country, territory,
CONTENTS
Preface ........................................................................................................ v
Food additives
a-Cyclodextrin ........................................................................................ 3
Benzoyl peroxide ................................................................................... 17
Hexose oxidase from Chondrus crispus expressed in
Hansenula polymorpha ...................................................................... 37
Lutein from Tagetes erecta L. ............................................................... 49
Peroxyacid antimicrobial solutions containing
1-hydroxyethylidene-1,1-diphosphonic acid ....................................... 87
Steviol glycosides .................................................................................. 117
D-Tagatose ............................................................................................. 145
Xylanases from Bacillus subtilis expressed in B. subtilis...................... 149
Zeaxanthin (synthetic) ........................................................................... 159
Safety evaluation of groups of related favouring agents
Introduction ............................................................................................ 191
Pyridine, pyrrole and quinoline derivatives............................................ 195
Aliphatic and alicyclic hydrocarbons...................................................... 235
Aromatic hydrocarbons .......................................................................... 291
Aliphatic, linear a,b-unsaturated aldehydes, acids and related
alcohols, acetals and esters .............................................................. 317
Monocyclic and bicyclic secondary alcohols, ketones and
related esters ..................................................................................... 385
Amino acids and related substances .................................................... 435
Tetrahydrofuran and furanone derivatives ............................................. 487
Phenyl-substituted aliphatic alcohols and related aldehydes
and esters .......................................................................................... 525
A natural constituent
Glycyrrhizinic acid .................................................................................. 561
Annexes
Annex 1 Reports and other documents resulting from
previous meetings of the Joint FAO/WHO Expert
Committee on Food Additives ..................................... 621
Annex 2 Abbreviations used in the monographs ....................... 631
Annex 3 Participants in the sixty-third meeting of
the Joint FAO/WHO Expert Committee on
Food Additives ............................................................. 633
Annex 4 Recommendations on compounds on the
agenda and further toxicological studies and
information required ..................................................... 637
Annex 5 Summary of the safety evaluation of secondary
components for favouring agents with minimum
assay values of less than 95% ................................... 649
iv CONTENTS
PREFACE
The monographs contained in this volume were prepared at the sixty-third
meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA),
which met at WHO Headquarters in Geneva, Switzerland, 817 June 2004. These
monographs summarize the safety data on selected food additives reviewed by
the Committee.
The sixty-third report of JECFA has been published by the World Health
Organization as WHO Technical Report No. 928. Reports and other documents
resulting from previous meetings of JECFA are listed in Annex 1. The participants
in the meeting are listed in Annex 3 of the present publication; a summary of the
conclusions of the Committee is given in Annex 4. Some of the substances listed
in Annex 4 were evaluated at the meeting only for specifcations. Annex 5 contains
a summary of the safety evaluation of secondary components for favouring agents
with minimum assay values of less than 95%.
Specifcations that were developed at the sixty-third meeting of JECFA have
been issued separately by FAO as Food and Nutrition Paper, No. 52, Addendum
12. The monographs in the present publication should be read in conjunction with
the specifcations and the report.
JECFA serves as a scientifc advisory body to FAO, WHO, their Member States,
and the Codex Alimentarius Commission, primarily through the Codex Committee
on Food Additives and Contaminants and the Codex Committee on Residues of
Veterinary Drugs in Foods, regarding the safety of food additives, residues of vet-
erinary drugs, naturally occurring toxicants and contaminants in food. Committees
accomplish this task by preparing reports of their meetings and publishing specif-
cations or residue monographs and toxicological monographs, such as those
contained in this volume, on substances that they have considered.
The toxicological monographs contained in the volume are based on working
papers that were prepared by Temporary Advisers. A special acknowledgement is
given at the beginning of each monograph to those who prepared these working
papers.
Many proprietary unpublished reports are unreferenced. These were voluntarily
submitted to the Committee by various producers of the food additives under
review, and in many cases represent the only data available on those substances.
The Temporary Advisers based the working papers they developed on all the
data that were submitted, and all of these reports were available to the Committee
when it made its evaluation. The monographs were edited by H. Mattock, Illkirch-
Graffenstaden, France.
The preparation and editing of the monographs included in this volume were
made possible through the technical and fnancial contributions of the Participating
Organizations of the International Programme on Chemical Safety (IPCS), which
supports the activities of JECFA.
The designations employed and the presentation of the material in this publica-
tion do not imply the expression of any opinion whatsoever on the part of the
v
organizations participating in the IPCS concerning the legal status of any country,
territory, city, or area or its authorities, or concerning the delimitation of its frontiers
or boundaries. The mention of specifc companies or of certain manufacturers
products does not imply that they are endorsed or recommended by the organiza-
tions in preference to others of a similar nature that are not mentioned.
Any comments or new information on the biological or toxicological properties
of the compounds evaluated in this publication should be addressed to: Joint WHO
Secretary of the Joint FAO/WHO Expert Committee on Food Additives, Interna-
tional Programme on Chemical Safety, World Health Organization, Avenue Appia,
1211 Geneva 27, Switzerland.
vi PREFACE
K2
FOOD ADDITIVES
K2

a-CYCLODEXTRIN (addendum)
First draft prepared by
Professor R. Kroes
1
, Dr P. Verger
2
and Dr J.C. Larsen
3
1
Institute for Risk Assessment Sciences, Utrecht University,
Soest, Netherlands;
2
Food risk analysis methodologies, National Institute for
Agricultural Research/National Institute for Agriculture Paris-Grignon,
Paris, France; and
3
Division of Toxicology and Risk Assessment, Danish Institute of Food
and Veterinary Research, Sborg, Denmark
Explanation............................................................................... 3
Biologicaldata.......................................................................... 4
Biochemicalaspects:absorption,distribution,
metabolism,andexcretion........................................... 4
Toxicologicalstudies.......................................................... 4
Specialstudies............................................................. 5
Skinirritationand/orsensitization......................... 5
Skinirritationandcorrosion.................................. 5
Ocularirritation...................................................... 5
Cellmembraneandintestinalpermeability........... 5
Digestibilityinvitro................................................ 6
Interactionwiththeabsorptionoflipophilic
nutrients.......................................................... 6
Interactionwiththeabsorptionofminerals........... 6
Impurities............................................................... 7
Observationsinhumans.................................................... 8
Studiesinhumanvolunteers....................................... 8
Digestibilityinhumans................................................. 8
Attenuationbya-cyclodextrinoftheglycaemic
responsetofoodcontainingstarch....................... 9
Intake........................................................................................ 9
Comments ............................................................................... 9
Evaluation ............................................................................... 13
References............................................................................... 13
1. EXPLANATION
a-Cyclodextrin (synonyms: cyclohexaamylose, cyclomaltohexaose, a-
Schardinger dextrin) is a non-reducing cyclic saccharide comprising six glucose
unitslinkedbya-1,4bonds.a-CyclodextrinwasevaluatedbytheCommitteeatits
ffty-seventh meeting (Annex 1, reference 154), when the Committee concluded
that, on the basis of the results of available studies with a-cyclodextrin and with
the structurally related compounds b-cyclodextrin (seven glucose units) and g-
cyclodextrin (eight glucose units), for which acceptable daily intakes (ADIs) had
K2
a-CYCLODEXTRIN
K2
been allocated, there was suffcient information to allocate anADI not specifed
fora-cyclodextrin.
At its ffty-seventh meeting, the Committee evaluated a-cyclodextrin on the
basisofknownusesundergoodmanufacturingpracticeasacarrierandstabilizer
for favours, colours, and sweeteners, as a water-solubilizer for fatty acids and
certain vitamins, as a favour modifer in soya milk, and as an absorbent in con-
fectionery.Theannular(doughnut-shaped)structureof a-cyclodextrinprovidesa
hydrophobiccavitythatallowstheformationofinclusioncomplexeswithavariety
ofnon-polarorganicmoleculesofappropriatesize,whilethehydrophilicnatureof
the outer surface of the cyclic structure causes such complexes to be soluble in
water.a-Cyclodextrinisproducedbytheactionofcyclodextringlucosyltransferase
and may contain residues of 1-decanol, which is used in the purifcation
process.
At its present meeting, the Committee evaluated a-cyclodextrin for use as a
foodingredient,suggestedbythemanufacturertobeadietaryfbre.Itisstressed
that the Committee only evaluated the safety of the estimated intake of a-cyclo-
dextrinresultingfromtheproposeduselevels.TheCommitteedidnotassessthe
effcacyofa-cyclodextrinusedasadietaryfbre.
2. BIOLOGICAL DATA
2.1 Biochemical aspects: absorption, distribution, metabolism,
and excretion
The biochemical aspects related to absorption, distribution, metabolism and
excretion were described in the previous monograph (Annex 1, reference 154),
andnonewrelevantdatainanimalswereavailable.a-Cyclodextrin,likeb-cyclo-
dextrin,isnotdigestedinthegastrointestinaltractbutisfermentedbytheintestinal
microfora. In germ-free rats, a-cyclodextrin is almost completely excreted in the
faeces, while g-cyclodextrin is readily digested to glucose by the luminal and/or
epithelial enzymes of the gastrointestinal tract. Overall, studies indicate that a-
cyclodextrincanbeabsorbedintactatalevelofapproximately1%fromthesmall
intestine. Absorbed intact a-cyclodextrin is excreted rapidly in the urine (Van
Ommen & de Bie, 1995). All the nondigested and non-absorbed a-cyclodextrin
reaches the microbially colonized segments of the gut, where the a-cyclodextrin
ring is readily opened by microbial enzymes (certain amylases and cyclodextri-
nase). The resulting linear malto-oligosaccharides are then further hydrolysed
andfermentedviawellestablishedmetabolicpathwaystoshort-chainfattyacids
(Antenucci & Palmer, 1984). Absorption of the metabolites of a-cyclodextrin
leadstoslowremoval,mainlyinexhaledcarbondioxide(CO
2
)

orintheurine(Van
Ommen&deBie,1995).
2.2 Toxicological studies
No new data on toxicology in animals treated orally became available since
therecentevaluationofa-cyclodextrin(Annex1,reference154).TheCommittee
concludedthattheacutetoxicityofa-cyclodextrinwaslow,butwhengivenbythe
a-CYCLODEXTRIN
K2
intraperitonealorintravenousrouteitcancauseosmoticnephrosis(alsodescribed
intheliteratureasresorptivevacuolization)athighdoses,whichmayleadtorenal
failure.Theresultsofshort-term(28-and90-day)studiesoftoxicityindicatedthat
a-cyclodextrinhaslittleeffectwhengivenorallytoratsordogs.Afteradministration
of a very high concentration of a-cyclodextrin in the diet (20%, corresponding to
a dose of 13.9g/kgbw per day in rats and 10.4g/kgbw per day in dogs), caecal
enlargement and associated changes were seen in both species. This effect is
likelytoresultfromthepresenceofahighconcentrationofanosmoticallyactive
substance in the large intestine. No studies of intravenous administration were
available to permit a comparison of the systemic toxicity of this compound with
thatofb-andg-cyclodextrin.
Studiesinmice,rats,andrabbitsgivena-cyclodextrinatconcentrationsofup
to 20% in the diet (corresponding to doses of 49.3, 20 and 5.67.5g/kgbw per
day,respectively)didnotindicateanyteratogeniceffects.Similarly,theresultsof
assays for genotoxicity were negative. No long-term studies of toxicity, carcino-
genicity, or reproductive toxicity have been conducted with a-cyclodextrin, but at
itsfftyseventhmeeting(Annex1,reference154)theCommitteeconcludedthat,
given the known fate of this compound in the gastrointestinal tract, such studies
werenotrequiredforanevaluation.
2.2.1 Special studies
(a) Skinirritationand/orsensitization
The potential of a-cyclodextrin to induce cutaneous delayed hypersensitivity
was examined in guinea-pigs (a control group of fve animals of each sex and a
treatedgroupof10animalsofeachsex),whichwereinducedintwosteps.First,
a3%solutionofa-cyclodextrinwithFreundcompleteadjuvantwasinjectedintra-
dermally.The controls received water with or without Freund complete adjuvant.
Oneweeklater,a30%dilutionofa-cyclodextrininvaselinewasappliedtopically
(controlsreceivedvaselineonly).Aftertwomoreweeks,achallengetreatmentwas
madebytopicallyapplyingvaselinewith0%(control),10%or30%a-cyclodextrin.
The challenge treatment did not provoke signs of hypersensitivity (erythema,
oedema)at24or48hafterthechallenge.Itwasconcludedthata-cyclodextrinis
notasensitizer(Prinsen,1992).
(b) Skinirritationandcorrosion
The potential of a-cyclodextrin to induce dermal irritation and corrosion was
examined in three albino rabbits. A mixture of a-cyclodextrin (0.5g) with water
(0.3g)wasappliedtotheshavenskinfor4h.Skinirritationscoreswererecorded
at 1, 24, 48 and 72h after removal of the test material. No sign of skin irritation
wereobservedatanytimeinanyanimal(Prinsen,1991a).
(c) Ocularirritation
Toexaminepotentialocularirritation,0.062gofa-cyclodextrinwasinstilledas
adrypowderintheconjunctivalcul-de-sacoftherighteyeofthreealbinorabbits.
a-CYCLODEXTRIN
K2
Thereactionwasexaminedat1,24,48and72hand7and14daysafteradmin-
istration. Different signs of acute ocular irritation were seen starting at 1h after
treatment.At 7 days after treatment, eye effects had cleared completely in one
rabbit,whereasischaemicnecrosisofthenictitatingmembrane,slightrednessand
slightswellingoftheconjunctivaewerestillobservedintheothertworabbits.At
14 days after treatment, these eye effects had also cleared completely. It was
concludedthatdrya-cyclodextrinpowderisirritatingbutnotcorrosivetotheeye
(Prinsen, 1990). Two groups of three rabbits received a-cyclodextrin in solution
(7.25%and14%,w/v),instilledintheconjunctivalcul-de-sacoftherighteye.The
ocularreactionswereexaminedafter1,24,48and72h.Thetreatmentscaused
slight redness and slight swelling of the conjunctivae in some animals. All eye
effectshadclearedcompletelyat24haftertreatment.Itwasconcludedthatsolu-
tions of a-cyclodextrin are not irritating and not corrosive to the eye (Prinsen,
1991b).
(d) Cellmembraneandintestinalpermeability
Effectsonthecellmembraneandonintestinalpermeabilityweredescribedin
the previous evaluation of a-cyclodextrin (Annex 1, reference 154). In vitro, a-
cyclodextrin, like b-cyclodextrin, sequestered components of the membranes of
erythrocytes,causinghaemolysis.Thethresholdconcentrationforthiseffectwas,
however,higherthanthatobservedforb-cyclodextrin.Similarly,ofthethreecyclo-
dextrinsa-cyclodextrinhadthesmallesteffectonabsorptioninsituinrats.
(e) Digestibilityinvitro
Early experiments on the digestibility of cyclodextrins by amylases in vitro
demonstratedthatpancreaticjuiceofdogsdoesnotcleavea-cyclodextrin(Karrer,
1923) and that salivary amylase leaves a-cyclodextrin intact, hydrolyses b-
cyclodextrinonlyveryslowly,buthydrolysesg-cyclodextrinatarateofabout1%
that for starch.At that time, a-cyclodextrin was called diamylose and tetraamy-
lose(French,1957).Recentstudiesinvitroshowedthathumansalivaryamylase,
likehumanorporcinepancreaticamylases,areunabletohydrolysea-cyclodextrin
andb-cyclodextrintoanymeasurableextent,butreadilyhydrolyseg-cyclodextrin
(Marshall&Miwa,1981;Kondoetal.,1990;McCleary,2002).
(f) Interactionwiththeabsorptionoflipophilicnutrients
It has been demonstrated that the solubility of retinol acetate and vitamin K1
inwaterishigherinthepresenceofa-cyclodextrin(Pitha,1981).
a-Cyclodextrin is known not to form complexes with vitamin D or vitamin E
(Pitha,1981).
(g) Interactionwiththeabsorptionofminerals
Thepossibilitythattheabsorptionofvitaminsandmineralsmightbeimpaired
bytheconsumptionofincreasedamountsofdietaryfbrehasbeenaddressedin
a-CYCLODEXTRIN
K2
severalreviews(e.g.Kelsay,1990;Rossanderetal.,1992;Gorman&Bowman,
1993;Gordonetal.,1995).Invariablyitwasconcludedthatdietaryfbre,atrecom-
mendedlevelsofintake,doesnotadverselyaffectthevitaminandmineralstatus
of the average consumer. For resistant starch this was demonstrated recently in
astudyinwhichratsandpigsreceiveddietswith6%nativestarchorretrograded
high-amylosestarch.Theingestionoftheresistantstarchdidnotsignifcantlyaf-
fect the absorption or retention of calcium, phosphorus, magnesium or zinc (De
Schrijver et al., 1999). In addition, the low viscosity of a-cyclodextrin and its
lackofanionicorcationicgroups,makeitunlikelythattheabsorptionofminerals
fromthesmallintestineswouldbeimpaired.
(h) Impurities
Theenzymecyclodextrin-glycosyltransferase,whichisusedintheproduction
of a-cyclodextrin, is derived from a non-genotoxic, non-toxigenic source and is
completelyremovedduringthepurifcationof a-cyclodextrin(Annex1,reference
154).
1-Decanol is used as complexant for the precipitation of a-cyclodextrin. 1-
Decanol has been used as a favour for many years (estimated intake, 728mg/
personperday)andhasbeenevaluatedpreviouslybytheCommittee(Annex1,
reference132).Nodataareavailableontheabsorption,distribution,metabolism
andexcretionof1-decanol;however,itisgenerallyassumedthatingestedaliphatic
primaryalcoholsareabsorbedandoxidizedtothecorrespondingaldehyde,which
is then rapidly oxidized to the acid. Acids with an even number of carbons are
metabolizedviab-oxidationtoacetyl-coenzymeA,whichthenentersthecitricacid
cycle(Annex1,reference132).
The safety of 1-decanol has been examined in studies of genotoxicity, acute
oraltoxicityandembryotoxicityandteratogenicity(inhalationandoraladministra-
tion).AnassayforgenemutationwithB.subtilisH17(rec
+
)andM45(rec
-
)using
17mg of 1-decanol per disk yielded a negative result (Oda et al., 1978, cited in
Annex1,reference132).
Theacuteoraltoxicityof1-decanolwasexaminedintwostudiesinrats.Median
lethal doses (LD
50
) of >5 and 12.8g/kgbw were reported (Henkel, K.G.A., un-
published data, Archive No 281; Br & Griepentrog, 1967). In mice, a LD
50
of
6.5g/kgbw was observed. In a study of embryotoxicity and teratogenicity in
Sprague-Dawley rats, the dams were exposed to 1-decanol by inhalation
(100mg/m
3
; 6h per day) on days 119 of gestation. No maternal toxicity was
observed. The reproductive outcome (number of resorptions, litter size, fetal
weights)wasnotadverselyaffectedbythetreatment,andtherewerenosignsof
fetotoxicityorteratogenicity(Nelsonetal.,1990).
Inastudyofembryotoxicityandteratogenicity,unspecifedrandom-bredalbino
ratsweregivenaseriesofprimaryalcohols,including1-decanol,byoraladminis-
tration. A group of 10 female rats received daily doses of 1-decanol of 400mg
(equalto2g/kgbwperday)mixedwith600mgofwaterbygavageondays115
ofgestation.Acontrolgroupof20ratsreceived1mlofwaterperdaybygavage.
Nosignsofmaternaltoxicitywerereported.Pre-andpostimplantationlosseswere
a-CYCLODEXTRIN
K2
signifcantlyincreasedwith1-decanol,butsizeandweightofthefetuseswasnot
impaired. No teratogenic activity was observed. It was concluded that all the
primaryalcohols(C1,C2,C4,C9,C10)testedincreasedthenumberofpre-and
postimplantation losses. 1-Decanol and nonanol were clearly less active than
ethanol or methanol. Retardation of fetal development was observed with all the
alcohols tested, except 1-decanol. None of the alcohols tested had teratogenic
activity(Barilyaketal.,1991).
2.3 Observations in humans
2.3.1 Studies in human volunteers
Inanearlystudyofthemetabolismofa-cyclodextrin,twopatientswithtype-2
diabetesreceived50gofa-cyclodextrinofunknownpurityperday.Thesubstance
wasgivenwithalow-carbohydratediet.Nauseawasnotedinonesubjectonone
outoftwoexperimentaldays,about1012minafteringestion.Otherside-effects
did not occur. The authors attributed this effect to an (unknown) impurity rather
thantoa-cyclodextrinitself(VonHoesslin&Pringsheim,1923).
In a subsequent series of experiments, a preparation of purifed cyclodextrin
(consistingmainlyofa-cyclodextrinwithsomeb-andg-cyclodextrin)wasgivenat
adoseof50to100g/day.Some,butnotall,volunteers(proportionnotspecifed)
reported nausea and, occasionally, diarrhoea. Urine analyses of four diabetic
patients (two of whom were presumably type-1 diabetics) were presented and
demonstratedthatingestionofthecyclodextrinpreparationdidnotleadtoanele-
vationofurinaryglucoseexcretion,aswasseenaftertheingestionofbread(Von
Hoesslin&Pringsheim,1927).
The gastrointestinal tolerance of a-cyclodextrin was examined in 12 healthy
malevolunteersinthecontextofastudyonitsglycaemiceffects.Asinglebolus
doseofa-cyclodextrinof25g(dissolvedin250mlofwater)wasadministeredto
men who had fasted overnight. One man reported diarrhoea and three others
reported abdominal discomfort. These effects were rated as mild and did not
preventthevolunteersfromfurtherparticipationinthestudy.
Theingestionof10gofa-cyclodextrin(dissolvedin250mlofwater)together
with100goffreshwhitebreadwasnotassociatedwithanyintestinalside-effects
inanyofthemen(Diamantis&Br,2002).
2.3.2 Digestibility in humans
In ileostomic subjects, more than 90% of an oral dose of b-cyclodextrin may
be recovered from the ileal effuent (Flourie et al., 1993). b-Cyclodextrin and a-
cyclodextrin are similarly resistant to the hydrolytic action of pancreatic amylase
invitro;itisthereforeexpectedthatthedigestibilityofa-cyclodextrininvivoisas
low as that of b-cyclodextrin. Direct proof for the low degree of digestibility of a-
cyclodextrin stems from a study in which 12 healthy male volunteers received
single doses of 25g of a-cyclodextrin, 50g starch (in the form of about 100g of
whitebread),andamixtureof50gofstarchand10gofa-cyclodextrin.Bloodwas
collectedatregularintervalsoveraperiodof3hforanalysisofglucoseandinsulin.
a-CYCLODEXTRIN
K2
Whereastheingestionof50gofstarchproducedtheexpectedriseinbloodcon-
centrationsofglucoseandinsulin,nosignifcantincreaseinbloodconcentrations
of glucose and insulin was noted after the intake of 25g of a-cyclodextrin
(Diamantis&Br,2002).
Twodiabeticsubjectsweregivena-cyclodextrin(ofunknownpurity)atadose
of50g/daywithalow-carbohydratediet.Noincreaseinurinaryglucoseexcretion
wasobserved,incontrasttothatobservedafterconsumptionof50gofwhitebread
(VonHoesslin&Pringsheim,1923).Similarresultswerenotedindiabeticpatients
(includingatleasttwotype-1diabetics)receivingmixedcyclodextrins(consisting
mainly of a-cyclodextrin with some b- and g-cyclodextrin) at a daily dose of 50
to100g(VonHoesslin&Pringsheim,1927).
2.3.3 Attenuation by a-cyclodextrin of the glycaemic response to food
containing starch
Diamantis & Br (2002) examined the ability of a-cyclodextrin to reduce the
glycaemicindex.Twelvehealthymalevolunteersreceived,onseparatedaysafter
overnightfasting,singledosesof25gofa-cyclodextrin,50gofstarch(intheform
of about 100g of fresh white bread) and a mixture of 50g of starch (bread) and
10g of a-cyclodextrin. Capillary blood was collected in regular intervals over a
periodof3hforanalysisofglucoseandinsulin.Theconsumptionof50gofstarch
produced the expected rise in blood concentrations of glucose and insulin. In
contrast,nosignifcantincreaseinbloodconcentrationsofglucoseandinsulinwas
notedaftertheintakeof25gofa-cyclodextrin.Afterintakeofstarch(bread)with
a-cyclodextrin, the glycaemic and insulinaemic responses were delayed and
reducedby55%comparedwiththoseobservedafterintakeofstarch(bread)only.
Similar observations were made with certain other types of soluble dietary fbre
(Br,2004).
Whileonlyafewstudieswitha-cyclodextrininhumansareavailable,studies
onothercarbohydratesoflowdigestibility(suchasinulin,fructooligosaccharides,
polydextrose, resistant (malto)dextrins and other oligosaccharides) provide addi-
tionalinformation.Thelargestnumberofstudiesisprobablyavailableforfructoo-
ligosaccharides and inulin. In a review of the safety data on fructans, including
dataonintestinaltoleranceinchildrenandadults(Carabin&Flamm,1999),itwas
concludedthatabdominalcomplaintswouldoccurinadultsafterasingledoseof
20g.Childrenofschoolagetoleratedsupplementationofthedietwithfructooli-
gosaccharidesatalevelof39g(singledose).Ingestionofasingledoseof10g
ofa-cyclodextrin(dissolvedin250mlofwater)togetherwith100goffreshwhite
breadwasnotassociatedwithanyintestinalside-effects.
3. INTAKE
Atitsffty-seventhmeeting(Annex1,reference154),theCommitteeestimated
thepotentialintakeofa-cyclodextrinfromitsknownfooduses.Thepredictedmean
intakeofa-cyclodextrinbyconsumers,basedonindividualdietaryrecordsforthe
USAandproposedmaximumlevelsofuseinavarietyoffoods,was1.7g/person
10 a-CYCLODEXTRIN
K2
perday.Forconsumersatthe90thpercentileofintake,thepredicteddailyintake
ofa-cyclodextrinwas3gperperson.
The intended use levels of a-cyclodextrin from its proposed new use as an
ingredientinanumberoffoodproductsrangefromamaximumof10g/kginnon-
alcoholicbeveragestoamaximumof100g/kginbakeryproducts.
Assuming that a-cyclodextrin would be added to all possible food categories
atthemaximumproposeduselevelsandusingthedataonEuropeandietfood
consumptionintheGlobalEnvironmentMonitoringSystemFoodContamination
MonitoringandAssessmentProgramme(GEMS/Food)database,theCommittee
calculated a total intake of a-cyclodextrin of 65g/person per day (see Table 1).
Thisestimateisveryconservativesinceitisunlikelythata-cyclodextrinwouldbe
consumedsimultaneouslyfromallsourcesonaregularbasis.
AnintakeassessmentwasprovidedbyAustraliaandNewZealandbasedon
anational1-dayrecallsurvey.Itwasassumedthata-cyclodextrinwouldbepresent
at the highest proposed concentrations in all foods for which use was intended.
The average dietary intake from intended uses was estimated to be 16g/person
perdayandthe95thpercentileofintakewasestimatedtoreach37g.
In order to estimate the potential intake of a-cyclodextrin in a single eating
occasion, the Committee used the GEMS/Food large portion database, which
containsthehighestfguresfor97.5thpercentileconsumption(eatersonly)reported
innationalsurveys.Thehighestestimatedpotentialingestionofa-cyclodextrinper
eatingoccasionisbetween19and38gforbreadonly,dependingontheproposed
uselevel.
Table 1. Simulation of exposure to a-cyclodextrin using the European GEMS/
Food
a
diet
Maximum Meanfood Exposure %oftotal
proposed consumption (g/day) exposure
uselevel (g/day)
(g/kg)
Cereals 50100 222 1122 1632
Sugar 150 107 16 23
Margarine 200 17 3.4 5
Stimulant 0.088 14 0.0012
Milk 25 336 8.4 12
Nonalcoholicbeverage 10 1500* 15 21
Total 65
a
GlobalEnvironmentMonitoringSystemFoodContaminationMonitoringand
AssessmentProgramme.
b
ConsumptionforsoftdrinksisnotavailableintheGEMS/Fooddatabase;therefore,this
fgureisanestimate.
a-CYCLODEXTRIN 11
K2
4. COMMENTS
Onlysmallquantities(1%orlessoftheadministereddose)ofintacta-cyclo-
dextrin are absorbed from the small intestine.Absorbed a-cyclodextrin is rapidly
excreted in the urine. a-Cyclodextrin, like b-cyclodextrin, is not digested in the
gastrointestinal tract but is fermented to short-chain fatty acids by the intestinal
microfora. These fatty acids are absorbed, oxidized, and eliminated largely as
exhaledCO
2
.
a-Cyclodextrinisnothydrolysedbyhumansalivaryandpancreaticamylases
invitro.Indirectproofthata-cyclodextrinisnotdigestedinhumansisdrawnfrom
experimentsshowingthattheintakeof25gofa-cyclodextrindoesnotleadtoan
increaseinbloodconcentrationsofglucoseandinsulin.
The results of short-term (28- and 90-day) studies of toxicity indicate that a-
cyclodextrinhasloworaltoxicityinratsanddogs.Afteradministrationofa-cyclo-
dextrinataveryhighconcentrationinthediet(20%,correspondingtoadoseof
13.9g/kgbwperdayinratsand10.4g/kgbwperdayindogs),caecalenlargement
and associated changes were seen in both species.This effect is likely to result
from the presence of a high concentration of an osmotically active substance in
thelargeintestine.
Studiesofembryotoxicityandteratogenicityinmice,rats,andrabbitsfeddiets
containinga-cyclodextrinataconcentrationofupto20%(correspondingtoadose
of49.3g/kgbwperdayinmice,20g/kgbwperdayinrats,and5.97.5g/kgbwper
dayinrabbits)didnotindicateanyadverseeffects.
a-Cyclodextrinisneitheranirritantnorasensitizerafterdermalapplication.
a-Cyclodextrinshowednoeffectsinassaysforgenotoxicityinvitroandinvivo.
Nolong-termstudiesoftoxicity,carcinogenicity,orreproductivetoxicityhavebeen
conducted with a-cyclodextrin, but the Committee reiterated its conclusion from
theffty-seventhmeeting(Annex1,reference154),statingthatsuchstudieswere
notrequiredfortheevaluation,inviewoftheknownfateofthiscompoundinthe
gastrointestinaltract.
Itispossiblethatthepotentialinteractionofa-cyclodextrinwithlipophilicnutri-
entsmightimpairtheirabsorption.Althoughthishasnotbeenstudiedspecifcally
fora-cyclodextrin,suchaneffectwasconsideredtobeunlikelybyanalogytothe
resultsofstudieswithb-cyclodextrin.Complexesbetweenfat-solublevitaminsand
b-cyclodextrin have been shown to have a greater bioavailability than uncom-
plexedforms.Inthiscontext,a-cyclodextrinisknowntoenhancethesolubilityof
retinolacetateandvitaminK1inwater,butdoesnotformcomplexeswithvitamin
DandvitaminE.
Itisalsoconsideredunlikelythattheconsumptionoflargeamountsofa-cyclo-
dextrinwouldimpairtheabsorptionofminerals,sinceitisknownthattheingestion
of resistant starch does not signifcantly affect the absorption or retention of
calcium, phosphorus, magnesium or zinc. Moreover,a-cyclodextrin is of low vis-
cosity,anditschemicalstructurelacksanionicorcationicgroups.
12 a-CYCLODEXTRIN
K2
A few studies in human volunteers indicate that fatulence, bloating, nausea
andsoftstoolsmayoccurinsomeindividualsuponingestionofa-cyclodextrinat
ahighdose.Thisisawell-knownphenomenonforcarbohydratesoflowdigestibil-
ity,particularlyifingestedinliquidformonanemptystomach.Itispartlycaused
byaninfuxofwaterinthesmallintestine(achievingisotonicity)andpartlybythe
ensuingfermentationprocessinthemoredistalpartsofthegut.Mildabdominal
discomfortoccurredinfouroutoftwelvemen,whohadfastedovernight,givena
singledoseof25gofa-cyclodextrininwater,whilenoeffectswerereportedafter
administration of 10g of a-cyclodextrin in water together with white bread. In
studieswithothercarbohydratesoflowdigestibility,suchasinulin,fructooligosac-
charides, polydextrose, resistant (malto)dextrins and other oligosaccharides,
abdominal complaints were reported after a single dose of 20g in adults, and
children of school age tolerated supplementation of the diet with fructooligosac-
charidesatasingledoseof39g.
Evaluationofpotentialimpurities
Theenzymecyclodextrin-glycosyltransferase,whichisusedintheproduction
of a-cyclodextrin, is derived from a nontoxigenic microorganism. The enzyme is
completelyremovedfroma-cyclodextrinduringpurifcationandisthereforeofno
safetyconcern.1-Decanol,whichisusedascomplexantfortheprecipitationofa-
cyclodextrin,maybepresentinthefnalproductataconcentrationof<20mg/kg.
For example, an assumed intake of a-cyclodextrin of 65g/person per day would
correspondtoanintakeof1-decanolof<1.3mg/personperday.Thisisnotasafety
concernbecause1-decanolisrapidlyoxidizedintheintestinalmucosatothecor-
respondingfattyacid,whichthenundergoesb-oxidation.
Intake
Atitsffty-seventhmeeting(Annex1,reference154),theCommitteeestimated
thepotentialintakeofa-cyclodextrinfromknownfooduses.Thepredictedmean
intakeofa-cyclodextrinbyconsumers,basedonindividualdietaryrecordsforthe
USAandmaximumproposedlevelsofuseinavarietyoffoods,was1.7g/person
perday.Forconsumersatthe90thpercentileofintake,thepredicteddailyintake
ofa-cyclodextrinwas3g.
The intended use levels from the proposed new use of a-cyclodextrin as an
ingredientinanumberoffoodproductsrangefromamaximumof10g/kginnon-
alcoholicbeveragestoamaximumof100g/kginbakeryproducts.
Assuming that a-cyclodextrin would be added to all possible food categories
at the maximum proposed use levels, and using the GEMS/Food database,
Europeandietfoodconsumptionfgures,theCommitteecalculatedatotalintake
ofa-cyclodextrinof65g/personperday.Thisestimateisveryconservativesince
it is unlikely that a-cyclodextrin would be consumed simultaneously from all
sourcesonaregularbasis.
An intake assessment based on a national 1-day recall survey was provided
by Australia and New Zealand. It was assumed that a-cyclodextrin would be
a-CYCLODEXTRIN 1
K2
present at the highest proposed concentrations in all foods for which use
was intended. The average dietary intake from intended uses was estimated to
be 16g/person per day and the 95th percentile of intake was estimated to
reach37g.
In order to estimate the potential intake of a-cyclodextrin in a single eating
occasion, the Committee used the GEMS/Food large portion database, which
containsthehighestfguresfor97.5thpercentileconsumption(eatersonly)reported
fromnationalsurveys.Thehighestestimatedpotentialingestionofa-cyclodextrin
per eating occasion is between 19 and 38g for bread only, depending on the
proposeduselevel.
5. EVALUATION
At its present meeting, the Committee evaluated the safety of a-cyclodextrin
based on its known use as food additive and on its proposed use as food
ingredient.
A very conservative assessment of international exposure to a-cyclodextrin
suggestedthatintakescouldreach65g/personperday,whilemorerealisticesti-
matesatanationallevelsuggestedthatintakeswerelikelytobe3050%ofthis
value.a-Cyclodextrinhasbeentestedinvariousstudiesinanimals,andnotoxicity
wasobservedatthehighestdosestested,whichwere10100timeshigherthan
thedifferentestimatesofpotentialintakebyhumans.
Withrespecttothepreviouslyevaluateduseofa-cyclodextrinasafoodaddi-
tive and the present consideration of a-cyclodextrin as a food ingredient, the
Committee concluded that there were no safety concerns at the proposed use
levelsandresultingpredictedconsumption.
Thefactthattheingestionof20gofa-cyclodextrinonasingleeatingoccasion
maycausegastrointestinaleffectsinhumansshouldbetakenintoaccountwhen
consideringappropriatelevelsofuse.
ThepreviouslyestablishedADInotspecifedforthefoodadditiveusesofa-
cyclodextrinasacarrierandstabilizerforfavours,colours,andsweeteners,asa
water-solubilizerforfattyacidsandcertainvitamins,asafavourmodiferinsoya
milk,andasanabsorbentinconfectionerywasretained.
6. REFERENCES
Antenucci,R.N.&Palmer,J.K.(1984)Enzymaticdegradationof a-andb-cyclodextrinsby
Bacteroidesofthehumancolon.J.Agric.Fd.Chem.,2,13161321.
Br A. (2004) Reducing the glycemic impact of food. A new role for some dietary fbres.
Submittedforpublication
Br,F.&Griepentrog,F.(1967)Diesituationindergesundheitlichenbeurteilungderaroma-
tisierungsmittelfurlebensmittel.MedizinundErnahrung,244251.
Barilyak,I.R.,Korkach,V.I.&Spitkovskaya,L.D.(1991)Theembryotoxiceffectsofcertain
monoatomicalcohols.Ontogenez,22,7175.
1 a-CYCLODEXTRIN
K2
Carabin,I.G.&Flamm,W.G.(1999)Evaluationofsafetyofinulinandoligofructoseasdietary
fber.Reg.Toxicol.Pharmacol.,0,268282.
De Schrijver, R., Vanhoof, K. & VandeGinste, J. (1999) Nutrient utilization in rats and pigs
fedenzymeresistantstarch.Nutr.Res.,1,13491361.
Diamantis,I.&Br,A.(2002)Effectofa-cyclodextrinontheglycemicindex(GI)andinsu-
linemicindex(II)ofstarchinhealthyhumanvolunteers.Unpublishedstudyreport.
Flourie,B.,Molis,C.,Achour,L.,Dupas,H.,Hatat,C.&RambaudJ.-C.(1993)Fateofbeta-
cyclodextrininthehumanintestine.J.Nutr.,12,676680.
French,D.(1957)TheSchardingerdextrins.Adv.Carbohydr.Chem.,12,189260.
Gordon,D.T.,Stoops,D.&Ratliff,V.(1995)Dietaryfberandmineralnutrition.In:Kritchevsky,
D. & Bonfeld, C. eds, Dietary Fiber in Health and Disease, St Paul, Minnesota, USA:
EaganPress,pp.267293.
Gorman, M.A. & Bowman, C. (1993) Position of theAmerican DieteticAssociation: health
implicationsofdietaryfber.J.Am.DieteticAssoc.,,14461447.
Karrer, P. (1923) Polysaccharide. XX. Zur Kenntnis polymerer Kohlenhydrate. Helv. Chim.
Acta,,402409.
Kelsay, J.L. (1990) Effects of fber on vitamin bioavailability. In: Kritchevsky, D. et al., eds,
Dietary fber. chemistry, physiology, and health effects, New York: Plenum Press, pp.
129135.
Kondo,H.,Nakatani,H.&Hiromi,K.(1990)Invitroactionofhumanandporcinea-amylases
oncyclo-maltooligosaccharides.Carbohydr.Res.,20,207213.
Marshall, J.J. & Miwa, I. (1981) Kinetic difference between hydrolyses of g-cyclodextrin by
humansalivaryandpancreatica-amylases.Biochem.Biophys.Acta,1,142147.
McCleary, B.V. (2002) Measurement of cyclodextrins as dietary fbre (using the resistant
starchformat).UnpublishedreportofDecember18,2002.
Nelson,B.K.,Brithwell,W.S.,Khan,A.,Krieg,E.F.&Hoberman,A.M.(1990)Developmental
toxicologyassessmentof1-octanol,1-nonanol,and1-decanoladministeredbyinhalation
torats.J.Am.Coll.Toxicol.,,9397.
Pitha, J. (1981) Enhanced water solubility of vitaminsA, D, E, and K by substituted cyclo-
amyloses.LifeSci.,2,307311.
Prinsen,M.K.(1990)Acuteeyeirritation/corrosionstudywitha-cyclodextrininalbinorabbits.
UnpublishedreportNo.V90.439/200069fromTNO-CIVOInstitutes,Zeist,Netherlands.
Prinsen, M.K. (1991a) Acute dermal irritation/corrosion study with a-cyclodextrin in albino
rabbits.UnpublishedreportNo.V91.552/210061fromTNONutritionandFoodResearch
Institute,Zeist,Netherlands.
Prinsen, M.K. (1991b) Acute eye irritation/corrosion studies with a-cyclodextrin in albino
rabbitsandexvivotestingofa-cyclodextrinintheenucleated-eyetestwithchickeneyes.
UnpublishedreportNo.V91.553/210069fromTNONutritionandFoodResearchInstitute,
Zeist,Netherlands.
Prinsen, M.K. (1992) Sensitization study with a-cyclodextrin in guinea pigs (maximization
test). Unpublished report No. V92.574/352063 from TNO Nutrition and Food Research
Institute,Zeist,Netherlands.
Rossander, L., Sandberg, A.-S. & Sandstrom, B. (1992) The infuence of dietary fber on
mineralabsorptionandutilisation.In:Schweizer,T.F.&Edwards,Ch.A.,eds,Dietaryfbre
acomponentoffood,London:Springer-Verlag,pp.197216.
a-CYCLODEXTRIN 1
K2
Van Ommen, B. & de Bie, A.Th.H.J. (1995) Oral and intravenous disposition study with
[14C]-a-cyclodextrin in rats. Unpublished report No. V94.389 from TNO Nutrition and
FoodResearchInstitute,Zeist,Netherlands.
Von Hoesslin, H. & Pringsheim, H. (1923) Zur physiologie der polyamylosen. II.
Glykogenbildungundtierischeverbrennung.Hoppe-SeylersZeitschriftfrPhysiologische
Chemie,11,168176.
VonHoesslin,H.&Pringsheim,H.(1927)Uberdieernahrungvondiabetikernmitpolyamy-
losen.Munch.Med.Wschr.,9596.
K2
17
BENZOYL PEROXIDE
First draft prepared by
Professor R. Kroes
1
, Dr M. DiNovi
2
and Professor J.R. Bend
3
1
Institute for Risk Assessment Sciences, Utrecht University, Soest,
Netherlands;
2
Offce of Food Additive Safety, Center for Food Safety and Applied
Nutrition, Food and Drug Administration, College Park, MD, USA; and
3
Department of Pharmacology and Toxicology, Faculty of Medicine and
Dentistry, University of Western Ontario, London, Ontario, Canada
Explanation............................................................................... 17
Biologicaldata.......................................................................... 18
Biochemicalaspects.......................................................... 18
Absorption,distribution,metabolism,and
excretionofbenzoylperoxide............................... 18
Effectsonotherbiochemicalparameters.................... 19
Toxicologicalstudies.......................................................... 20
Acutetoxicity................................................................ 20
Short-termstudiesoftoxicity....................................... 21
Long-termstudiesofcarcinogenicity........................... 22
Reproductivetoxicity:developmentaltoxicity.............. 24
Genotoxicityandothercellulareffects........................ 25
Observationsinhumans.................................................... 25
Carcinogenicityinworkersexposedinindustry
orinpatientstreatedforacne............................... 25
Reproductivetoxicity.................................................... 27
Immuneresponse........................................................ 27
Technologicaldata.................................................................... 28
Secondaryeffectsoftreatmentoffoodwith
benzoylperoxide.......................................................... 28
Degradationproductsofbenzoylperoxide.................. 28
Destructionofessentialnutrients................................ 29
Productionoftoxiccompounds................................... 29
Intake........................................................................................ 29
Comments ............................................................................... 30
Evaluation ............................................................................... 31
References............................................................................... 31
1. EXPLANATION
Benzoylperoxideisusedasableachingagentinfour,inmilkforproduction
of cheeses and in whey from the manufacture of cheeses in which annatto and
carotenoidpigmentsarepresent.Atitspresentmeeting,theCommitteeevaluated
thesafetyofbenzoylperoxideatamaximumconcentrationof100mgperkgused
asableachingagentinwhey.
18 BENZOYL PEROXIDE
K2
Atitsseventhmeeting(Annex1,reference7),theCommitteeevaluatedbenzoyl
peroxideusedasableachingagentinfour,andconcludedthattreatmentoffour
withbenzoylperoxideatconcentrationsofupto40mgperkgoffourwasaccept-
able.Atthatmeeting,theCommitteenotedthatwhenbenzoylperoxideisusedas
ableachingagentinfour,itreactswithoxidizableconstituentsofthefourandis
almosttotallyconvertedtobenzoicacid;anyremainingtracesofbenzoylperoxide
are further reduced during the baking process and converted into benzoic acid.
Onthisbasis,theissuesrequiringconsiderationforuseofbenzoylperoxideasa
bleachingagentweredeterminedtobethepresenceofsmallamountsofbenzoic
acidinbreadandbakeryproducts,thepossibleeffectsofoxidativetreatmenton
thenutritionalvalueoffour,andthepossibleformationofharmfulsubstancesor
anti-metabolites.
Attheffty-ffthmeetingoftheCommittee(Annex1reference149),theevalu-
ation of the nutritional and toxicological implications of treatment of foods with
benzoyl peroxide, with respect to potential effects on proteins, vitamins, antioxi-
dants and physiologically important lipids, was postponed, owing to lack of
information.
Benzoylperoxideismanufacturedbythereactionofbenzoylchloride,sodium
hydroxide and hydrogen peroxide. During cheese-making or whey-drying, nearly
all(>91%)benzoylperoxideisconvertedtobenzoicacid.
Concerningresiduesofbenzoicacid,attheforty-frstmeetingoftheCommittee
(Annex1,reference107)agroupacceptabledietaryintake(ADI)of05mg/kgbw
for benzoic acid and its calcium, potassium and sodium salts, benzyl acetate,
benzylalcohol,benzaldehydeandbenzylbenzoatewasestablished,andthiswas
maintainedbytheCommitteeatitsforty-sixthmeeting(Annex1,reference122).
At its ffty-ffth meeting (Annex 1, reference 149), the Committee noted that the
intakeofbenzoicacidfromfoodstreatedwithbenzoylperoxideshouldbeconsid-
eredtogetherwithintakefromotherdietarysourcesofbenzoatesinthegroupADI
of05mg/kgbw.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution, metabolism, and excretion of
benzoyl peroxide
Whenbenzoylperoxideisusedasaprocessingaidinfoodpreparation,much
ofitisconvertedtobenzoicacidduringheattreatmentorstorage(Annex1,refer-
ence 7). Absorption of any residual benzoyl peroxide after oral ingestion may
occur;however,mostingestedbenzoylperoxidewillbeconvertedtobenzoicacid
(Nencki&Zaleski,1899).Anyremainingbenzoylperoxidethatisabsorbedislikely
tobesubjecttothefrst-passeffectintheliverandbemetabolized.
Changetal.(1977)demonstratedthat91.7%of
14
C-labelledbenzoylperoxide
wasconvertedto[
14
C]benzoicacidafterreactionwithwhey;inaddition,7%ofthe
radiolabel became tightly bound to nondialysable (6.84%) or dialysable (0.6%)
BENZOYL PEROXIDE 19
K2
wheycomponents,andthisboundradiolabelwasrecoveredas[
14
C]benzoicacid
after hydrolysis with hydrochloric acid (6mol/l) and extraction. Minor amounts of
hydroxybenzoicacids,phenolandphenylbenzoatewerealsoformeduponreac-
tion of [
14
C]benzoyl peroxide with whey; only a very small amount of unreacted
benzoyl peroxide remained.This investigation verifed that residual benzoic acid
inwheyaftertheuseofbenzoylperoxidetobleachthisfoodstuffisanissuetobe
considered.
Animals
Benzoylperoxidemaybemetabolizedbycleavageoftheperoxidebond,result-
ing in benzoyloxyl radicals (Swauger et al., 1991). These radicals can either
degrade to phenyl radicals and carbon dioxide, or remove hydrogen from mole-
culessuchasnicotinamideadeninedinucleotide,reduced(NADH)toformbenzoic
acid.Peroxidasescatalysetheinsertionoftwohydrogenions(donatedbyNADH)
between the two oxygen atoms of hydrogen peroxide to form two molecules of
water(Devlin,1993).Benzoylperoxideislikelytobeconvertedtobenzoicacidby
thesamemechanism;thus,benzoicacidisthemajorstablemetaboliteofbenzoyl
peroxide produced by keratinocytes (Nacht et al., 1981; Rothman & Pochi,
1988).
After metabolism of benzoyl peroxide, the major product, benzoic acid, is
excretedintheurine,eitherasbenzoate(Rothman&Pochi,1988)orasaconju-
gate with glycine (benzoyl glycine; hippuric acid) (Life Science Research Offce,
1980).
Humans
Inastudyusinghumankeratinocytesinvitro,alkylradicaladductswereformed
when benzoyl peroxide was incubated with 5,5-dimethyl-1-pyrroline-N-oxide
(Kensleretal.,1988),consistentwithformationofbenzoyloxylandphenylradicals
(Hazlewood & Davies, 1996). Production of free radicals occurs in freshly har-
vestedandculturedkeratinocytesatnontoxicconcentrationsofbenzoylperoxide
(Iannoneetal.,1993).
2.1.2 Effects on other biochemical parameters
Kaul&Khanduja(1999)foundthatbenzoylperoxidestimulatestheformation
of superoxide anion radicals in murine peritoneal macrophages in vitro. Benzoyl
peroxide also increased the accumulation of diacylglycerol in these cells, with a
concurrentreleaseofcholinemetabolites.Eluantsfromdentalresindiskscontain-
ing benzoyl peroxide were found to cause a decrease in the free fatty acid pool
andanincreaseindiacylglycerolformationinhamsteroralepithelialcellsinvitro
(Lefebvreetal.,1996).Thepresenceofbenzoylperoxideoritsmetabolicproducts
couldstimulatephospholipaseCand/orD,whichwouldresultinhigherlevelsof
diacylglycerols that could alter cellular growth. Diacylglycerols have been shown
toactivateproteinkinaseCandstimulatetheformationofsuperoxideanionradi-
calsbyinfammatorycells.Thus,withtheaccumulationofdiacylglycerol,thepro-
20 BENZOYL PEROXIDE
K2
ductionofsuperoxideanionradicalsbyinfammatorycellsisalsolikelytoincrease
(Kaul&Khanduja,1999).
Manystudieshavedemonstratedtheinvolvementoffreeradicalsandreactive
oxygenspecies,suchassuperoxideanionradicals,intheprocessoftumourpro-
motion (Zhang & Mock, 1992; Kaul & Khanduja, 1999). Infammatory cells, such
asneutrophilsandmacrophages,aremajorsourcesofsuperoxideanionradicals
during the promotional phase of skin carcinogenesis. In a study by Odukoya &
Shklar(1984),chronicinfammationwithinfltratesoflymphocytesandhistiocytes,
butnotumours,waspresentinthecontrolgroupofhamstersadministeredbenzoyl
peroxideonly,withoutacarcinogenicinitiator.InastudybydeReyetal.(1994),
treatment with benzoyl peroxide produced an increase in mast cells in the
dermis.
Evidence that benzoyl peroxide generates free radicals involved in tumour
promotion is supported by observations that exogenous antioxidants (e.g. butyl-
atedhydroxytoluene,disulframandascorbicacid)haveageneralinhibitoryeffect
on the incidence of skin tumours in studies of co-carcinogenesis. Free radical
scavengers,suchasglutathioneandn-acyldihydroxylaminesalsohaveaninhibi-
toryeffect(Slaga,1995).
Benzoylperoxidehasbeenshowntobindcovalentlytoprotein,butnottoDNA,
and benzoyloxyl radicals likely covalently bind to macromolecules in a similar
manner.BenzoicaciddoesnotbindtoeitherproteinorDNAundersimilarcondi-
tions (Swauger et al., 1990). Potassium ion-activated phosphatase and sodium
and potassium ion transporting ATPase (Na
+
- and K
+
-ATPase) are inhibited by
benzoylperoxideinrabbitdentalpulpinvitro(Abikoetal.,1978;Konoetal.,1981).
Haemolyticeffectshavealsobeenreported(Fujisawa,1978).
2.2 Toxicological studies
2.2.1 Acute toxicity
Mouse
Evidenceconcerningtheacutetoxicityofbenzoylperoxideinmicehasbeen
summarized by the Life Science Research Offce (1980). The acute oral toxicity
of benzoyl peroxide is low, with a median lethal dose (LD
50
) of 2127mg/kgbw
(Antonyuk,1969).Studiestodeterminetoxicityafterintraperitonealinjectionwere
notconsideredtoberelevanttothedeterminationofthesafetyofbenzoylperoxide
asafoodadditive(LifeScienceResearchOffce,1980).
Rats
Benzoyl peroxide also has low acute oral toxicity in rats, with reported LD
50

valuesintherangeof4006400mg/kgbw.Whenlethaldoseswereadministered,
animalsdisplayedcentralnervoussystemdepressionwithin3040minanddeath
occurredwithin24h(LifeScienceResearchOffce,1980).
BENZOYL PEROXIDE 21
K2
Rabbits
InDraizetestsforirritationconductedbyWazeter&Goldenthal(1973)inNew
Zealand white rabbits, benzoyl peroxide was not found to be a dermal irritant;
however, it did cause transient ocular irritation. Conjunctivitis and swelling were
noted,butnoulcerationsoropacities.Benzoylperoxidedustirritatedtheeyesof
albinorabbitswhennotwashedoutwithin5minafteradministration,whiledermal
applicationofa10%solutionofbenzoylperoxideinpropyleneglycolcausedslight
tomoderateerythemainguinea-pigs(IARC,1985).
Humans
Inhumans,benzoylperoxideisknowntoproducedermalirritationandsensi-
tizationreactions,particularlyaftermultipletopicalapplicationsforacnetreatment
(Zesch,1986;Kraus,1995;NationalToxicologyProgram,2002).
2.2.2 Short-term studies of toxicity
Mice
Ina4-monthstudyoftoxicity(Antonyuk,1969),miceweregivenbenzoylper-
oxideatadosecorrespondingto2,5or10%ofthecalculatedLD
50
(148mg/kg)
byintraperitonealadministration.Decreasedbody-weightgainswerenotedineach
group,butnodeathswereobserved.
Rats
Twoshort-termstudiesoftoxicitywithbenzoylperoxideadministeredbyintra-
peritonealinjectionwereconducted.
In a 4-month study, rats given benzoyl peroxide at a dose of 7.5,18.7 or
37.3mg/kgbwperdaysurvivedtheobservationperiod.Adecreaseinbody-weight
gainwasmeasured(Antonyuk,1969).
Mkhitaryan et al. (1974) demonstrated that concentrations of a-tocopherol in
thebraindecreasedinratsgivenbenzoylperoxideatadoseof48mg/kgbwper
day.
Dogs
Dogsfedfourtreatedwithbenzoylperoxidefor12weeksshowednoevidence
oftoxicity(Newelletal.,1947).Thefourwastreatedwith8gofbenzoylperoxide
per 45kg (corresponding to a dose of approximately 5mg/kgbw per day). After
treatment,thefourwassteamedbeforebeinggiventothedogs.Concentrations
of benzoyl peroxide were not determined after steaming. In a very similar study
(Radomskietal.,1948),threedogswerefedfourtreatedwithbenzoylperoxide
(one ounce of benzoyl peroxide per pound of four, i.e. 28g per 454g, equal to
aproximately15.6mg/kgbwperday)for6weeksapparentlydidnotshowsignsof
toxicity. Again, concentrations of benzoyl peroxide were not determined after
steaming.
22 BENZOYL PEROXIDE
K2
2.2.3 Long-term studies of carcinogenicity
Mice
The results of studies of carcinogenicity in mice fed with benzoyl peroxide in
mice, as cited by Kraus et al. (1995) and the InternationalAgency for Research
onCancer(IARC,1999),aresummarizedinTable1.Manyofthesestudieswere
previouslyreviewedbyIARC(IARC,1985,1999).
Benzoyl peroxide was not carcinogenic in one dietary study in which groups
of25maleand25femalemicereceivedbenzoylperoxideatadoseof28,280or
2800mg/kgofdiet(equaltoadoseof4.2,42and420mg/kgbw)for80weeks.In
the same study, subcutaneous injection of a single dose of benzoyl peroxide of
50mgpermouse(equaltoadoseofapproximately2500mg/kgbw)didnotlead
totumourformation.Inthesestudies,thefnalconcentrationofbenzoylperoxide
inthedietgiventotheanimalswasnotdetermined(Sharratetal.,1964).
The carcinogenicity of benzoyl peroxide administered by dermal application
hasbeenthoroughlyevaluatedinmice.Sixteenstudies(duration,2080weeks)
gavenegativeresults.Inalmostallthesestudies,benzoylperoxidewasadminis-
teredatdosesvaryingfrom20to40mg,appliedtopicallyonetothreetimesper
week, except in one study in which benzoyl peroxide was applied six times per
week(Sharratetal.,1964).
Twostudiesgavepositiveresults.Inthefrst,benzoylperoxidecausedasta-
tisticallysignifcantincreaseinskintumours(8outof20mice),ofwhich5outof
20weresquamouscellcarcinomas(Kurokawaetal.,1984).
Inthesecondstudy,whichusedatransgeniclineofmicewithgeneticallyiniti-
ated skin, the incidence of papillomas in heterozygous males was 0/5, 0/5, 3/5,
4/5at0,1,5or10mgofbenzoylperoxide,respectively.Ingroupsofthreehomo-
Table 1. Results of studies of carcinogenicity in mice fed with
benzoyl peroxide
Strain No.per Route; Dose Duration Result Reference
group exposure (weeks)
Albino 25M, Oral,dietary; 28,280, 80 Notumours Sharratetal.
25F adlib 2800mg/kg observed (1964)
ofdiet
Albino 25M, Subcutaneous; 50mg 80
a
Notumours Sharratetal.
25F singledose observed (1964)
Unknown 30 Subcutaneous; 0.53% >12 Notumours Oppenheimer
implantation observed etal.(1955)
F,female;M,male.
a
Animalswereapparentlygivenasingledoseandthenobservedforapproximately80
weeks.
BENZOYL PEROXIDE 23
K2
zygousmice,anincreasedincidenceofpapillomaovertimewasnoticedinfemales
receiving 5 or 10mg of benzoyl peroxide topically, twice per week. Since these
micewerepronetodevelopskintumours,thisstudysupportsthepromotingnature
ofbenzoylperoxide(Spaldingetal.,1993).
In light of the negative results reported by most of the available studies, the
positiveresultsobtainedinasinglestudyaresurprising,andprobablyshowthat
the mice used were extremely sensitive to skin irritation and the development of
skintumours.
In a summary of all studies in mice treated with benzoyl peroxide by topical
application to the skin (one to two times per week, at doses ranging from 10 to
40mg)afterinitiationwithcarcinogens,benzoylperoxidewasshowntobeapro-
moter of skin tumours in most cases, although different strains showed different
sensitivities(Krausetal.,1995).
Rats
The results of studies of carcinogenicity with benzoyl peroxide in rats have
beensummarizedbyKrausetal.(1995).
In albino rats given benzoyl peroxide at a dose of 28, 280 or 2800mg/kg of
diet (equal to approximately 1.9, 19 or 190mg/kgbw for males, and 2.3, 23 and
230mg/kgbwforfemales)for120weeks,nocarcinogeniceffectwasrevealed;the
incidence of malignant and/or benign tumours was not different between treated
groupsandcontrols.Therewasasignifcantincreaseintestisatrophyinthemales
atthehighestdose,whichaccordingtotheauthors,wasprobablyduetovitamin
Edefciency.Body-weightgainsinfemalesatthehighestdoseandinthemales
attheintermediatedoseweresignifcantlyreduced.Theauthorsspeculatedthat
theseweightdepressionsofabout10%werecausedbymarginalnutritionaldef-
ciencies,becauseanincreasedintakeoffoodreversedthephenomenon.Inthese
studies,theconcentrationofbenzoylperoxideinthefnaldietgiventotheanimals
wasnotdetermined(Sharratetal.,1964).
Benzoylperoxidewasnotcarcinogenicinthreestudiesinatleastthreediffer-
ent strains of rat treated by subcutaneous administration; however, the single
subcutaneous injection apparently administered in the study by Sharrat et al.
(1964)isnottypicalforalong-termstudyoftoxicityorcarcinogenicity.
Hamsters
Benzoylperoxidewasfoundnottobecarcinogenicinhamsterswhenapplied
dermallyatadoseof160mg,threetimesperweekfor16months.However,when
7,12-dimethylbenz[a]anthracene (DMBA) was administered as a single dose of
10mg/kgbw by gavage, followed by 80 or 160mg of benzoyl peroxide applied
topicallyonthedermis,anincreaseindermalmelanoticfoci,consideredtobea
precursorofmelanotictumours,andanincreaseinmelanotictumourswerefound
(Schweizer,1987).
24 BENZOYL PEROXIDE
K2
Eight male and eight female hamsters were painted in the cheek pouch with
an0.1%solutionofDMBAinmineraloilfor10weeks,threetimesperweek.After
a subsequent non-treatment period of 6 weeks, the animals were painted, three
timesperweek,witha40%solutionofbenzoylperoxideinacetone(approximately
20mgeachtime).Sixhamstersservedascontrolsandweretreatedwithbenzoyl
peroxideonly.Inbothgroupsinfammatorychangesandhyperkeratosisoccurred.
InthehamsterspretreatedwithDMBA,carcinomainsituandepidermoidcarcino-
mas were found in all animals at the application site, while no lesions were
observedinorgansexaminedhistopathologically(heart,lungs,liverandkidneys)
(Odukoya&Shklar,1984).
Twenty-two male hamsters were painted with a 0.5% solution of DMBA in
mineraloil,followedby27weeksofpaintingwitha40%solutionofbenzoylper-
oxideinacetone,threetimesperweek.Acontrolgroupofsixhamsterswastreated
only with benzoyl peroxide, using a similar dose and schedule. In the hamsters
pretreated with DMBA, severe dysplastic changes, carcinoma in situ and early
invasive squamous cell carcinomas were noticed in a nonspecifed number of
animals.Thecontrolanimalstreatedonlywithbenzoylperoxideshowedacantho-
sis,ulcerationandinfammationofthepaintedareas,withseveredysplasiainone
animal. Unexpectedly, benzoyl peroxide caused a reduction in DMBA-induced
g-glutamyltranspeptidase(GGT)fociintheliver(Zhang&Mock,1992).
Thus benzoyl peroxide acts as a promoter for oral, topical and dermal carci-
nogenesis in hamsters, and this is consistent with benzoyl peroxide acting as a
promoterinstudiesofcarcinogenicityinmouseskin(seeabove).
In a 120-week study of carcinogenicity, mice and rats given diets containing
benzoylperoxidedidnotshowanincreaseintheincidenceoftumours,although
a signifcant decrease in body weight (10%) was measured in females at the
highestdose(230mg/kgbwperday)andinmalesattheintermediatedose(19mg/
kgbwperday),butnotinmalesatthehighestdose(190mg/kgbwperday).On
thebasisofthesedataandtheconcentrationsofbenzoylperoxideaddedtothe
diet(upto2000mg/kgofdiet),theCommitteedecidedthatthetreatmentofwhey
withbenzoylperoxidewouldnothaveanadverseeffectonitsnutritionalvaluenor
resultintheformationofharmfulsubstancesoranti-metabolitesinthewhey.
Duringitsdeliberations,theCommitteealsoconsideredthepotentialadverse
effectsofoxidationproductsofbixinandnorbixin(carotenoidscontainedinannatto)
formed from benzoyl peroxide, but found no evidence that this was a safety
concern. In an evaluation by IARC, it was concluded that benzoyl peroxide was
not classifable as to its carcinogenicity to humans (IARC Group 3) (IARC,
1985).
2.2.4 Reproductive toxicity: developmental toxicity
Chickens
Benzoyl peroxide was dissolved in acetone at doses of 0, 0.05, 0.10, 0.21,
0.42,0.83,and1.7mmolandinjectedintotheairchamberof3-day-oldeggsfrom
white Leghorn chickens, 30 eggs per dose group. There was a dose-related
BENZOYL PEROXIDE 25
K2
increase in early embryonic deaths at all except the lowest dose. The dose
response curve was fat at the three higher doses, which indicates saturation of
penetration.Only1/80controlsweremalformed;however,therateofmalformation
was increased in all treatment groups and varied from 13 to 33%, without an
apparentdoseresponserelationship(Korhonenetal.,1984;IARC,1985).
2.2.5 Genotoxicity and other cellular effects
Theresultsofstudiesofgenotoxicitywithbenzoylperoxidearesummarizedin
Table2.
Ascanbeconcludedfromthedatainthistable,benzoylperoxideisnotmuta-
genic,itinhibitscellularcommunication,anditcancausesingle-strandbreaksin
DNA.
WhilebenzoicaciddidnotproduceDNAdamageinacell-freesystemutilizing
FX-174 plasmid DNA in the presence of copper, benzoyl peroxide did produce
DNA damage under these conditions. However, there was no apparent covalent
bindingofbenzoylperoxidetoDNA(Swaugeretal.,1991).
2.3 Observations in humans
2.3.1 Carcinogenicity in workers exposed in industry or in patients
treated for acne
Epidemiological and clinical studies were carried out to determine whether
exposuresofworkerstobenzoylperoxideduringindustrialuseorofacnepatients
treatedwithbenzoylperoxidewereassociatedwithcarcinogenicity.Thesestudies
have been reviewed by IARC (IARC, 1985, 1999) and by Kraus (1995). Topical
preparationsofbenzoylperoxidehavebeenusedinthetreatmentofacneformore
than30years,withnoreportsofadverseeffectsthatcouldberelatedtocarcino-
genicity.Adverse effects are usually limited to dermal irritation and sensitization
reactions(Krausetal.,1995).
A population-based casecontrol study of acne treatments as risk factors for
skin cancer of the head and neck was performed in Canada. Women and men
aged 1051 years or 1056 years, respectively, were asked to fll out question-
nairesrelatingtoalistofwidelyusedmedicationsforthetreatmentofacne.The
responserateforparticipationforthe964caseswas91%,andforthe3856con-
trols was 80%. Of the respondents, 92.3% had basal cell carcinoma, 4.8% had
squamouscellcarcinoma,and2.9%hadmelanoma.Benzoylperoxidehadappar-
ently been used in the treatment of acne for 9% of the cases and 10.1% of the
controls. The odds ratio for use of benzoyl peroxide was 0.8 (95% confdence
interval(CI),0.51.3)forallcasesofskincanceroftheheadandneckcombined;
therewasnoassociationwithuseofbenzoylperoxide(Hoganetal.,1991).
2.3.2 Reproductive toxicity
Althoughnostudiesinpregnantwomenhavebeenperformed,yearsofclinical
useofbenzoylperoxideinpreparationsusedforthetreatmentofacneappearto
26 BENZOYL PEROXIDE
K2
Table 2. Results of studies of genotoxicity with benzoyl peroxide
End-point Testsystem Concentration Results Reference
/dose
In vitro
Reverse S. typhimurium, NS Negative
b
LittonBionetics,
mutation TA1535, Inc.(1975)
TA1537,
TA1538
Reverse Saccharomyces NS Negative
b
LittonBionetics,
mutation cerevisiae, Inc.(1975)
strainD4
Reverse S. typhimurium, 2500mg/ml Negative
b
Ishidate(1980)
mutation TA100,TA1535,
TA1537,TA98,
TA92,TA94
Reverse S. typhimurium, 100mg/plate Negative
b
Dillonetal.
mutation TA100,TA102, (1998)
TA104,TA97a
Chromosomal Chinesehamster 200mg/ml Negative
c
Ishidate(1980)
aberration lungcells
Aneuploidy Chinesehamster 200mg/ml Negative
c
Ishidate(1980)
lungcells
DNAsingle- Humanbronchial 242mg/ml Positive
c
Saladinoetal.
strandbreaks epithelialcells (1985)
andDNA
proteincross-
links
Increasein Syrianhamster 242mg/ml Positive
c
Mikalsen&
intercellular embryocells Sanner
communication (1994)
Inhibitionofgap- Primarymouse 40mg/ml Positive
c
Jansenetal.
junctional keratinocytes (1996)
intercellular
communication
Inhibitionofgap- Initiatedprimary 10mg/ml Positive
c
Jansen&
junctional mouse Jongen
intercellular keratinocytes (1996)
communication
Inhibitionof Chinesehamster 0.11.5mg/ml Positive,dose- Slagaetal.
metabolic V79cells dependent
d
(1981)
cooperation
Sisterchromatid Chinesehamster NS Positive,dose- Jarventaus
exchange ovarycells dependent etal.(1984)
responsewith (abstract)
metabolic
activation;
negative
without
metabolic
activation
Inhibitionof Human 0.53.6mg/ml Positive,dose- Lawrenceetal.
metabolic keratinocytes dependent
d
(1984)
cooperation
BENZOYL PEROXIDE 27
K2
Table 2. (contd)
End-point Testsystem Concentration Results Reference
/dose
DNAdamage Cell-freesystem 1mmol/l Negative
d
Swaugeretal.
usingFX-174 (1991)
plasmidDNA 0.11mmol/l+ Positive
d
copper(Cu)
In vivo
Dominant Mice 62mg/kgbw
e
Negative
c
Epsteinetal.
lethalmutation (1972)
NS,notstated.
a
Lowesteffectivedoseorhighestineffectivedose.
b
Withorwithoutmetabolicactivation,sourcenotstated.
c
Withmetabolicactivation;nottestedwithoutmetabolicactivation(notapplicableinthe
caseofthetestfordominantlethalmutationinmiceinvivo).
d
Withoutmetabolicactivation;nottestedwithmetabolicactivation.
e
Singledose,administeredintraperitoneally.
indicate that benzoyl peroxide causes no detrimental reproductive effects in
humans.Sincebenzoylperoxideabsorbedaftertopicaladministrationismetabo-
lizedtobenzoicacidintheskinandsubsequentlyexcretedasbenzoicacidoras
aconjugateofglycine,adversesystemiceffectsareunlikelytooccur(Rothman&
Pochi,1988).
2.3.3 Immune response
Incidencesofallergenicresponseshavebeendocumentedinworkersexposed
tobenzoylperoxideusedasableachingagentinfour.Ayoungmalebakerworking
withfourtreatedwithbenzoylperoxidesufferedforayearwithasthmaticwheezing
and severe dermatitis of the face, neck, shoulders, and arms. When the baker
substituted unimproved wheat four for that treated with benzoyl peroxide, the
allergic reactions disappeared. Two years later, when he was again exposed to
fourtreatedwithbenzoylperoxide,thebakerpromptlydevelopeddermatitis(Baird,
1945).
Leyden & Kligman (1977) reported that benzoic acid was not sensitizing in a
seriesofpatientswhoweresensitivetobenzoylperoxide.Positivepatchtestswith
benzoylperoxidewerereportedin38outof400bakerstested(Grosfeld,1951).
In a study by Haustein el al. (1985), benzoyl peroxide was only a weak allergen
butastrongirritant;only11outof155patientsexhibitedintolerancetotheprepa-
rationandofthose,10wereabletocontinueuseofthepreparation.
Benzoylperoxideiswidelyusedasatopicalagent,particularlyinthetreatment
ofacne,butalsoforotherskindiseases,suchaschronicskinulcers,tineapedis,
and tinea versicolor (Hogan, 1991; IARC, 1999). Dermatologists have reported
reactionsamongpatientsreceivingvarioustopicalpreparationsofbenzoylperox-
ideforthetreatmentofacne;however,thereportedincidencesofcontactsensiti-
zationtobenzoylperoxidevariedwidelyamongthevariousinvestigators.
28 BENZOYL PEROXIDE
K2
The reported incidence of positive patch test reactions varied from 0 to 76%
(Hogan, 1991). Leyden & Kligman (1977) reported a high incidence of contact
sensitization with benzoyl peroxide.These investigators applied squares of cloth
saturatedwitheither5%or10%benzoylperoxidegelto25patientsforfveperiods
of48h.Thesensitizationratewas76%amongthesesubjects,regardlessofdose.
The highest incidence (76%) of an allergenic response was reported in patients
receivingbenzoylperoxideathighconcentrations,appliedunderocclusivepatches
totreatchroniclegulcers(Agathos&Bandmann,1984).However,theincidence
of positive patch tests does not appear to increase with the duration of use of
benzoyl peroxide, and most patients exhibiting a reaction were able to continue
usingpreparationscontainingbenzoylperoxide(Hogan,1991).
Inadouble-blindstudywith196patientswithacne,onegroupwastreatedwith
a placebo while three groups were treated with different lotions each containing
5.5%benzoylperoxide.Thelotionswereappliedonetofourtimesdailyfor4weeks
and left on the skin for at least 34h each time. None of the patients exhibited
dermalsensitization,norwereanysignifcantsystemiceffectsobservedduringthe
study(Ede,1973).
InastudybyPooleetal.(1970),40%ofadultvolunteersbecamesensitized
toanointmentcontaining1%sulfurand10%benzoylperoxide.Theinvestigators
appliedthepreparationninetimesfor24h,withinaperiodof3weeks.Theprepa-
rationwasreappliedaftera2-weekinterval.Asaresultofthischallenge,25out
of69subjectsexhibitedseveredermalsensitizationreactions.Twomonthsafter
thefrstapplications,tensubjectswhohadexhibitedonlymoderatereactivitywere
rechallengedwiththeointmentandallreactedseverely(Pooleetal.,1970).
In conclusion, most studies and clinical experience have demonstrated that
benzoyl peroxide is a sensitizer when used in the treatment of acne, and that
benzoylperoxidecanbeasevereirritant.
3. TECHNOLOGICAL DATA
3.1 Secondary effects of treatment of food with benzoyl peroxide
Inevaluatingthehealthaspectsofbenzoylperoxide,anysecondary,possibly
deleteriouseffectsthatmightresultfromitsuseinfoodsshouldalsobeconsidered.
Threepossibleeffectsofsuchactioninclude:theformationofharmfuldegradation
products; the destruction of essential nutrients; and the production of toxic sub-
stancesfromthefoodcomponents(LifeScienceResearchOffce,1980).
3.1.1 Degradation products of benzoyl peroxide
Asindicatedearlier,benzoylperoxideinfoodisrapidlyandalmostcompletely
converted to benzoic acid during processing. This results in an increase in the
benzoic acid content of the treated food that is roughly equal to two molecules
ofbenzoicacidpermoleculeofbenzoylperoxideemployed.Thedirectadditionof
benzoicacidandsodiumbenzoatetofoodisapproximatelytwotothreetimesthis
BENZOYL PEROXIDE 29
K2
amount(SubcommitteeonreviewoftheGRASList,1972).Furthermore,benzoic
acidisnaturallyfoundinseveralfoods,includingfruit,spices,milkproducts,meats,
and beverages (Van Straten, 1977). A daily intake of 46g of benzoic acid in
humans causes no toxic symptoms, apart from slight gastric irritation (Goodman
&Gilman,1975).Atitsffty-ffthmeeting(Annex1,reference149),theCommittee
reaffrmedthattherewassuffcientinformationonthetoxicityofbenzoicacidand
relatedcompoundstomaintaintheearlierestablishedADIof05mg/kgbwperday
ofbenzoicacidequivalents.
3.1.2 Destruction of essential nutrients
BenzoylperoxidereducesthevitaminAcontentofproductscontainingfat.As
whey is essentially fat-free, the treatment of whey is not affected by this
problem.
Nodataareavailableonthefateofotheressentialnutrientsinfoodsbleached
withbenzoylperoxide,althoughresultsobtainedinstudieswithhydrogenperoxide
mayberelevantinthisconnection.Treatmentofmilkfor24hat30C,orfor30min
at 51C with 0.3% hydrogen peroxide almost completely destroyed the small
amountsofascorbicacidanda-tocopherolpresent(Luck,1958a;1958b).These
treatments had no effect on thiamin, ribofavin, or pyridoxine. No reduction in
methionine content was noted when fsh protein concentrates were treated with
1.25%hydrogenperoxideat50Cfor20min,andonlyaslightreduction(8%)after
treatment with 5% hydroperoxide (Rasekh, 1972). It should be noted that these
latter concentrations of hydrogen peroxide are two orders of magnitude greater
thanthoseusedintheproposedbleachingofwheywithbenxoylperoxide.
3.1.3 Production of toxic compounds
It is possible that benzoyl peroxide might react with various constituents in
whey.Changetal.(1977)reportedthattherateofdecompositionofbenzoylper-
oxideinwheyfollowedfrst-orderkinetics,suchthattheratedependedonthesize
ofthebenzoylperoxideparticlesandtheagitationvelocity.Moreover,theyaffrmed
thatthepHofwheyhadlittleeffectonthedecompositionrateofbenzoylperoxide
and that benzoic acid was the major product. Minor amounts of hydroxybenzoic
acids,phenylbenzoate,phenol,andbenzoylperoxidewerealsofound.
4. INTAKE
Asnotedabove,mostofthebenzoylperoxideusedinthebleachingtreatment
of whey is converted to benzoic acid. Subsequent processing will further reduce
any traces of benzoyl peroxide that might remain in the whey that is used as a
food ingredient. If any benzoyl peroxide were ingested, it would be subjected to
further destruction in the gastrointestinal tract and by tissue peroxidases.There-
fore,themajorquestionrequiringconsiderationistheacceptabilityofsmallamounts
of benzoic acid being added to the diet by the consumption of food products to
whichbleachedwheyhasbeenadded.
30 BENZOYL PEROXIDE
K2
In the Food and Agricultural Organization of the United Nations (FAO) food
balance sheet for the year 2000, it was reported that 89 million tonnes of whey
are produced annually worldwide. Estimates based on the production fgures in
theFAOSTAT2000foodbalancesheettablessuggestthat<15%oftheworlds
wheyproductionwouldbesubjecttothisbleachingprocess.Theworldwidecon-
sumptionofwhey,bothbleachedandunbleached,was0.8kg/personperyearand
thehighestconsumptionwas15.4kg/personperyearintheUSA.Thelatterwould
resultinatotaldailyexposuretobenzoicacidof0.151540mgofbenzoicacid
peryear,oradailyexposureof0.01mg/kgbw(forapersonwithabodyweightof
60kg),assumingcompleteconversionofbenzoylperoxide.
5. COMMENTS
Almostallthebenzoylperoxideusedinfoodprocessingisconvertedtobenzoic
acid during heat treatment or storage. While traces of benzoyl peroxide may be
present in the processed food, most, if not all, of the benzoyl peroxide ingested
willbedegradedtobenzoicacidintheintestine.Itislikelythatanybenzoylper-
oxide absorbed will be metabolized to benzoic acid in the liver. Finally, benzoic
acidwillbeexcretedintheurine,eitherasbenzoateorasaconjugatewithglycine.
Onthisbasis,themajorissuestobeconsideredwhenbenzoylperoxideisused
asableachingagentinwheyarethepresenceofsmallamountsofbenzoicacid
residuesandthepotentialnutritionaleffectsonwhey.
Duringthemetabolismofbenzoylperoxide,superoxideanionradicalsmaybe
produced. The low concentration of radicals formed will not, however, saturate
superoxidedismutaseanddonotposeasafetyconcern.
Clinical studies have shown that benzoyl peroxide can be a severe dermal
irritant, and is a dermal sensitizing agent in humans. The short-term studies of
toxicity that are available are of limited quality. Benzoyl peroxide did not cause
signifcanttoxicityinratsormiceafterrepeatedintraperitonealinjection.Benzoyl
peroxide has been shown to cause single-strand breaks in DNA and to disrupt
intercellular communication in vitro. However, it was not mutagenic and did not
bindcovalentlytoDNA.Benzoylperoxidewasnotcarcinogenicaftersubcutaneous
or after dermal application. Benzoyl peroxide was shown to be a promoter in
assaysforinitiationpromotioninmicetreateddermally.
Inalong-termstudyofcarcinogenicity,theincidenceoftumoursdidnotincrease
inratsandmicereceivingdietscontainingbenzoylperoxide.Theseandadditional,
althoughlimited,dataindicatethatitisunlikelythattreatmentoffoodwithbenzoyl
peroxidewillhaveaneffectonthenutritionalvalueofwhey,orresultintheforma-
tionofharmfulsubstances.
Epidemiological and clinical studies did not fnd an association between the
incidence of skin cancer in industrial workers or acne patients and exposure to
benzoyl peroxide. Adverse effects were usually limited to dermal irritation and
sensitizationreactions.
BENZOYL PEROXIDE 31
K2
Intake
IntheFAOfoodbalancesheetfortheyear2000,itwasreportedthat89million
tonnesofwheyareannuallyproducedintheworld.Estimatesbasedonthepro-
ductionfguresintheFAOSTAT2000foodbalancesheettablessuggestthat<15%
of the worlds whey production would be subject to this bleaching process. The
worldwideconsumptionpercapitaofwhey(bothbleachedandunbleached)was
0.8kg per year, and the highest consumption per capita was 15.4kg per year in
the USA. This results in a total daily exposure to benzoic acid of 0.01mg/kgbw
(fora60kgperson),assumingcompleteconversionofbenzoylperoxide.
6. EVALUATION
TheCommitteeconsideredtheacceptabilityofsmallamountsofbenzoicacid
residuesaddedtothedietbytheconsumptionoffoodproductscontainingbleached
whey.
Assuming that 15% of cheese whey were bleached, the per capita intake of
benzoicacidwasestimatedtobe0.01mg/kgbwperday.TheCommitteeconcluded
that this was a minor contribution to the total dietary intake of benzoic acid, for
whichagroupADIwasestablishedattheforty-frstmeeting,andthattreatmentof
wheywithbenzoylperoxideatamaximumconcentrationof100mg/kgdidnotpose
asafetyconcern.
The Committee restated its conclusion from the ffty-frst meeting (Annex 1,
reference122)thatitwaspossiblethattheintakeofbenzoicacidfromalldietary
sources by some consumers could exceed the ADI, and concluded that more
preciseintakedatawererequiredtoestimatethenumberofsuchconsumersand
themagnitudeanddurationofintakesthataregreaterthantheADI.
7. REFERENCES
Abiko,Y.,Kono,Y.,Matsubayashi,H.,Honda,M.&Takiguchi,H.(1978)Effectofcomposite
resin materials on potassium-activated phosphatase activity in dental pulp. IRCS Med.
Sci.,6,169.
Agathos,M.&Bandmann,H.J.(1984)Benzoylperoxidecontactallergyinlegulcerpatients.
Contact Dermatitis,11,316317.
Antonyuk, O.K. (1969) Toxicity of benzoyl peroxide and triphenyl phosphate. Gig. Primen.
Polim. Mater. lzdelii Nikh1,311313.
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32 BENZOYL PEROXIDE
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K2
37
HEXOSE OXIDASE FROM chondruscrispus EXpRESSED In
hansenulapolymorpha
First draft prepared by
M.E. van Apeldoorn
1
, M.E.J. pronk
1
, Dr. C. Leclercq
2
, C. Le Donne
2
,
Dr. Z. Olempska-Beer
3
and Dr. D. Hattan
3
1
Centre for Substances and Integrated Risk Assessment, national Institute
for public Health and the Environment, Bilthoven, netherlands;
2
Research Group on Food Safety Exposure Analysis, national Research
Institute for Food and nutrition, Rome, Italy; and
3
Center for Food Safety and Applied nutrition, Food and Drug
Administration, College park, MD, USA
Explanation............................................................................... 37
Geneticmodifcation........................................................... 38
Productcharacterization.................................................... 38
Biologicaldata.......................................................................... 39
Biochemicalaspects.......................................................... 39
Toxicologicalstudies.......................................................... 39
Acutetoxicity................................................................ 39
Short-termstudiesoftoxicity....................................... 39
Long-termstudiesoftoxicityandcarcinogenicity....... 40
Genotoxicity................................................................. 41
Reproductivetoxicity.................................................... 42
Specialstudies............................................................. 42
Dermalirritation..................................................... 42
Ocularirritation...................................................... 42
ToxicologicaldataonLTABandCTAB................. 42
Observationsinhumans.................................................... 43
Intake ..................................................................................... 43
Comments ............................................................................... 45
Evaluation ............................................................................... 46
References............................................................................... 46
1. EXpLAnATIOn
Theenzymepreparationunderevaluationcontainstheactiveenzymehexose
oxidase (D-hexose: oxygen 1-oxidoreductase), which has not been evaluated
previously by the Committee. Hexose oxidase is an enzyme that catalyses the
oxidationofC6sugarstotheircorrespondinglactones,withtheconcomitantforma-
tionofhydrogenperoxide.HexoseoxidasehasthehighestaffnityforD-glucose
andD-galactose.
The hexose oxidase is produced from a nonpathogenic and nontoxigenic
genetically modifed strain of the yeast Hansenula polymorpha containing the
hexose oxidase gene derived from the red alga Chondrus crispus. The enzyme
activityisexpressedinhexoseoxidaseunits(HOXU).
38 HEXOSE OXIDASE FROM chondruscrispus
K2
Hexoseoxidasecanbeusedasaprocessingaidintheproductionofarange
offoodsatdosesof150200HOXU/kgoffood(typical)to5002500HOXU/kgof
food (maximum). The commercial preparation contains 0.2mg of total organic
solids(TOS)perHOXU.Thetechnologicalfunctionsofhexoseoxidasearedough
strengthening,curdformation,oxygenscavenging,anddecreasingtheformation
oftheproductsoftheMaillardreaction.
1.1 Genetic modifcation
ThegeneencodinghexoseoxidasewasderivedfromtheredalgaC. crispus,
whichisnotknowntobepathogenicortoxigenic.C. crispushasalonghistoryof
use in food in Asia and is one of the sources of carageenan, which has been
evaluated previously as a food additive by the Committee (Annex 1, references
32,137).Asyntheticgenewasconstructed,basedonthehexoseoxidasecDNA
from C. crispus, that was adapted for expression in yeast. The synthetic gene
encodes a hexose oxidase with the same amino acid sequence as that of the
native C. crispus enzyme. The synthetic gene was combined with regulatory
sequences, promoter and terminator, derived from H. polymorpha, and inserted
intothecommonly-usedplasmidpBR322.TheURA3genefromSaccharomyces
cerevisiaeandtheHARS1sequencefromH. polymorphawerealsoinsertedinto
theplasmid.TheURA3geneservesasaselectablemarkertoidentifycellscon-
taining the transformation vector. The native pBR322 plasmid contains genes
encoding proteins that confer resistance to ampicillin and tetracycline. These
geneswereremovedduringtheconstructionofthetransformationvector.
In order to develop the H. polymorpha production strain, the wild-type strain
ATCC34438wassubjectedtochemicalmutagenesis.Astrainrequiringuracilfor
growth(uracilauxotroph)wasusedasahoststrain.Thestrainwastransformed
withthehexoseoxidasetransformationvector.Thetransformedstrainwasfurther
improvedbymutagenesisusingultravioletlight(UVmutagenesis)andusedasthe
hexoseoxidaseproductionstrain.AlltheintroducedDNAiswell-characterizedand
would not result in the production of any toxic or undesirable substances. The
productionstrainisstablewithrespecttotheintroducedDNA.
1.2 product characterization
Hexose oxidase is produced by submerged fermentation of a pure culture of
theH. polymorphaproductionstrain.Theenzymeisproducedintracellularlyand,
upon cell disruption with lauryl trimethyl ammonium bromide (LTAB), is released
intothefermentationbrothandissubsequentlyseparatedfromtheyeastcellsand
subjectedtoultrafltrationanddiafltrationtoobtainconcentratedhexoseoxidase.
Itisthenspray-driedontoasuitablefood-gradecarrier,suchaswheatstarch,to
formmicrogranulesthatareoff-whitetobrownishincolour.SmallamountsofLTAB
maybepresentinthefnalproduct.Theenzymeistypicallydenaturedduringheat
treatment, and thus is no longer active in the fnal food product as eaten. The
enzyme preparation conforms to theGeneral specifcations for enzyme prepara-
tions in food processing(Annex1,reference156).
HEXOSE OXIDASE FROM chondruscrispus 39
K2
2. BIOLOGICAL DATA
2.1 Biochemical aspects
Noinformationwasavailable.
2.2 Toxicological studies
ToxicologicalstudieshavebeenperformedwiththeenzymepreparationsFerm
sample I, Ferm sample II, HOX-TOX-3-99, HOX-TOX-1 and HOX-TOX-4, all of
whichareyellow,water-solubleturbidliquidconcentratesproducedfromfermenta-
tion of the recombinant production organism. As hexose oxidase is produced
intracellularlyinthehost,cellsaremadepermeableafterfermentationtorelease
theenzyme.Intheenzymepreparationstestedthiswasdonebymechanicaldis-
ruption(FermsampleI),treatmentwithcetyltrimethylammoniumbromide(CTAB;
FermsampleII),treatmentwithLTAB(HOX-TOX-3-99andHOX-TOX-1),ortreat-
mentwithhexanol(HOX-TOX-4).ThepreferredtreatmentwaswithLTAB.Owing
tocarry-overofLTABintotheenzymepreparation,itispossiblethatsmallquanti-
ties of this quaternary ammonium compound might be present in the fnal food
product.
2.2.1 Acute toxicity
Studiesofacutetoxicityhavebeenperformedwithtwoenzymepreparations,
designated as Ferm sample I and Ferm sample II, with enzyme contents of 300
and 400HOXU/ml, respectively. The studies followed OECD test guideline 420
(fxeddosemethod,1992),andwerecertifedforcompliancewithgoodlaboratory
practice (GLP) and quality assurance (QA). The results are summarized in
Table1.
2.2.2 Short-term studies of toxicity
Rats
In a range-fnding study that was certifed for compliance with GLP and QA,
groups of fve male and fve female Sprague Dawley rats (aged 56 weeks)
received enzyme preparation HOX-TOX-1 (enzyme content, 500HOXU/g; TOS
content, 8.57%) at a dose equivalent to 0, 500, 1250, or 5000HOXU/kgbw per
Table 1. Acute toxicity of hexose oxidase
Enzymepreparation Species Sex Route LD
50
(mg/kgbw) References
FermsampleI Rat M,F Oral >2000 Kaaber(2000a)
FermsampleII Rat M,F Oral >2000 Kaaber(2000b);Cook
&Thygesen(2003)
F,female;M,male.
40 HEXOSE OXIDASE FROM chondruscrispus
K2
daybyoralgavagefor2weeks.Thevehiclewassterilewater.Allvisiblesignsof
ill health and behavioural changes were recorded daily. Body weight and food
consumption were recorded once per week. At termination of treatment, the
animalswereweighedandmacroscopicexaminationswereperformed.
One male rat receiving the intermediate dose and one female receiving the
highestdoseshowedhaemorrhagesinthethymus.Thesewereconsideredtobe
incidental fndings. No adverse effects were observed at up to and including the
highest dose of 5000HOXU/kgbw per day (Glerup, 2000; Cook & Thygesen,
2003).
Groups of ten male and ten female Sprague Dawley rats (aged 56 weeks)
were given enzyme preparation HOX-TOX-3-99 (enzyme content, 500HOXU/g;
TOS content, 9.55%
1
, LTAB content, 1130mg/g) at a dose equivalent to 0, 500,
1250, or 5000HOXU/kgbw by gavage (in sterile water), daily for 13 weeks.The
studywasperformedaccordingtoOECDtestguideline408(1998),andwascerti-
fedforcompliancewithGLPandQA.Allvisiblesignsofillhealthorbehavioural
changes were recorded daily, as were morbidity and mortality. Once per week,
bodyweightandfoodconsumptionwererecorded,anddetailedclinicalobserva-
tionswereperformedoutsidethecage.Allanimalswereexaminedbyophthalmos-
copyatthestartoftheexperiment,andanimalsinthegroupreceivingthehighest
dose and in the control group were re-examined before termination, as were
animals in the groups receiving the lowest and the intermediate dose when this
wasindicated.Inweek11or12,allanimalswereexaminedforsensoryreactivity
todifferenttypesofstimuli,gripstrength,andmotoractivity.Atterminationoftreat-
ment,bloodsampleswerecollectedfromallanimalsforhaematologyandclinical
chemistrydeterminations.Atnecropsy,amacroscopicexaminationwasperformed
on all animals, and absolute and relative (to body weight) weights of 11 organs
weredetermined.Microscopywascarriedoutonabout35organsandtissuesfrom
allanimalsinthecontrolgroupandatthehighestdose,onallorgansandtissues
fromanimalsdyingorsacrifcedduringthestudy,andonallgrosslesionsfromall
animals.
Afewincidencesofhaemorrhagesinthethymusandmandibularlymphnodes
wereattributedtobloodsamplingbeforenecropsyandwerenotconsideredtobe
relatedtotreatment.Noadverseeffectswerenotedonanyotherparameterexam-
ined. The no-observed-effect level (NOEL) was 5000HOXU (equivalent to an
intake of TOS of 955mg/kgbw per day), the highest dose tested (Glerup, 2001;
Cook&Thygesen,2003).
2.2.3 Long-term studies of toxicity and carcinogenicity
Noinformationwasavailable.
1
Theoretical value; since ash content was not known, it was assumed that all dry matter
wasorganicmaterial.
HEXOSE OXIDASE FROM chondruscrispus 41
K2
2.2.4 Genotoxicity
The results of two studies of genotoxicity with hexose oxidase in vitro are
summarizedinTable2.
In the frst study, which followed OECD test guideline 471 (1997) and was
certifed for compliance with GLP and QA, the enzyme preparation tested was
designated as HOX-TOX-399 (enzyme content, 500HOXU/g; TOS content,
9.55%;LTABcontent,1130mg/g).Inthesecondstudy,whichfollowedOECDtest
guideline473(1997),alsocertifedforcompliancewithGLPandQA,theenzyme
preparationtestedwasHOX-TOX-1(enzymecontent,500HOXU/g;TOScontent,
8.57%).
Table 2. Studies of genotoxicity with hexose oxidase in vitro
Enzyme End-point Testsystem Concentration Results References
preparation
HOX-TOX Reverse S. typhimurium 505000mg/plate, Negative
a
Edwards
399 mutation TA98,TA100, S9.Solvent: (2001a);
TA102, steriledistilled Cook&
TA1535, water. Thygesen
TA1537 (2003)
HOX-TOX Chromosomal Human Firstexperiment: Negative
b
Edwards
1 aberration lymphocytes 75,150,and (2001b);
300mg/ml,-S9; Cook&
150,300,and Thygesen
600mg/ml,+S9. (2003)
Second
experiment:
9.4,18.8,and
37.5mg/ml
-S9;150,300,
and600mg/ml
+S9.No
solventused
a
WithandwithoutmetabolicactivationfromS9(9000gsupernatantofratliver),using
thetreat-and-platemethod(toavoidanyproblemsthatmighthavebeencausedhad
thetestsubstancecontainedsignifcantconcentrationsofbioavailablehistidine).
Cytotoxicitywasobservedatthehighestortwohigherdoses.
b
WithandwithoutmetabolicactivationfromS9.Inthefrstexperiment,thecellcultures
weretreatedfor3hwithandwithoutS9andwereharvested17hlater.Reductionsin
meanmitoticindexwereobservedwithoutS9(to75,83and46%ofvaluesforthe
negativecontrolat75,150and300mg/ml,respectively)andwithS9(to81and47%of
valuesforthenegativecontrolat300and600mg/ml,respectively,butnotat150mg/ml).
Inthesecondexperiment,thecellswereexposedcontinuouslyfor20hwithoutS9and
thenharvested;withS9,cellsweretreatedfor3handharvested17hlater.WithoutS9,
reductionsinthemeanmitoticindexof61,58and26%thatforthenegativecontrol
wereobservedat9.4,18.8and37.5mg/ml,respectively.WithS9,themeanmitoticindex
was95,93and57%thatofthenegativecontrolat150,300and600mg/ml,respectively.
42 HEXOSE OXIDASE FROM chondruscrispus
K2
2.2.5 Reproductive toxicity
Noinformationwasavailable.
2.2.6 Special studies
(a) Dermal irritation
Astudyofprimarydermalirritationinrabbitswasperformedwiththeenzyme
preparation designated HOX-TOX-4 (enzyme content, 360HOXU/g; dry matter
content, 3.6%). The study followed OECD test guideline 404 (1992), and was
certifed for compliance with GLP and QA. An occluded application of 0.5ml of
HOX-TOX-4wasappliedtotwotestsitesonthecloselyclippeddorsalskinofthree
female New Zealand white rabbits for 4h. Two other clipped dorsal skin sites
remained untreated. After removal of the test substance, the treated skin was
washed with lukewarm water and mild soap, and skin reactions were assessed
1,24,48and72hlater.Noskinreactionswereobservedinanyoftheanimalsat
anytime-point(Bollen,2002a).
(b) Ocular irritation
A study of acute ocular irritation in rabbits was performed with HOX-TOX-4.
ThisstudyfollowedOECDtestguideline405(1987),andwascertifedforcompli-
ancewithGLPandQA.ThreefemaleNewZealandwhiterabbitsreceivedasingle
ocularinstillationof0.1mlofHOX-TOX-4inthelefteye.Therighteyeremained
untreated and served as a control. No ocular reactions were observed in any of
theanimalsat1,24,48and72hafterinstillation(Bollen,2002b).
(c) Toxicological data on LTAB and CTAB
A literature search did not reveal any toxicological data on LTAB (CETOX,
1999).TheonlydataavailableonLTABwereonLTABasaresidueintheenzyme
preparation HOX-TOX-3-99. As such, it was implicitly tested in an Ames test
(Edwards, 2001a) and in a 13-week study of toxicity (Glerup, 2001), and did not
causeadverseeffects.Moreinformationwasavailableonthecloselyrelatedqua-
ternaryammoniumcompoundCTAB.CTABispoorlyabsorbedfromthegastroin-
testinaltract.CTABdidnotshowmutagenicactivityintheAmestest,andthevery
similarcompoundcetyltrimethylammoniumchloride(CTAC)alsotestednegative
in the Ames test, as it did in assays for chromosomal aberrations in Chinese
hamsterV79cellsinvitro andcelltransformationinSyriangoldenhamsterembryo
cellsinvitro.InpregnantratsgivenCTABorallyatadoseof50mg/kgbwperday
(thehighestdose)ondays514ofgestation,reducedfetalsurvivalandincreased
incidenceofresorptionsiteswereobserved.TheNOELwas25mg/kgbwperday
(Anonymous,1997;CETOX,1999).
In a 1-year study of toxicity, rats given drinking-water containing CTAB at a
doseof45mg/kgbwperday(thehighestdose)onlyshowedareductioninbody
weight. The NOEL was 20mg/kgbw per day (Isomaa et al., 1976; Anonymous,
1997;CETOX,1999).
HEXOSE OXIDASE FROM chondruscrispus 43
K2
2.3 Observations in humans
Noinformationwasavailable.
3. InTAKE
Hexoseoxidasecanbeusedasanalternativetoglucoseoxidaseinthebaking
industrytostrengthendoughand,inasimilarway,inthepastaandnoodleindus-
triestoproduceafrmerstructure.Hexoseoxidasecanalsobeusedinfoodsfor
which the browning Maillard reactions that normally occur with heating are not
desirable, and in cheese and tofu manufacture to improve curd formation. It is
claimedthattheenzymecanalsofunctionasanoxygenscavengerinsaucesand
dressingstoimproveappearanceandshelf-life(Cook&Thygesen,2003).Hexose
oxidasemaythereforebeusedintheproductionofabroadrangeoffoods,includ-
ingmilkandmilkproducts(e.g.cheese,yoghurts,creams,wheyproteinconcen-
trate),cheeseandcheeseproducts(e.g.tofu),grainproducts(e.g.bread,pasta),
potatoes (e.g. fried potatoes), eggs (e.g. powder of egg white), condiments, and
salad dressings (e.g. mayonnaise, ketchup). The typical use levels of hexose
hoxidase range from a minimum of 150200HOXU/kg of food, to a maximum of
5002500HOXU/kgoffood,accordingtofoodapplications.
The enzyme is typically denatured during heat treatment of foods, such as
bakingorpasteurization(dataprovidedsuggestthatitisnotstableattemperatures
above30C)andisthereforenolongeractiveinthefnalproductaseaten.Itcan
thusberegardedasaprocessingaid.
Thedailyintakeresultingfromthecombinedconsumptionofseveralfoodsin
a worst-case situation was estimated. Based on the addition of high-level (90th
percentile)intakesforeachseparatefoodcategory,theestimatedcombinedintake
levelswere42and43HOXU/kgbwperdayonthebasisoffoodconsumptiondata
fromtheUSAandDenmark,respectively.Thisapproachassumesthathigh-level
consumersoflargequantitiesofonefoodarealsohigh-levelconsumersofallthe
others. It is, however, very unlikely that an individual has a high intake of many
food categories. In order to refne the intake estimates, an alternative approach
wasused,whichwasdevelopedwithinSCOOPTask4.2(EuropeanCommission,
1998). This approach is applicable when the number of food categories under
considerationislimited.Itassumesthatanindividualmighthaveahighconsump-
tionoffoodintwocategoriesandanaverageconsumptionoffoodsinothercatego-
ries.Averageandhighpotentialintakesofintakewerecalculatedusingdatafrom
the North/South Ireland Food Consumption Survey (Irish Universities Nutrition
Alliance, 2001) and the maximum recommended dosages per food category
(seeTable3).
The two highest (95th percentile) potential intakes estimated were
482.5HOXU/person from white breads and rolls and 380HOXU/person from
wholemeal and brown breads, corresponding to a total of 14.4HOXU/kgbw per
dayfora60kgperson([482.5+380]/60).Themeanpotentialintakefromtherest
ofthefoodcategorieswas437.5HOXU/person,equivalentto7.3HOXU/kgbwper
day for a 60kg person. The combined intake for all food categories is thus
44 HEXOSE OXIDASE FROM chondruscrispus
K2
Table 3. Average and high potential intake of hexose oxidase (in HOXU) per
food category based on data on food consumption from Ireland
Foodgroup Average 95th Maximum Average 95th
foodintake percentile dosage intakeof percentile
(kg/person) foodintake (HOXU/kg HOXU/ intakeof
(kg/person) offood) person HOXU/
person
Rice,pasta, 0.020 0.086 2500 50.0 215.0
fours,grains,
starches
Savouries(e.g. 0.024 0.094 2500 60.0 235.0
pizzas)
Whitebreads, 0.078 0.193 2500 195.0 482.5
a
rolls
Wholemeal, 0.045 0.152 2500 112.5 380.0
a

brownbreads,
rolls
Otherbreads 0.015 0.061 2500 37.5 152.5
(e.g.scones,
croissants)
Ready-to-eat 0.019 0.064 2500 47.5 160.0
breakfast
cereals
Biscuits 0.014 0.047 2500 35.0 117.5
Cakes,pastries 0.017 0.064 2500 42.5 160.0
andbuns
Othermilks(e.g. 0.005 0.010 2000 10.0 20.0
processed
milks)
Creams 0.002 0.009 2000 4.0 18.0
Cheeses 0.012 0.039 2000 24.0 78.0
Yoghurts 0.016 0.089 2000 32.0 178.0
Ice-creams 0.007 0.034 2000 14.0 68.0
Milkpuddings 0.006 0.036 2000 12.0 72
(e.g.rice
pudding,
custards)
Eggs,egg 0.017 0.054 500 8.5 27.0
dishes
Lowfatspreads 0.004 0.027 500 2.0 13.5
Otherspreading 0.012 0.004 500 6.0 20.0
fats
Chipped,fried 0.059 0.178 500 29.5 89.0
androasted
potatoes
Soups,sauces, 0.046 0.151 500 23.0 75.5
miscellaneous
foods
FromIrishUniversitiesNutritionAlliance(2001)
HOXU,hexoseoxidaseunits.
a
ThevaluesinboldcorrespondtothecategoryleadingtothehighestintakeofHOXUat
the95thpercentileoffoodintake.
HEXOSE OXIDASE FROM chondruscrispus 45
K2
estimated to be 14.4 + 7.3 = 22HOXU/kgbw per day, equal to an intake ofTOS
of4mg/kgbwperday.
LTAB is used as a processing aid during the production of the enzyme and
thus may be carried over in the enzyme preparation and be present in the fnal
foodproduct.LTABmaybepresentinthefnalenzymepreparationataconcentra-
tionof0.0050.05mg/g.Basedontheadditionofhighlevels(90thpercentiles)of
intake for each separate food category and taking the maximum recommended
enzyme dosage and maximum content of LTAB residue, the combined intake
ofLTABwasconservativelyestimatedtobe5.35mg/kgbwperday,onthebasisof
food consumption data from Denmark. On the basis of the SCOOP approach,
describedabove,theestimatedintakeofenzyme,22HOXU/kgbwperday,results
inanintakeofLTABof2.7mg/kgbwperday(221000/4000.05)foranenzyme
preparationwithaspecifcactivityof400HOXU/g.
4. COMMEnTS
Toxicological studies were performed with water-soluble turbid liquid enzyme
test concentrates, designated Ferm sample I, Ferm sample II, HOX-TOX-3-99,
HOX-TOX-1andHOX-TOX-4.Theseenzymepreparationswerenotacutelytoxic
whentestedinrats,norirritatingtotheskinoreyeofrabbits,normutagenicinan
assayformutationsinbacteriainvitronorclastogenicinanassayforchromosomal
aberrations in mammalian cells in vitro. In a 2-week range-fnding study in rats
treated with HOX-TOX-1 by gavage and in a 13-week study in rats treated by
gavagewithHOX-TOX-399(containingnotonlyhexoseoxidasebutalsoLTAB),
nosignifcanttreatment-relatedeffectswereseenatuptoandincludingthehighest
doseof5000HOXU/kgbwperday(equivalenttoanintakeofTOSof955mg/kgbw
per day). This highest dose, which also represents an exposure to LTAB at
11.3mg/kgbw per day, is therefore considered to be the NOEL. No toxicological
data on LTAB only were available. The closely-related quaternary ammonium
compoundCTABwasnotmutagenicinanassayformutationsinbacteriainvitro.
Ina1-yearstudyoftoxicitywithCTABinrats,theonlyeffectobservedwasreduced
body-weightgain;theNOELwas20mg/kgbwperday.
Neither H. polymorpha nor C. crispus have been associated with
allergenicity.
Aconservativeestimateoftheintakeofhexoseoxidasewhenusedatmaximum
dosageintheproductionofallpotentialfoodcategoriesis22HOXU/kgbwperday
(equivalent to an intake of TOS of 4mg/kgbw per day). When this intake is
compared with the NOEL of 5000HOXU (equivalent to an intake of TOS of
955mg/kgbwperday),thehighestdosetestedinthe13-weekstudyoforaltoxicity,
the margin of safety exceeds 200. The concomitant intake of LTAB present at
maximumconcentrationsofresidueinallpotentialfoodcategorieswasestimated
tobe2.7mg/kgbwperday.WhenthisintakeiscomparedwiththeNOELforLTAB
of11.3mg/kgbwperdayinthe13-weekstudyoforaltoxicityandwiththeNOEL
fortheclosely-relatedsubstanceCTABof20mg/kgbwperdayina1-yearstudy
oftoxicityinrats,themarginofsafetyisatleast4000.
46 HEXOSE OXIDASE FROM chondruscrispus
K2
5. EVALUATIOn
The Committee allocated an ADI not specifed to hexose oxidase from the
recombinantstrainofHansenula polymorphawhenusedintheapplicationsspeci-
fed and in accordance with good manufacturing practice. The Committee con-
cluded that the presence of LTAB at the concentrations observed in the enzyme
preparationwouldnotposeasafetyconcerntoconsumers.
6. REFEREnCES
Anonymous(1997)Finalreportonthesafetyassessmentofcetrimoniumchloride,cetrimo-
niumbromide,andsteartrimoniumchloride.Int. J. Toxicol.,16,195220.
Bollen,L.S.(2002a)HOX-TOX-4Primaryskinirritationintherabbit.Unpublishedreport
No.47939fromScantox,LilleSkensved,Denmark.SubmittedtoWHObyDaniscoUSA
Inc.,Ardsley,NY,USA.
Bollen,L.S.(2002b)HOX-TOX-4Acuteeyeirritation/corrosionstudyintherabbit.Unpub-
lishedreportNo.47940fromScantox,LilleSkensved,Denmark.SubmittedtoWHOby
DaniscoUSAInc.,Ardsley,NY,USA.
CETOX(1999)CTAB/LTABpreliminarysafetyevaluationforuseinenzymefoodapplica-
tion.Unpublishedreport03/12/99-MS/ing99-258fromCentreforIntegratedEnvironment
and Toxicology, Hrshold, Denmark. Submitted to WHO by Danisco USA Inc.,Ardsley,
NY,USA.
Cook, M.W. & Thygesen, H.V. (2003) Safety evaluation of a hexose oxidase expressed in
Hansula polymorpha.Fd Chem. Toxicol.,41,523529.
Edwards, C.N. (2001a) HOX-TOX-3-99 Ames test. Unpublished report No. 42119 from
Scantox, Lille Skensved, Denmark. Submitted to WHO by Danisco USA Inc., Ardsley,
NY,USA.
Edwards,C.N.(2001b)HexoseoxidaseIn vitromammalianchromosomeaberrationtest
performed with human lymphocytes. Unpublished report No. 39720 from Scantox, Lille
Skensved,Denmark.SubmittedtoWHObyDaniscoUSAInc.,Ardsley,NY,USA.
EuropeanCommission(1998)ScientifcCo-OperationonQuestionsRelatingtoFood.Devel-
opmentofmethodologiesforthemonitoringoffoodadditiveintakeacrosstheEuropean
Union.Task4.2fnalreport(SCOOP/INT/REPORT/2).Brussels.
Glerup, P. (2000) Hexose oxidase A two week dose-range fnding study in rats. Unpub-
lished report No. 39143 (including frst amendment ofAugust 2001) from Scantox, Lille
Skensved,Denmark.SubmittedtoWHObyDaniscoUSAInc.,Ardsley,NY,USA.
Glerup,P.(2001)Hexoseoxidasea13-weekoral(gavage)toxicitystudyinrats.Unpub-
lishedreportNo.40232fromScantox,LilleSkensved,Denmark.SubmittedtoWHOby
DaniscoUSAInc.,Ardsley,NY,USA.
Isomaa, B., Reuter, J. & Djupsund, B.M. (1976) The subacute and chronic toxicity of
cetyltrimethyl-ammoniumbromide(CTAB),acationicsurfactant,intherat.Arch. Toxicol.,
35,9196.
Irish Universities Nutrition Alliance (2001) North/South Ireland Food Consumption Survey.
Availablefromhttp://www.iuna.net/survey2000.htm
HEXOSE OXIDASE FROM chondruscrispus 47
K2
Kaaber,K.(2000a)FermsampleIacuteoraltoxicitystudyintherat.Unpublishedreport
No.36523fromScantox,LilleSkensved,Denmark.SubmittedtoWHObyDaniscoUSA
Inc.,Ardsley,NY,USA.
Kaaber,K.(2000b)FermsampleIIacuteoraltoxicitystudyintherat.Unpublishedreport
No.36524fromScantox,LilleSkensved,Denmark.SubmittedtoWHObyDaniscoUSA
Inc.,Ardsley,NY,USA.
K2
K2
49
LUTEIN FROM TAGETESERECTA L.
First draft prepared by
Professor M.C. Archer,
1
Professor H. Ishiwata,
2
and Professor R. Walker
3
1
Department of Nutritional Sciences, University of Toronto, Toronto,
Ontario, Canada
2
Seitoku University, Chiba, Japan
3
School of Biomedical and Molecular Sciences, University of Surrey,
Guildford, Surrey, England
Explanation............................................................................... 49
Biologicaldata.......................................................................... 50
Biochemicalaspects.......................................................... 50
Absorption,distribution,andexcretion........................ 50
Absorption............................................................. 50
Pharmacokineticstudies....................................... 53
Biotransformation......................................................... 58
Effectsonenzymesandotherbiochemical
parameters............................................................ 58
Toxicologicalstudies.......................................................... 60
Acutetoxicity................................................................ 60
Short-termtoxicity........................................................ 60
Long-termstudiesoftoxicityandcarcinogenicity....... 62
Genotoxicity................................................................. 63
Reproductivetoxicity.................................................... 66
Multigenerationstudies......................................... 66
Developmentaltoxicity.......................................... 66
Specialstudies............................................................. 67
Cardiovasculareffects........................................... 67
Immuneresponses................................................ 67
Oculartoxicity........................................................ 69
Dermalandocularirritation................................... 69
Observationsinhumans.................................................... 70
Clinicalstudies............................................................. 70
Epidemiologicalstudies............................................... 71
Intake ..................................................................................... 72
Concentrationsinfoods..................................................... 72
Dietaryintake..................................................................... 72
Comments ............................................................................... 73
Evaluation ............................................................................... 75
References............................................................................... 76
1. EXPLANATION
Lutein((all-E,3R,3R,6R)-b,e-carotene-3,3-diol),anaturallyoccurringxantho-
phyll pigment, is an oxygenated carotenoid that has no provitamin A activity. It
occurs with the isomeric xanthophyll zeaxanthin in many foods, particularly
50 LUTEIN FROMTAGETESERECTA L.
K2
vegetablesandfruit.Itisusedasafoodcolourandnutrientsupplementinawide
rangeofapplicationsatconcentrationsrangingfrom2to330mg/kg.
Xanthophylls obtained from Tagetes erecta L. (marigold) petals were consid-
ered by the Committee at its thirty-frst meeting (Annex 1, reference 77). At
that time, tentative specifcations were prepared, but no toxicological data were
available and no toxicological evaluation was made. Tagetes extract, containing
xanthophylls at low concentrations, was again considered by the Committee at
its ffty-ffth and ffthy-seventh meetings (Annex 1, references 149, 154) and
therevisedtentativespecifcations(Annex1,reference151)weresupersededby
full specifcations (Annex 1, reference 156).At the present meeting, information
was received on Tagetes preparations with a high lutein content (>80%), which
hadbeenusedinanumberoftoxicologicalstudies.Thesestudieswerereviewed
in the safety assessment and allocation of an acceptable daily intake (ADI) for
thisproduct.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution, and excretion
(a) Absorption
Xanthophyllsmaybeingestedineitherfreeoresterifedforms,althoughnon-
esterifed lutein is the subject of the present evaluation. Before absorption, the
estersarehydrolysedbypancreaticesterasesandlipasessuchthatonlythefree
formsarefoundinthecirculation(Wingerathetal.,1995).Oncereleasedfromthe
foodmatrixasalipidemulsion,likeothernon-polarlipids,thesecompoundsmust
be solubilized within micelles in the gastrointestinal tract to permit absorption by
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
HO
CH
3
CH
3
CH
3
OH
Figure 1. Chemical structure of lutein
Figure 2. Chemical structure of zeaxanthin
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
HO
CH
3
CH
3
OH CH
3
LUTEIN FROMTAGETESERECTA L. 51
K2
mucosalcells(Erdmanetal.,1993).Thetransferofcarotenoidsfromlipidemulsion
droplets to mixed micelles depends on their hydrophobicity, as well as pH and
concentration of bile acid (Tyssandier et al., 2001). Other carotenoids, such as
lycopene and xanthophylls, can impair the transfer of b-carotene, but neither b-
carotene nor other xanthophylls affect the transfer of lutein (Tyssandier et al.,
2001). The more polar carotenoids, such as the xanthophylls, are preferentially
solubilized on the surface of lipid emulsion droplets and micelles, while the less
polar carotenoids are incorporated into the core area (Borel et al., 1996). This
facilitatesthetransferofcompoundslikeluteinandzeaxanthinfromthelipiddrop-
letstotheaqueousphase.Indeed,ithasbeendemonstratedthatxanthophyllsare
morereadilyincorporatedintomicellesthanareothercarotenoids(Garrettetal.,
1999,Garrettetal.,2000).
The absorption of carotenoids, including lutein, is potentially affected by the
foodmatrixinwhichthecarotenoidsareconsumed,dietaryfat,andthepresence
ofothercarotenoidsinthediet(reviewedinCastenmiller&West,1998;Zaripheh
&Erdman,2002).
The absorption of carotene is higher when fat is present in the diet
(Roodenburg et al., 2000) and lower in disease-induced cases of malabsor-
ptionoffat(Erdmanetal.,1993).Thepresenceoffatinthesmallintestinestim-
ulatesthesecretionofbileacidsfromthegallbladderandimprovestheabsorption
of carotenoids by increasing the size and stability of micelles, thus allowing a
greater amount of carotenoids to be solubilized. Absorption of carotenoids by
mucosalcellsisbelievedtooccurbypassivediffusion(Hollander&Ruble,1978).
Afteruptakeintomucosalcells,carotenoidsareincorporatedintochylomicrons
and released into the lymphatics. When mucosal cells are sloughed off, carot-
enoidsthathavebeentakenupbythecellsbutnotyetincorporatedintochylomi-
cronsarelostintothelumenoftheintestine.Thecarotenoidswithinthechylomicrons
are transported to the liver where they are distributed between the lipoprotein
fractions. In contrast to the less polar carotenoids, a signifcant fraction of the
xanthophylls is carried in the blood stream by high-density lipoprotein (HDL)
(Romanchiketal.,1995).
The availability of lutein from a diet of mixed vegetables has been shown to
be67%relativetoathatfromadietsupplementedwithcrystallinelutein(vanhet
Hof et al., 1999a). In another study, the relative bioavailability of lutein and
b-carotene from various spinach products was compared with that from supple-
ments containing 6.6mg of lutein plus 9.8mg of b-carotene. The values ranged
from4554%forluteintoonly5.19.3%forb-carotene(Castenmilleretal.,1999).
Processing, such as mechanical homogenization or heat treatment, has been
showntoincreasetheavailabilityofb-caroteneinvegetablesby18to600%(van
het Hof et al., 2000). There is evidence, however, that disruption of the matrix
affects the bioavailability of carotenoids differentially, possibly because of
differencesintheirlipophiliccharacter.Forexample,theplasmaconcentrationof
luteinwasincreasedbyabout14%whenspinachwasconsumedchoppedrather
thanwhole,whilethatofb-carotenewasnotaffected(vanhetHofetal.,1999b).
The matrices of formulated natural or synthetic carotenoids (e.g. water-
dispersible beadlets, crystalline powders, oils suspensions etc.) and whether
52 LUTEIN FROMTAGETESERECTA L.
K2
the compounds are esterifed or non-esterifed may clearly affect availability
(Swansonetal.,1996;Boileauetal.,1999).
Thepresenceofdietaryfbremayexplain,atleastinpart,thelowavailability
of carotenoids from plant foods. It has been suggested that fbre interferes with
micelle formation by partitioning bile salts and fat in the gel phase of the fbre.
Riedl et al. (1999) tested the effects of pectin, guar, alginate, cellulose or wheat
branontheavailabilityofluteininsixhealthyfemalevolunteers.Allthefbressig-
nifcantlyreducedtheplasmaconcentrationsoflutein,witharangeof40to74%.
Anotherstudy,however,indicatedthatpectinhadnoeffectonserumconcentra-
tions of lutein after administration of a diet supplemented with liquefed spinach
(Castenmilleretal.,1999).
Because the absorption of carotenoids occurs via incorporation into mixed
micelles,ingestionoffataffectstheiravailability.Theamountofdietaryfatrequired
to ensure absorption of carotenoids seems to be low (35g/meal), although it
depends on the physico-chemical characteristics of the carotenoids ingested. In
one experiment, the plasma concentration of lutein, added as esters, was about
100%higherwhenluteinwasconsumedwith35goffatthanwith3goffat(van
hetHofetal.,2000).Thelowamountoffatmayhavelimitedthesolubilizationof
luteinestersand/orthereleaseofesterasesandlipases(Roodenburgetal.,2000).
Bioavailabilityofcarotenoidsisalsoaffectedbytheabsorbabilityofthedietaryfat
(Boreletal.,1998).Sterolandstanolestersapparentlyhavenoeffectonabsorp-
tionoflutein(Raeini-Sarjazetal.,2002).Eggyolkisasourceofhighlybioavailable
zeaxanthinandlutein.Thelipidmatrixoftheeggyolk,containingcholesterol,tria-
cylglycerols and phospholipids, provides a vehicle for the effcient absorption of
xanthophylls(Handelmanetal.,1999).
Interactions between carotenoids may decrease absorption. Competition for
absorptionmayoccuratthelevelofmicellarincorporation,intestinaluptake,lym-
phatictransportoratmorethanonelevel.Alternatively,simultaneousingestionof
variouscarotenoidsmayinduceanantioxidant-sparingeffectintheintestinaltract,
resulting in increased levels of uptake of the protected carotenoids. It has been
demonstratedthatinthepresenceoflargeamountsofb-carotene,chylomicrons
preferentiallytakeupxanthophyllsratherthanb-carotenefromtheintestinallumen
(Grtneretal.,1996).Aninhibitoryeffectofdietaryluteinontheabsorptionofb-
carotene has been observed when the carotenoids were measured in plasma
lipoproteins (van den Berg, 1998; van den Berg & van Vliet, 1998). In another
study,healthyvolunteersweregivensingleoraldosesof15mgofluteinderived
from marigold extract either alone or together with 15mg of b-carotene derived
from palm oil. The inclusion of b-carotene reduced the area under the curve of
concentrationtime(AUC)forluteinto5461%ofthatforluteinadministeredalone
(Kostic et al., 1995). In the same study, while lutein appeared to slow the initial
absorptionofb-carotene,luteindidnothaveanysignifcanteffectontheplasma
concentrationofb-caroteneatthemainpeakorontheAUCvalueforb-carotene.
Indeed, lutein enhanced the AUC value for b-carotene in subjects whose AUC
value for b-carotene only was the lowest. In a similar study to investigate the
interactionsbetweenb-caroteneanddietarylutein,healthymalesubjectsoncon-
trolleddietsweregivencapsulescontainingpurifedb-caroteneatahighdailydose
LUTEIN FROMTAGETESERECTA L. 53
K2
(12 or 30mg/day, corresponding to 0.2 or 0.5mg/kgbw per day) for 6 weeks.
Plasmaconcentrationsofluteininthegroupreceivingb-caroteneweredecreased
compared with baseline and were signifcantly lower than the levels reported in
controlgroupsgivenaplacebo(Micozzietal.,1992).Anotherstudyshowedthat
the post-prandial appearance of vegetable-borne carotenoids in chylomicrons is
competitive, but that this did not affect the plasma concentrations of the carot-
enoids after 3 weeks of feeding (Tyssandier, et al., 2002). Van den Berg (1999)
has concluded that, in general, long-term supplementation with b-carotene has
limited or no effect on plasma or serum concentrations of other carotenoids.
However, in the a-Tocopherol and b-Carotene Cancer Prevention Study Grou
(ATBC Study), a total of 29133 male Finnish smokers aged 5069 years were
given daily supplements of 20mg of b-carotene (0.3mg/kgbw per day) for an
average of 6.7 years. Signifcantly decreased serum concentrations of lutein (no
changes in concentrations of zeaxanthin) were observed in comparison with
groups that did not receive supplements containing b-carotene (Albanes et al.,
1997).
Incontrasttotheinteractionsobservedbetweenluteinandb-caroteneduring
absorption,supplementationwithlycopene(5mg/dayfromwholetomatoes,tomato
juice,orgelcapsulescontainingtomatooleoresin)reportedlyhadnoeffectonthe
plasmaconcentrationsofluteinorzeaxanthinina6-weekinterventionstudyin22
healthyfemalevolunteers(Bhm&Bitsch,1999).
Inadditiontothefactorsalreadydescribed,theisomericform(cis versus trans)
of the carotenoids may affect their absorption. Lutein and zeaxanthin occur in
naturepredominantlyintheall-transconfguration.Smallamountsofcisisomers
of each carotenoid, however, have been isolated from human serum (Krinsky et
al.,1990;Khachiketal.,1999).Itisnotknownwhetherthepresenceofcisisomers
in human serum is exclusively due to their selective uptake and absorption from
thediet,orwhethertheyaretheproductofinvivoisomerizationofall-translutein/
zeaxanthin in the presence of gastric acids. Snodderly et al. (1990) investigated
themajorcarotenoidpigmentsintheplasmaandinacommon,non-purifeddiet
formacaquesandsquirrelmonkeys.Inthediet,bothluteinandzeaxanthinwere
abundantintheall-trans,the9-cis,andthe13-cisisomers.Intheplasma,however,
the9-cisisomerwasrarelydetectable,whilethe13-cisisomerwasfoundinhigher
proportions than in the diet. These results suggest that either the isomers are
absorbedselectively,orthatisomerizationprocessesoccurintheanimalgut.
A number of non-dietary factors also affect the availability of carotenoids,
including exposure to tobacco smoke, alcohol consumption, intestinal parasites,
malabsorption diseases, liver and kidney diseases, hormone status, poor intake
of iron, zinc and protein, gastric pH and hyperthyroidism (Albanes et al., 1997;
Williamsetal.,1998;Patrick,2000;Alberg,2002).
(b) Pharmacokinetic studies
Pharmacokineticstudieswithluteinhavebeenperformedinmice,rats,cows
andhumans.
54 LUTEIN FROMTAGETESERECTA L.
K2
Mice
Inastudydesignedtoinvestigatetheuptakeofluteinandzeaxanthin,groups
of36BALB/cmicereceiveddietscontaininganextractofmarigoldpetalsforup
to 28 days (Park et al., 1998a). Based on data on food intake and body weight,
intakesofluteinforeachgroupcorrespondedtoapproximately0,75,150,300,or
600mg/kgbwperday,whileintakesofzeaxanthinwereapproximately0,1.0,2.0,
4.0,or8.0mg/kgbwperday,respectively.Sixmicepergroupwerekilledondays
0, 3, 7, 14, 21 or 28. Body, liver and spleen weights did not differ between the
treatedgroupthroughouttheexperiment.Plasmauptakeofluteinandzeaxanthin
(analysedtogetherinallcases)wasrapidandreachedamaximum(about3mmol/l)
byday3ofdosing(thefrsttime-pointexaminedafterthestartofdosing)anddid
notdifferbetweengroupsthereafter.Untilday3therewasalsoarapidincrease
inconcentrationsofluteinandzeaxanthinintheliverandspleen,withcontinued,
although small, increases to day 28. The liver was considered to be the major
storageorganforluteinandzeaxanthin.
Rats
The absorption, distribution and excretion and plasma kinetics of [
14
C]lutein
given as a single oral dose at 2mg/kgbw were investigated in groups of three
female RoRo SPF rats per time-point (Wendt et al., 2000). The synthetic radio-
labelledcompoundwasdilutedwithnonradiolabelledluteinpurifedfrommarigold
petals, and was formulated as a beadlet containing an emulsion of gelatin and
vegetable oil. Lutein was rapidly absorbed from the intestinal tract, resulting in
peak plasma concentrations within 4h after dosing.About 80% of the dose was
recoveredfromthefaecesand11%fromtheurinewithin96hafterdosing.Ofthe
radiolabelexcreted,80%wasrecoveredwithin24h.Lowtissueconcentrationsof
radiolabelindicatedthatluteinand/oritsmetabolitesdidnotaccumulate.Withthe
exception of the intestinal tract, kidneys and liver, radiolabel was present in all
tissuesatalltimesat<0.01%oftheadministereddose.Residualradioactivityin
thecarcassanddissectedtissueswasnegligible(0.23%).Fromthedataonexcre-
tion,absorptionwasestimatedtohavebeen11.3%.
Thepharmacokineticsandtissuedistributionof[
14
C]luteinwereinvestigatedin
groups of fve male and fve female Wistar rats, following a single dose of 2 or
20mg/kgbwadministeredbygavage(Froescheisetal.,2001).Beforeadministra-
tion of [
14
C]lutein, the rats had been maintained on a diet containing 2 or 20mg/
kgbwperdaynonradiolabelledluteinfor2weekstoestablishsteady-statecondi-
tions.Thenonradiolabelledluteinwasadministeredinthedietasabeadletformu-
lation.Theabsorptionofthe[
14
C]luteinwasrapid,withpeakplasmaconcentrations
reached within 3 to 4 h at either dose. The pharmacokinetics of lutein were not
linear with a tenfold increase in dose resulting in an increase of approximately
twofold in the maximum plasma concentration of radioactivity. Elimination from
plasmawasnotcompleteby48h.At4hafterdosing,themajorityoftissueshad
beenexposedtolowlevelsofradiolabelledlutein,maximumtissueconcentrations
havingbeenreachedatthistime-point.Highestconcentrationswerefoundinthe
liverandgastrointestinalmucosa.Concentrationsofluteinat96hafterdosingwere
LUTEIN FROMTAGETESERECTA L. 55
K2
belowthelimitofdetectioninalltissuesexaminedexcepttheliver.Therewasno
evidence for accumulation of lutein in any tissue. Most radiolabelled lutein was
eliminatedinthefaeces(>90%and>65%oftheadministereddoseformalesand
females, respectively, within 48h of dosing), with urinary and biliary excretion
accounting for <6% and <2% of the administered dose. There was negligible
(<0.1%) recovery of radiolabel from expired air. Excretion was slightly more
prolonged in females than males. Increasing the dose had minimal effects on
the absorption or the rates and routes of excretion. There were no signifcant
differencesbetweenmalesandfemales.
Plasmaandliverconcentrationsofluteinwereassessedaspartofa4-week
study of toxicity, which complied with good laboratory practice (GLP). Groups of
sixmaleandsixfemaleWistarratsreceivedcrystallineluteinformulatedasbead-
lets(actualdosesachievedwere85115%oftargetdoses)atdietarydosesof0,
2, 6, 20, 60, 200, or 600mg/kgbw per day (Buser et al., 1999; Simpson, 1999).
The lutein was extracted from marigold petals. There was a dose-dependent,
almostlinearincreaseinplasmaconcentrationsoflutein.Atenfoldincreaseindose
between 20 and 200mg/kgbw resulted in a two- to threefold increase in plasma
concentrations. Plasma steady state conditions were reached by day 3 (plasma
concentrationswerebelowthelimitofdetectionatthelowestdose,andtherewere
insuffcientdataatthenexthigherdoseof6mg/kgbwperday).Livertissuedeter-
minations revealed a dose-dependent increase in concentrations of lutein, and
suggested that saturation was reached at 200mg/kgbw per day. There were no
relevant sex differences in plasma concentrations, but the liver content of lutein
was1.53-foldhigherinfemalesthaninmalesinthethreegroupsreceivingthe
highestdose.
Theabsorptionofluteinwasinvestigatedingroupsof15maleweanlingFischer
344ratsmaintainedondietssupplementedwithluteinfor8weeks(Jenkinsetal.,
2000).Lutein(extractedfrommarigolds)wasformulatedwith2.2%vitaminE(a-
and g-tocopherols) in beadlets delivering lutein at a dose of 0, 2.1 4.3, 8.6, 17.8
or 34.7mg/day. The apparent absorption of lutein was estimated to range from
28.7%athighestdoseto43.1%atthelowestdose,basedonintakeandthefaecal
excretionoflutein.Thelimitedabsorptionathigherintakeswasreportedlydue,in
part, to factors such as solubility (i.e. limited capacity for micellar incorporation).
Thereweresignifcantlyincreasedplasmaconcentrationsofluteininanimalsfed
the two higher doses, and increases in liver and spleen concentrations of lutein
withincreasingdietaryintake.Therelativedistributionofluteinbetweentheliver
andspleenwasapproximately95and5%,respectively,withnoluteindetectedin
theheart,lung,kidney,testes,orbrain,theonlyotherorgansexamined.
Cows
Sixcalveswerefedmilkreplacerfor1week,andthenweregivenasingleoral
doseof20mg(about0.4mg/kgbw)ofcrystallineluteinfrommarigoldpetals(con-
taining small amounts of zeaxanthin) in olive oil (Bierer et al., 1995).The calves
showedincreasedplasmaconcentrationsofluteinthatpeakedat12hafterdosing,
declinedrapidlythereafter,andlevelledoutatapproximately72h.
56 LUTEIN FROMTAGETESERECTA L.
K2
Humans
Concentrationsofluteinandzeaxanthininserumandtissueshavebeenshown
to be quite variable (Boileau et al., 1999), but to increase, as expected, with
increasedintakeeitherfromdietarysourcesorfromsupplements(e.g.Hammond
etal.,1997;Landrumetal.,1997a,1997b;Carrolletal.,1999;Mlleretal.,1999;
Tucker et al., 1999; Berendschot et al., 2000; Johnson et al., 2000; Curran-
Celantano et al., 2001; Olmedilla et al., 2001; Schalch et al., 2001; Bone et al.,
2003).Inapopulation-basedstudy,Bradyetal.(1996)reportedthatlowerserum
concentrations of lutein and zeaxanthin are generally associated with males,
smoking,youngerage,lowerHDLcholesterol,greaterconsumptionofethanoland
higher body mass index. Carotenoids are present in variable amounts in many
tissues such as kidneys, buccal mucosal cells and adrenal glands, but the main
sitesofstorageareadiposetissueandliver(Parker,1996).Asinserum,b-carotene,
luteinandlycopenearethemaincarotenoidsfoundintissues,althoughb-crypto-
xanthin and zeaxanthin are also present in signifcant amounts (Boileau et al.,
1999).Theeyeingeneral,andtheretinainparticular,containextraordinarilyhigh
concentrationsofzeaxanthinandlutein(Boneetal.,1993).Othercarotenoidsare
presentinonlytraceamountsintheretinaandlens(Khachiketal.,1997a,1997b,
Yeumetal.,1999,Bernsteinetal.,2001).Zeaxanthinandluteinarethepigments
responsibleforthecolourationofthemaculalutea(yellowspot)(Landrum&Bone,
2001).Inhumans,theadministrationofluteinorluteinestersextractedfrommari-
goldpetalsatdosesof0.2to0.5mg/kgbwhasbeenshowntoresultinaccumulation
ofluteininthemacula,asevidencedbyanincreaseinthemacularpigmentdensity
(Landrumetal.,1997a,1997b;Berendschotetal.,2000;Duncanetal.,2002).
Plasmaconcentrationsofluteinandzeaxanthinweremeasuredinasmallpilot
studyingroupsofeightvolunteers(fourmenandfourwomen)afterdailysupple-
mentationwithcapsulescontainingeither4.1mgofcrystallinelutein(with0.34mg
ofzeaxanthin)or20.5mgoflutein(with1.7mgofzeaxanthin)for42days(Cohn
etal.,2001).Subjectsweremonitoredforafurther25daysafterthedosingphase.
Steady-stateconcentrationsofxanthophyllswerereachedbetweendays38to43
(0.06mmol/land0.13mmol/lforthelowestandhighestdoses,respectively).Dose-
normalized incremental maximum and average steady-state concentrations of
lutein and zeaxanthin were found to be comparable, indicating that they have
similarbioavailability.Theeliminationhalf-lifewascalculatedtobeapproximately
57daysforeithercompound.
In studies of the absorption of lutein administered as a supplement in single
doses,anumberofinvestigatorshavenotedconsiderableinterindividualvariability
intheeffciencyofabsorption,onthebasisofplasmaconcentrationsofluteinfol-
lowingsupplementation(Kosticetal.,1995;Burri&Neidlinger,2000;Torbergsen
&Collins,2000).
When absorption of lutein was measured in the triacylglycerol-rich fraction of
thebloodofthreemenandthreewomenfedwithastandardmealafteranover-
nightfastandgivenluteinatadoseof31.2mg(about0.4mg/kgbw,basedonthe
mean body weight of 75.4kg in these individuals), peak concentrations of lutein
wereobserved2hafterdosing(ONeill&Thurnham,1998).Thispeakconcentra-
tionoccurredearlierthanforb-caroteneorlycopene.
LUTEIN FROMTAGETESERECTA L. 57
K2
Serum levels of lutein were measured in eight adult subjects (males and
females)givensingledosesof0.5mmol/kgbw(about0.3mg/kgbw,orabout17mg
per60kgadult)ofcrystallineluteinfrommarigoldextractincornoil(Kosticetal.,
1995).Ameanpeakserumconcentrationofabout0.7mmol/lwasreachedat16h
afterdosing,followedbyamoderatedeclinetoabout50%ofthepeakinthesub-
sequent 120h, then a slow decline to baseline levels at 440h. b-Carotene was
showntoreducetherateofabsorptionoflutein.Similarly,plasmaconcentrations
of radiolabelled lutein from an algal source measured in four women given 3mg
of[
13
C]lutein(0.05mg/kgbwfora60kgadult)showedameanpeakconcentration
ofabout0.007mmol/lthatwasreachedbetween11and16hafterdosing,followed
byamoderatedeclinetoabout50%ofthepeakconcentrationinthesubsequent
100h, then a slow decline to baseline levels within about 500h (Yao et al.,
2000).
In three studies, repeated dosing of lutein from marigold petals formulated
eitherincapsulesormixedinoilresultedindose-dependentincreasesinplasma
concentrations of lutein that were observed at least 7 days after dosing at 10 to
20mg/day.Inoneofthesestudies,dosesof4or20mgwereadministeredto16
healthyvolunteersfor42days(basedonmeanbodyweightsforeachgroup,the
doses were about 0.1 and about 0.3mg/kgbw per day, respectively) (Schalch et
al.,2001).Plasmaconcentrationsofluteinwereobservedtobeaboutthreeand
abouteighttimesgreaterthanthoseinuntreatedcontrols.Plasmaconcentrations
werenearlyatbaselinelevels25daysaftertheendofdosingforthegroupreceiv-
ing the lower dose, but were still noticeably greater than baseline in the group
receivingthehigherdose.Atthelowerdose,subjectscontinuedtoshowincreasing
plasmaconcentrationsofluteinuptoday42,butmanysubjectsatthehigherdose
showedpeakconcentrationsatearliertime-points.Similarly,threemalesreceiving
dosesof10mglutein/dayforatotalof18days(about0.2mg/kgbwperdaybased
on a 60kg adult) showed four- to fvefold increases in plasma concentrations of
luteincomparedwithbaselinebyday7ofdosing(Khachiketal.,1995a).Inanother
study,fvemaleandthreefemalesubjectsreceived15mgoflutein/dayfor7days
(about 0.25mg/kgbw per day, based on a 60kg adult) (Torbergsen & Collins,
2000).Theluteinwasincapsuleformandcontained80%all-trans-luteinand20%
cis-luteins.Byday7,plasmaconcentrationsofluteinhadincreasedtwo-tothree-
fold compared with baseline levels, decreasing to near-baseline levels after 3
weeksofwash-out.
Thekineticsofcarotenoiddepletionandeliminationhavebeeninvestigatedin
19healthywomen(Burrietal.,2001)and12healthymen(Rocketal.,1992)fed
controlled low-carotenoid diets for approximately 10 and 13 weeks, respectively.
Infemales,thedeclineinserumconcentrationsofallcarotenoids(includinglutein
andzeaxanthin)followedapparentfrst-orderkinetics,andapproachedasteady-
statelevelafterabout30days,indicatinganeliminationhalf-lifeofabout6days.
Theauthorsanalysedthesedataandderivedhalf-livesforluteinandzeaxanthin
of76and38days,respectively(Burrietal.,2001).Inmales,thereweresignifcant
decreasesinconcentrationsofallcarotenoids,includingluteinandzeaxanthin,up
todays1415,followedbyaslowerdeclinetodays6364thatmaybeindicative
oftwopools,withonepoolhavingamorerapidrateofturnover.Concentrations
ofluteinandzeaxanthininthefnalsampleofplasma(days6364)averaged40%
58 LUTEIN FROMTAGETESERECTA L.
K2
oftheinitialconcentration,andthemeanplasmadepletionhalf-lifeforluteinand
zeaxanthin (combined) was estimated by the authors to be between 33 and
61days.
In a study of 41 patients with biliary and pancreatic diseases and 14 healthy
controls,therewasanon-statisticallysignifcanttrendtowarddecreasedconcen-
trations of lutein in plasma and bile in diseased patients compared with controls
(Leoetal.,1995).Itisclear,however,thatcarotenoids,includinglutein/zeaxanthin,
undergoappreciablebiliarysecretion.Inaddition,itwasnotedthatbiliaryconcen-
trationsofcarotenoidsrefectplasmaconcentrationsinbothnormalandpathologi-
calconditions.Interferencewithbiliarysecretiondidnotresultinplasmaretention
ofcarotenoids.
2.1.2 Biotransformation
Anumberofcompoundsderivedfromluteinandzeaxanthinhavebeenidenti-
fed in human serum (Figure 3) (reviewed by Khachik et al., 1995a). These are
called metabolites, but they are undoubtedly formed by chemical rather than
enzymicreactions.Themetabolitesresultprincipallyfromthreetypesofreactions
involvingtheendgroupsofthesecarotenoidsoxidation,reductionanddouble-
bondmigration(Figure3).Luteinandzeaxanthincanexistinanequilibriuminvolv-
ingtheintermediatecarotenoid3-epilutein.AllylicoxidationofluteinatC3results
in the formation of oxolutein B that can exist in equilibrium with lutein and 3epi-
lutein through reduction reactions. 3-Epilutein and zeaxanthin can also exist in
equilibrium through reversible double-bond migration. Thus, presence of 3-
epilutein in human serum may be due to conversion of lutein and/or zeaxanthin.
Acid-catalyseddehydrationisanotherreactionofcarotenoidswith3-hydroxy-eend
groups. Lutein is believed to undergo dehydration in stomach acid to form 3-
hydroxy-3,4-didehydro-b,g-carotene and 3-hydroxy-2,3-didehydro- b,e-carotene
(anhydroluteins)thathavebeenisolatedfromserum.Inadditiontotheirpresence
inhumanserum,thesemetaboliteshavealsobeendetectedinbreastmilkaswell
as retinal extracts (Khachik et al., 1995b; Khachik et al., 1997a, 1997b, 1997c).
Thetoxicologicalimportanceofthesecompoundsisnotknown.
2.1.3 Effects on enzymes and other biochemical parameters
The xanthophylls are precursors of retinol. Indeed, they have been shown to
havelittleornoactivityassubstratesofb-carotene-15,15-dioxygenase,although
theyareabletoinhibittheconversionofb-carotenetoretinol(Ershovetal.,1993;
van Vliet et al., 1996; Grolier et al., 1997). However, in a rat model, Weiser &
Korman(1993)showedthatthexanthophyllshavesmallbutsignifcantprovitamin
A activity (45% of the activity of b-carotene), probably via a vitamin A-sparing
effect.Furthermore,Weiser&Kormanreportedthatdietaryzeaxanthinisableto
induceduodenal15,15-dioxygenaseactivityin1-day-oldchicks.
Theeffectsoflutein(oleoresinextractedfrommarigoldpetals)oncytochrome
P450andglutathione-S-transferaseenzymeactivityintheliver,lung,kidney,and
smallintestineweremeasuredinmaleWistarratsafter16daysofdietarysupple-
mentation(Jewell&OBrien,1999).Groupsofeightanimalsreceived(i)abasal
LUTEIN FROMTAGETESERECTA L. 59
K2
dietthathadnotbeensupplementedwithlutein(negativecontrols);or(ii)abasal
dietsupplementedwithluteintoprovideadoseofapproximately45mg/kgbwper
day;or(iii)abasaldietsupplementedwith3-methylcholanthrene(3-MC)(positive
controls). There were no changes in feed intake, body-weight gains, or organ
weightsafterdietarysupplementationwithlutein.Asexpected,luteincontentwas
increased in the tissues examined. However, cytochrome CYP450 activities
ethoxyresorufn-O-deethylation (EROD), methoxyresorufn-O-demethylation
(MROD), benzyloxyresorufn-O-dearylation (BROD) and pentoxyresorufn-O-
depentylation (PROD) were not induced in the liver, kidneys, or lungs after
supplementationwithlutein,andBRODactivitywassignifcantlydecreasedinthe
lung.CYP450enzymeactivitieswereundetectableinthesmallintestine.Supple-
mentation with lutein had no effect on glutathione-S-transferase activity or gluta-
thionelevelsinanyofthetissuesexamined.
Inanotherstudy,theeffectsofdietaryexposuretolutein(extractedfrommari-
goldpetals)onxenobioticmetabolizingenzymesintheliverwereinvestigatedin
maleSPFWistarrats(Gradeletetal.,1996).Twogroupsofsixanimalsreceived
diets containing corn oil (control) or 10% lutein oleoresin (mixed with corn oil) at
300mg/kgofdietfor15days.Basedonreferencebodyweightanddataonfeed
intake (Blackburn, 1988), this corresponded to a dose of lutein of approximately
HO
(P)
OH
(3R,3'R,6'R)-Lutein
Natural Lutein
HO
(P)
O
(3R,6'R)-3-Hydroxy-
b,e-caroten-3'-one
Oxolutein B
OH
HO
(P)
(3R,3'S,6'R)-Lutein
3'-Epilutein
HO
(P)
OH
(3R,3'R)-Zeaxanthin
HO
(P)
OH
(3R,3'S,meso)-Zeaxanthin
P=
OH
HO
(P)
(3S,6S,3'S,6'S)-e,e-carotene-
3,3'-diol (Lactucaxanthin)
O
(P)
O
(6S,6'S)-e,e-carotene-3,3'-dione
O
HO
(P)
(6S,3'S,6'S)-3'-Hydroxy-
e,e-carotene-3-one
HO
(P)
OH
(3R,6S,3'S,6'S)-e,e-carotene-
3,3'-diol
Double Bond
Migration
Reduction Oxidation
Reduction
Oxidation
Reduction
Oxidation
Oxidation
Reduction
Oxidation
Reduction
Double Bond
Migration
Double Bond
Migration
Double Bond
Migration
Figure 3. Proposed reactions of lutein and zeaxanthin in humans
AdaptedfromKhachiketal.(1997a)
60 LUTEIN FROMTAGETESERECTA L.
K2
2.8mg/kgbw per day. Total microsomal P450 and associated cytochrome c
reductase activities, as well as EROD, MROD, PROD, BROD, erythromycin N-
demethylase (ERDM) and N-nitrosodimethylamine N-demethylase (NDMAD), p-
nitrophenol-and4-hydroxybiphenylUDPglucuronosyltransferases(4NP-UGTand
4-HBT-UGT),andtotalcytosolicglutathione-S-transferaseactivityweremeasured.
Therewerenochangesinfeedintake,bodyweights,ororganweightsthroughout
the15-dayfeedingperiod.Luteinwaspresentinlivermicrosomes(approximately
0.3nmol/mg of protein), but did not induce any signifcant changes in any of the
phaseIorphaseIIenzymes.
In humans, plasma concentrations of lutein have been negatively associated
withtheactivityofCYP1A2(caffeinetest),aliverenzymeinvolvedinthemetabolic
activationofanumberofhumancarcinogens(LeMarchandetal.,1997).
2.2 Toxicological studies
2.2.1 Acute toxicity
Inastudyofacutetoxicity,whichcompliedwithGLP,groupsofthreefemale
orthreemaleHanIbm:WISTratsweregivenlutein(purifedplantextractcontaining
7085%lutein)atadoseof2000mg/kgbwbyoralgavageinapolyethyleneglycol
(PEG300)vehicle.Nodeathsoccurredduringthestudyandnoothertreatment-
relatedeffectswereobserved.Onthebasisofthelackoflethalityobservedinthis
study,themedianlethaldose(LD
50
)fororally-administeredluteininratswasesti-
matedtobe>2000mg/kgbw(Pfannkuchetal.,1999).
2.2.2 Short-term toxicity
Mice
Inastudydesignedtoinvestigatetheabsorptionofdietarylutein,groupsof36
BALB/cmiceweregivendietscontainingluteinestersfrommarigoldextract(37%
lutein esters and 0.5% zeaxanthin esters) for up to 28 days. Based on reported
foodintakeandbodyweightdata,dailyintakesofluteincorrespondedtoapproxi-
mately0,75,150,300,or600mg/kgbwrespectively,and,takingintoaccountthe
composition of the marigold extract, daily intakes of zeaxanthin corresponded to
approximately 0, 1.0, 2.0, 4.0, or 8.0mg/kgbw, respectively. Six mice per group
werekilledondays0,3,7,14,21and28.Nodifferencesinbody,liver,orspleen
weightsamongtreatmentgroupswerereported,andtherewerenosignifcantdif-
ferencesinfeedintakethroughouttheexperimentalperiod(Parketal.,1998a).
Rats
Inastudyoforaltoxicity,whichcompliedwithGLP,groupsofsixmaleandsix
femaleWistarratsweregivendietscontainingluteinfrommarigoldpetalsattarget
doses of 0, 2, 6, 20, 60, 200, or 600mg/kgbw per day in a beadlet formulation
containinggelatinandvegetableoilfor28days.Actualdosesbasedonfoodcon-
sumptionwere85115%ofthetargetdoses.Furthermore,onthebasisofaccom-
panyinganalysesofplasmaconcentrationsatweeks2and4anddeterminations
LUTEIN FROMTAGETESERECTA L. 61
K2
ofluteincontentintheliverattermination,whichshoweddose-dependentincreases
inconcentrationsoflutein,itwasconsideredthatratshadbeenadequatelyexposed
to lutein in this study (Buser et al., 1999). No exposure-related mortality was
reported.Therewerenoeffectsofdietaryluteinonbodyweights,feedconsump-
tion,haematology,clinicalchemistryparameters,organweights(selectedorgans),
orgrosspathologyatnecropsy.Histopathologicalfndingswerelimitedtohistiocyte
fociinthemesentericlymphnodesofsomeanimals(particularlyfemales)atthe
highest dose.The foci were characterized as discrete subcapsular aggregations
ofmacrophageswithabundantcytoplasmcontainingvaryinglevelsofredgranular
material.Thesefociwereconsideredtoberelatedtophysiologicaluptakeoflutein,
and were not considered by the investigators to be refective of specifc target
organ toxicity. The mesenteric lymph nodes from animals in the other treated
groupswerecomparabletothoseofanimalsinthecontrolgroup.Theno-observed-
effect level (NOEL) in this 28-day study in rats was 600mg/kgbw per day, the
highestdosetested(Simpson,1999).
TheoraltoxicityofluteinwasevaluatedinWistarratsina13-weekstudythat
complied with GLP. Rats received target doses of lutein (from marigold petals
containing about 79 and 5% lutein and zeaxanthin, respectively) of 0, 2, 20, or
200mg/kgbw per day formulated as beadlets incorporated into the diet. Actual
doseswere99101%oftargetdosesforplaceboandtreatedanimals.Background
concentrationsofluteinandzeaxanthininthedietwere0.51.4and0.20.4mg/kg,
respectively. There were 10 animals of each sex per group at the lowest and
intermediate doses, and 15 animals of each sex per group for the control group
andatthehighestdose,owingtotheinclusionof5animalsofeachsexpergroup
for a 4-week recovery phase. There were reported to be no treatment-related
deaths, clinical signs, or adverse effects. There were no signifcant changes in
body-weightgain,feedintake,orhaematologyorclinicalchemistryparameters.A
batteryoftestsforneurotoxicityandophthalmoscopicobservationsdidnotindicate
any changes attributable to treatment. Mean absolute organ weights were not
differentbetweentreatmentandcontrolgroupsineitherthemainstudyorinthe
animalsintherecoveryphase.Therewerenotreatment-related,biologicallysig-
nifcantmacroscopicormicroscopicabnormalitiesreportedinanyoftheanimals,
exceptforoccasionalincidencesofvacuolationintheliverandtubulardegenera-
tion/regenerationinthekidneyofsomecontrolandtreatedfemales.Theincidence
and severity of these lesions in treated females was not dose-related, and they
wereconsideredtobeassociatedwithbloodsampling.Analysesofbloodsamples
showedthepresenceoflutein(andzeaxanthin)atdose-relatedconcentrationsin
theplasmaandliver,anditwasconsideredthatanimalswereadequatelyexposed
tothexanthophyllsundertheconditionsofthestudy.TheNOELinthis13-week
studyinratswas200mg/kgbwperday(208mgoflutein+zeaxanthin/kgbwper
day), the highest dose tested (Pfannkuch et al., 2000a; Pfannkuch et al., 2001;
Krugeretal.,2002).
Monkeys
InastudythatcompliedwithGLPandthatwasdesignedprimarilytoinvesti-
gate the ocular effects of long-term exposure to lutein, cynomologus monkeys
62 LUTEIN FROMTAGETESERECTA L.
K2
aged47yearsweregivenlutein(extractedfrommarigoldpetals)atdosesof0,
0.2, or 20mg/kgbw per day by gavage for 52 weeks. The solutions for gavage
were prepared by dispersing beadlets in water. The control was prepared using
beadletsthatcontainednolutein.Thereweretwomalesandtwofemalesineach
group, with an additional male and female included in the group receiving the
highest dose, which were designated for examination at 6 months. All animals
survived the treatment period. All animals at 20mg/kg per day showed orange/
yellowdiscolourationofthefaecesfromday2ofthestudyonwards(attributedto
treatmentwithlutein).Therewasnoeffectonoverallmeanbody-weightgainand
onoverallgroupmeanfeedintakeineitherofthetreatmentgroups.Therewere
no treatment-related changes in haematology, blood chemistry, or urine analysis
measurements. There were no changes in data on electrocardiogram waveform
orbloodpressurethatcouldberegardedasbeingrelatedtotheadministrationof
lutein.Therewerenotreatment-relatedorganweightchanges.Mostoftheanimals
showeddarkyellow-colouredmesentericfatatinterimsacrifceandgoldenyellow
mesentericfatatterminalsacrifce(attributedtotreatmentwithlutein).Histopatho-
logicalexaminationsrevealednotreatment-relatedfndings.Comprehensiveoph-
thalmicexaminationsshowednoevidenceofadversechangesrelatedtotreatment.
Animals showed a dose-related increase in plasma and liver concentrations of
lutein.Luteinwasthusconsideredtobewelltolerated.TheNOELinthis52-week
studyincynomologusmonkeyswas20mg/kgbwperday,thehighestdosetested
(Pfannkuchetal.,2000b,2000c;Pfannkuch,2001).
2.2.3 Long-term studies of toxicity and carcinogenicity
No experimental lifetime bioassays or studies of carcinogenicity have been
conductedwithlutein.However,luteinhasbeenreportedtohavechemopreventive
effects in a number of models of tumours in mice and rats (reviewed by Nishino
et al., 2000).The dosing period in all these studies was of shorter duration than
thatinthe13-weekstudyoforaltoxicityinrats,describedabove.
The chemopreventive effects of lutein have been examined in models of
tumours in mice and rats given lutein in the diet or by gavage, respectively. In
B6C3F
1
mice initiated with the colon carcinogen 1,2-dimethylhydrazine and then
givendietscontaining0.05%lutein(purity,95%)for8weeks,thenumberofputa-
tive preneoplastic aberrant crypt foci was signifcantly decreased compared with
micenotgivenlutein(Kimetal.,1998).
Inanotherstudyonformationofaberrantcryptfoci,Sprague-Dawleyratswere
giventhreeintrarectaldosesofmethylnitrosoureainweek1andlutein(unspecifed
source)atadailydoseof6,1.2,or0.24mgbygavageduringweeks2and5.The
formation of aberrant crypt foci was signifcantly reduced at all doses (Narisawa
etal.,1996).
InamodelofbreastcancerinwhichBALB/cmicewereinoculatedwithmouse
mammary tumour cells, then given diets containing lutein esters from marigold
petals for 70 days, the incidence of tumours was decreased and tumour latency
wasincreasedinanimalsfedwithluteinatlowlevels(0.002or0.02%)whilehigher
levels(0.2or0.4%)werelesseffective(Parketal.,1998b).
LUTEIN FROMTAGETESERECTA L. 63
K2
Inasimilarexperimentusingatransplantablemurinemammarytumour,itwas
demonstrated that mice consuming diets containing lutein esters from marigold
petals at a level of 0.1 or 0.4% for 3 weeks had signifcantly fewer tumours and
increased tumour latency than did controls, and the effect was dose-dependent
(Chewetal.,1996).
Inallthesestudies,luteinshowednoeffectonbodyweightorfeedintake.One
studyshowedthatluteindidnotaffectspleenweights(Parketal.,1998b),although
decreased liver weights without accompanying histopathological changes were
reportedinanotherstudy(Kimetal.,1998).
2.2.4 Genotoxicity
Anumberofstudiesthatevaluatedthepotentialgenotoxicityofluteininvitro
and in vivo are summarized inTable 1. In these studies, there was no evidence
of genotoxicity. The concentrations and doses in some of the studies were
consideredtobelow,butthemaximumfeasibledoseswereused.
InadditiontothetestsreportedinTable1,Luteinwasevaluatedformutagenic
activity in (Ames) assays for reverse mutation using both the plate incorporation
and the preincubation methods (Kruger et al., 2002). Salmonella typhimurium
strainsTA1535,TA97,TA98,TA100,andTA102,withandwithoutmetabolicactiva-
tion(S9fractionfromratliver)wereused.Thedoserangeoflutein(frommarigold
petals containing 79% lutein and 5% zeaxanthin) tested was 15.81580mg/plate
in the preincubation and the plate incorporation assays. In addition, lutein in a
beadletformulation(10%)wastestedatconcentrationsof15815800mg/platein
boththeplateincorporationandpreincubationassays.Noincreasesinthenumber
ofmutantcolonieswereobservedforanyofthefvetesterstrainsaftertreatment
with lutein or lutein as a 10% beadlet, demonstrating that neither formulation is
mutagenicinS. typhimuriumstrains.Positivecontrolsverifedthesensitivityofthe
strainsandtheactivityoftheS9mix.Nocytotoxiceffectsasevidencedbyareduc-
tionofbackgroundcolonynumberswereapparentforanyofthestrainswiththe
possible exception of TA102 in the absence of the metabolic activation system.
However, it should be noted that precipitation of the test compound frequently
occurredatconcentrationsof50mg/plateorhigher.Thus,the10%beadletformula-
tionwasnotevaluatedatthehighestconcentrationof15800mg/plateduetopre-
cipitationandthe1580mg/platedosewasdiscardedfortheplateincorporationtest
plates.
Theabilityofluteintomodulatethegenotoxiceffectsofknownmutagenshas
alsobeeninvestigatedinvariousstudiesthataresummarizedinTable2.
ItisclearfromTable2thatluteininhibitstheactivityofknownmutagens,both
invitro(bacterialtestsystems),andinvivo(micronucleusassay).Rauscheretal.
(1998)proposedthattheantimutageniceffectsofluteinmaybecausedbyinhibi-
tionofmetabolicactivationofthemutagens.
64 LUTEIN FROMTAGETESERECTA L.
K2
Table 1. Studies of genotoxicity with lutein
End-point Testsystem Concentration/dose Results Reference
In vitro
Bacterialreverse S. typhimurium 0.1mlofdiluted Negative Yoshikawa
mutation(Ames) TA98 eggplantfruit etal.(1996)
assaywith juiceextracts
metabolic containinglutein
activation inthemethanol
fromS9 layer
Bacterialreverse S. typhimurium 25,50,75,or100ml Negative Rauscher
mutation(Ames) TA98and ofsolventextracts etal.(1998)
assaywith TA100 fromfruitsand
metabolic vegetables
activation Humanperipheral containinglutein;
fromS9 blood isolatedlutein
lymphocytes alsotested
Chromosome Negative Chtelatetal.
aberration 10%luteinina (2002a)
beadlet
formulation:
3.9125mg/mlfor
3or24hinthe
absenceof
metabolic
activation,orfor3
and5hinthe
presenceof
microsomesfrom
phenobarbital-
and5,6-
benzofavone-ore-
treatedrats,
respectively
In vivo
Micronucleus MaleNMRImice 180mg/kgbwperos Negative Rauscher
formation etal.(1998)
Micronucleus Rat 45,89,or178mg Negative Chtelatetal.
formation 10%lutein (2002b)
beadlets/kgbwon
2consecutivedays
Cometassay Human Subjectsfromwhom Negative Collinsetal.
lymphocytes lymphocyteswere (1998)
obtainedhad
consumed15mg
supplemental
lutein/dayfor12
weekswith
measurementat
16weeks
S9,9000gsupernatantfromratliver.
LUTEIN FROMTAGETESERECTA L. 65
K2
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66 LUTEIN FROMTAGETESERECTA L.
K2
2.2.5 Reproductive toxicity
(a) Multigeneration studies
Nomultigenerationstudieswereavailable.
(b) Developmental toxicity
In a GLP-compliant study of developmental toxicity, three groups of female
Sprague-Dawleyrats(matedwhentheywereaged1013weeks)weregivendiets
mixedwithbeadletscontaining10%lutein(frommarigoldextract;79%lutein,5%
zeaxanthin)fromday6today20ofgestation(Edwardsetal.,2002).Thetarget
dosesofluteinwere250,500,and1000mg/kgbwperday,whiletheactualdoses
administeredasmeasuredfromdataondietaryintakewere252,535,and1118mg/
kgbwperday.Afourthgroupof25matedfemalesreceiveddietcontainingplacebo
beadlets(nozeaxanthinorlutein).Animalsatthelowestandintermediatedoses
receivedplacebobeadletsinadditiontothebeadletscontaininglutein,toensure
similarity in the total concentration of beadlets received by all treatment groups.
Therewasnoevidenceofaneffectofluteininthedams,buttherewasaninverse
dose-related reduction in food consumption and in both maternal and fetal body
weightatthelowestandintermediatedoses.Studyinvestigatorsattributedthese
fndingstodecreasedpalatabilityofthedietatthesedoses(thelowestdosewould
havecontainedthehighestquantityoftheplacebobeadletswhichwereconsidered
to be less palatable than the beadlets containing lutein). In keeping with this
hypothesis, maternal and fetal body weights in the group receiving the highest
dose(noplacebobeadlets)werecomparabletothoseforhistoricalcontrols.There
werenoeffectsonpre-orpostimplantation,embryo-fetalsurvival,orsexratios.
Fetuseswereexaminedforvisceralandskeletalabnormalitiesandsofttissue
changes.Therewasaninversedose-relatedincreaseinformsofreducedossifca-
tion,butthedegreeofossifcationinthefetusesatthehighestdosewassimilar
tothatforhistoricalcontrols.Thesefndingswereconsideredtobeinkeepingwith
thematernalfndingsofdecreasedfoodconsumptioninthecontrolgroupandat
thelowestdose.
There were no adverse effects of treatment with lutein on the incidence of
externalorskeletalabnormalitiesinanygroup.Minorvisceralabnormalitieswere
observedinoneortwofetusesineachofthetreatedgroups,buttheincidenceof
these changes was similar to that for historical controls and was therefore not
considered to be treatment-related. There was a slight, dose-related increase in
the incidence of rudimentary extra lumbar ribs in the groups receiving the inter-
mediate and highest doses. However, these fndings were not considered to be
oftoxicologicalsignifcanceowingtotheknownreversibilityofthisminorskeletal
fnding. Analyses of blood samples showed dose-dependent increases in mean
totalplasmaconcentrationsofluteinondays7and16ofgestation.Meanplasma
concentrationswereapproximately80%higheronday16ofgestationthanonday
7.Thesedataindicatethatanimalswereadequatelyexposedtoluteinthroughout
the experimental period. The NOEL in this study of embryotoxicity/teratogenicity
inratswas1000mg/kgbwperday,thehighestdosetested.
LUTEIN FROMTAGETESERECTA L. 67
K2
2.2.6 Special studies
(a) Cardiovascular effects
Theeffectofsupplementationwithcrystallinelutein(0.2%byweightinthediet)
onatheroscleroticlesionformationhasbeenassessedinapolipoproteinE(ApoE)-
nullmiceandlow-densitylipoprotein(LDL)receptor-nullmice(Dwyeretal.,2001).
Groupsof10femalesofeachstrainreceivedeitherdietsupplementedwithlutein
orbasaldietfor8weeks.Basedonreporteddataonbodyweightandreference
intake(Blackburn,1988),theadministereddoseofluteincorrespondedtoapproxi-
mately500mg/kgbwperday.Supplementationwithluteinwaswelltolerated,and
there were no adverse effects on body-weight gain throughout the 8 weeks of
exposure.Supplementationwithluteinsignifcantlyreducedthesizeofatherscle-
rotic lesions inApoE- and LDL receptor-null mice, and signifcantly reduced the
leveloflipidhydroperoxides,thelevelsofvery-low-densitylipoprotein(VLDL)plus
intermediate-densitylipoprotein(IDL),andlysisoferythrocytesinApoE-nullmice.
NosignifcantchangeswereobservedinLDLorHDLlevels.Onthebasisofthese
fndings, it was concluded that increased intake of lutein protected against the
progressofearlyatherosclerosis,possiblyviaanpathwayinvolvingantioxidants.
(b) Immune responses
Mice
Theeffectsofluteinonmitogen-inducedlymphoproliferation,cytotoxicity,and
interleukin-2(IL-2)production)wereinvestigatedinBALB/cmicegivendietscon-
taining 0.1 or 0.4% lutein esters from marigold extracts (37% lutein esters and
0.5%zeaxanthinesters)for2or4weeks(Chewetal.,1996).Basedonreported
dataonbodyweightandfeedintake,aswellasthecompositionofthemarigold
extract, these doses corresponded to approximately 200 and 803mg/kgbw per
day for lutein, and 2.7 and 10.9mg/kgbw per day for zeaxanthin. No signifcant
treatment-related differences in body-weight gain or feed intake were reported.
Dietary lutein enhanced phytohaemagglutinin-induced lymphocyte proliferation,
buthadnoeffectonIL-2productionorlymphocytecytotoxicity.
Theeffectsofluteinonantibodyproductionwereinvestigatedingroupsof812
young C57B/6J mice primed with T-dependent antigens (sheep erythrocytes)
(Jyonouchi et al., 1994). Mice were injected intraperitoneally with 0.5ml of lutein
in RPMI cell culture medium supplemented with calf serum (10
6
mol/l; purity,
>97%;510
-10
mol/mouse)1hbeforepriming,andwereeuthanized5dayslater.
Basedonreferencedataonbodyweight(Blackburn,1988),thiscorrespondedto
a dose of lutein of approximately 31.5mg/kgbw. Spleen cells were isolated and
preparedfortheplaqueformationcellassay,andspleenweightsandcellnumbers
were recorded. Exposure to lutein via intraperioneal injection had no effect on
spleenweights.However,comparedwithcontrolanimals,miceexposedtolutein
had signifcantly enhanced plaque formation (i.e. enhanced antibody formation),
and a signifcantly increased number of immunoglobulin M (Ig-M)- and Ig-G-
secretingcells,suggestingthatluteinmaymodulatehumoralimmuneresponses
toT-dependentantigens.
68 LUTEIN FROMTAGETESERECTA L.
K2
The possible effects of lutein on the expression of the pim-1 gene, which is
involvedinearlyactivationofTcellsandcellsofotherlineages,wereinvestigated
in BALB/c mice fed diets containing 0, 0.02, or 0.4% lutein for 14 days (Park et
al., 1999). Based on reference data on body weight and feed intake (Blackburn,
1988),luteinwasreceivedatadoseofapproximately0,40,or780mg/kgbwper
day,respectively.Analysesofsamplesofplasmaandlivercollectedafter14days
revealeddose-dependentincreasesinconcentrationsoflutein.Noexternalsigns
oftoxicitywerenotedinanyofthetreatedmice.Splenocytesisolatedfrommice
fedwithluteinwereculturedinthepresenceofconcanavalinA(ConA),showed
adose-dependentincreaseinsteady-statelevelsof pim-1mRNA.Thisisapoten-
tialmechanismthroughwhichluteinmaymodulateimmunefunction.
Cats and dogs
In similar experiments, the effects of diets containing lutein (crystalline, from
marigoldpetalscontainingabout77%luteinandabout5%zeaxanthin)onhumoral
andcell-mediatedimmuneresponseswereinvestigatedinfemaletabbycats(Kim
etal.,2000a)andfemalebeagledogs(Kimetal.,2000b).Ineachstudy,animals
(56perspecies)receivedbasaldietssupplementedwithluteinat0,1(catsonly),
5, 10, or 20 (dogs only) mg/day for 12 weeks. In the cats, these intakes corre-
spondedtodosesofluteinofapproximately0,0.7,3.5,or7.1mg/kgbwperday,
and of zeaxanthin of approximately 0, 0.05, 0.25, and 0.5mg/kgbw per day. In
dogs,theseintakescorrespondedtodosesofluteinof0,0.4,0.9,or1.75mg/kgbw
per day, and of zeaxanthin of 0, 0.03, 0.06, and 0.12mg/kgbw per day. In dogs
only, blood was collected from weeks 13 to 17 to determine changes in plasma
concentrations of immunoglobulin following second and third challenges. Dietary
supplementation with lutein increased plasma concentrations of lutein in each
experimentalanimalmodel,butdidnotsignifcantlyaffectchangesinbodyweights.
Incats,therewasasignifcantdose-relatedincreaseindelayed-typehypersensi-
tivity response to vaccine, but not to concanavalin A (Con A), and signifcantly
enhancedConA-andpokeweedmitogen(PWM)-stimulatedproliferationofperiph-
eralbloodmononuclearcells.Atthehighestdose(10mg/day),thepercentagesof
CD4
+
and CD21
+
cells were signifcantly elevated at week 12, and in the groups
fedluteinat1and10mg/day,concentrationsofIgGweresignifcantlyhigherfrom
weeks 8 to 12. In dogs, supplementation with lutein signifcantly increased the
delayed-typehypersensitivityresponsetovaccineandphytohaemagglutinin(PHA),
andsignifcantlyincreasedmitogen(PHA,ConA,andPWM)-stimulatedprolifera-
tion of peripheral blood mononuclear cells. The percentages of cells expressing
CD5, CD4, CD8 and major histocompatibility complex class II molecules were
signifcantlyincreased,andtheproductionofIgGsignifcantlyincreasedafterthe
secondantigenicchallenge.TherewerenodifferencesinIL-2productionincats
or dogs throughout the experimental periods. These results suggest that dietary
lutein stimulated both cell-mediated and humoral immune responses in cats
anddogs.
LUTEIN FROMTAGETESERECTA L. 69
K2
(c) Ocular toxicity
The long-term ingestion of canthaxanthin at high doses has been shown to
leadtoaccumulationandcrystallizationintheretinaofhumans(Arden&Barker,
1991)andmonkeys(Goralczyketal.,1997),andthequestionhasthereforearisen
astowhetherluteinbehavessimilarly.Theeffectsofluteinontheeyewereinves-
tigatedinaGLP-compliantstudyincynomolgusmonkeystreatedviaoralgavage
withdailydosesoflutein(frommarigoldpetals)asa10%beadletformulationfor
52 weeks (Pfannkuch et al., 2000c; Pfannkuch, 2001).The cynomolgus monkey
was chosen since it was shown to be an excellent model for investigating
the induction and dose-dependency of carotenoid crystal formation in the retina
(Goralczyk et al., 1997; Goralczyk, 2000; Goralczyk et al., 2002). Groups of two
monkeys of each sex were given lutein at a dose of 0 (placebo beadlet), 0.2, or
20mg/kgbw per day; an additional male and female were included in the group
receiving the highest dose. One male and one female at the highest dose were
killedafter26weeks(6months).Occasionalretinalchanges,suchasinclusions
inthemacula,wereobservedinsomegroupsofanimals,includingcontrols,and
wereconsideredtobeunrelatedtotreatment.Overall,comprehensiveophthalmic
examinations (ophthalmoscopy and biomicroscopy examinations, fundus photo-
graphy,electroretinography(consideredtobeaverysensitiveproceduretodetect
earlysignsofgeneralizedretinaldegeneration),andpost-mortemexaminationsof
the right retina (including macroscopic inspection, microscopic pathology under
polarizedandbrightlightforperipheralretinaandmacula,confocalmicroscopyof
themaculaandhistopathologicalexaminationoftheperipheralretinal)showedno
evidenceoftreatment-relatedadversechanges,includingnoevidenceforformation
of crystals in the eyes during or after 52 weeks of treatment with lutein. Dose-
dependent increases in concentrations of lutein were reported in the peripheral
retina. In the central retina and lens, lutein content was markedly increased in
animalsatthehighestdose,buttherewasnoevidenceforcrystallinedeposits.It
was concluded that cynomolgus monkeys treated with lutein for 52 weeks in at
dosesof0.2and20mg/kgbwperdaydidnotshowtoxiceffectsontheeye.
(d) Dermal and ocular irritation
Crystalline lutein (76%) extracted from marigold petals was tested in a study
ofprimaryskinirritationinthreeadultNewZealandwhiterabbits(Csato&Braun,
1999). The primary irritation score for lutein was 0.00 (maximum potential score
is 8.0) and it was classifed as not irritating to rabbit skin in this study, which
compliedwithGLP.
Crystalline lutein (76%) extracted from marigold petals was also tested in a
study of primary eye irritation in three adult New Zealand white rabbits (Csato &
Arcelin,1999).Theprimaryirritationscoreforluteinwas0.11(maximumpotential
scoreis13.0)anditwasclassifedasnotirritatingtotherabbiteyeinthisstudy,
whichcompliedwithGLP.
70 LUTEIN FROMTAGETESERECTA L.
K2
2.3 Observations in humans
2.3.1 Clinical studies
Therehavebeenanumberofstudiesdesignedtoinvestigatethepharmaco-
kineticsofluteinandzeaxanthinthatdidnotnecessarilyincludesafetyend-points,
butalsodidnotreportanyadverseeffectsofthexanthophylls(seesections2.1.1
and2.1.2).Furthermore,arelativelylargenumberofstudiesinhumanshasexam-
ined correlations between dietary intake of lutein or zeaxanthin, the effects of
dietarysupplements,orserumconcentrationsofluteinorzeaxanthinandtheinci-
denceofage-relatedmaculardegeneration,macularpigmentdensityorcatarac-
togenesis with varying results (Eye Disease CaseControl Study Group, 1993;
Seddonetal.,1994;Mares-Perlmanetal.,1995a,1995b;Khachiketal.,1997b;
Beatty et al., 1999; Chasan-Taber et al., 1999; Lyle et al., 1999a, 1999b; Pratt,
1999; Richer, 1999; Bone et al., 2000; Johnson et al., 2000; Gale et al., 2001;
Schalchetal.,2001;Boneetal.,2003;Galeetal.,2003).Thesestudieswillnot
be reviewed here, but in many cases, weak inverse associations were found,
although it is apparent that the protective effect of lutein or zeaxanthin against
age-related macular degeneration or cataract formation remains unproven.
Of importance here, however, is that none of these studies reported adverse
effectsoflutein/zeaxanthin,includingoculartoxicity,althoughinsomecasesdiets
or supplements containing lutein/zeaxanthin at high concentrations were
consumed.
Afewotherclinicalstudieshavebeenperformedthatarerelevanttothesafety
evaluationofluteininhumans.
Inamulticentretrial,90healthyvolunteersweregivenlutein(mixedesterforms
extractedfrommarigoldpetals)at15mg/day,correspondingtoadoseof0.25mg/
kgbwperday,for20weeks(Olmedillaetal.,2002).After20weeks,therewasan
elevationinserumconcentrationsoflutein(aboutfvefold)andserumzeaxanthin
(abouttwofold).Bloodsamplestakenafterfasting(collectedfromasubpopulation
of approximately 16 male and female Spanish subjects at baseline, each month
duringthesupplementationperiod,and3monthsaftersupplementation)showed
no changes in haematological or biochemical parameters or in total cholesterol,
HDL-cholesterol,orLDL-cholesterollevels.Carotenodermia,ayellowishdiscolou-
ration of the skin that is considered to be a harmless, reversible effect of high
intake of carotenoids (Institute of Medicine, 2000), was observed in 40% of the
Spanishcohortfollowingtheinterventionperiod.Carotenodermiawasnotobserved
in the other cohorts from the Netherlands, Northern Ireland, or the Republic of
Ireland(Olmedillaetal.,1997;Granadoetal.,1998;Olmedillaetal.,2001).
In a study of the effects of 2-week exposures to the different carotenoids,
includinglutein,fromfoods(tomatojuice,carrotjuice,spinach)(Mlleretal.,1999),
it was found that daily ingestion of 11.3mg of lutein in a liquid spinach powder
preparation administered daily with meals was well tolerated by all subjects (23
healthyvolunteers).Therewerenosignifcantchangesinbloodconcentrationof
haemoglobin,leukocytes,orserumelectrolytes(sodium,potassium,chloride).
LUTEIN FROMTAGETESERECTA L. 71
K2
In a study designed to examine the effect of the food matrix on the bioavail-
ability of carotenoids, measurements of serum concentrations of cholesterol and
triacylglycerol were included (Castenmiller et al., 1999). After administration of
dietscontainingcrystallineluteinfrommarigoldpetalextracts(suspensioninveg-
etableoilwithb-carotene)at6.6mg/dayfor3weeks,nosignifcantdifferencesin
serumconcentrationsofcholesterolortriacylglycerolwerereportedinnon-obese,
nonsmoking,normolipidaemicmenandwomen(aged1858years)comparedwith
valuesforcontrols.
Inadouble-blind,parallel,placebo-controlledinterventionstudytoinvestigate
the effects of signifcant elevations in plasma concentrations of lutein on fasting
plasmafattyacidprofles,healthynonsmokingmalesreceiveddailysupplementa-
tion with lutein (lutein-rich marigold extract, encapsulated) at 15mg/day (10 sub-
jects) or placebo (encapsulated corn oil) (11 subjects) for 26 days (Wright et al.,
1999). Blood samples were taken before treatment (baseline) and on day 28 for
analysisofconcentrationsoflong-chainfattyacid.Supplementationwithluteinfor
4weekshadnoeffectonindividualfattyacids(14:0,16:0,16:1,18:0,18:1,18:2,
18:3,20:3,20:4,20:5,22:6),totalfattyacids,totalsaturated(S),totalunsaturated
(U),monounsaturated(M),orpolyunsaturated(P)fattyacids,oronratiosofU:S,
P:S,andM:Sfattyacids.
2.3.2 Epidemiological studies
Mostepidemiologicalstudiesonxanthophyllshaveaddressedthehypothesis
that intake of these compounds is inversely related to development of cancer.A
number of such studies has suggested that dietary xanthophylls may protect
againstthedevelopmentofavarietyofcancersincludingthoseoftheoesophagus,
colon, breast, prostate and lung (e.g. Le Marchand et al., 1995; Freudenheim et
al.,1996;Zhangetal.,1997;Franceschietal.,2000;Levietal.,2000;Luetal.,
2001;Nkondjock&Ghadirian,2004),althoughrecentstudiesonbreastandlung
cancerhaveindicatedthatthesecompoundsarenotprotective(Terryetal.,2002;
Mannistoetal.,2004).
A recent large prospective study examined the relationship between serum
concentrations of carotenoids and subsequent risk of developing cancers of the
stomach and upper digestive tract in a region of China with epidemic rates of
oesophageal and gastric cancer (Abnet et al., 2003). There was an association
betweentheincidenceofgastricnon-cardiacancerandtheserumconcentrations
of lutein/zeaxanthin derived from normal dietary sources. These observations,
however,areonlycorrelativeand,indeed,theresultsofaDutchcohortstudyhave
suggested that dietary intake of lutein/zeaxanthin is not associated with risk of
gastriccancer(pathologicaltypenotspecifed),althoughintakesofretinalandb-
carotene were positively associated with risk of this cancer (Botterweck et al.,
2000).
Inviewofthestructuralsimilaritiesbetweenxanthophyllsandb-carotene,the
Committeeconsideredtheoutcomeoftwotrialsthatshowedthatsupplementation
withb-caroteneincreasesriskoflungcancerinheavysmokers;onestudyinvolved
theadministrationofb-caroteneat30mg/dayplus25000IUofretinylpalmitatein
72 LUTEIN FROMTAGETESERECTA L.
K2
18314smokers,formersmokersandworkersexposedtoasbestos(Omennetal.,
1996), while in the second study, b-carotene at 20mg/day with or without 50mg
of a-tocopherol was given to 29133 male smokers (The a-Tocopherol and b-
Carotene Cancer Prevention Study Group, 1994). However, in the light of the
negative results in studies of genotoxicity and the absence of tumour-promoting
activityoflutein,itwasconsideredthattheseinterventionstudieswithb-carotene
werenotappropriatefortheriskassessmentforlutein.
Theresultsofanumberofepidemiologicalstudies,includingdescriptive,cohort
and casecontrol studies, suggest that carotenoid-rich diets are associated with
reduced risk of cardiovascular disease (reviewed in Institute of Medicine, 2000).
Furthermore,noadverseoutcomeshavebeenreportedbetweenincreasedserum
levelsofluteinandzeaxanthinandriskofsubsequentmyocardialinfarction(Street
etal.,1994).Recentepidemiologicalfndings,aswellasthosefromstudiesinvitro
andinmousemodels,supportthehypothesisthatincreaseddietaryintakeoflutein
protectsagainstthedevelopmentofearlyatherosclerosis(Dwyeretal.,2001).
3. INTAKE
3.1 Concentrations in foods
Adatabaseofconcentrationsofcarotenoids,includingluteinandzeaxanthin,
in120foodswasassembledbyMangelsetal.(1993),andwasupdatedbyHolden
etal.(1999).Itshouldbenotedthatthecarotenoidcontentoffoodishighlyvariable
and depends on a number of factors, including geographical area and growing
conditions,cultivarorvariety,processingtechniques,preparationandlengthand
conditionsofstorage(Holdenetal.,1999,andreferencestherein).Majorsources
oflutein/zeaxanthinareleafygreenvegetables(e.g.rawspinach,11.9mg/100g),
corn (boiled, 1.8mg/100g) and green vegetables such as broccoli (raw,
2.4mg/100g), Brussels sprouts (boiled, 1.3mg/100g), green beans (boiled,
0.7mg/100g), and peas (canned, 1.3mg/100g). Although it is not a major part
of the diet in western Europe and North America, kale has the highest lutein/
zeaxanthincontentofallfoodsanalysed(raw,39.5mg/100g).
3.2 Dietary intake
Dietaryrecalldatafrom1102adultwomenparticipatinginthe1986Continuing
SurveyofFoodIntakebyIndividualsindicatemeanintakesoflutein/zeaxanthinof
1.3mg/day with a total carotenoid intake of 6mg/day (Chug-Ahuja et al., 1993).
Food frequency data from 8341 adults participating in the 1992 National Health
InterviewSurveyindicatethatmeanintakesofluteinformenwere2.2mg/dayand
for women 1.9mg/day (Nebeling et al., 1997). The Nutritional Factors in Eye
Disease Study reported mean dietary intakes of lutein/zeaxanthin of 0.70.8mg/
day(VandenLangenbergetal.,1996).Inapooledanalysisofsevencohortstudies
designedtoassesstheeffectofdietarycarotenoidsonriskoflungcancer,intakes
oflutein/zeaxanthinwereenergy-adjustedusingthepredictedintakeof2100kcal/
dayformenand1600kcal/dayforwomen(Mannistoetal.,2004).Foodconsump-
tion was assessed at baseline using a validated dietary questionnaire for each
LUTEIN FROMTAGETESERECTA L. 73
K2
studypopulation.Forthesesevenpopulations,themeanintakeoflutein/zeaxan-
thinformenandwomencombinedwas3.7mg/day(range,16mg/day).
Theestimatedmeanand90th-percentileconsumptionofluteinandzeaxanthin
inasurveyofsamplefoodswere1.71and3.01mg/dayrespectivelyintheUnited
StatesofAmerica(USA)(DSMNutritionalProducts,2004)(Table3).Simulations
considering proposed food use levels in the total population of the USA resulted
in estimated mean and 90th-percentile intake of lutein by all users of 7.3 and
13.4mg/dayrespectively(DSMNutritionalProducts,2004)(Table3).Krugeretal.
(2002)estimatedtheintakeoflutein/zeaxanthinintheUSAusingdietaryrecords.
Themeanand90th-percentileintakesoflutein/zeaxanthinwere3.83and7.29mg/
day respectively, and 0.91 and 1.77mg/day respectively from crystalline lutein
product (Table 3). Intake of lutein in 1543 Canadians (aged 1865 years), esti-
matedby24hrecall,was1.41and0.57mg/day(meanandmedian,respectively)
(Johnson-Down,2002)(Table3).Intakeofluteinin76women(aged5065years)
fromtheUnitedKingdom(UK),estimatedbythedeterminationofbothfoodintake
andconcentrationsofluteinwas0.92mg/day(Scottetal.,1996)(Table3).
Formulations of lutein/zeaxanthin are also available as dietary supplements,
buttherearenoreliableestimatesofintakefromthesesources.
4. COMMENTS
Inrats,peakconcentrationsofradiolabelintheplasmaandtissuesoccurred
about 4h after a single oral dose of [
14
C]lutein. Most of the radiolabel was elimi-
nated via the faeces within about 2 days; very low urinary and biliary excretion
indicated that there was poor absorption from the intestinal tract. Based on data
onfaecalexcretion,theabsorptionofluteinwasabout3040%whenadministered
to rats in the diet as beadlets containing vitamin E (the beadlet formulation was
usedtoenhancethestabilityoflutein).Tenfoldincreasesindose,intherangeof
2to200mg/kgbw,resultedintwo-tothreefoldincreasesinplasmaconcentrations,
indicatingreducedabsorptionathigherdoses.Steady-stateplasmaconcentrations
ofluteinwerereachedbyabout3daysafterthestartofdietaryadministrationof
luteintorats,indicatingthatthehalf-lifeofluteinisabout1day.
Inhumans,peakplasmaorserumconcentrationsofluteinoccurredat1116h
afteradministrationofasingleoraldose.Duringdailysupplementationwith20mg
of lutein/day, steady-state plasma concentrations were reached within about 30
days.Thisisconsistentwithaneliminationhalf-lifeofabout57days.
The food matrix, including its fbre and lipid contents, and the concentrations
ofothercarotenoidsinthedietmayinfuencetheextentofabsorptionofcarotenoid
compounds. The relative absorption of lutein from a mixed vegetable diet was
lower than from a diet containing pure lutein.A mixed preparation of lutein and
zeaxanthindidnotinfuencetheabsorptionofb-carotene.
LuteinhasanoralLD
50
of>2000mg/kgbwinrats.Ina13-weekstudyinrats,
luteinadministeredatoraldosesofupto200mg/kgbw,thehighestdosetested,
caused no treatment-related effects. In a 52-week study designed primarily to
investigate possible adverse effects on the eye in monkeys, lutein was adminis-
74 LUTEIN FROMTAGETESERECTA L.
K2
Table 3. Estimated daily intake of lutein (Tagetesextract)
Country Estimated Targetyear Method/ Standards Reference
dailyintake compound foruse
(mg/person
perday)
New Nodata 1997 Aslutein, Foodcolour, submittedby
Zealand GMP*food GMP/lowest NewZealand
consumption possible forthe
level Committeeat
its63rd
meeting
USA 1.71 19941996, Food-usesand DSMNutritional
(mean), 1998 food Productsand
3.01 consumption NeminFoods,
(90th
a
) amount,all LC,forthe
person, Committeeat
including its63rd
zeaxanthin Meeting(from:
7.3 19941996, Food-usesand Dietary
(mean), 1998 food Reference
Intakes,
Instituteof
Medicine,
2000)
13.4 consumption
(90th) amount,all
users,
including
zeaxanthin
USA 0.91 19941996 From84% Krugeretal.
(mean), pure (2002)
1.77 crystalline
(90th) product,
lutein+
zeaxanthin
3.83 2000 Dietary
(mean), guidelines,
7.29 naturallutein
(90th) and
zeaxanthin
Canada 1.41 Sept1997 24-hfood Johnson-Down
(mean), Jul1998 recall,lutein, etal.(2002)
0.57 foradults
(median) (aged1865
years)
UK 0.92 Nov1988 Determination Scottetal.
(women Oct1989 ofbothfood (1996)
aged intakeand
5065 concentration
years) oflutein
EU:EuropeanUnion;GMP:Goodmanufacturingpractice.
a
90thpercentile.
LUTEIN FROMTAGETESERECTA L. 75
K2
teredatadoseof0.2or20mg/kgbwperdaybygavage.Thisstudywasperformed
becauseadverseoculareffectshadbeenseenwithcanthaxanthin(Annex1,refer-
ences 78, 89, 117). There were no treatment-related effects on a wide range of
toxicological end-points. Furthermore, comprehensive ophthalmic examinations,
including electroretinography, showed no evidence of treatment-related adverse
changes.
Nolong-termstudiesoftoxicityorcarcinogenicitywereundertaken.
Lutein gave negative results in several studies of genotoxicity in vitro and in
vivo.AlthoughtheCommitteenotedthatthedosesusedinthesetestswerelow,
itrecognizedthatmaximumfeasibledoseswereused.Therewasnoevidencefor
tumourpromotingactivityinanimalmodels.
Inastudyofdevelopmentaltoxicitywithluteininrats,therewasnoevidence
fortoxicityatdosesofupto1000mg/kgbwperday,thehighestdosetested.
Ina20-weekmulticentreinterventiontrialwithluteininhealthyhumansubjects,
therewerenochangesinhaematologicalorbiochemicalparametersaftercontinu-
ous daily doses of lutein of 15mg (0.25mg/kgbw, assuming a body weight of
60kg).Therehasbeenarelativelylargenumberofstudiesinhumansthathave
examinedcorrelationsbetweenmaculardegenerationanddietaryintakeoflutein
orzeaxanthin,intakesviadietarysupplements,orserumconcentrations.Although
thesestudiesweredesignedtolookforoculareffects,whereclinicalorbiochemical
parameters were also examined, no adverse effects of these xanthophylls were
reported.
Intake
DataondietaryintakefromanumberofstudiesinNorthAmericaandtheUK
indicate that intake of lutein from natural sources is in the range of 12mg/day
(approximately 0.010.03mg/kgbw per day). Simulations considering proposed
levels of use as a food ingredient resulted in an estimated mean and 90th-
percentile intake of lutein plus zeaxanthin of approximately 7 and approximately
13mg/day, respectively. Formulations containing lutein and zeaxanthin are also
availableasdietarysupplements,buttherewerenoreliableestimatesofintakes
fromthesesources.
5. EVALUATION
In several studies of toxicity, including developmental toxicity, no adverse
effects were documented in animals, including monkeys, or humans.Taking into
accountdatashowingthatluteinwasnotgenotoxic,hadnostructuralalert,didnot
exhibittumour-promotingactivity,andisanaturalcomponentofthebody(theeye),
theCommitteeconcludedthattherewasnoneedforastudyofcarcinogenicity.
Luteinhassomestructuralsimilaritiestob-carotene,whichhasbeenreported
toenhancethedevelopmentoflungcancerwhengivenasasupplementtoheavy
smokers.Theavailabledataindicatedthatluteininfoodwouldnotbeexpectedto
havethiseffect.TheCommitteewasunabletoassesswhetherluteinintheform
ofsupplementswouldhavethereportedeffectinheavysmokers.
76 LUTEIN FROMTAGETESERECTA L.
K2
The52-weekstudyinmonkeyswasdesignedtoevaluateoculareffects,and
although there were no adverse toxicological effects at the highest dose tested
(20mg/kgbwperday),thisstudywasconsideredtobeinappropriatefortheestab-
lishmentofanADI,inviewofthemuchhigherdosesusedinseveralotherstudies
and found to be without effect. The available comparative toxicokinetic data for
humansandratsindicatedthatthestudiesoftoxicityinratscouldbeusedtoderive
anADI. The Committee concluded that the best study for this purpose was the
90-daystudyinrats.AnADIof02mg/kgbwwasestablishedbasedontheNOEL
of200mg/kgbwperday(thehighestdosetestedinthisstudy)andasafetyfactor
of100.
AlthoughtheADIwasbasedontheresultsofashort-termstudy,thesupporting
data and lack of effects at much higher doses in some studies (e.g. a study of
developmentaltoxicity),indicatedthatthesafetyfactorof100wasappropriate.
In view of the toxicological data and structural and physiological similarities
betweenthexanthophyllsluteinandzeaxanthin,theCommitteedecidedtoinclude
zeaxanthinintheADI(02mg/kgbw)forlutein,whichhadastrongertoxicological
database, and to make this a groupADI for the two substances.This groupADI
doesnotapplytootherxanthophyll-containingextractswithaluteinorzeaxanthin
contentlowerthanthatcitedinthespecifcations.
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K2
87
PEROXYACID ANTIMICROBIAL SOLUTIONS CONTAINING
1-HYDROXYETHYLIDENE-1,1-DIPHOSPHONIC ACID (HEDP)
First draft prepared by
Dr A. Mattia
1
, Dr R. Merker
1
, Dr S. Choudhuri
1
, Dr M. DiNovi
1

and Professor R. Walker
2
1
Division of Biotechnology and GRAS Notice Review, Offce of Food
Additive Safety, Center for Food Safety and Applied Nutrition, Food and
Drug Administration, College Park, MD, USA; and
2
School of Biomedical and Life Sciences, University of Surrey, Guildford,
Surrey, England
Explanation............................................................................... 88
Compositionofantimicrobialsolutions.............................. 89
Residuesofcomponentsofantimicrobialsolutions.......... 89
Biologicaldata.......................................................................... 89
Biochemicalaspects.......................................................... 90
Absorption,distributionandexcretion......................... 90
Biotransformation......................................................... 91
Toxicologicalstudies.......................................................... 92
Acutetoxicity................................................................ 92
Short-termstudiesoftoxicity....................................... 93
Long-termstudiesoftoxicityand
carcinogenicity....................................................... 94
Genotoxicity................................................................. 95
Reproductivetoxicity.................................................... 95
Specialstudy:skeletaleffectsindogs........................ 97
Environmentalstudies........................................................ 98
Microbiologicalaspects...................................................... 98
Roleofcomponentsinantimicrobialsolutions............ 98
Studiesofantimicrobialeffcacy.................................. 99
SolutionA.............................................................. 99
SolutionB.............................................................. 100
SolutionC.............................................................. 102
SolutionD.............................................................. 102
Observationsinhumans.................................................... 103
Intake ..................................................................................... 103
Residuesonfoods............................................................. 103
Internationalestimatesofintake........................................ 104
Nationalestimatesofintake............................................... 105
Non-foodusesofHEDP.................................................... 107
Studiesonthequality,nutritionalvalue,orother
propertiesoffoodtreatedwithantimicrobial
solutions....................................................................... 108
Thiobarbituricacidandfattyacidproflesofmeat
andpoultryproducts.................................................... 108
Theeffectofthepotentialreactivityofhydrogen
peroxideandperoxyaceticacidonmeatand
poultryproducts........................................................... 108
88 PEROXYACID ANTIMICROBIAL SOLUTIONS
K2
Nutritionalteststodeterminetheeffectsof
peroxyaceticacidandhydrogenperoxide
onfruitandvegetables................................................ 108
Comments ............................................................................... 109
Evaluation ............................................................................... 112
References............................................................................... 113
1. EXPLANATION
The Committee considered the safety of antimicrobial solutions that are pre-
paredfromaceticacidandoctanoicacid(singlyorincombination),togetherwith
hydrogenperoxide,andusing1-hydroxyethylidene-1,1-diphosphonicacid (HEDP)
asasequestrantorstabilizer.Preparationsthatarereadyforusealsocontainas
activecompoundstheperoxyformsofbothacids.Beforeuse,concentratedsolu-
tionsaredilutedtoachievetargetconcentrationsoftotalperoxyacidrangingfrom
80to200mg/kg.Theseantimicrobialsolutionsareintendedforuseascomponents
ofwashsolutionsonfreshpoultryandmeatandinwashwaterforfreshandpro-
cessedfruitsandvegetables.Afterbeingappliedinprocesswater,theyarelargely
eliminatedbydrainage,furtherwashingandtrimmingofproducts,andevaporation.
Thesafetyoftheantimicrobialsolutionswasthereforeassessedonacomponent-
by-component basis, considering the potential residue of each component or its
breakdownproductsinfoodasconsumed.
Atitsseventeenthmeeting(Annex1,reference32),theCommitteeallocated
anacceptabledailyintake(ADI)notlimited
1
toaceticacidanditspotassiumand
sodiumsalts.ThisADIwasretainedattheforty-ninthmeeting(Annex1,reference
131) when the Committee evaluated a group of favouring agents (saturated ali-
phatic acyclic linear primary alcohols, aldehydes, and acids) that included acetic
acid.
Atitsforty-ninthmeeting,theCommitteeevaluatedoctanoicacidforuseasa
favouring agent as part of the group of saturated aliphatic acyclic linear primary
alcohols,aldehydes,andacids,andconcludedthatoctanoicacidposednosafety
concerns at intakes of up to 3800mg/person per day (or 63mg/kg bw per day,
assumingabodyweightof60kg).
Atitstwenty-fourthmeeting(Annex1,reference53),theCommitteeevaluated
hydrogen peroxide as a preservative and sterilizing agent for use in milk. While
anADIwasnotallocated,theCommitteenotedthathydrogenperoxideshouldbe
usedonlywhenbettermethodsofmilkpreservationwerenotavailable.
Peroxyacetic acid and peroxyoctanoic acid, and HEDP have not been previ-
ouslyevaluatedbytheCommittee.
Atitspresentmeeting,theCommitteeconsideredanumberofstudiesonthe
antimicrobialeffcacyofperoxyacidsolutions,thetoxicityofHEDP,andtheeffects
1
A term no longer used by the Committee, which has the same meaning as ADI not
specifed.
PEROXYACID ANTIMICROBIAL SOLUTIONS 89
K2
ofperoxyacidsolutionsonfoodqualityandnutritionalvalue.TheCommitteealso
evaluatedestimatesoftheintakeoftheindividualcomponentsinthesesolutions
forconsiderationinthesafetyevaluation.
1.1 Composition of antimicrobial solutions
Thecompositionoffourantimicrobialsolutions,AD,aredescribedinTable1.
Theconcentratedsolutionsaredilutedbeforeusetoachievetargetconcentrations
oftotalperoxyacidrangingfrom80to200mg/kg.
In manufacturing each antimicrobial solution, measured quantities of each
component are added in a specifc order. Hydrogen peroxide reacts with acetic
acidtoformperoxyaceticacid,whichitthushelpstostabilize.Hydrogenperoxide
also reacts with octanoic acid, when present, to form peroxyoctanoic acid. The
resultisanequilibriumsolutioncontainingperoxyaceticacid,aceticacid,hydrogen
peroxide,HEDP,andinsomecases,octanoicacidandperoxyoctanoicacid.The
concentrationofperoxyacidscontinuestoincreasefor713daysaftermanufac-
ture. HEDP is needed to ensure the stability of the solution since peroxy com-
poundsareinherentlyunstable.Onceequilibriumisachieved,thesolutionremains
relativelystableatroomtemperatureforupto1year.Themainchemicalreactions
thatoccurintheequilibriumsolutionsareshowninFigure1.
1.2 Residues of components of antimicrobial solutions
Afterapplication,theantimicrobialsolutionsandtheircomponentsarelargely
lost due to drainage, further washing, trimming and evaporation. Residues of
hydrogen peroxide, peroxyacetic acid, or peroxyoctanoic acid on food rapidly
decompose into water, oxygen, acetic acid and octanoic acid (Figure 1). Small
amountsofaceticacid,octanoicacidandHEDPwillremainonthetreatedcom-
modities. Intake assessments for the components of the antimicrobial solutions
aredescribedinsection3.
2. BIOLOGICAL DATA
Antimicrobial mixtures are equilibrium mixtures that are diluted in water prior
totheiruseinprocessingfood.Hydrogenperoxideinthesemixtureswilldissociate
intowaterandoxygen.AlthoughtheirstabilityisenhancedbyHEDP,bothperoxy-
acetic acid and peroxyoctanoic acid are also inherently unstable and will break
downintoaceticacidandoctanoicacid,respectively.Lowresiduallevelsofthese
simpleorganicacidspresentonfoodwouldposenoconcern.Noresiduesofper-
oxyaceticacidorperoxyoctanoicacidinthesemixtureswereexpectedtoremain
ontreatedfoods.Thus,theperoxidecomponentsoftheperoxyacidantimicrobial
mixtures did not pose toxicological concerns for the uses being considered at
presentandthefocusofthebiochemicalandtoxicologicalaspectsofthissafety
evaluationwasHEDP.
90 PEROXYACID ANTIMICROBIAL SOLUTIONS
K2
Table 1. Composition of four antimicrobial solutions (AD) and maximum
concentration of components in ready-to-use solutions (after dilution)
Component Weightofeachcomponentin Maximumconcentrationof
thesolutionatequilibrium
a
eachcomponentinthe
(%) solutionafterdilution
b
(mg/kg)
A B C D A B C D
Aceticacid 40.6 49.4 32.0 42.0 985 2000 208
d
NS
Peroxyaceticacid 12 12.2 15.0 12.0 213
c
220
c
80 80
Hydrogenperoxide 6.2 4.5 11.1 4.0 110 150 59 59
Octanoicacid 3.2 8.8 0.0 10.0 74 300 0 NS
Peroxyoctanoicacid 0.8 1.4 0.0 3.4 14
c
25
c
0 NS
1-Hydroxy-ethylidene-1, 0.6 0.6 0.9 0.6 13 13 4.8
d
4.8
1-diphosphonicacid
(HEDP)
Water 36.6 23.1 41.0 28.0
NS,notstated.
a
Atequilibrium,whichoccurs713daysaftermanufacture,dependingonthetemperature
atwhichthesolutionisstored.
b
SolutionsAandBaredilutedtoachieveatargetconcentrationoftotalperoxyacidof
200mg/kg;toaccountforvariations,maximumvaluesassumethatusewillresultina
concentrationoftotalperoxyacidsof220mg/kg.SolutionsCandDaredilutedtoachieve
atargetconcentrationoftotalperoxyacidof40mg/kg;toaccountforvariations,
maximumvaluesassumethatusewillresultinaconcentrationoftotalperoxyacidsof
80mg/kg.
c
Concentrationoftotalperoxyacidasperoxyaceticacid=[220]+[(22/160)76];thereis
variationofupto10%inthemeasuredconcentrationofperoxyaceticacid,dueto
differencesinequipmentformeasurementanddispensing.
d
Theoreticalvalue(notbasedonanalysis).
2.1 Biochemical aspects
2.1.1 Absorption, distribution and excretion
Caniggia&Gennari(1977)publishedaconcisereportontheintestinalabsorp-
tion and kinetics of
32
P-labelled EHDP (disodium ethane-1-hydroxy-1, 1 diphos-
phonate;disodiumetidronate;referredtoasHEDPinthismonograph)inhumans.
Ten volunteers were given HEDP at an oral dose of 20mg/kg (the carrier dose)
togetherwith40mCi(1480kBq)of[
32
P]HEDP.After6days,7090%oftheadmin-
istereddosewasfoundinthefaeces.SevenothersubjectsweregivenEHDPat
anoraldoseof100mg(thecarrierdose)togetherwith20mCi(740kBq)of[
32
P]HEDP
intravenously.Sixdaysafterintravenousadministration,3550%oftheradiolabel
administered was excreted unchanged in the urine, with negligible [
32
P]HEDP
foundinthefaeces.Therewasarapiddeclineintheconcentrationof[
32
P]HEDP
in the plasma; after 6 days, <0.03% of the administered dose remained in the
plasma.Althoughonlylimitedinformationwaspublishedinthisreport,theresults
suggestedthatorallyadministeredHEDPispoorlyabsorbedinhumans,andthat
HEDPmayaccumulateoutsideoftheblood.
PEROXYACID ANTIMICROBIAL SOLUTIONS 91
K2
2.1.2 Biotransformation
Michael et al. (1972) studied the absorption, distribution, and metabolism of
HEDPinrats(n=3or4),rabbits(n=3),dogs(n=2or3)andmonkeys(n=3)
afteroraladministrationof
14
C-labelledHEDPviaintragastriccannula.Thedoses
administeredrangedfrom10to50mg/kgbw.Theauthorsfoundthatabout90%
oftheadministereddosewasexcretedinthefaecesoftheadultanimals.Absorp-
tion,whichoccurredinthestomach,was<10%inrats,rabbitsandmonkeys,and
ranged from about 14% in older dogs to 21% in young dogs. Consistent with a
possibleage-dependenteffect,absorptionwassomewhatgreaterinweanlingrats.
SomeratshadbeenpreviouslyfedHEDPaspartoftheirdiet,butsuchprecondi-
tioningdidnotaffectabsorption.Theauthorsattemptedtoidentifymetabolitesin
biologicalsamplesobtainedfromratsanddogs,buttheyreportedthattherewas
nometabolismofHEDPineitherspecies.Inallspecies,abouthalftheabsorbed
dosewasexcretedunchangedintheurineandtherestwasdepositedinthebone,
where its half-life in rats was demonstrated to be about 12 days. The results of
thesestudies,conductedinavarietyofspecieswithsmallnumbersoftestanimals,
wereconsistentwiththedataobtainedfromasmallsampleofhumanvolunteers.
Collectively,thedataindicatedthatabsorptionofHEDPfromthegastrointestinal
tract is very limited and its metabolism is negligible. Negligible metabolism of
O
H
3
C
Acetic acid
Peroxyacetic acid
2H
2
O
2
2 H
2
O
2 H
2
O + O
2
2 2 2
OH
O
H
3
C OH
O
H
3
C O OH
+ +
O
C
7
H
15
C
7
H
15
C
7
H
15
Octanoic acid
Hydrogen peroxide
Peroxyoctanoic acid
2H
2
O
2
2 H
2
O
2 H
2
O + O
2
2 H
2
O + O
2
2 2 2
OH
O
OH
O
O OH
2 H
2
O
2
+ +
Figure 1. Main chemical reactions in the equilibrium solutions
92 PEROXYACID ANTIMICROBIAL SOLUTIONS
K2
systemic HEDP could have been due to the fact that carbonphosphorus (CP)
bondsarediffculttobreak.
2.2 Toxicological studies
2.2.1 Acute toxicity
Theavailablemedianlethaldose(LD
50
)valuesforHEDPadministeredorally
are summarized in Table 2. Two of these studies, in which high doses of HEDP
wereassociatedwithkidneydamageinratsandrabbits,aredescribedmorefully
below.
Rats
Groups of 10 male and 10 female fasted Charles River CD rats were given
HEDP(asdisodiumetridronate,thedisodiumsaltofHEDP)bystomachtube,at
oneoffourdosesselectedonthebasisofanassumedtoxicityanddoseresponse
curve.TheLD
50
wasdeterminedtobe1.34g/kgbw.Amongthesurvivinganimals
that had received a higher dose (1.60 or 1.14g/kg bw) of disodium etidronate, 3
outof10werefoundtohavelightgrey,granularkidneys.Microscopicevaluation
revealeddamagetothenephritictubules.Thekidneysofallanimalsreceivingthe
lowestdose(0.814g/kgbw)alsoshowedmildtubularchanges.Thekidney:body
weightratiosoftheanimalsatthetwohigherdosesweresignifcantlyhigherthan
thoseofanimalsat0.814g/kgbw,aswellasbeinghigherthanthenormalrange
(Nixonetal.,1972).
Rabbits
The same procedure was used to determine LD
50
values in New Zealand
rabbits.ItwasfoundthatthesusceptibilityofrabbitstotheacuteeffectsofHEDP
(asdisodiumetridronate,thedisodiumsaltofHEDP)isafunctionofage,weight,
and sex.The LD
50
values ranged from 0.581 to 1.14g/kg bw, and were lower in
malesthaninfemales,andlowerinmatureanimals(bodyweight,>3300g)than
immatureanimals(bodyweight,about2500g).Nounusuallesionswerereported
uponmicroscopicanalysis.About50%ofthesurvivinganimals(inallgroups)were
found to have kidney lesions indicative of chronic interstitial nephritis; however,
Table 2. Acute toxicity of HEDP administered orally
Species Sex LD
50
(g/kgbw) Reference
Rat Notspecifed 2.40 MonsantoMSDS
Rat M,F 1.34 Nixonetal.(1972)
Rabbit M,F 0.5811.14 Nixonetal.(1972)
Dog M,F 84.80
a
Nixonetal.(1972)
Rat M,F 3.13 YoungerLaboratories
(1965)
F,female;M,male.
a
Valuewasestimated,duetoemesisinsomedogs.
PEROXYACID ANTIMICROBIAL SOLUTIONS 93
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chronicinterstitialnephritisiscommonlyfoundintherabbitanditisthushardto
determinewhetherthefndingwasrelatedtotreatment(Nixonetal.,1972).
Dogs
The administration of HEDP (as disodium etridronate, the disodium salt of
HEDP) produced an immediate emetic response in some dogs, and it was thus
not possible to clearly defne an LD
50
value. On the basis of early deaths and
necropsyofanimalsfoundinamoribundconditionathigherdoses(1.010.0g/kg),
however,theLD
50
wasestimatedtobeabout1.0g/kg(Nixonetal.1972).
2.2.2 Short-term studies of toxicity
Rats
Ina91-dayfeedingstudy,groupsof20maleand20femaleCharlesRiverCD
rats were fed diets containing HEDP (disodium monohydrate salt) at 0, 0.2, 1.0,
or5.0%(equivalenttodosesof0,100,500,and2500mg/kgbwperday).Owing
toseveremortalityandweightlossobservedat5.0%,thestudyofthatgroupwas
terminated after 1 week.After conclusion of the study (91 days for groups at 0,
0.2,and1.0%,and1weekforthe5.0%group),fvemalesandfvefemalesfrom
eachgroupwererandomlyselectedfornecropsy.Histopathologicallesionsand/or
alterations of blood parameters were observed and appeared to be associated
withgastritis.At5.0%,gastrointestinalerosionswereobservedandthekidney:body
weight ratio was high (1.48% and 1.55% for females and males, respectively)
comparedwithcontrols(1.11%).Notreatment-relatedchangeswereobservedin
histopathological lesions or blood haematological values at 0.2% or 1.0%. The
kidney:body weight ratio in females at 1.0% was slightly higher (at 0.82%) than
controls (0.64%). All other parameters measured in the study were normal and
similar to those in the controls. Hence, the no-observed-effect level (NOEL) was
1.0%,equivalenttoadoseof500mg/kgbwperday(Nixonetal.,1972).
A 90-day feeding study in rats was designed to assess the toxicity of HEDP
(crystalline sodium salt characteristic of DEQUEST2010 phosphonate in the
nature and amount of by-products and impurities). Groups of 15 male and 15
female rats were given HEDP at a dietary concentration of 0, 3000, 10000 or
30000mg/kg of feed (equivalent to 0, 150, 500, or 1500mg/kg bw per day) of
HEDP.Bodyweights,foodconsumptionandmortalityweredeterminedweekly.At
45 and 90 days, haematology and clinical chemistry parameters were assessed
and urine analysis was performed. The animals were necropsied at the end
of the study and histopathological examinations were performed on tissues from
animalstreatedatthehighestdoseonly.Ahighlevelofmortalitywasobservedat
30000mg/kg.This fnding might be related to the ingestion of HEDP, although it
was possibly the result of trauma induced by blood collection. At 30000mg/kg,
body-weightgainwasinhibitedinmales.Atthehighestdosetested,haematology
revealed signifcant changes, including increased erythrocyte counts in males,
decreasedhaemoglobinconcentrationanderythrocytevolumefractionsinmales
andfemales,anddecreasedleukocytecountsattheendofthestudyinfemales
only. The lesions observed in histopathological examinations, which were con-
94 PEROXYACID ANTIMICROBIAL SOLUTIONS
K2
ductedonanimalstreatedatthehighestdoseonly,weredescribedbythepatholo-
gist as being typical of the controls.At 10000mg/kg (500mg/kg bw per day), no
adverse effects were noted in any parameters measured in this study; however,
histopathologicalexaminationswerenotconductedonratsinthegroupsgiventhe
twolowerdoses.TheNOELwas500mg/kgbwperday(IndustrialBio-TestLabo-
ratories,Inc.,1975a).
Dogs
In a 90-day study of toxicity designed to test the effects of HEDP (crystalline
sodiumsaltcharacteristicofDEQUEST2010phosphonateinnatureandamount
of by-products and impurities), groups of four male and four female beagle
dogs(aged5monthsatthestartofthestudy)weregivenHEDPataconcentration
of 0, 1000, 3000, or 10000mg/kg of diet (equivalent to a dose of 0, 25, 75, or
250mg/kgbwperday).Foodandwaterwereavailableadlibitum.Bodyweights
and food consumption were recorded weekly. Haematology and blood chemistry
parameterswereassessedandurineanalysiswasconductedatthebeginningof
the study and at 56 and 85 days. At the end of the study, organ weights were
determinedandgrossandhistopathologicalexaminationswereconducted.There
werenoadverseeffectsofthetestmaterialonbodyweight,althoughfoodintake
in females at the intermediate and highest doses was decreased compared with
that of the controls. No deaths were reported. Small changes in haematological
parameters (increased erythrocyte counts and decreased mean corpuscular
volume)andvariationsinbloodchemicalparameters(serumpotassiumandmag-
nesium concentrations in males and females, respectively) were noted, but the
effectswereinconsistentandwerenotattributedtotreatment.Increasednumbers
ofleukocytesandcrystalswerefoundintheurineofdogsfromalltreatmentgroups
atthefnalanalysis.However,thiswasnotconsideredtobeasignifcantfnding
sincenochangesinthegenitourinarysystemwereobservedonmicroscopy.Some
differences were noted in organ weights, including increased brain weights in
females at the intermediate and highest doses and increased thyroid weights in
males at the highest dose. These differences, which were not associated with
microscopicchangesintheseorgans,werenotconsideredtoberelatedtotreat-
ment. There were no gross or histopathological changes reported for any of the
tissues or organs examined in this study.The NOEL was 250mg/kg bw per day
(IndustrialBio-TestLaboratories,Inc.,1975b).
2.2.3 Long-term studies of toxicity and carcinogenicity
Long-term studies to address the toxicity or carcinogenic potential of HEDP
werenotavailableforthisevaluation.ThepublicationbyNixonetal.(1972)refers
todataobtainedinchronicteststhatweretobepublishedelsewhere.However,
aliteraturesearchfailedtorevealanydataresultingfromtraditionallong-termtests
of toxicity in animals. One study on the skeletal effects of HEDP administered
subcutaneouslytobeagledogsforapproximately1or2yearsisdescribedbelow
(see2.2.6,specialstudy).
PEROXYACID ANTIMICROBIAL SOLUTIONS 95
K2
2.2.4 Genotoxicity
ThepotentialgenotoxicityofHEDPwasassessedusinganassayforreverse
mutation in which fve strains of S. typhimurium (TA98,TA100,TA1535,TA1537
and TA1538), were tested, with and without metabolic activation provided by rat
liver microsomes, at doses of 0.001, 0.01, 0.1, 1, 5 or 10ml/plate (in water).The
testarticlewasacommercialproductthatcontained60%HEDPinaqueoussolu-
tion.Aberrationsinthebackgroundlawnwereobservedatconcentrationsof5and
10ml/plate, indicating toxicity at the two highest concentrations tested for all fve
strains of Salmonella. The tester strains responded as expected to solvent and
positivecontrols.Undertheconditionsoftheassay,thetestarticlewasnotmuta-
genic(MonsantoCo.,1977).
The potential genotoxicity of HEDP was also assessed by thymidine kinase
(Tk) gene forward mutation assay in mouse lymphoma L5178Y cells, with and
withoutmetabolicactivationprovidedbyratlivermicrosomes;atdosesof0.064,
0.125, 0.250, 0.500, or 0.600ml/ml in the absence of microsomal enzymes and
0.125,0.250,0.500,0.600and0.800ml/mlinthepresenceofmicrosomalenzymes.
The test article, a commercial product that contained 60% HEDP in an aqueous
solution,wasthesameasthattestedintheassayforreversemutation,butwas
inthiscasedilutedindimethylsulfoxide(DMSO).Atconcentrationsof0.5ml/ml,
cytotoxicitywasobservedthatwasgreaterinthepresenceofmicrosomalenzymes.
Inthefrstofthetwotrials,althoughcontrolvaluesforspontaneousmutagenesis
werehigherthanexpected,theincidenceofmutagenesiscausedbyHEDPatthe
highest concentration tested was more than 2.5 times that for the controls for
spontaneous mutagenesis with microsomal activation. In the second trial, the
incidence of spontaneous mutagenesis was not elevated and the incidence of
mutagenesiscausedbyHEDPatthehighestconcentrationtestedwasabouttwice
thatforthecontrolsforspontaneousmutagenesis.Thepositivecontrolsgavethe
expectedresults.Undertheconditionsoftheassay,thetestarticledidnotinduce
forwardmutationinthemouselymphomaassay(LittonBionetics,Inc.,1978).
2.2.5 Reproductive toxicity
Rats
Inacombinedtwo-generationstudyofreproductivetoxicityandteratogenicity,
fvegroupsof22femaleand22maleweanlingCharles-Riverratsweregiventhe
disodium salt of HEDP (disodium etidronate) at a dietary concentration of 0, 0.1
or0.5%(equivalenttoadoseof0,50or250mg/kgbwperday),eithercontinu-
ously or only on days 615 of gestation for two generations. Reproductive end-
pointsandoffspringparameterswereanalysedintheF
1a
,F
1b
,andF
2a
litters.The
thirdlitteroftheF
1
generation(F
1c
)andthesecondlitterofthesecondgeneration
(F
2b
)wereusedinteratologicalexaminations.Duringtheteratologyphase,halfof
theanimalsineachgroupweresacrifcedatday13andtheothersatday21of
gestation.Body-weightgainsweresimilarforallgroupsinbothgenerations,and
theoverallconceptionratewas90%,indicatingthatthecompounddidnotinterfere
withspermatogenesisorovulation.Inthefrstgeneration,atthehighestdose,the
numberofpubsborninthefrstlitter(F
1a
)wasreducedandtherewasanincrease
96 PEROXYACID ANTIMICROBIAL SOLUTIONS
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instillbornpupsinthesecondlitter(F
1b
).Therateofmortalityinpupsafterbirth
waslowandtheweightsofpupsatday4andatweaningwerethesame.Terato-
logicalexaminationofthethirdlitter(F
1c
)showednodifferencesinresorptionsor
implantationsinfemalessacrifcedatday13(corporaluteawerenotcounted)and
nodifferencesinlivefetuses,corporalutea,orimplantationat21days.Atday21,
however,signifcantresorptionswerereportedinthecontrols.Inthesecondgen-
eration,thefrstlitters(F
2a
)weresmallerthanthelittersinthefrstgeneration,but
therewerenootherdifferencesinreproductiveparameters.Duringtheteratology
phase,nodifferencesincorporalutea,implantations,orresorptionswerenotedin
ratssacrifcedat13days.Incontinuallyfedratssacrifcedat21days,thenumber
of implantations was reduced and corpora lutea formation was depressed at the
highestdose.Adecreaseinthenumberoflivefetusesatthehighestdose,signif-
cantonlyinratsfedduringgestation,wasalsoobserved.Theincidenceofdefec-
tive pups was similar to that in control animals and the study authors concluded
that disodium etidronate was not teratogenic in rats at either dose tested. The
NOEL,basedonreducedlittersizeanddecreasednumberofpupsatthehighest
dose,was50mg/kgbwperday(Nolen&Buehler,1971).
Rabbits
Inacombinedstudyofreproductivetoxicityandteratogenicityinrabbits,two
separate experiments were conducted. In the frst experiment, four groups of 25
virgin New Zealand white rabbits were given HEDP (as an aqueous solution of
disodiumetidronate)atadoseof0,100,250or500mg/kgbwperdayviaintuba-
tionondays216ofgestation.Therabbitswereinseminated(day1)anddosing
commencedbeforeimplantation(day7).After45consecutivedosesofHEDPat
500mg/kgbwperday,thepregnantrabbitsdied.Fourrabbitssurvivedthreedaily
dosesofHEDPat500mg/kgbwperdayandtheseanimalssubsequentlyreceived
HEDPat250mg/kgfortherestofthestudy.Atadoseof100mg/kgbwperday,
HEDPcauseda68%reductionintheconceptionrateinrabbits.Owingtotoxicity
at500mg/kgbwperdayandareducedconceptionrateatthelowestdosetested
(i.e.100mg/kg bw per day), a second experiment was performed in which the
highestdosetestedwas100mg/kgbwperday.
Inthesecondexperiment,fourgroupsof25virginNewZealandwhiterabbits
weregivenHEDP(disodiumetidronate)atadoseof0(water),25,50,or100mg/kg
bw per day in the diet or 100mg/kg by gavage, on days 216 of gestation. An
additional untreated control group was also used in this study. Reproductive
parametersandoffspringmalformationswereanalysed.Theauthorsreportedno
statistical differences in food consumption or body-weight gain, although these
datawerenotpresented.However,theauthorsnotedthatrabbitsgiventhehighest
dose(100mg/kgbwperday)consumedtheleastamountoffoodandgainedthe
leastweight.Theconceptionrateindamsgivendisodiumetidronateatadoseof
100mg/kg bw per day by gavage or in the diet was 90% or 95%, respectively,
indicatingnoeffectonconceptionornidation.Nodifferenceswereobservedinthe
numbers of corpora lutea, resorptions, or live fetuses. Fetuses from dams given
disodium etidronate at a dose of 100mg/kg bw per day by gavage were signif-
cantlysmallerthanthosefromuntreatedcontrols.Nodifferencesinthedefective
PEROXYACID ANTIMICROBIAL SOLUTIONS 97
K2
fetuses in treated groups compared with the controls were reported. Very few
skeletaldefectswereseen,althoughvariationsinthenumberofribsandsterne-
brae occurred in up to 50% of the rabbit fetuses. The study authors stated that
thesevariationsintheribsandsternebraewerenotteratogeniceffects(Cozens,
1965).Theythusconcludedthattherewerenotreatment-relatedadverseeffects
on reproduction parameters and that disodium etidronate is not teratogenic in
rabbits(Nolen&Buehler,1971).
Nolan&Buehler(1971)speculatedthattheeffectsonconceptionrateinrabbits
given HEDP (disodium etidronate) at a dose of 100mg/kg per day in their frst
experimentmayhaveresultedfromstresscausedbygavage,becauseareduction
in conception rate was not observed in their second experiment. In the second
experiment,however,theauthorsreportedareductioninfetalweightswithHEDP
atadoseof100mg/kgbwperdayadministeredbygavage,whichtheyattributed
to slightly larger litters. Although, not statistically signifcant, decreases in food
consumptionandbody-weightgainindamsatthesamedosewerereportedinthe
second experiment. On the basis of decreased fetal weights, the NOEL was set
conservativelyat50mg/kgbwperday(Nolan&Buehler,1971).
2.2.6 Special study: skeletal effects in dogs
In a long-term study to determine the skeletal effects of HEDP, adult female
beagledogs(aged34yearsatthestartofthestudy)weregivenHEDPatadose
of0,0.1,0.5,2,5or10mg/kgbwperdayviasubcutaneousinjectionfordifferent
timesrangingfrom1to2years.Therewere10dogsinthecontrolgroupandfve
dogsineachtreatmentgroup.Dogsatthetwolowerdoses(0.1and0.5mg/kgbw
per day) were treated for 2 years. Dogs at 5mg/kg bw per day were sacrifced
after13.5months,whiledogsat2and10mg/kgbwperdayweresacrifcedafter
12 months.At the two lower doses, there was a slight reduction in osteoblastic
activity,reductioninthepercentageofbonesurfaceswithactivemineralization,a
reduction in mineralization rates, and a reduction in resorption spaces, but
no change in osteoid seam width. There were no treatment-related fractures at
0.1mg/kgbw,butradiographicstudiesindicatedthattheincidenceoffractureswas
slightlyincreasedat0.5mg/kgbwperday.Profoundeffectsonboneparameters
were observed at doses of 210mg/kg bw per day. The number of resorption
spaces was reduced and mineralization activity was blocked to the extent that
osteoidseamsbecamethickened.Atthesehigherdoses,theincidenceoffractures
was markedly increased and fractures were radiologically apparent after 912
months.Healingoffractures,whentheyoccurred,wasinhibitedatdosesofHEDP
of>0.5mg/kgbwperday.TheauthorssuggestedthathighdosesofHEDPdidnot
cause any permanent change in the skeleton that would interfere with fracture
healing.This study indicates that HEDP caused profound effects on the skeletal
systemthataredose-relatedanddependentontheperiodoftreatment,butthat
theeffectsarereversible(Floraetal.,1981).
Floraetal.(1981)alsopointedoutthatoraladministrationofthedisodiumsalt
ofHEDPatadoseof5mg/kgbwperdayforupto6monthsisrecommendedfor
thetreatmentofPagetdiseaseinhumans.Theauthorsindicatedthatthedoseof
HEDP that resulted in the development of spontaneous fractures in dogs was
98 PEROXYACID ANTIMICROBIAL SOLUTIONS
K2
about10timeshigherthanthedoserecommendedforextendeduseinhumans.
Thisisbasedontheassumptionthatgastrointestinalabsorptionoforallyadmin-
isteredHEDPwouldoccuratarateof1%inhumans.Thus,aorallyadministered
dose of HEDP of 5mg/kg bw per day would be expected to lead to a systemic
dose of 0.05mg/kg bw per day in humans, or 3mg/day for an adult with a body
weightof60kg.
2.3 Environmental studies
HEDPcanalsoundergophotolysistoacetateandphosphatewithinafewdays
(Steber & Wierich, 1986). In distilled water and in the presence of calcium, no
photodegradation of HEDP was observed, but the addition of Fe(III) and Cu(II)
resultedinrapidphotodegradation(Fischer,1993).Thus,aftertheuseoftheanti-
microbial solutions, residual HEDP in foods may undergo photolysis before the
treatedfoodsareconsumed.
2.4 Microbiological aspects
2.4.1 Role of components in antimicrobial solutions
Differentantimicrobialwashsolutionsareaddedtowatertospray,wash,rinse,
dip,coolorotherwiseprocessmeat,poultry,andfreshaswellasprocessedfruits
and vegetables. The solutions are used to inhibit the growth of Salmonella sp.,
Campylobacter jejuni,Listeria monocytogenes,andEscherichia coli O157:H7,and
spoilageanddecayorganismsontheproductorsurfacetobetreated(Table3).
Peroxyaceticacid(alsoreferredtoasperaceticacid)isthemajoractiveingredi-
ent in all of the antimicrobial wash solutions. The effect of peroxyacetic acid is
Table 3. The intended uses of four antimicrobial wash solutions
a
Solution Product/surfacetobetreated Function
A Poultrycarcasses,parts,and AntimicrobialeffcacyagainstSalmonella
organs sp.,C. jejuni,L. monocytogenes, E. coli
O157:H7andspoilageorganismson
poultry
B Meatcarcasses,parts,trims, AntimicrobialeffcacyagainstSalmonella
andorgans sp.,L. monocytogenes,E. a coli O157:H7
andspoilageorganismsonmeat
C Post-harvest,fresh-cut,and Antimicrobialeffcacyagainstspoilageand
furtherprocessedfruitsand decayorganismsontreatedfruitsand
vegetables,including vegetablesandinprocesswater
processwater
D Furtherprocessedfruitsand AntimicrobialeffcacyagainstS. a javiana,
vegetables L. monocytogenes,E. coli O157:H7,
spoilageanddecayorganismsonfurther
processedfruitandvegetablesurfaces.
a
SeeTable1forthecompositionofthesesolutions.
PEROXYACID ANTIMICROBIAL SOLUTIONS 99
K2
similartothatofotherantimicrobialagentsthatfunctionasoxidizingagents,and
which attack multiple cell sites and can disrupt the chemiosmotic balance. A
recentlypublishedsummary(Kitis,2004)statedthatperaceticacidwasidentifed
to have antimicrobial properties as early as 1902; that it had often been used in
cold sterilization procedures for medical instruments and had been found to be
bactericidalat0.001%,fungicidalat0.003%andsporicidalat0.3%;andthatithad
been used in the production of gnotobiotic (germ-free) animals. This publication
also proposed that the antimicrobial action of peracetic acid may result from the
oxidationofproteinsand,inparticular,theirsulfhydrylbonds.Alternatively,perace-
tic acid may disrupt the chemosmotic functions of outer membrane lipoproteins
andoxidizenitrogenousbasesinDNA,resultingincelldeath.Peraceticacidwas
compared favourably with chlorine-based compounds; it was proposed that its
antimicrobialeffcacywassimilaranditsdecompositiontotheenvironmentallysafe
productsaceticacid,waterandoxygenprovidesanadvantageoverchlorine-based
products.
Octanoicacidalsocontributestotheeffcacyoftheseantimicrobialsolutions.
ApublicationbySunetal.(2002)concludesthatatlowerpH,caproate(C6:0)and
caprylicacid(C8:0,thealternativenameforoctanoicacid)inhibitmicrobialgrowth.
In addition, octanoic acid functions as a surfactant to aid in wetting hydrophobic
surfaces,particularlyonmeat.
While acetic acid and hydrogen peroxide are known to have antimicrobial
effects,theireffectswithinthesesolutionsareminimal.Aceticacidandhydrogen
peroxideare,however,inequilibriumwiththeperoxyaceticacid,sotheirpresence
iscriticalfortheantimicrobialeffectsoftheperoxyaceticacid.Peroxyoctanoicacid
doesnothaveantimicrobialactivity.Itispresentinthesolutionbecauseitispro-
duced when octanoic acid reacts with hydrogen peroxide. HEDP has no anti-
microbialeffects.Itfunctionsasastabilizerinthesesolutionsbypreventingmetal
ionsfromcatalysingthebreakdownofperoxyaceticacidandhydrogenperoxide.
2.4.2 Studies of antimicrobial effcacy
Laboratoryandin-plantstudiesweredoneonfourantimicrobialwashsolutions,
describedassolutionsA,B,CandDinTables1and3,todemonstratethereduc-
tionofmicrobesfortheintendeduseofeachsolution.Overall,theresultsofthese
testsindicatemodestreductionsinthenumberofsurfacemicrobesonpoultryand
meat.Inwashwaterforfreshandprocessedfruitsandvegetables,greaterreduc-
tionsinconcentrationsofmicrobeswereobserved.Theresultsofstudiesthatwere
availableforthisevaluationaredescribedbelow.
(a) Solution A
TheproposeduseofantimicrobialwashsolutionAisforadditiontowaterused
forsprayingorsubmerging,orsprayingfollowedbysubmergingevisceratedpoultry
carcasses. Tests were done to compare specimens treated with water with
those treated with the test substance. Thus, the key result is the net reduction
beyond that found with water only. There were three groups, a group that was
submersion-chilled,agroupthatwassprayed,andagroupthatwassprayed,then
100 PEROXYACID ANTIMICROBIAL SOLUTIONS
K2
submerged. The mean log
10
reductions using United States Department ofAgri-
culture procedures for carcass processing are listed in Table 4. These results
indicatethatamodestnetreductionofuptoaboutlog
10
0.8canbeachievedfrom
thesetreatments(unpublisheddatafromthesubmitter).
Asecondsetoftestswasperformedonpathogens(Listeria monocytogenes,
Salmonella typhimurium, andEscherichia coli O157:H7)ondifferentchickenparts
(carcasses, wings, and livers). Net log
10
reductions in pathogens varied from a
modesttoaconsiderableamount(fromlog
10
0.32to0.75forS. typhimurium, from
log
10
1.13 to 2.11 for L. monocytogenes, and from log
10
0.82 to 3.17 for E. coli
O157:H7).
(b) Solution B
TheproposeduseofantimicrobialwashsolutionBisforaddingtowaterused
forsprayingbeefcarcasses.Thesolutionwasdilutedappropriatelyandaddedto
waterforsprayingbeef.Threeseparatetestrunswereconducted.Inthefrsttest,
10 randomly selected carcasses were selected; in the second test, 2930 ran-
domly selected carcasses were selected, and in the third, 128 carcasses were
selected in-plant. In all tests, the carcasses were aseptically sampled by tissue
excision,eitherbeforetreatment,aftertreatment,oratfnalinspection,thenserially
dilutedandplated.Thenumberofcoloniesformed(CFU/cm
2
)forallaerobicbac-
teria(totalaerobicplatecounts),coliforms,andE. coliweredetermined.Forthese
trials,reductionsrangedfromlog
10
0.434(standarddeviation(SD),1.083)to1.05
(SD, 0.495) for samples taken immediately after treatment and from log
10
0.246
(SD, 1.221) to 0.573 (SD, 0.567) at the fnal inspection. In essence, the values
indicated that a modest, but highly variable, initial reduction of microorganisms
was followed by some renewed microbial growth or acquisition of more
microbes.
When pathogens were inoculated onto beef, reductions in the numbers of
microbesweremodest,approximatelylog
10
0.5to1.0morethanreductionsafter
washingwithwateronly.ThespecifcresultsaresummarizedinTable5.Therela-
tivereductionsreportedweremodest,rangingfromlog
10
0.5to1.3(unpublished
datafromthesubmitter).
Microbial contamination primarily occurs on the surface of meats. Various
sprayinganddippingmethods,usuallytransientinnature,areemployedtoremove
surfacebacteria.Althoughseveralchemicalsareemployedinthesemethods,the
levels of reduction of microbes, with respect to resident bacteria and specifc
pathogens,aretypicallylow.Inarecentpublication,theuseofoneoftheseprod-
uctswascomparedwithothermethods(Gill&Badoni,2004);theresultsindicated
that use of a solution containing 0.02% peroxyacetic acid was associated with
modest reductions in the number of pathogens from log
10
0.5 to 1.0 l compared
with meat treated with water only, but reductions after treatment with lactic acid
werelog
10
>1.
PEROXYACID ANTIMICROBIAL SOLUTIONS 101
K2
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102 PEROXYACID ANTIMICROBIAL SOLUTIONS
K2
(c) Solution C
TheproposeduseofantimicrobialwashsolutionCisforadditiontowaterused
for processing vegetables for post-harvest, fresh-cut, and further processed fruit
andvegetables.Peroxyaceticacidataconcentrationof1030mg/kgwasadded
towaterforprocessingvegetables.Therewasareductionofupto4-log(log
10
4)
intherelativeconcentrationsofmicroorganismsfoundinthetreatedwashwater,
compared with the untreated wash water; this correlated with the amount of
residualperoxyaceticacid(Table6)(unpublisheddatafromthesubmitter).
(d) Solution D
TheproposeduseofantimicrobialwashsolutionDisforreductionofcontami-
nation,eitherfororganismsonsurfacesorforcross-contaminationinwashwater,
onthesurfaceofprocessedfruitandvegetables.Totestthereductionofcontami-
nation,tomatosurfaceswereinoculatedwithE. coli O157:H7,L. monocytogenes,
andS.javianaandtreatedwitheithertapwaterorTsunami200.Theresultsindi-
catedthatsolutionDeffectivelyreducednumbersofthesepathogens(Table7).
Totestfortheeliminationofcross-contamination,cherrytomatoeswereinocu-
latedwiththesametargetpathogens,whichwereallowedtoattachfor24h.Inocu-
lated and non-inoculated cherry tomatoes were then submerged in solution D
(Tsunami 200) or in tap water. The non-inoculated tomatoes were removed to a
Table 6. Mean log
10
reductions in microorganisms found in water treated with
antimicrobial wash solution C relative to untreated water
Residualperoxyaceticacid(mg/kg) Log
10
reduction(log
10
CFU)
<3 2
1030 24
4050 56
Fromunpublisheddatafromthesubmitter.
CFU,colony-formingunits.
Table 5. Mean log
10
reductions in specifc pathogens inoculated onto beef
washed with water or with antimicrobial wash solution B
Pathogen Averagelog
10
reduction Relativelog
10
reduction,
Water SolutionB
solutionBrelativetowater
L. monocytogenes 0.7 1.22 0.52
S. typhimurium 0.32 1.62 1.3
E. coli 0.4 1.48 1.08
Fromunpublisheddatafromthesubmitter.
PEROXYACID ANTIMICROBIAL SOLUTIONS 103
K2
neutralizing solution, which was vortexed to remove bacteria from the tomatoes,
thendilutedandplated.Therewasareductionofgreaterthan2-log(log
10
2)inall
three pathogens transferred by cross-contamination (unpublished data from the
submitter).
2.5 Observations in humans
ThedisodiumsaltofHEDP,whichisknownclinicallyassodiumetidronate,is
usedtotreatPagetdisease,whichisanidiopathicdiseasecharacterizedbyaccel-
eratedbonemetabolism.Fracturesandotherabnormalitiesofbonearecommon
in patients with Paget disease. Due to its high affnity for solid-phase calcium
phosphate,HEDPpreventshydroxyapatitecrystalgrowthanddissolutiononcrystal
surfacesofbone.Itsmechanismofaction,however,isnotfullyunderstood.
The recommended dose of sodium etidronate is 510mg/kg bw given orally
oncedailyfor6monthsorless,or1120mg/kgbwperdayfor3monthsorless.
Doses in excess of 20mg/kg bw per day are not recommended.The dose must
bereducedincasesofrenalinsuffciency.Sodiumetidronateisgenerallywelltoler-
ated and the incidence of side-effects is low (Center for Drug Evaluation and
Research,2001;PhysiciansDeskReference,2004).Initialtherapywithadoseof
5mg/kgbwperdayofsodiumetidronateappearstomaximizebeneftsforpatients
with Paget disease while minimizing possible adverse effects (Canfeld et al.,
1977).
Numerousabstracts/citationsaddressingtheuseofHEDPincancertherapy,
osteoporosis, nuclear imaging, and hypercalcaemia associated with malignancy,
and other disorders of calcium and phosphorus balance have been published.
Such studies were not considered to be relevant to food safety and are beyond
thescopeofthisassessment.
3. INTAKE
3.1 Residues on foods
The use of the four solutions of peroxyacid in antimicrobial water washes for
the processing of meat, poultry, fruits, and vegetables results in predictable
Table 7. Mean log
10
reductions in pathogens on tomato surfaces treated with
water only or with antimicrobial wash solution D
Pathogen Pathogensontomatosurfaces(log
10
CFU)
Water SolutionD Log
10
reduction
L. monocytogenes 4.73 0.00 4.73
E. coli 5.00 0.87 4.13
S. javiana 2.62 0.00 2.62
Fromunpublisheddatafromthesubmitter.
104 PEROXYACID ANTIMICROBIAL SOLUTIONS
K2
residuesontreatedfoods.Thehydrogenperoxideinthesolutionandtheperoxy-
aceticandperoxyoctanoicacidsformedinsituareinherentlyunstable,especially
in the presence of oxidizable organic material. Therefore, there would be no
expectedresiduesofthesesubstancesontreatedfoods.Aceticandoctanoicacid
present in the solution and as by-products from the corresponding peroxyacids
wouldbeexpectedtoremainonanytreatedfoodsthatarenotwashedorfurther
processedaftertreatment,aswouldHEDP,whichisstableandnon-reactiveunder
theconditionsofuse.
Acetic and octanoic acids are components of many foods and are also used
as favouring agents in foods. The Committee has previously evaluated both of
thesesubstances.Theminorresiduesofthesesubstancesremainingontreated
foods result in exposures that are insignifcant in comparison to those from con-
sumption of foods containing the substances naturally, or as added favouring
agents.Themeanintakeofoctanoicacidfromfoodsconsumedaspartofthediet
intheUSAwasestimatedtobeapproximately200mg/day.Ahighlyconservative
estimateofexposureforoctanoicacidof1.9mgperdayresultingfromtheuseof
the antimicrobial solutions was noted by the Committee. This estimate was pre-
paredemployingWHOGlobalEnvironmentMonitoringSystemFoodContami-
nation Monitoring andAssessment Programme (GEMS/Food) international diets.
Intake of acetic acid was not explicitly analysed, but its use in and on foods
(vinegar) would result in a greater food exposure than that from octanoic acid.
Exposure to these common food acids was not further considered in this
evaluation.
HEDP is expected to remain on foods that are treated with the antimicrobial
washesandnotfurtherwashed,processed,orcooked.TheCommitteeconsidered
submittedinformationconcerningresiduesofHEDPonfoods.Studieswerecon-
ducted with meats, poultry, fruits, and vegetables, each treated with one of the
solutions under typical conditions of use. The foods were allowed to drain, but
were not further processed or cooked. It was assumed that all additional weight
inthemeatstreatedwasattributabletotheantimicrobialwash;theconcentration
of HEDP in the solutions was used to determine the residual concentration of
HEDP present in the meat. Poultry was further treated to recover any HEDP
present. Fruit and vegetables were washed with deionized water to recover the
residualHEDP.Forvegetables,lower-andupper-boundestimatesofintakewere
madebasedonthedifferingsurfaceareasofthetreatedfoods.Broccoli,avegeta-
ble with a high surface area, provided the data for the upper-bound estimates,
while tomato was used to provide the lower-bound estimates. Furthermore, for
processedfruitandvegetables,itwasassumedthateachwouldbetreatedtwice;
beforecuttingorprocessingandagainafterwards.Thus,themeasuredresidues
weredoubled,assumingnolossfromeithertreatment.Theresultsarereportedin
Table8(unpublisheddatafromthesubmitter).
3.2 International estimates of intake
TheCommitteeconsideredinternationalestimatesofintakeofHEDP,prepared
usingfoodinformationtakenfromtheGEMS/Foodregionaldietsandthedataon
HEDPresiduesfromTable8.Theintakeofeveryfoodthatcouldbetreatedwith
PEROXYACID ANTIMICROBIAL SOLUTIONS 105
K2
HEDP was combined with the appropriate residue concentration for each of the
fve regional diets. Two estimates were prepared for each region; one using the
residueconcentrationforavegetablewithalowsurfaceareaandtheotherusing
theresidueconcentrationforavegetablewithahighsurfacearea.Itwasassumed
that there would be no reduction in HEDP residues after washing or cooking.
Further,itwasassumedthatallfruitandvegetableswouldbetreatedthreetimes
withtheantimicrobialsolutionwithnoloss;onceontherawcommodityandtwice
duringfurtherprocessing.
ThehighestestimateofintakewasfromtheEuropeandiet;3.6mg/kgbwper
dayfortheupper-boundestimateusingamodelforavegetablewithahighsurface
area.AlltheestimatesaresummarizedinTables9and10.
3.3 National estimates of intake
TheCommitteeconsideredthreenationalestimatesofintake.Thefrstwasa
totaldietstudyfromtheCzechRepublic.Thetworemainingstudieswerebased
onindividualdietaryrecordsintheUSAandtheUK,respectively.
Completedin1995,theCzechtotaldietstudyconsidered160foodstuffs.The
foodswerepreparedusingstandardrecipes.Eachfoodthatmightbetreatedwith
theantimicrobialsolutionwasconsidered,withafoodintakematchedtoanHEDP
concentrationfromTable8.Lower-boundandupper-boundestimatesweremade
using the data for vegetables with low surface area and data for the vegetables
with high surface area separately. Average daily food consumption values
for the Czech Republic were used. The lower-bound estimate of exposure was
0.405mg/kg bw per day and the upper-bound estimate was 2.224mg/kg bw per
day.
TheestimatesfromtheUSAandtheUKweremadeinasimilarmanner.For
each individual surveyed, all foods that could have been treated with the antimi-
crobialsolutionwereconsidered.TheappropriateconcentrationofHEDPresidue
was multiplied by the intake of each food and the total intake of HEDP for each
Table 8. Residues of HEDP in treated foods
Typeoffoodtreated ResidueofHEDP(mg/kg,ppb)
Meats
Carcasses 58
Parts/trim 161
Poultry 198
Fruitandvegetables(singletreatment)
Lowsurfacearea 4.2
Highsurfacearea 67.5
Fruitsandvegetables(doubletreatment)
Lowsurfacearea 8.4
Highsurfacearea 135
Fromunpublisheddatafromthesubmitter.
106 PEROXYACID ANTIMICROBIAL SOLUTIONS
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Table 9. International estimates of intake of HEDP (lower bound)
GEMS/ Food HEDP IntakeofHEDP(mg/kgbwperday)inGEMS/
Food residue Foodregionaldiet
code (mg/kg,ppb)
Middle Far Africa Latin Europe
East East America
VR75 Roots 12.6 0.013 0.023 0.067 0.033 0.051
VD70 Pulses 12.6 0.005 0.004 0.004 0.005 0.003
VD70 Nuts 12.6 0.003 0.011 0.007 0.012 0.006
VD70 Vegetablefat 12.6 0.008 0.003 0.005 0.005 0.008
HS93 Spices 12.6 0.001 0.001 0.000 0.000 0.000
HS93 Vegetables 12.6 0.049 0.038 0.016 0.032 0.078
PE112 Fruit 12.6 0.043 0.018 0.020 0.057 0.045
MO105 Offal 68 0.005 0.002 0.003 0.007 0.014
MO105 Meat 68 0.042 0.037 0.027 0.053 0.176
PM110 Poultry 198 0.102 0.044 0.018 0.083 0.175
PO111 Poultryoffal 198 0.000 0.000 0.000 0.001 0.001
PF111 Poultryfat 198 0.010 0.004 0.002 0.008 0.017
MF95 Mammalian 68 0.001 0.002 0.001 0.005 0.009
fat
Totalintake 0.321 0.222 0.114 0.211 0.753
Table 10. International estimates of intake of HEDP (upper bound)
GEMS/ Food HEDP IntakeofHEDP(mg/kgbwperday)inGEMS/
Food residue Foodregionaldiet
code

(mg/kg,ppb)
Middle Far Africa Latin Europe
East East America
VR75 Roots 202.4 0.208 0.366 1.084 0.537 0.816
VD70 Pulses 202.4 0.083 0.067 0.060 0.078 0.041
VD70 Nuts 202.4 0.043 0.169 0.115 0.194 0.101
VD70 Vegetablefat 202.4 0.136 0.048 0.079 0.074 0.130
HS93 Spices 202.4 0.008 0.010 0.006 0.002 0.002
HS93 Vegetables 202.4 0.786 0.604 0.260 0.508 1.254
PE112 Fruit 202.4 0.689 0.288 0.319 0.915 0.716
MO105 Offal 68 0.005 0.002 0.003 0.007 0.014
MO105 Meat 68 0.042 0.037 0.027 0.053 0.176
PM110 Poultry 198 0.102 0.044 0.018 0.083 0.175
PO111 Poultryoffal 198 0.000 0.000 0.000 0.001 0.001
PF111 Poultryfat 198 0.010 0.004 0.002 0.008 0.017
MF95 Mammalian 68 0.001 0.002 0.001 0.005 0.009
fat
Totalintake 2.153 1.676 1.994 2.515 3.623
PEROXYACID ANTIMICROBIAL SOLUTIONS 107
K2
foodwascalculatedforeachindividual.Themeanand90th-percentileintakesfor
thewholepopulationwerecomputedfromtheindividualrecords.Thedataonfood
intakefromtheUSAweretakenfromtheUSADepartmentofAgricultureContinu-
ingSurveyofFoodIntakesbyIndividuals,19946,1998.Thedataonfoodintake
from the UK were taken from the Ministry of Agriculture, Food, and Fisheries
DietaryandNutritionalSurveyofBritishAdults,19867.Hereagain,lower-bound
and upper-bound estimates were made using the data for vegetables with low
surfaceareaandvegetableswithhighsurfaceareaseparately.
The mean estimate of intake for the USA was 0.357 (lower bound) or 2.235
(upperbound)mg/kgbwperday.Thecorrespondingintakesforindividualsatthe
90th percentile of consumption were 0.740 and 4.706mg/kg bw per day, respec-
tively.ThemeanestimateofintakefortheUKwas0.243(lowerbound)or1.795
(upper bound) mg/kg bw per day. The corresponding intakes for individuals at
the 90th percentile of consumption were 0.458 and 3.263mg/kg bw per day,
respectively.
Table 11 summarizes the estimates of intake of HEDP used in antimicrobial
washsolutions.
3.4 Non-food uses of HEDP
HEDPisusedasananti-scalingagentforwatertreatmentandinboilers.The
regulatorylimitforthisuseintheUSAis25mg/l.However,HEDPisknowntobe
usedintherestoftheworld,includingChina.Itisalsousedasadrugfortreat-
ment of Paget disease (a disease of excessive bone turnover) and in some
over-the-counter cosmetic and pharmaceutical formulations. The Environmental
ProtectionAgencyintheUSAhasestimatedexposuretoHEDPfromtheseuses
to be no more than 6mg/kg bw per day, including 0.04mg/kg bw per day from its
useonfood(EnvironmentalProtectionAgency,1998).TheCommitteenotedthat
thisestimateofexposureforfoodusesofHEDPwasmuchlessconservativethan
that evaluated herein, assuming that cooking and further processing of treated
foodswouldresultinconcentrationsofHEDPofnogreaterthan1mg/kgonfood
asconsumed.
Table 11. Estimates of intakes of HEDP used in
antimicrobial wash solutions
Estimates Exposure(mg/kgbwperday)
GEMS/Food 3.6(Europeanregionaldiet)
CzechTotalDietStudy 2.224(mean)
USAdietaryrecords 4.706(90thpercentile,upper
bound)
UKdietaryrecords 3.263(90thpercentile,upper
bound)
108 PEROXYACID ANTIMICROBIAL SOLUTIONS
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4. STUDIES ON THE QUALITY, NUTRITIONAL VALUE, OR OTHER
PROPERTIES OF FOOD TREATED WITH ANTIMICROBIAL
SOLUTIONS
4.1 Thiobarbituric acid and fatty acid profles of meat and
poultry products
The antimicrobial wash solution identifed as solution A was added to water
usedforsprayinganddippingpoultrycarcassesinastudytodeterminewhether
the treatment resulted in signifcant differences in thiobarbituric acid (TBA) and
fatty acid profles of raw or cooked poultry products. No differences were found
whencomparedwithtreatmentwithwater(Ecolab,Inc.,2000).
Samples of fresh beef were exposed to antimicrobial wash solution B, which
containstotalperoxyacidsataconcentrationof200mg/kg,todeterminewhether
the treatment resulted in signifcant differences in TBA and fatty acid profles of
cookedanduncookedmeat(Ecolab,Inc.,1999a,1999b).Cookingtoaninternal
temperatureof175FincreasedtheTBAvaluebyeightfoldrelativetouncooked
samples.NodifferencesinTBAorfattyacidproflescomparedwithtreatmentwith
water were found.There was a slight difference (p = 0.54) in values for myristic
acidbetweenrawmeatandcookedmeattreatedwithperoxyacid;thiswasattrib-
utedtocookingorvariationinthemeatsamplestested.
The reagentTBA is commonly used to determine the extent to which animal
and vegetable fats and oils (including fatty acids, their esters, and related sub-
stances) are oxidized. Thus TBA values provide a measure of rancidicity. The
results of testing for TBA and the determinations of fatty acid profles described
above suggested that treating poultry or meat with solutions A or B did not
adverselyimpactthequalityoftreatedpoultryormeatproducts,respectively.
4.2 The effect of the potential reactivity of hydrogen peroxide and
peroxyacetic acid on meat and poultry products
In a study by Upendraroa et al. (1972), vegetable oils placed in contact with
30% or 60% hydrogen peroxide and 5% or 17% peroxyacetic acid for 210h
underwent epoxidation. Other studies that used high concentrations of hydrogen
peroxideandlongperiodsofcontactwerefoundintheliteratureandmainlyindi-
cated potential reactions of hydrogen peroxide with other food components.The
low concentrations of the components of antimicrobial solutions in ready-to-use
wash solutions and sprays, and the transient nature of their contact with food is
expectedtopreventpotentialoxidationreactionsfromoccurringonfood.Thelow
reactivity potential of solutions C and D has been confrmed in a study of their
effects,undertheintendedconditionofuse,onfruitandvegetables(Ecolab,Inc.,
1995).
4.3 Nutritional tests to determine the effects of peroxyacetic acid and
hydrogen peroxide on fruit and vegetables
A study was conducted to determine the effects of peroxyacetic acid and
hydrogen peroxide on the nutrient content of fruit and vegetables (Ecolab, Inc.,
PEROXYACID ANTIMICROBIAL SOLUTIONS 109
K2
1995).Tomatoes,potatoes,andbroccoliwerepreparedforconsumption,exposed
tosolutionC(containingperoxyaceticacidat80mg/kgandhydrogenperoxideat
59mg/kg)for5min(worse-caseconditionsofexposure),thenrinsed.Controland
treatedsampleswereanalysedforeffectsonb-caroteneandvitaminC,nutrients
chosenforanalysisbecauseoftheirsusceptibilitytooxidationandotherdegrada-
tionreactions.Therewasnoeffectonb-carotenecontentintomatoesorbroccoli.
TherewasnoeffectonvitaminCinpotatoesorbroccoli.Therewasatreatment-
relateddecreaseof37%intheascorbicacidcontentoftomatoes,whichoccurred
inconjunctionwithanequivalentincreaseindehydroascorbicacidcontent.Thus,
the active content of vitamin C (Sabry et al., 1958) in tomatoes was unchanged
(unpublished data from the Pillsbury Company).These results indicated that the
useofantimicrobialwashsolutionConfreshfruitsandvegetableswouldnotbe
expectedtoadverselyaffecttheirnutrientcontent.
5. COMMENTS
Antimicrobialsolutionsareequilibriumsolutionsthataredilutedinwaterbefore
use in food processing. Hydrogen peroxide in these solutions will dissociate into
waterandoxygen.Bothperoxyaceticacidandperoxyoctanoicacidarealsoinher-
entlyunstableandwillbreakdownintoaceticacidandoctanoicacid,respectively,
althoughtheirstabilityisenhancedbyHEDP.Lowresidualamountsofthesesimple
organic acids present on food at the time of consumption would pose no safety
concern. It is not expected that residues of peroxyacetic acid or peroxyoctanoic
acidfromthesesolutionswillbepresentontreatedfoodsatthetimeofconsump-
tion.Theperoxidecomponentsoftheperoxyacidantimicrobialsolutionsthuspose
no toxicological concerns with regard to the uses considered by the Committee.
TheCommitteeconcludedthatHEDP,whichsequestersmetalions,therebysta-
bilizing the peroxy compounds in peroxyacid antimicrobial solutions, is the only
componentofpotentialtoxicologicalconcern.
DatareviewedbytheCommitteeindicatedthatabsorptionofHEDPfromthe
gastrointestinaltractisverylimitedandthatitsmetabolismisnegligible.Thelimited
amount of data available to the Committee suggested that absorption may be
related to age and species. The skeleton is the target site for the disposition of
HEDPinallspecies.
HEDPdidnotshowevidenceofmutagenicactivityinassaysinfvestrainsof
Salmonella or in an assay for mutation in mouse lymphoma L51718 Tk
+/-
cells,
withandwithoutmetabolicactivationfrommammalianmicrosomes.
Intwo90-daystudiesoftoxicity,ratswerefeddietscontainingHEDPatdoses
rangingfrom100to2500mg/kgbwperday.Thehighestdosetestedineachstudy
(i.e.1500or2500mg/kgbwperday)causedmortalityandsignsoftoxicity,butno
effectswerereportedatlowerdosesineitherstudy.TheNOELwas500mg/kgbw
perdayinbothstudies.
Ina90-daystudyoftoxicityindogs,HEDPwasadministeredorallyatadose
equivalentto0,25,75,or250mg/kgbwperday.Noadverseeffectsattributable
to treatment were reported and the NOEL for HEDP was 250mg/kg bw per day.
The Committee also evaluated the results of a long-term study to determine the
110 PEROXYACID ANTIMICROBIAL SOLUTIONS
K2
skeletal effects of daily subcutaneous injections of HEDP administered to adult
femaledogsforvaryingperiodsrangingfrom1to2years.Someeffectsonbone
parameterswereobservedatalldoses.Profoundskeletaleffectswereassociated
with the administration of daily subcutaneous doses of HEDP of 210mg/kg bw
per day for 1 year. Spontaneous bone fractures were slightly increased in dogs
given daily subcutaneous doses of 0.5mg/kg bw for 2 years, but no permanent
skeletalchangeswereobservedatthisdoseandhealingwasnormal.Nofractures
wereobservedatadailysubcutaneousdoseof0.1mg/kgbwafter2years.Assum-
ingthat1020%oftheadministereddosewereabsorbedfromthegutindogs,a
subcutaneousdoseof0.1mg/kgbwperdaywouldcorrespondtoanoraldoseof
0.51mg/kg per day. In considering these studies, the Committee noted that 90
daysmightnotbelongenoughtoobserveskeletaleffectsindogsandthatthere
might be differences in the disposition of HEDP in bone that are related to the
routeofadministration.
Inacombinedtwo-generationstudyofreproductivetoxicityandteratogenicity,
rats were given HEDP (disodium salt) in the diet at concentrations equivalent to
0,50or250mg/kgbwperdayeitherduringtheirlifetimeoronlyondays615of
gestation, for two generations. No fetal abnormalities indicative of a teratogenic
effectwerereportedateitherdosetested.HEDPwasembryotoxicwhenadminis-
tered at a dose of 250mg/kg bw per day during organogenesis. The NOEL for
HEDPwas50mg/kgbwperday.
TheeffectsofHEDP(disodiumsalt)weredeterminedinacombinedstudyof
reproductivetoxicityandteratogenicityinrabbits.Twoexperimentswereperformed
because of the observation of toxicity at the lowest and highest doses, adminis-
teredbygavage,inthefrstexperiment.Inthesecondexperiment,rabbitsreceived
HEDP at a dose of 0, 25, 50, or 100mg/kg bw per day in the diet, or 100mg/kg
bwperdaybygavage.FetusesfromdamsreceivingHEDPatadoseof100mg/kg
bw per day by gavage were signifcantly smaller than those from untreated con-
trols. No fetal abnormalities indicative of a teratogenic effect in rabbits were
observedineitherexperiment.TheNOELwas50mg/kgbwperday.
Use of HEDP to treat Paget disease
ThedisodiumsaltofHEDP,knownclinicallyassodiumetidronate,isadminis-
teredorallyatastartingdoseof5mg/kgbwperday,fornotlongerthan6months,
totreatpatientswithPagetdisease.Pagetdiseaseisanidiopathicdiseasechar-
acterized by accelerated bone metabolism; fractures and other abnormalities of
thebonearecommoninpatientswithPagetdisease.Owingtoitshighaffnityfor
solid-phase calcium phosphate, HEDP prevents the growth and dissolution of
hydroxyapatite crystals on crystal surfaces of bone. The mechanism of action,
however,isnotfullyunderstood.
Antimicrobial effcacy
InformationavailabletotheCommitteeindicatedthatsolutionsofperoxyacetic
acidenhancetheactionofwatersprayedonfoodsurfacestoreducenumbersof
bacteria. While reductions in numbers of microbes were demonstrated, some of
PEROXYACID ANTIMICROBIAL SOLUTIONS 111
K2
thedataprovidedsuggestthattheresultsofreplicatetestswereratherinconsis-
tent,withstandarddeviationsclosetoorgreaterthanthevalueofthereductions
themselves. Testing of food surfaces showed modest reductions in numbers of
microbes,wheneitherendogenousmicroorganisms(representedbytotalaerobic
platecounts)orinoculated(spiked)pathogens(commonlyL. monocytogenes, E.
coli O157:H7,andsomeSalmonellaserotypes)weremonitored.Datafromlabora-
toryandin-planttestsindicatedthattheuseofthesesolutionswouldminimizethe
possibilityofcross-contamination,althoughtheyareunabletoremovealladherent
viablebacteriafromfoodsurfaces.
TheCommitteedidnotfurtherconsidertheantimicrobialeffcacyofperoxyacid
antimicrobialsolutionscontainingHEDP.
Intake
TheCommitteeevaluatedestimatesofintakeofeachcomponentusedinthe
peroxyacid solutions on the basis of residual amounts anticipated to be present
ontreatedfoodatthetimeofconsumption.Consistentwithwhatwasknownabout
the chemistry of peroxy compounds, no residues of hydrogen peroxide, peroxy-
acetic acid, or peroxyoctanoic acid were anticipated to be present on foods that
havebeenwashedin,sprayedwith,orotherwisetreatedusingthesesolutions.
Aceticandoctanoicacidpresentinthesolutionsandasby-productsfromthe
correspondingperoxyacidswouldbeexpectedtoremainonanytreatedfoodsthat
are not washed or further processed after treatment. The Committee noted that
theestimateofexposuretooctanoicacidresultingfromtheuseoftheantimicrobial
solutions,1.9mg/day,washighlyconservative.Themeanintakeofoctanoicacid
fromfoodsconsumedaspartofthedietintheUSAwasestimatedtobeapproxi-
mately200mg/day.Intakeofaceticacidwasnotexplicitlyanalysed,butitsusein
andonfoods(vinegar)wouldresultinagreaterexposurethanthatfromtheuse
of peroxyacid antimicrobial solutions. The Committee did not further consider
exposuretothesecommonfoodacids.
HEDPisexpectedtoremainonfoodsthataretreatedwithantimicrobialsolu-
tionsandthatarenotfurtherwashed,processed,orcooked.Thehighestestimate
of intake of HEDP prepared using GEMS/Food diets was that for the European
diet:3.6mg/kgbwperdayfortheupper-boundestimateusingamodelforvegeta-
bleswithahighsurfacearea.TheCommitteealsoconsiderednationalestimates
ofintakefromtheCzechRepublic,theUSA,andtheUK.Theupper-boundesti-
mateofintakewas2.2mg/kgbwperdayfortheCzechRepublic.Themeanand
90thpercentileupper-boundestimatesofintakefortheUSAwere2.2and4.7mg/kg
bw per day, respectively. The mean and 90th percentile upper-bound estimates
of intake for the UK were 1.8mg/kg bw per day and 3.3mg/kg bw per day,
respectively.
The Committee was aware of the non-food uses of HEDP. It is used as an
anti-scalant for water treatment and in boilers worldwide (the regulatory limit for
thisuseis25mg/lintheUSA).HEDPisalsousedasadrugtotreatPagetdisease,
andinsomeover-the-countercosmeticandpharmaceuticalformulations.TheUSA
EnvironmentalProtectionAgencyestimatedthatexposuretoHEDPfromallthese
112 PEROXYACID ANTIMICROBIAL SOLUTIONS
K2
useswasnotmorethan6mg/kgbwperday,including0.04mg/kgbwperdayfrom
its use on food (Environmental ProtectionAgency, 1998). The Committee noted
that this estimate of exposure resulting from food uses of HEDP was much less
conservativethanthatusedinthepresentevaluation.
Assessment of the effects on food quality and nutritional value
Limiteddataonthequalityandnutritionalvalueoffoodstreatedwithperoxyacid
antimicrobialsolutionswereprovidedtotheCommittee.Studieswereconducted
to determine whether treatment of foods with peroxyacid antimicrobial solutions
resultedinsignifcantdifferencesinconcentrationsofthiobarbituricacid(ameasure
ofrancidity),orinfatty-acidprofletestingofraworcookedpoultryproductsand
freshbeefsamples,whencomparedwithtreatmentwithwateronly.Nodifferences
werefound.
The Committee was aware that studies in the literature indicated potential
reactions of hydrogen peroxide with components of food. The Committee noted
that such studies are typically conducted using high concentrations and long
periods of exposure and that, under the conditions of their intended use, the
potentialreactivityofperoxyacidantimicrobialsolutionsisexpectedtobelimited.
Studies available to the Committee confrmed the low potential reactivity of two
peroxyacidantimicrobialsolutionsindiluteready-to-usesolutionsthatareinbrief
contactwithfruitsandvegetables.
A study was conducted to determine the effects of peroxyacetic acid and
hydrogenperoxideonthecontentofb-caroteneandvitaminCintomatoes,pota-
toesandbroccoli.Thesefoodswerepreparedforconsumptionusingworst-case
exposureconditions,i.e.peroxyaceticacidat80mg/kgandhydrogenperoxideat
59mg/kg for 5min, and then rinsed. When treated samples were compared with
controls,therewerenoeffectsontheb-carotenecontentoftomatoesorbroccoli,
onthevitaminCcontentofpotatoesorbroccoli,orontheactivevitaminCcontent
oftomatoes.
Onthebasisoftheavailabledata,theCommitteeconcludedthatperoxyacid
antimicrobial solutions are unlikely to have an adverse effect on food quality or
nutritionalvalue,withregardtotheusesconsideredbytheCommittee.
6. EVALUATION
The Committee considered the safety, on a component-by-component basis,
of antimicrobial solutions containing HEDP and three or more of the following
components:peroxaceticacid,aceticacid,hydrogenperoxide,octanoicacidand
peroxyoctanoic acid. These solutions are intended to be diluted before use to
achieveperoxyacidconcentrationsintherangeof80to220mg/kg.TheCommittee
concluded that the peroxy compounds in these solutions (hydrogen peroxide,
peroxyaceticacidandperoxyoctanoicacid)wouldbreakdownintoaceticacidand
octanoicacid,andthatsmallresidualquantitiesoftheseacidsonfoodsatthetime
of consumption would not pose a safety concern. Therefore, the Committee
focused its evaluation on the residues of HEDP that are expected to remain on
PEROXYACID ANTIMICROBIAL SOLUTIONS 113
K2
foods treated, in accordance with manufacturers instructions, with peroxyacid
antimicrobialsolutionsthatcontainHEDPatupto<1%.
The Committee compared the highest estimate of intake of HEDP from the
uses of peroxyacid antimicrobial solutions considered by the Committee (i.e.
0.004mg/kg bw per day) with the starting oral dose used to treat Paget disease
(i.e.5mg/kgbwperday)andnotedthatthemarginofexposureis>1000.Onthe
basisofthismarginofexposure,theconservativenatureoftheestimatesofintake
ofHEDP,andtheavailabletoxicitydata,theCommitteeconcludedthatHEDPdoes
not pose a safety concern at the concentrations of residue that are expected to
remainonfoods.
TheCommitteenotedthattheuseofperoxyacidantimicrobialsolutionsdoes
not replace the need for good hygienic practices in handling and processing of
food.
7. REFERENCES
Caniggia,A. & Gennari, C. (1977) Kinetics and intestinal absorption of
32
P-EHDP in man.
Calc. Tiss. Res.,22,428429.
Cozens,D.D.(1965)AbnormalitiesoftheexternformandoftheskeletonintheNewZealand
whiterabbit.Food Cosmet. Toxicol.,3,695700.
Canfeld,R.,Rosner,W.,Skinner,J.,McWhorter,J.,Resnick,L.,Feldman,F.,Kammerman,
S., Ryan, K., Kunigonis, M. & Bohne, W. (1977) Diphosphonate therapy of Pagets
diseaseofbone.J. Clin. Endocrinol. Metab.,44,96106.
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New and Generic DrugApprovals: 19982004 (http://www.fda.gov/cder/approval/d.htm)
andDidronel(etidronatedisodium)(http://www.fda.gov/cder/foi/label/2002/17831slr052lbl.
pdf).
Ecolab,Inc.(1995)ReactivitystudyforPOAAandhydrogenperoxidewithfruitsandvege-
tables.UnpublishedreportdatedFebruary78,1995fromtheEcolabResearchCenter,
MendotaHeights,Minnesota,USA.SubmittedtoWHObyEcolab,Inc.
Ecolab, Inc. (1999a) Effect of KX-6110 on fatty acid/lipid profle of red meat. Unpublished
report dated October 6, 1999 from the Ecolab Research Center, Mendota Heights,
Minnesota,USA.SubmittedtoWHObyEcolab,Inc.
Ecolab, Inc. (1999b). Effect of KX-6110 on thiobarbituric acid (TBA) values for red meat.
UnpublishedreportdatedOctober26,1999fromtheEcolabResearchCenter,Mendota
Heights,Minnesota,USA.SubmittedtoWHObyEcolab,Inc.
Ecolab, Inc. (2000). Effect of KX-6145 on thiobarbituric acid level and fatty acid profle.
UnpublishedreportdatedSeptember1222,2000fromtheUniversityofArkansasPoultry
Processing Research Facility, Fayetteville, Arkansas, USA and the Ecolab Research
CenterinMendotaHeights,Minnesota,USA.SubmittedtoWHObyEcolab,Inc.
Environmental Protection Agency (1998) Hydroxyethylidine diphosphonic acid: exemption
fromtherequirementofatolerance,Fed. Regist.,63,2825328258.
Fischer,K.(1993)DistributionandeliminationofHEDPinaquatictestsystems.Water Res.,
27,485493.
Flora,L.,Hassing,G.S.,Cloyd,G.G.,Bevan,J.A.&Parftt,A.M.(1981)Thelong-termskel-
etaleffectsofEHDPindogs.Metab. Bone Dis. Rel. Res.,4&5,289300.
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Gill,C.O.&Badoni,M.(2004)Effectsofperoxyaceticacid,acidifedsodiumchloriteorlactic
acid solutions on the microfora of chilled beef carcasses. Int. J. Food Microbiol., 91,
4350.
Industrial Bio-Test Laboratories, Inc. (1975a) 90-day sub-acute oral toxicity study with
DEQUEST2010inalbinorats.UnpublishedreportNo.622-05545,datedMay22,1975,
to Monsanto Co. from Industrial Bio-Test Laboratories, Inc., Northbrook, Illinois, USA.
SubmittedtoWHObyEcolab,Inc.Areportofanauditofthis90-daystudyinrats,con-
ductedbyBoozAllen&Hamilton,Inc.,FlorhamPark,NJ,USA,anddatedNovember7,
1978,wasavailable.
Industrial Bio-Test Laboratories, Inc. (1975b) 90-day sub-acute oral toxicity study with
DEQUEST2010inbeagledogs.UnpublishedreportNo.611-05544,datedMay22,1975,
to Monsanto Co. from Industrial Bio-Test Laboratories, Inc., Northbrook, Illinois, USA.
SubmittedtoWHObyEcolab,Inc.Reportsofanauditofthis90-daystudyindogs,con-
ducted by Booz Allen & Hamilton, Florham Park, NJ, USA, and dated September 28,
1978andMarch7,1979,wereavailable.
Kitis, M. (2004) Disinfection of wastewater with peracetic acid: a review. Environ. Int., 30,
4755.
Litton Bionetics, Inc. (1978) Mutagenicity evaluation of Bio-77-401 FA 65184 in the mouse
lymphomaforwardmutationassay(LBIprojectNo.20839).UnpublishedreporttoMon-
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Michael,W.R.,King,W.R.&Wakim,J.M.(1972)Metabolismofdisodiumethane-1-hydroxy-
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Pharmacol.,21,503515.
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Monsanto,Co.(1977)Mutagenicityplateassay:Dequest2010(medicalprojectNo.LF-76-
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IndustrialChemicalsCo,St.Louis,Missouri,USA.
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andbroccoliwithandwithouttreatmentwithOXY-15.UnpublishedreporttoEcolab,Inc.
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afterpartialhydrolysisbycalfpregastriclipase.Chem. Biol. Interact.,140,185198.
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the epoxidation techniques of vegetable oils. Fette Seifen Anstrichmittel, 76,
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No.Y-65-74).UnpublishedreporttoMonsantoCo.fromF.M.Younger,YoungerLabora-
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K2
K2
117
STEVIOL GLYCOSIDES
First draft prepared by
Dr D.J. Benford
1
; Dr M. DiNovi
2
and Dr. J. Schlatter
3
1
Food Standards Agency, London, United Kingdom;
2
Division of Biotechnology and GRAS Notice Review, Offce of Food
Additive Safety, Center for Food Safety and Applied Nutrition, Food and
Drug Administration, College Park, MD, USA; and
3
Food Toxicology Section, Swiss Federal Offce of Public Health;
Zrich, Switzerland
Explanation............................................................................... 117
Biologicaldata.......................................................................... 118
Biochemicalaspects.......................................................... 118
Absorption,distributionandexcretion......................... 118
Biotransformation......................................................... 121
Effectsonenzymesandotherbiochemical
parametersinvitro................................................ 123
Toxicologicalstudies.......................................................... 127
Short-termstudiesoftoxicity....................................... 127
Long-termstudiesofcarcinogenicity........................... 127
Genotoxicity................................................................. 128
Reproductivetoxicity.................................................... 129
Specialstudy:effectsonhumanmicrofora................ 129
Observationsinhumans.................................................... 134
Intake ..................................................................................... 135
Introduction......................................................................... 135
Useinfoods....................................................................... 135
Internationalestimatesofintake........................................ 135
Nationalestimatesofintakeofsteviolglycosides............. 136
Summaryofintakes........................................................... 138
Comments ............................................................................... 138
Evaluation ............................................................................... 140
References............................................................................... 141
1. EXPLANATION
Steviol glycosides are natural constituents of the plant Stevia rebaudiana
Bertoni,amemberoftheCompositaefamily.TheleavesofS. rebaudiana Bertoni
contain at least ten different glycosides, the major constituents being stevioside
and rebaudioside A. The material evaluated at the present meeting contains
not less than 95% glycosylated derivatives of steviol, primarily stevioside,
rebaudiosidesAandCanddulcosideA(Figure1),withminoramountsofrubuso-
side,steviolbioside,andrebaudiosidesB,D,EandF(Figure2).
118 STEVIOL GLYCOSIDES
K2
At its ffty-frst meeting (Annex 1, reference 149), the Committee evaluated
toxicological data on stevioside and the aglycone steviol. The Committee noted
severalshortcomingsintheavailableinformationandrequestedthatspecifcations
should be developed to ensure that the material tested is representative of the
materialofcommerce.Furtherinformationwasrequiredonthenatureofthesub-
stance tested, on the metabolism of stevioside in humans and on the activity of
steviolinsuitablestudiesofgenotoxicityinvivo.
Thereisnosinglecommonortrivialnameincommonusagefortheevaluated
mixture of glycosylated derivatives of steviol.At its thirty-third meeting (Annex 1,
reference83),theCommitteedevelopedguidelinesfordesignatingtitlesforspeci-
fcation monographs. According to these guidelines, the title of a monograph
should,insuchcircumstances,beselectedfromtheavailablescientifc,common
andtrivialnames.Thenamechosenmustbenonproprietaryandshouldbeasci-
entifcally accurate description of the substance. In addition, the name should
communicatetotheconsumeranaccuratedescriptionofthesubstance,withinthe
scopeofexistingnamesforfoodadditives.Atitspresentmeeting,theCommittee
establishedthattheevaluatedmaterialofcommerceforwhichspecifcationswere
developed should be known as steviol glycosides. The Committee reviewed
additionalbiochemicalandtoxicologicaldataonthemajorglycosylatedderivatives
ofsteviolandontheaglycone,steviol.
Thismonographdescribesthenewdataonsteviolglycosidesdiscussedatthe
presentmeeting,togetherwithsummariesofthekeytoxicologicaldataonstevio-
sideevaluatedbytheCommitteeatitsffty-frstmeeting.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution and excretion
Atitsffty-frstmeeting(Annex1,reference149),theCommitteenotedthatin
ratstreatedorallysteviosideisnotreadilyabsorbedfromtheuppersmallintestine
but is hydrolysed to the aglycone, steviol, before absorption from the gut. New
informationonabsorptioninin-vitromodelsandinratswasavailableatthepresent
meeting.
Intestinaltransportofstevioside(1mmol/l),rebaudiosideA(1mmol)andsteviol
(30mmol/l-1mol/l)hasbeeninvestigatedinaCaco-2cellmonolayermodel.The
integrityofthemonolayerwasverifedwithfuorescein.Transportofsteviosideand
rebaudiosideAwasverylow(apparentpermeabilitycoeffcients,0.1610
-6
and
0.11 10
-6
cm/s, respectively). Steviol was transported more effectively, with a
higherapparentpermeabilitycoeffcientforabsorptivetransport(44.510
-6
cm/s)
thanforsecretorytransport(7.9310
-6
cm/s)ataconcentrationof100mmol/l.At
concentrationsof300mmol/land1mol/l,steviolslightlycompromisedtheintegrity
ofthemonolayersduringtransport(Geunsetal.,2003a).
The intestinal absorption of a Stevia mixture and the aglycone steviol was
investigated using everted gastrointestinal sacs from four male Sprague-Dawley
STEVIOL GLYCOSIDES 119
K2
O
HO H
2
C
OH
OH
O
CH
2
O
O
CH
2
OH
OH
OH
OH
H
CH
3
H
H
3
C
CO
O
O
CH
2
OH
OH
OH
OH
O
HO H
2
C
OH
O
CH
2
O
O
CH
2
OH
OH
OH
OH
H
CH
3
H
H
3
C
CO
O
O
CH
2
OH
OH
OH
OH
O
O
CH
2
OH
OH
OH
OH
Stevioside Rebaudioside A
O
HO H
2
C
OH
O
CH
2
O
O
C H
3
OH
OH
OH
H
CH
3
H
H
3
C
CO
O
O
CH
2
OH
OH
OH
OH
O
O
CH
2
OH
OH
OH
OH
O
HO H
2
C
OH
OH
O
CH
2
O
O
OH
OH
OH
H
CH
3
H
H
3
C
CO
O
O
CH
2
OH
OH
OH
OH
CH
3
Rebaudioside C Dulcoside A
Figure 1. Structures of the major steviol glycosides
120 STEVIOL GLYCOSIDES
K2
O
HOH
2
C
OH
OH
O
CH
2
H
CH
3
H
H
3
C
CO
O
O
CH
2
OH
OH
OH
OH
OH
O
HOH
2
C
OH
OH
O
CH
2
O
O
CH
2
OH
OH
OH
OH
H
CH
3
H
H
3
C
COOH
Steviolbioside Rubusoside
O
HOH
2
C
OH
O
O
O
CH
2
OH
OH
OH
OH
H
CH
3
H
H
3
C
COOH
O
O
CH
2
OH
OH
OH
OH
Rebaudioside B
Figure 2. Structures of minor steviol glycosides
STEVIOL GLYCOSIDES 121
K2
rats.TheSteviamixturecontainedrebaudiosideA(28.8%),rebaudiosideC(25.2%),
stevioside(17.0%)anddulcosideA(10.2%).Theevertedsacswereincubatedin
Steviamixture(0.5mg/ml)orsteviol(0.1mg/ml)for30min.Transportofsalicyclic
acid (10mg/ml) was used to confrm that the sacs were functional. Steviol was
transportedinboththeduodenumjejunumandtheileum(76%and95%ofsali-
cyclic acid transport, respectively). The steviol glycosides were poorly absorbed
from the Stevia mixture, with more than 93% remaining in the mucosal fuid
(Koyamaetal.,2003a).
AbsorptionoftheSteviamixturedescribedabovewasalsoinvestigatedinvivo
infourmaleSprague-Dawleyrats.Steviamixture(in2%gumarabic)wasadmin-
isteredatadoseof125mg/kgbw.Steviolwasnotdetectedinplasmaat1h,but
wasdetectedatincreasingconcentrationsbetween2hand8h,whentheconcen-
trationreachedapeakofabout5mg/ml.Incontrast,thepeakplasmaconcentration
of steviol (18.31mg/ml) was observed 15min after a single oral administration of
steviol(45mg/kgincornoil).Thesedoseswereapproximatelyequimolarforsteviol
(Koyamaetal.,2003a).
Similarly, in male Sprague-Dawley rats given a single oral dose of stevioside
(purity,95%)at0.5g/kgbw,lowconcentrationsofsteviolweredetectedinplasma
forthefrst8h,followedbyarapidincreasetoaconcentrationofabout1000ng/ml
at24h.Thisstudyusedahighlysensitivemethodfordetectionofsteviol,butdid
notexaminelevelsofsteviosideorothermetabolites(Wangetal.,2004).
One study has reported detectable levels of stevioside, but not steviol, in
plasmaafteradministrationofaSteviaproduct.GroupsofmaleSprague-Dawley
ratsweregivenT100sunstevia95%(containing70%stevioside)atadoseof0.5
or 2g/kgbw by gavage. Stevioside was detected in plasma 5min after dosing.
There was considerable variation between animals, with the time to maximum
plasmaconcentrationvaryingfrom10to300min.Clearancedidnotdiffersignif-
cantlybetweenthedoses.Reportedplasmahalf-liveswere10.68.7and6.7
3.7h at 0.5 and 2.0g/kgbw, respectively.At 48h, 5.716.9% and 16.7% of the
total administered dose of stevioside was receovered in the faeces and urine,
respectively.Steviolwasdetectedinfaecescollectedupto48h,butnotinplasma
sampledupto24hafterdosing(limitofdetection,1mg/ml)(Sung,2002).
2.1.2. Biotransformation
Incubation of stevioside (purity, >96%; concentration, 50mg/l) with chicken
excretaunderanaerobicconditionsfor24hresultedina20%conversionofstevio-
sideintosteviol(Geunsetal.,2003b).
Faecal bacterial suspensions from eleven healthy volunteers (six men and
fvewomen)wereincubatedunderanaerobicconditionswith40mgofstevioside
(purity, 85%) and 40mg of rebaudiosideA (purity, 90%) for 72h. Stevioside and
rebaudioside A were completely hydrolysed to the aglycone steviol within 10
and24h,respectively.Amongculturesofcoliforms,bifdobacteria,enterococciand
bacteroides,onlythebacteroideswereabletohydrolysethesecompounds.The
dataindicatedthatbothglycosideswereinitiallyhydrolysedtosteviolbioside(this
occurredmoreslowlywithrebaudiosideA),andthesteviolbiosidewasthenrapidly
122 STEVIOL GLYCOSIDES
K2
metabolized to steviol. Steviol remained unchanged during the 72h incubation,
indicating that bacterial enzymes are not able to cleave the steviol structure
(Gardanaetal.,2003).
Human faecal metabolism of Stevia compounds was investigated in pooled
faecal homogenates obtained from fve healthy Japanese male volunteers. The
materials tested were Stevia mixture (main components: rebaudiosideA, stevio-
side, rebaudioside C, dulcoside A), its a-glucose derivative, referred to as
enzymatically modifed Stevia (main components: a-glucosylrebaudioside A, a-
glucosylstevioside, a-glucosylrebaudioside C, a-glucosyl dulcosideA), rebaudio-
side A, stevioside, steviol, rebaudioside C, dulcoside A, rebaudioside B,
rubusoside, a-monoglucosylrebaudioside A and a-monoglucosylstevioside. After
incubation of the faecal homogenates under anaerobic conditions for 24h, the
Stevia mixture, glycosides and a-glucose derivatives were all rapidly degraded.
Steviosidewashydrolysed,withsuccessiveremovalofglucoseunitsviarubuso-
side, to the aglycone steviol. The metabolism of a-monoglucosylstevioside was
similar to that of stevioside after a-deglucosylation. For rebaudioside there were
two pathways, a major pathway in which rebaudioside A was hydrolysed via
stevioside to steviol, and a minor pathway that suggested that rebau-
dioside A is metabolized via rebaudioside B to steviol. The metabolism of a-
monoglucosylrebaudioside A was similar to that of rebaudioside A after
a-deglucosylation.Nodegradationofsteviolwasobservedoverthe24hincubation
period. The authors concluded that steviol was the only fnal product of the
metabolismofStevia-relatedcompounds,includingenzymaticallymodifedStevia
inhumanintestinalmicrofora,andthattherewerenoapparentspeciesdifferences
intheintestinalmetabolismofSteviamixturebetweenratsandhumans(Koyama
etal.,2003b).
Metabolismofsteviol(puritynotspecifed)inratsandhumanshasbeeninves-
tigatedusingpooledhumanlivermicrosomalpreparationsfromfvemaleandfve
femaledonors,andfromratlivermicrosomalpreparationswiththesameprotein
content.Metaboliteformationrequiredanicotinamideadeninedinucleotidephos-
phate,reduced(NADPH)-generatingsystem,indicatingcytochromeP450(CYP)-
dependentmetabolism.Themetabolicprofleobtainedwithhumanlivermicrosomal
fractionswassimilartothatobtainedwithratlivermicrosomalpreparations;mass
spectrometric analysis indicated the presence of two dihydroxy metabolites and
four monohydroxy metabolites. One additional monohydroxy metabolite was
detected with the rat preparation. The liver microsomal clearance of steviol was
approximatelyfourtimeslowerinhumansthaninrats(Koyamaetal.,2003a).
Hamstersweregivenstevioside(puritynotspecifed)atadoseof1g/kgbwby
gavage and metabolites were measured in the plasma, urine and faeces at 3,
24and24h,respectively.Thesamplesweretreatedwithglucuronidase/sulfatase
to hydrolyse conjugated metabolites. Steviol-16,17a-epoxide, stevioside, 15 a-
hydroxysteviolandsteviolbiosideweredetectedintheplasma,urineandfaeces.
In addition, isosteviol was detected in the urine and faeces, and steviol was
detectedinthefaeces(Hutapeaetal.,1999).
STEVIOL GLYCOSIDES 123
K2
Chickens were given stevioside (purity, >96%) at a dose of 643 or 1168mg/
kgbw by intubation. Most of the stevioside was recovered unchanged in excreta
in the 2448h after administration, and only about 2% was converted to steviol.
Neithersteviosidenorsteviolweredetectedintheblood.Sixteenbroilerchickens
andfourlayinghenswerealsogivensteviosideatadoseof667mg/kgoffeedfor
14 and 10 days, respectively. Most of the stevioside was untransformed in the
excreta,withabout21.5%and7.3%beingconvertedtosteviolbybroilerchickens
orlayinghens,respectively.Nosteviosideorsteviolwasdetectedinthebloodor
intheeggs(Geunsetal.,2003b).
Six female pigswere given stevioside (purity, >96%) at a dose of 1.67g/kg of
feed for 14 days (equivalent to approximately 70mg/kgbw per day). Steviol, but
notstevioside,wasdetectedinthefaeces,indicatingbacterialmetabolismofste-
viosidetosteviol.Nosteviosideorsteviolwasdetectedintheblood.Theauthors
concludedthatsteviosidewascompletelyconvertedtosteviolandsuggestedthat
thepossibleuptakefromthecolonwasverylow(Geunsetal.,2003a).
Metabolismofsteviosidebyhumanvolunteershasbeeninvestigatedinacol-
laborativestudyconductedinBelgiumandItaly.InItaly,ninehealthymen(aged
2050 years) were given capsules containing 375mg of stevioside (purity not
specifed)afteranovernightfast.Lowconcentrationsofsteviosideweredetected
intheplasmaofsevenofthesubjects,withamaximumof0.1mg/ml.Peakplasma
concentrations occurred at 60 to 180min after dosing. Steviol glucuronide
was detected in fve of the men. No free steviol, steviol-16,17a-epoxide, 15a-
hydroxysteviol or 15-oxo-steviol was detected. Steviol glucuronide was detected
intheurineofallmen,andlowconcentrationsofsteviosidewerealsopresentin
the urine of two men. Free steviol or its unconjugated metabolites were not
detected.Onlyfreesteviolwasdetectedinthefaeces.InBelgium,fvemaleand
fvefemalevolunteers(aged242years)wereeachgivenninedosesof250mg
ofstevioside(purity,>97%;impuritiesbeingotherSteviaglycosides)at8hintervals
onthreesuccessivedays.Nosteviosideorfreesteviolwasdetectedintheblood.
Afterhydrolysiswithb-glucuronidase/sulfatase,steviolwasdetectedatconcentra-
tionsrangingfrom0.7to21.3mg/ml,withpeakconcentrationsoccurringatvarying
times up to 5h. Similarly, stevioside and conjugated steviol were detected in the
urineat24h.Theonlycompounddetectedinthefaeceswasfreesteviol.Thedif-
ferencesbetweenthetwostudieswereconsideredtobeduetothedifferentdoses
ofsteviosideadministeredandthedifferentdetectionlimitsoftheanalyticalmethod
forstevioside.Thetotalrecoveryofsteviolmetabolitesvariedbetween22%and
86%oftheadministereddailydoseofstevioside(meantotalrecovery,52.127%)
(Geuns&Pietta,2004).
ThemajormetabolitesofsteviolglycosidesareshowninFigure3.
2.1.3 Effects on enzymes and other biochemical parameters in vitro
In isolated aortic rings from normal rats, stevioside (purity not stated) at a
concentration of 10
-8
to 10
-5
mol/l caused a concentration-dependent relaxation
of vasopressin-induced vasoconstriction when incubated in medium containing
calcium, but not in calcium-free medium. The results of studies in a rat aortic
124 STEVIOL GLYCOSIDES
K2
CH
2
H
CH
3
H
H
3
C
CO OH
OH
H
CH
3
H
H
3
C
CO OH
OH
O
CH
2
Steviol Steviol-16,17-epoxide
CH
2
H
CH
3
H
H
3
C
COOH
OH
OH
O
H
CH
3
H
H
3
C
COOH
CH
3
15 -hydroxysteviol Isosteviol
CH
2
CH
3
O
HOOC
CH
3
OH
15-oxo-steviol
Figure 3. Metabolites of steviol glycosides
STEVIOL GLYCOSIDES 125
K2
smoothmusclecellline(A7r5)indicatedthatthiswasduetoinhibitionofthestimu-
latoryeffectsofvasopressinonintracellularcalciumions(Ca
2+
).Steviosidedidnot
inhibit calcium ionophore (A23187)-induced Ca
2+
infux.The effects of stevioside
werenotinhibitedbymethyleneblue.Theauthorsconcludedthatthevasorelax-
ationeffectofsteviosidewasmediatedmainlythroughinhibitionofCa
2+
infuxand
wasnotrelatedtonitricoxide(Leeetal.,2001;Liuetal.,2003).
The role of potassium channels in the vasodilator effect of isosteviol (purity
not stated) was investigated in isolated aortic rings prepared from Wistar rats.
Isosteviol at concentrations of 10
-8
to 10
-5
mol/l relaxed the vasopressin-induced
vasoconstriction in a concentration-dependent manner. Potassium chloride, and
inhibitorsspecifcfortheATP-sensitivepotassiumchannel,inhibitedthevasodilator
effect of isosteviol. Methylene blue failed to modify the vasodilation produced by
isosteviol,suggestingthatnitricoxidedidnotplayarole.Theauthorsconcluded
thatvasodilationinducedbyisosteviolwasrelatedtotheopeningofthecalcium-
activatedandATP-sensitivepotassiumchannels(Wongetal.,2004).
Stevioside (purity, 95%) and steviol (purity, 90%) at concentrations of 10
-9
to
10
-3
mol/lenhancedinsulinsecretioninisolatedmousepancreaticisletsandina
pancreatic-b-cell line (INS-1). The maximal effect was observed with steviol at
10
-6
mol/landwithsteviosideat10
-3
mol/l.Theinsulinotropiceffectwasdependent
ontheconcentrationofglucose(Jeppesenetal.,2000).
Subsequentstudiesindicatedthatsteviosideat10
-3
mol/lenhancedtheinsulin
contentoftheINS-1cells,partlybyinductionofgenesinvolvedinglycolysis.Ste-
viosideupregulatedtheexpressionoftheliver-typepyruvateandacetyl-coenzyme
A (CoA)-carboxylase and downregulated the expression of carnitine palmitoyl-
transferase 1 (CPT-1), long-chain acyl-CoA dehydrogenase, cytosolic epoxide
hydrolase and 3-oxoacyl-CoA thiolase. In addition, stevioside improved nutrient
sensingmechanisms,increasedcytosoliclong-chainfattyacyl-CoAanddownregu-
lated phosphodiesterase 1 (PDE1). Steviol showed similar effects (Jeppesen et
al.,2003).
The effect of stevioside (purity, 95%) on the transepithelial transport of p-
aminohippurate was investigated in isolated S
2
segments of the rabbit proximal
renal tubules. Stevioside (0.70mol/l) in the tubular lumen had no effect on the
transportofp-aminohippuratetransport,butwhenpresentinthebathingmedium
it inhibited transport by 2535%; the inhibitory effect was gradually abolished
after stevioside was removed. Stevioside had no effect on Na
+
/K
+
-activated
ATPase activity or cellATP content. The authors concluded that stevioside at a
pharmacological concentration of 0.7mol/l inhibits transepithelial transport of p-
aminohippurate by interfering with the basolateral entry step, the rate-limiting
stepfortransepithelialtransport.Thelackofeffectofsteviosideontransepithelial
transport of p-aminohippurate on the luminal side and the reversible inhibitory
effectonthebasolateralsideindicatedthatsteviosidedidnotpermanentlychange
p-aminohippurate transport and would not be expected to harm renal tubular
functionatnormallevelsofintakeinhumans(Jutabhaetal.,2000).
126 STEVIOL GLYCOSIDES
K2
Rats
Groups of normotensive Wistar-Kyoto rats, spontaneously hypertensive rats,
deoxycorticosterone acetate-salt sensitive rats and renal hypertensive rats were
givenstevioside(puritynotstated)atadoseof50,100,200or400mg/kgbwper
daybyintraperitonealinjectionfor1to10days.Treatmentwithsteviosideresulted
in signifcantly decreased blood pressure in all strains of rat, and the effect per-
sisted throughout the 10 days of treatment. Decreased blood pressure was also
observedinmature,spontaneouslyhypertensiveratsgivendrinking-watercontain-
ing 0.1% stevioside.Administration of drinking-water containing 0.1% stevioside
alsoslowedtheage-relatedprogressiveincreaseinbloodpressurethatoccursin
ratsofthisstrain(Hsuetal.,2002).
InGoto-Kakizakirats(whichareusedasanon-obeseanimalmodeloftype-2
diabetes), the intravenous administration of stevioside (purity, 96%) at a dose of
200mg/kgbwresultedinsuppressedplasmaglucagon,increasedinsulinresponse
andsuppressedtheresponsetoaglucosetolerancetest(incrementalarea-under-
the-curve:stevioside,64850mol/l120min;control,95885mol/l120min).
In normal Wistar rats, insulin concentrations were increased without altering the
bloodglucoseresponseorglucagonconcentrations(Jeppesen,2002).
InGoto-Kakizakiratsgivendrinking-watercontainingstevioside(purity,>99.6%)
atadoseof25mg/kgbwperdayfor6weeks,anantihyperglycaemiceffect was
observed, with enhanced insulin response and suppressed glucagon concentra-
tions, and a pronounced suppression of systolic and diastolic blood pressure
(Jeppesen,2003).
Insulin-sensitiveleanZuckerratsandinsulin-resistantobeseZuckerratswere
given stevioside (purity not stated) at a dose of 200 or 500mg/kgbw by oral
gavage, 2h before an oral test for glucose tolerance. There was no effect on
plasmaglucose,insulinorfreefattyacidconcentrationsineithertheleanorobese
groups.Atthehigherdose,steviosideenhancedwhole-bodysensitivitytoinsulin
in the lean and obese rats, as shown by a decreased insulin incremental area
underthecurveandglucoseinsulinindex.Noeffectwasobservedafteradminis-
trationofsteviosideat200mg/kgbw.
In vitro, stevioside at concentrations of 0.010.1mol/l was found to enhance
insulin-stimulated glucose transport in type 1 soleus and type llb epitrochloearis
muscle of both lean and obese Zucker rats. Higher concentrations of stevioside
inhibited the insulin-stimulated transport of glucose. The authors concluded that
onepotentialsiteofactionofsteviosidewastheskeletalmuscleglucosetransport
system(Lailerdetal.,2004).
Dogs
In healthy mongrel dogs, nasogastric administration of stevioside (purity not
stated)atadoseof200mg/kgbwresultedinaloweringofbloodpressurethatwas
maximal at 90min, returning to baseline by 180min. A more rapid decrease in
blood pressure was observed after intravenous injection of stevioside at 50mg/
kgbw, with the maximum decrease at 510min. In dogs with renal hypertension
STEVIOL GLYCOSIDES 127
K2
inducedbyligationoftheleftrenalartery,intravenousadministrationofstevioside
at20160mg/kgbwresultedinadose-dependentdecreaseinsystolicanddiastolic
bloodpressure.Noeffectwasobservedat10mg/kgbw(Liuetal.,2003).
2.2 Toxicological studies
2.2.1 Short-term studies of toxicity
Chickens
Sixteenbroilerchickensandfourlayinghensweregivendietscontainingste-
vioside(purity,>96%)ataconcentrationof667mg/kgoffeedfor14and10days,
respectively.Nosignifcantdifferenceswerefoundinfeedintake,body-weightgain
andfeedconversion(Geunsetal.,2003b).
2.2.2 Long-term studies of carcinogenicity
In a study discussed by the Committee at its ffty-frst meeting, groups of 50
maleand50femaleFischer344.DuCrjratsweregivenaccessadlibitumtodiets
containingstevioside(purity,95.6%;steviosidewasaddedtothepowdereddiet,
whichwasthenpelleted)ataconcentrationof0,2.5or5%(equaltodosesof0,
970and2000mg/kgbwperdayformales,and0,1100and2400mg/kgbwperday
forfemales)for104weeks.Thedoseswereselectedonthebasisoftheresults
of a 13-week study. Thereafter, all of the groups were maintained on basal (0%
stevioside)dietfor4weeks.Allsurvivingratswerekilledinweek108.Thebody-
weightgainofthetreatedanimalswasslightlydepressed,andadoseresponse
relationship was seen in males (2.3% and 4.4%) and females (2.4% and 9.2%)
at the lowest and highest doses, respectively. Food consumption did not differ
betweenthegroups.Thefnalsurvivalrateofmalesreceivingdietscontaining5%
stevioside was signifcantly decreased (60%) compared with that of the controls
(78%).Absolute weights of the kidney were decreased in males and females at
thehighestdose;however,therewasnosignifcanthistopathologicalevidenceof
neoplasticornon-neoplasticlesionsattributabletotreatmentinanyorganortissue,
exceptforadecreasedincidenceofmammaryadenomasinfemalesandareduced
severity of chronic nephropathy in males.The authors concluded that stevioside
wasnotcarcinogenicinFischer344ratsundertheexperimentalconditionsused
(Toyodaetal.,1995,1997).
Theeffectsofstevioside(puritynotstated)havebeeninvestigatedinmodels
oftwo-stageskincarcinogenesisinmice.Groupsof15maleICRmicewereiniti-
ated by topical application of 7,12-dimethylbenz[a]anthracene (DMBA; 100mg).
Promotiontreatmentcommenced1weeklater,andinvolvedtopicaladministration
of12-O-tetradecanoylphorbol-13-acetate(TPA;1mg)twiceperweekfor20weeks.
Topicaladministrationofstevioside(68mg)1hbeforetheTPAresultedinasignif-
cantdecreaseinthepercentageofanimalswithpapillomasat10and15weeks,
andinthenumberofpapillomaspermouseat15and20weeks.Inasimilarstudy,
groupsof15femaleSENCARmicewereinitiatedbyadministrationofperoxynitrite
(33.1mg)followedbypromotionwithTPA,twiceperweekfor20weeks.Administra-
tionofdrinking-watercontaining0.0025%steviosidefrom1weekbeforeto1week
128 STEVIOL GLYCOSIDES
K2
after initiation inhibited tumour formation. There was a statistically signifcant
decreaseinthepercentageofanimalswithpapillomasat10and15weeks,and
in the number of papillomas per mouse at 10, 15 and 20 weeks (Konoshima &
Takasaki,2002).
2.2.3 Genotoxicity
StudiesofgenotoxicitywithpurifedSteviaextractanditsmajorcomponents,
stevioside and rebaudioside A, reviewed by the Committee at its ffty-frst and
present meetings, are summarized in Table 1. These compounds gave negative
results in vitro and in vivo. Studies of genotoxicity with steviol and other Stevia-
derivedcompoundsaresummarizedinTable2.
Steviol and its oxidative derivatives steviol-16,17-epoxide, 15-oxo-steviol,
steviol methylester and 13,16-seco-13-oxo-steviol methylester induced forward
mutationsinS. typhimuriumTM677inthepresence,butnotintheabsence,ofa
metabolicactivationsystem.Themetabolizingsystemdecreasedthemutagenicity
of steviol methylester 8,13-lactone. The results for 15 a-hydroxy-steviol, steviol
methylesterand13,16-seco-13a-hydroxy-steviolmethylesterwerenegativeinthis
assay(Teraietal.,2002).
SteviolgavenegativeresultsinassaysforcellmutationandDNAdamagein
culturedcells(Ohetal.,1999;Sekihashietal.,2002).
Steviol(purity,>99%)hasbeeninvestigatedintwoindependentstudiesofDNA
damageusingthecometassay.Inonestudy,groupsoffourmaleBDF1micewere
givensteviolatadoseof0,250,500or2000mg/kgbwandtheliver,stomachand
colonwereexaminedforthepresenceofcomets.Inthesecondstudy,groupsof
fourmaleCRJ:CD-1miceweregivensteviolatadoseof0,500,1000or2000mg/
kgbw and the liver, kidney, colon and testes were examined for the presence of
comets. In both studies, groups of animals were sacrifced at 3h and 24h after
dosingandmethylmethanesulfonate(MMS)wasusedasapositivecontrol.There
were no signifcant differences in DNA migration distance in any of the organs
examined. MMS induced a positive response in all organs examined in both
studies(Sekihashietal.,2002).
Steviol (purity, about 90%) has also been tested in assays for induction of
micronucleiformationinthebonemarrowofSyriangoldenhamsters,Wistarrats
and Swiss albino mice. Groups of 20 male and 20 female animals were given
steviol at a dose of 4000mg/kgbw (hamsters) or 8000mg/kgbw (rats and mice)
bygavage.Fiveanimalsineachgroupwerekilled24,30,48and72hafterdosing.
An additional group, which served as a positive control, was treated with cyclo-
phosphamide and sacrifced at 30h. There were no signifcant increases in the
frequencies of micronucleated polychromatic erythrocytes (PCEs) in any of the
groupstreatedwithstevioside.TheratioofPCEstonormochromaticerythrocytes
(NCEs)wassignifcantlyreducedinthefemalehamstersat72haftertreatment,
andinfemaleratsandmiceat48hand72h.ThePCE:NCEratiodidnotchange
inmaleanimals.Cyclophosphamideinducedapositiveresponse(Temcharoenet
al.,2000).
STEVIOL GLYCOSIDES 129
K2 T
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e
e

I
s
h
i
d
a
t
e

e
t

a
l
.

(
1
9
8
4
)

a
b
e
r
r
a
t
i
o
n

f
b
r
o
b
l
a
s
t
s
C
h
r
o
m
o
s
o
m
e

C
H
L
/
I
U

C
h
i
n
e
s
e

h
a
m
s
t
e
r

l
u
n
g

R
e
b
a
u
d
i
o
s
i
d
e

N
S

1
.
2

5
5

m
g
/
m
l

N
e
g
a
t
i
v
e
a

N
a
k
a
j
i
m
a

(
2
0
0
0
a
)

a
b
e
r
r
a
t
i
o
n
s

f
b
r
o
b
l
a
s
t
s

A
130 STEVIOL GLYCOSIDES
K2
T
a
b
l
e

1
.

(
c
o
n
t
d
)
E
n
d
-
p
o
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n
t

T
e
s
t

s
y
s
t
e
m

M
a
t
e
r
i
a
l

P
u
r
i
t
y

(
%
)

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o
n
c
e
n
t
r
a
t
i
o
n

o
r

d
o
s
e

R
e
s
u
l
t

R
e
f
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r
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n
c
e
I
n

v
i
v
o
M
u
t
a
t
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n

D
.

m
e
l
a
n
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g
a
s
t
e
r

M
u
l
l
e
r

s
t
r
a
i
n

S
t
e
v
i
o
s
i
d
e

N
S

2
%

i
n

f
e
e
d

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e
g
a
t
i
v
e
b

K
e
r
r

e
t

a
l
.

(
1
9
8
3
)
D
N
A

d
a
m
a
g
e

M
a
l
e

B
D
F
1

m
o
u
s
e

s
t
o
m
a
c
h
,

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t
e
v
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a

e
x
t
r
a
c
t

S
t
e
v
i
o
s
i
d
e
,

2
5
0

2
0
0
0

m
g
/
k
g

N
e
g
a
t
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v
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c

S
e
k
i
h
a
s
h
i

e
t

a
l
.

(
2
0
0
2
)

(
c
o
m
e
t

a
s
s
a
y
)

c
o
l
o
n
,

l
i
v
e
r

5
2
;

r
e
b
a
u
d
i
o
s
i
d
e

A
,

2
2
D
N
A

d
a
m
a
g
e

M
a
l
e

d
d
Y

m
o
u
s
e

s
t
o
m
a
c
h
,

S
t
e
v
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a

N
S

2
0
0
0

m
g
/
k
g

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e
g
a
t
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v
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c

S
a
s
a
k
i

e
t

a
l
.

(
2
0
0
2
)

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c
o
m
e
t

a
s
s
a
y
)

c
o
l
o
n
,

l
i
v
e
r
,

k
i
d
n
e
y
,

b
l
a
d
d
e
r
,

l
u
n
g
,

b
r
a
i
n
,

b
o
n
e

m
a
r
r
o
w
M
i
c
r
o
n
u
c
l
e
u
s

d
d
Y

m
o
u
s
e

b
o
n
e

m
a
r
r
o
w

S
t
e
v
i
o
s
i
d
e

N
S

6
2
.
5

2
5
0

m
g
/
k
g

N
e
g
a
t
i
v
e
b

O
h

e
t

a
l
.

(
1
9
9
9
)

f
o
r
m
a
t
i
o
n

a
n
d

r
e
g
e
n
e
r
a
t
i
n
g

l
i
v
e
r
M
i
c
r
o
n
u
c
l
e
u
s

B
D
F
1

m
o
u
s
e

b
o
n
e

m
a
r
r
o
w

R
e
b
a
u
d
i
o
s
i
d
e

N
S

5
0
0

2
0
0
0

m
g
/
k
g

b
w

N
e
g
a
t
i
v
e
d

N
a
k
a
j
i
m
a

(
2
0
0
0
b
)

f
o
r
m
a
t
i
o
n

p
e
r

d
a
y

f
o
r

d
a
y
s
N
S
,

n
o
t

s
p
e
c
i
f
e
d
.
a

W
i
t
h

a
n
d

w
i
t
h
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u
t

m
e
t
a
b
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l
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c

a
c
t
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v
a
t
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o
n

(
s
o
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r
c
e

n
o
t

s
p
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c
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f
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d

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n

o
r
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)
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d
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.
d

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l
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3
0

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f
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m
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e

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t
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t

m
e
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c
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n
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f

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t
h

m
e
t
a
b
o
l
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c

a
c
t
i
v
a
t
i
o
n
.
STEVIOL GLYCOSIDES 131
K2
T
a
b
l
e

2
.

S
t
u
d
i
e
s

o
f

g
e
n
o
t
o
x
i
c
i
t
y

w
i
t
h

s
t
e
v
i
o
l

a
n
d

o
t
h
e
r

S
t
e
v
i
a
-
d
e
r
i
v
e
d

c
o
m
p
o
u
n
d
s
E
n
d
-
p
o
i
n
t

T
e
s
t

s
y
s
t
e
m

M
a
t
e
r
i
a
l

P
u
r
i
t
y

(
%
)

C
o
n
c
e
n
t
r
a
t
i
o
n
/
d
o
s
e

R
e
s
u
l
t

R
e
f
e
r
e
n
c
e
I
n

v
i
t
r
o
R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
A
9
8

S
t
e
v
i
o
l

N
S

2
0

m
g
/
p
l
a
t
e

N
e
g
a
t
i
v
e
a

S
u
t
t
a
j
i
t

e
t

a
l
.

(
1
9
9
3
)

a
n
d

T
A
1
0
0
R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
A
9
7
,

S
t
e
v
i
o
l

9
9

m
g
/
p
l
a
t
e

N
e
g
a
t
i
v
e
a

M
a
t
s
u
i

e
t

a
l
.

(
1
9
9
6
)

T
A
9
8
,

T
A
1
0
0
,

T
A
1
0
2
,

T
A
1
0
4
,

T
A
1
5
3
5

a
n
d

T
A
1
5
3
7
F
o
r
w
a
r
d

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
M
6
7
7

S
t
e
v
i
o
l

N
S

1
0

m
g
/
p
l
a
t
e
h

N
e
g
a
t
i
v
e

M
a
t
s
u
i

e
t

a
l
.

(
1
9
9
6
)

0
.
5

1
0

m
g
/
p
l
a
t
e
f

P
o
s
i
t
i
v
e
F
o
r
w
a
r
d

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
M
6
7
7

S
t
e
v
i
o
l

N
S

1
0

m
g
/
p
l
a
t
e
h

N
e
g
a
t
i
v
e

P
e
z
z
u
t
o

e
t

a
l
.

1
0

m
g
/
p
l
a
t
e
f

P
o
s
i
t
i
v
e

(
1
9
8
5
)
F
o
r
w
a
r
d

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
M
6
7
7

S
t
e
v
i
o
l

N
S

N
S

P
o
s
i
t
i
v
e
f

T
e
r
a
i

e
t

a
l
.

(
2
0
0
2
)
F
o
r
w
a
r
d

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
M
6
7
7

S
t
e
v
i
o
l
-
1
6
,
1
7
-
e
p
o
x
i
d
e

N
S

N
S

P
o
s
i
t
i
v
e
f

T
e
r
a
i

e
t

a
l
.

(
2
0
0
2
)
F
o
r
w
a
r
d

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
M
6
7
7

1
5
a
-
h
y
d
r
o
x
y
s
t
e
v
i
o
l

N
S

N
S

N
e
g
a
t
i
v
e
a

T
e
r
a
i

e
t

a
l
.

(
2
0
0
2
)
F
o
r
w
a
r
d

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
M
6
7
7

1
5
-
o
x
o
-
s
t
e
v
i
o
l

N
S

N
S

P
o
s
i
t
i
v
e
f

T
e
r
a
i

e
t

a
l
.

(
2
0
0
2
)
F
o
r
w
a
r
d

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
M
6
7
7

S
t
e
v
i
o
l

m
e
t
h
y
l
e
s
t
e
r

N
S

N
S

P
o
s
i
t
i
v
e
f

T
e
r
a
i

e
t

a
l
.

(
2
0
0
2
)
F
o
r
w
a
r
d

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
M
6
7
7

1
6
-
o
x
o
-
s
t
e
v
i
o
l

N
S

N
S

N
e
g
a
t
i
v
e
a

T
e
r
a
i

e
t

a
l
.

(
2
0
0
2
)

m
e
t
h
y
l
e
s
t
e
r
F
o
r
w
a
r
d

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
M
6
7
7

1
3
,
1
6
-
s
e
c
o
-
1
3
-
o
x
o
-

N
S

N
S

P
o
s
i
t
i
v
e
f

T
e
r
a
i

e
t

a
l
.

(
2
0
0
2
)

s
t
e
v
i
o
l

m
e
t
h
y
l
e
s
t
e
r
F
o
r
w
a
r
d

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
M
6
7
7

1
3
,
1
6
-
s
e
c
o
-
1
3
a
-

N
S

N
S

N
e
g
a
t
i
v
e
a

T
e
r
a
i

e
t

a
l
.

(
2
0
0
2
)

h
y
d
r
o
x
y
-
s
t
e
v
i
o
l

m
e
t
h
y
l
e
s
t
e
r
F
o
r
w
a
r
d

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
M
6
7
7

S
t
e
v
i
o
l

m
e
t
h
y
l
e
s
t
e
r

N
S

N
S

P
o
s
i
t
i
v
e
e

T
e
r
a
i

e
t

a
l
.

(
2
0
0
2
)

8
,
1
3
-
l
a
c
t
o
n
e
G
e
n
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

S
t
e
v
i
o
l

9
9

6
2
5

1
2
5
0

m
g
/

p
l
a
t
e
h

P
o
s
i
t
i
v
e

M
a
t
s
u
i

e
t

a
l
.

(
1
9
9
6
)

(
u
m
u
)

T
A
1
5
3
5
/
p
S
K
1
0
0
2

1
2
5
9

2
5
0
0

m
g
/
p
l
a
t
e
f

P
o
s
i
t
i
v
e
G
e
n
e

m
u
t
a
t
i
o
n

B
.

s
u
b
t
i
l
i
s

H
1
7

r
e
c
+
,

S
t
e
v
i
o
l

9
9

1
0

m
g
/
d
i
s
k

N
e
g
a
t
i
v
e
a

M
a
t
s
u
i

e
t

a
l
.

(
1
9
9
6
)

M
4
5

r
e
c
-
G
e
n
e

m
u
t
a
t
i
o
n

C
h
i
n
e
s
e

h
a
m
s
t
e
r

l
u
n
g

S
t
e
v
i
o
l

9
9

4
0
0

m
g
/
m
l
f

P
o
s
i
t
i
v
e

M
a
t
s
u
i

e
t

a
l
.

(
1
9
9
6
)

f
b
r
o
b
l
a
s
t
s
132 STEVIOL GLYCOSIDES
K2
T
a
b
l
e

2
.

(
c
o
n
t
d
)
E
n
d
-
p
o
i
n
t

T
e
s
t

s
y
s
t
e
m

M
a
t
e
r
i
a
l

P
u
r
i
t
y

(
%
)

C
o
n
c
e
n
t
r
a
t
i
o
n
/
d
o
s
e

R
e
s
u
l
t

R
e
f
e
r
e
n
c
e
G
e
n
e

m
u
t
a
t
i
o
n

M
o
u
s
e

l
y
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STEVIOL GLYCOSIDES 133
K2
In a study with limited reporting, available in Korean, groups of fve partially
hepatectomizedddYmiceweregivensteviol(puritynotstated)atanoraldoseof
0, 50, 100 or 200mg/kgbw. Steviol had no signifcant effect on the numbers of
micronucleatedhepatocytes.AgroupofmicetreatedwithmitomycinC,thepositive
control,didshowapositiveresponse(Ohetal.,1999).
2.2.4 Reproductive toxicity
Atitsffty-frstmeeting,theCommitteereviewedanumberofstudiesofrepro-
ductiveanddevelopmentaltoxicitywithsteviosideandSteviaextractsandnoted
thatadministrationofstevioside(purity,9096%)atdosesofupto2500mg/kgbw
perdayinhamstersand3000mg/kgbwperdayinratshadnoeffect.TheCom-
mitteealsonotedthat,althoughanaqueousinfusionofS. rebaudiana administered
orallytofemaleratswasreportedtocauseasevere,long-lastingreductioninfertil-
ity,thecontraceptiveeffectofSteviawasprobablynotduetostevioside.Stevioside
(purity, 95.6%) had neither teratogenic nor embryotoxic effects at doses of up to
1000mg/kgbwperdayinratstreatedbygavage.Atitspresentmeeting,theCom-
mitteereviewedtwoadditionalstudies.
Rats
Ten male Wistar rats (aged 2530 days) were each given 2ml of a crude
aqueousextractofS. rebaudiana(correspondingto0.67gofdriedleavesperml),
by gastric intubation, daily for 60 days.Ten control animals received saline only.
Therewerenosignifcanteffectsonfoodconsumptionorbody-weightgain.Animals
treated with Stevia extract showed decreased relative weights of the cauda
epididymides,seminalvesiclesandtestes,accompaniedbyareductioninplasma
concentrationoftestosteroneandinnumbersofspermatazoainthecaudaepidi-
dymidis. The fructose content of the prostate and seminal vesicle was also
decreased, which was considered by the author to be caused at least in part by
adefciencyintestosteronestimulation(Melis,1999).
Chickens
Onday7ofincubation,fertilebroilereggswereinjectedwith0.084.00mgof
stevioside (purity, >96%) or 0.0251.25mg of steviol (purity, >98%). The chicks
wereexaminedathatchingand1weeklater.Therewerenoeffectsonembryonic
mortality, body weight, malformations or body-weight gain during the frst week
afterhatching.Nosteviosideorsteviolwasdetectedinthebloodofthehatchlings
sacrifcedatage1day(Guensetal.,2003c).
2.2.5 Special study:effects on human microfora
Fortymgofstevioside(purity,85%)and40mgofrebaudiosideA(purity,90%)
wereincubatedfor72hunderanaerobicconditionswith40mloffaecalbacterial
suspensionsfromelevenhealthyvolunteers(sixmenandfvewomen).Stevioside
and rebaudiosideA did not signifcantly infuence the composition of faecal cul-
tures. However, stevioside caused a weak inhibition of the growth of anaerobic
134 STEVIOL GLYCOSIDES
K2
bacteria, while rebaudiosideA caused a weak inhibition of the growth of aerobic
bacteria,particularlycoliforms(Gardanaetal.,2003).
2.3 Observations in humans
Inamulticentrerandomized,double-blind,placebo-controlledtrialofhyperten-
sive Chinese men and women (aged 2875 years), 60 patients were given
capsules containing 250mg of stevioside (purity not stated) three times per day,
corresponding to a total intake of 750mg of stevioside per day (equivalent to
12.5mg/kgbwperday,assuminganaveragebodyweightof60kg)andfollowed
upatmonthlyintervalsforoneyear.Forty-sixpatientsweregivenaplacebo.After
3months,systolicanddiastolicbloodpressureinmenandwomenreceivingste-
vioside decreased signifcantly and the effect persisted over the year. Blood bio-
chemistryparameters,includinglipidsandglucose,showednosignifcantchanges.
Threepatientsreceivingsteviosideandonereceivingtheplacebowithdrewfrom
the study as a result of side-effects (nausea, abdominal fullness, dizziness). In
addition,fourpatientsreceivingsteviosideexperiencedabdominalfullness,muscle
tenderness,nauseaandastheniawithinthefrstweekoftreatment.Theseeffects
subsequently resolved and the patients remained in the study (Chan et al.,
2000).
A follow-up multicentre randomized, double-blind placebo-controlled trial was
conductedinhypertensiveChinesemenandwomen(aged2075years).Eighty-
fve patients were given capsules containing 500mg of stevioside (purity not
stated)threetimesperday,correspondingtoatotalintakeof1500mgofstevioside
perday(equivalentto25mg/kgbwperday,assuminganaveragebody-weightof
60kg). Eighty-nine patients were given a placebo. Three patients in each group
withdrewduringthecourseofthestudy.Therewerenosignifcantchangesinbody
massindexorbloodbiochemistryparametersthroughoutthestudy.Inthegroup
receivingstevioside,meansystolicanddiastolicbloodpressurewassignifcantly
decreasedcomparedwiththebaseline,commencingfromabout1weekafterthe
startoftreatment.After2years,6outof52patients(11.5%)inthegroupreceiving
steviosidehadleftventricularhypertrophycomparedwith17of50patients(34%)
in the group receiving the placebo (p < 0.001). Eight patients in each group
reported minor side-effects (nausea, dizziness and asthenia), which led two
patients in each group to withdraw from the study. Four patients in the group
receivingsteviosideexperiencedabdominalfullness,muscletenderness,nausea
andastheniawithinthefrstweekoftreatment.Theseeffectssubsequentlyresolved
andthepatientsremainedinthestudy(Hsiehetal.,2003).
Inapairedcross-overstudy,12patientswithtype-2diabetesweregiveneither
1g of stevioside (stevioside, 91%; other Stevia glycosides, 9%) or 1g of maize
starch (control group), which was taken with a standard carbohydrate-rich test
meal.Bloodsamplesweredrawnat30minbeforeandfor240minafteringestion
ofthetestmeal.Steviosidereducedpostprandialbloodglucoseconcentrationsby
anaverageof18%andincreasedtheinsulinogenicindexbyanaverageof40%,
indicatingbenefcialeffectsonglucosemetabolism.Insulinsecretionwasnotsig-
nifcantly increased. No hypoglycaemic or adverse effects were reported by the
STEVIOL GLYCOSIDES 135
K2
patients or observed by the investigators. Systolic and diastolic blood pressure
wasnotalteredbysteviosideadministration(Gregersenetal.,2004).
Forty-eighthyperlipidaemicvolunteerswererecruitedtoarandomized,double-
blindtrialdesignedtoinvestigatethehypolipidaemicandhepatotoxicpotentialof
steviolglycosideextract.Theextractusedinthisstudywasaproductcontaining
stevioside(732%),rebaudiosideA(242%)andotherplantpolysaccharides
(3%). The subjects were given two capsules, each containing 50mg of steviol
glycoside extract or placebo, twice daily (i.e. 200mg/day, equivalent to 3.3mg/
kgbwperdayassuminganaveragebodyweightof60kg),for3months.Onevol-
unteerreceivingplacebo,andthreevolunteersreceivingsteviolglycosidefailedto
completethestudyforpersonalreasons,notrelatedtoadversereactions.Atthe
end of the study, both groups showed decreased serum concentrations of total
cholesterol and of low density lipoproteins.Analyses of serum concentrations of
triglycerides,liver-derivedenzymesandglucoseindicatednoadverseeffects.The
authors questioned the subjects compliance with the dosing regime, in view of
thesimilarityofeffectbetweentreatmentandplacebo(Anonymous,2004a).Ina
follow-up study, 12 patients were given steviol glycoside extract in incremental
doses of 3.25, 7.5 and 15mg/kgbw per day, for 30 days per dose. Preliminary
resultsindicatednoadverseresponsesinbloodandurinebiochemicalparameters
(Anonymous,2004b).
3. INTAKE
3.1 Introduction
The Committee evaluated information on exposure to steviol glycosides sub-
mittedbyJapanandChina.AdditionalinformationwastakenfromareportonS.
rebaudiana Bertoni plants and leaves that was prepared for the European Com-
mission by the Scientifc Committee on Food (European Commission, 1999).All
oftheintakeresultsarepresentedintermsofequivalentsofsteviol,basedona
conversionof40%fromsteviolglycosides.
3.2 Use in foods
Steviol glycosides are used to sweeten a number of foods in China, Japan,
and South America. Table 3 summarizes the information submitted to the
Committee.
ItisalsoknownthatStevia leavesareusedtoprepareasweetenedteaina
numberofcountriesthroughouttheworld.Theconcentrationsofsteviolglycosides
intheseteasarelikelytobelowerthanthosereportedinTable3.
3.3 International estimates of intake
The WHO Global Environment Monitoring System Food Contamination
Monitoring and Assessment Programme (GEMS/Food) database was used by
the Committee to prepare international estimates of intake of steviol glycosides
136 STEVIOL GLYCOSIDES
K2
(assteviol).Itwasassumedthatsteviolglycosideswouldreplaceallsweeteners
usedinorasfood,refectingtheminimumreportedrelativesweetnessofsteviol
glycosidesandsucroseof200:1.TheestimatesareshowninTable4.
Theseestimatesareconservativeinthatitisveryunlikelythatauserofsteviol
glycosides would replace all commodity sweeteners found in their diets (WHO,
2003).
3.4 National estimates of intake of steviol glycosides
Japan submitted an estimate of intake of steviol glycosides per capita based
onthetotaldemandforsteviolglycosidesinJapan,estimatedat200tonnesper
year.Theestimateassumedapopulationof120millionpersonsandanaverage
body weight of 50kg. The resulting estimate of intake of steviol glycosides (as
steviol)was0.04mg/kgbwperday.
Additionally, the Japanese submission included two maximum consumption
estimates for steviol glycosides. These assumed that 10% of all added sugar in
thedietsofJapanortheUSAwouldbereplacedbysteviolglycosides,ataratio
of200:1,basedonsweetness.TheconsumptionofsugarinJapanwastakenas
25kg/person per year, while that in the USA was 125 pounds/person per year
(57kg/person per year). The average body weight for both Japan and the USA
was assumed to be 50kg. The resulting estimates of maximum consumption of
steviol glycosides (as steviol) were 0.3mg/kgbw per day for Japan and 0.6mg/
kgbwperdayfortheUSA.TheCommitteeconcludedthattherewasnoevidence
to suggest that only 10% of sugar consumed would be replaced. Therefore, the
Committeecalculatedmaximumintakesbasedonthereplacementofallsugarin
diets in Japan and the USA, resulting in estimates of 3mg/kgbw per day for a
50kg consumer in Japan and 5mg/kgbw per day for a 60kg consumer in the
USA.
Table 3. Food use levels of steviol glycosides
reported to the Committee
Foodtype Maximumuselevelreported(mg/kg)
Beverages 500
Desserts 500
Yogurt 500
Coldconfectionery 500
Sauces 1000
Pickles 1000
Delicacies 1000
Sweetcorn 200
Bread 160
Biscuits 300
STEVIOL GLYCOSIDES 137
K2 T
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138 STEVIOL GLYCOSIDES
K2
ThesubmissionfromChinacontainedinformationontheannualproductionof
steviol glycosides. It was reported that up to 1000 tonnes were produced each
year, with 200 tonnes retained for domestic consumption. In view of the larger
populationinChinathaninJapanortheUSA,theCommitteenotedthatanyesti-
mates prepared using these data would result in lower exposures than those
reportedabove.
3.5 Summary of intakes
Table 5 contains a summary of the intakes of steviol glycosides evaluated or
derivedbytheCommittee.
The Committee concluded that the replacement estimates were highly con-
servative and that intake of steviol glycosides (as steviol) would be likely to be
2030%ofthesevalues.
4. COMMENTS
Afteroraladministration,steviolglycosidesarepoorlyabsorbedinexperimental
animalsandinhumans.
Intestinalmicroforametabolizesteviolglycosidestotheaglycone,steviol,by
successivehydrolyticremovalofglucoseunits.DatareviewedbytheCommittee
at its current and ffty-frst meetings (Annex 1, reference 149) indicated that this
processissimilarinratsandhumans.ThehydrolysisofrebaudiosideAtosteviol
wasslowerthanthatofstevioside.Inhumanstreatedorallywithstevioside,small
amountsofsteviolweredetectedintheplasma,withconsiderableinterindividual
variability. The major route by which steviol is metabolized in humans in vivo
appears to be via conjugation with glucuronide and/or sulfate. Studies with liver
microsomalpreparationsindicatedthatsteviolisalsometabolizedtoanumberof
hydroxyanddihydroxyderivativesviaCYP-dependentpathways.
Table 5. Summary of estimates of intakes of steviol glycosides (as steviol)
Estimate Intake(mg/kgbwperday)
GEMS/Food(international)
a
1.33.5(60kgperson)
Japan,percapita 0.04
Japan,maximumconsumption
b
3
USA,maximumconsumption
b
5
GEMS/Food,WHOGlobalEnvironmentMonitoringSystemFoodContamination
MonitoringandAssessmentProgramme.
a
InternationalreferstotheinternationalestimatespresentedinTable4.
b
Theseestimateswerepreparedinparalleltothosefortheinternationalestimates:itwas
assumedthatalldietarysugarsindietsinJapanandtheUSAwouldbereplacedby
steviolglycosides,ataratioof200:1.
STEVIOL GLYCOSIDES 139
K2
Steviosideand/orsteviolaffectedavarietyofbiochemicalparametersinmodels
in vitro, indicating possible mechanisms of antihypertensive and antiglycaemic
effectsthatinvolvemodulationofionchannels.Highconcentrations(e.g.1mmol/l)
of stevioside were required to produce a maximal increase in insulin secretion,
while steviol was effective at a concentration that was about three orders of
magnitudelower.Steviosidealsoaffectedavarietyofbiochemicalparametersin
different animal species in vivo, mostly with parenteral administration; these
studies were considered by the Committee to be of limited relevance to dietary
exposure.
No new long-term studies of toxicity or carcinogenicity were available at the
presentmeeting.Atitsffth-frstmeeting,theCommitteenotedthatoraladministra-
tionofstevioside(purity,95.6%)atadietaryconcentrationof2.5%,equalto970
and1100mg/kgbwperdayinmaleandfemalerats,respectively,for2yearswas
not associated with toxicity. Reduced body-weight gain and survival rate were
observedwithsteviosideatadietaryconcentrationof5%.Inanewstudy,stevio-
sidewasfoundtoinhibitthepromotionofskintumoursbyTPAinamodelofskin
carcinogenesisinmice.
The Committee reviewed new data on genotoxicity that, considered together
withdatareviewedbytheCommitteeatitsffth-frstmeeting,allowedanumberof
conclusionstobedrawn.SteviosideandrebaudiosideAhavenotshownevidence
ofgenotoxicityinvitroorinvivo.Steviolandsomeofitsoxidativederivativesshow
clearevidenceofgenotoxicityinvitro,particularlyinthepresenceofametabolic
activationsystem.However,studiesofDNAdamageandmicronucleusformation
in rats, mice and hamsters in vivo indicate that the genotoxicity of steviol is not
expressedatdosesofupto8000mg/kgbw.
Onenewstudyofdevelopmentaltoxicitywasavailableatthepresentmeeting.
Adverse effects on the reproductive apparatus, which could be associated with
impairedfertility,wereobservedinmaleratsgivenacrudeextractofS. rebaudiana,
atadose correspondingto1.34gofdriedleaves.However,atitsffth-frstmeeting,
theCommitteereviewedanumberofstudiesofreproductiveanddevelopmental
toxicitywithstevioside(purity,90%or96.5%).Dosesofupto2500mg/kgbwper
day in hamsters and 3000mg/kgbw per day in rats had no effect in studies of
reproductivetoxicity.Noteratogenicorembryotoxiceffectswereobservedinrats
givensteviosideatadoseofupto1000mg/kgbwperdaybygavage.TheCom-
mitteeconsideredthattheadversereproductiveeffectsassociatedwithadministra-
tion of aqueous extracts of S. rebaudiana, noted at the present and ffty-frst
meeting,wereunlikelytobecausedbysteviolglycosides.
Steviosideisbeinginvestigatedasapotentialtreatmentforhypertensionand
diabetes.Administrationofsteviosideatadoseof750or1500mgperdayfor324
months resulted in decreased blood pressure in hypertensive patients, with no
adverse effects. These studies, in a limited number of subjects, provided some
reassurancethatsteviosideatadoseofupto25mg/kgbwperday(equivalentto
10mg/kgbw per day expressed as steviol) for up to 2 years shows no evidence
of signifcant adverse effects in these individuals.There is no information on the
effectsofrepeatedadministrationofsteviosideonbloodpressureinnormotensive
individuals.Asmallstudyin12patientswithtype-2diabetesshowedthatasingle
140 STEVIOL GLYCOSIDES
K2
doseof1gofsteviosidereducedpostprandialglucoseconcentrationsandhadno
effectonbloodpressure.
TheCommitteeevaluatedinformationonintakeofsteviolglycosides,submitted
by Japan and China. Additional information was available from a report on S.
rebaudiana Bertoni plants and leaves that was prepared for the European Com-
missionbytheScientifcCommitteeonFood.Alltheintakeresultsarepresented
intermsofequivalentsofsteviol,basedonaconversionof40%fromthesteviol
glycoside,stevioside(relativemolecularmass:steviol,318,stevioside,805).
The Committee used the GEMS/Food database to prepare international esti-
matesofintakeofsteviolglycosides(assteviol).Itwasassumedthatsteviolgly-
cosideswouldreplacealldietarysugars,atthelowestreportedrelativesweetness
ratioforsteviolglycosidesandsucrose,200:1.Theintakesrangedfrom1.3mg/
kgbwperday(Africandiet)to3.5mg/kgbwperday(Europeandiet).
The Committee evaluated estimates of per capita intake derived from disap-
pearance (poundage) data supplied by Japan and China. The Committee also
evaluated estimates of intake of steviol glycosides based on the replacement of
alldietarysugarsinthedietsforJapanandtheUSA.Theseresultsaresumma-
rizedinTable5.
The Committee concluded that the replacement estimates were highly con-
servative and that intake of steviol glycosides (as steviol) would be likely to be
2030%ofthesevalues.
5. EVALUATION
TheCommitteenotedthatmostofthedatarequestedatitsffty-frstmeeting,
e.g.dataonthemetabolismofsteviosideinhumans,andontheactivityofsteviol
insuitablestudiesofgenotoxicityin vivo,hadbeenmadeavailable.
TheCommitteeconcludedthatsteviosideandrebaudiosideAarenotgenotoxic
in vitro or in vivo and that the genotoxicity of steviol and some of its oxidative
derivativesinvitroisnotexpressedinvivo.Theno-observed-effectlevel(NOEL)
for stevioside was 970mg/kgbw per day in a long-term study evaluated by the
Committeeatitsffty-frstmeeting.
TheCommitteenotedthatsteviosidehasshownsomeevidenceofpharmaco-
logicaleffectsinpatientswithhypertensionorwithtype-2diabetesatdosescor-
responding to about 12.525mg/kgbw per day (equivalent to 510mg/kgbw per
day expressed as steviol).The evidence available at present was inadequate to
assesswhetherthesepharmacologicaleffectswouldalsooccuratlowerlevelsof
dietary exposure, which could lead to adverse effects in some individuals (e.g.
thosewithhypotensionordiabetes).TheCommitteethereforedecidedtoallocate
atemporaryacceptabledailyintake(ADI),pendingsubmissionoffurtherdataon
thepharmacologicaleffectsofsteviolglycosidesinhumans.
A temporary ADI of 02mg/kgbw was established for steviol glycosides,
expressedassteviol,onthebasisoftheNOELforsteviosideof970mg/kgbwper
day(or383mg/kgbwperdayexpressedassteviol)inthe2-yearstudyinratsand
STEVIOL GLYCOSIDES 141
K2
asafetyfactorof200.Thissafetyfactorincorporatesafactorof100forinter-and
intraspecies differences and an additional factor of 2 because of the need for
furtherinformation.TheCommitteenotedthatthistemporaryADIonlyappliesto
productscomplyingwiththespecifcations.
TheCommitteerequiredadditionalinformation,tobeprovidedby2007,onthe
pharmacological effects of steviol glycosides in humans. These studies should
involve repeated exposure to dietary and therapeutic doses, in normotensive
and hypotensive individuals and in insulin-dependent and insulin-independent
diabetics.
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145
d-TAGATOSE (addendum)
First draft prepared by
Dr D.J. Benford
1
and Dr M.C. de Figueiredo Toledo
2
1
Food Standards Agency, London, England; and
2
State University of Campinas, Campinas, Brazil
Explanation............................................................................... 145
Biologicaldata.......................................................................... 146
Toxicologicalstudies:long-termstudiesoftoxicity
andcarcinogenicity............................................................... 146
Intake ..................................................................................... 147
Comments ............................................................................... 147
Evaluation ............................................................................... 148
References............................................................................... 148
1. EXPLANATION
D-Tagatose is a ketohexose, an epimer of D-fructose isomerized at C4. It is
obtainedfromD-galactosebyisomerizationunderalkalineconditionsinthepres-
ence of calcium. Its properties permit its use as a bulk sweetener, humectant,
texturizerandstabilizer.
D-Tagatose was evaluated by the Committee at its ffty-ffth, ffty-seventh and
sixty-frstmeetings(Annex1,references149,154and166).Atitsffty-ffthmeeting,
theCommitteeconcludedthatD-tagatosewasnotgenotoxic,embryotoxicortera-
togenic.Italsoconcludedthatanacceptabledailyintake(ADI)couldnotbeallo-
cated for D-tagatose because of concern about its potential to induce glycogen
depositionandhypertrophyintheliverandtoincreasetheconcentrationsofuric
acidinserum.Atitsffty-seventhmeeting,theCommitteeevaluatedtheresultsof
fourstudiesinexperimentalanimals,theresultsofastudyinvolunteersandsome
publications concerning the increased concentration of uric acid in serum after
intake of D-tagatose and other substances. The Committee decided to base its
evaluation on the human data reviewed in the course of these two meetings.A
no-observed-effectlevel(NOEL)of0.75g/kgbwperdaywasidentifedina28-day
studyinwhichnoeffectswereobservedinhumansreceivingthreedosesof15g
of D-tagatose per day.AnADI of 080mg/kg bw for D-tagatose was established
onthebasisofthisNOELandasafetyfactorof10.
Atitssixty-frstmeeting,theCommitteereviewedtheresultsoftwonewstudies
oftoxicityinrats,andoftwonewstudiesofplasmaconcentrationsofuricacidin
humanvolunteers;thesestudiesweresubmittedbythesponsorwitharequestfor
are-evaluationofD-tagatose.
TheCommitteeconcludedthatthe2-yearstudyinratsdemonstratedthatthe
previously-reported liver glycogen deposition and hypertrophy did not result in
146 d-TAGATOSE
K2
histopathological changes after long-term administration of D-tagatose, and thus
addressed the concerns expressed at the ffty-ffth meeting. However, this study
alsoidentifednewfndings,namelyincreasedadrenal,kidneyandtestesweights.
TheCommitteeconsideredthatthesechangesmighthavebeencausedbyhigh
osmoticloadresultingfromthehighdietarydosesadministered,butthiscouldnot
be confrmed in the absence of histopathological examination of these tissues.
Pendingprovisionoftheresultsofhistopathologicalexamination,theCommittee
confrmedthatthehumandataprovidedthemostrelevantbasisforassessingthe
acceptableintakeofD-tagatose.
ResultsofastudyinhyperuricaemicindividualsindicatedthattheNOELidenti-
fedfornormalindividualswasalsoapplicabletothisvulnerablegroup.TheCom-
mittee considered that a safety factor of 3 would be appropriate to allow for
interindividualvariation.Inviewoftheadditionaluncertaintyregardingthenature
oftheeffectsobservedintheadrenals,kidneysandtestesinthe2-yearstudyin
rats,theCommitteeconcludedthattheADIshouldbetemporaryandappliedan
additionalsafetyfactorof2.ThepreviousADIwasremoved,andtheCommittee
allocated a temporaryADI for D-tagatose of 0125mg/kg bw on the basis of the
NOELof0.75g/kgbwperdayandasafetyfactorof6.
TheCommitteeconsideredthatthetemporaryADIdidnotapplytoindividuals
with hereditary fructose intolerance resulting from defciency of 1-phosphofructo-
aldolase(aldolaseB)orfructose1,6-diphosphatase.
The Committee requested information on the histological examination of the
adrenals,kidneysandtestesoftheratsfromthe2-yearstudyby2006.Thisinfor-
mation was provided to the Committee for evaluation at its present meeting,
together with additional data on the risk to individuals with hereditary fructose
intolerance.
2. BIOLOGICAL DATA
2.1 Toxicological studies: long-term studies of toxicity
and carcinogenicity
TheCommitteeconsideredanaddendumtoareportthathadbeendiscussed
atitssixty-frstmeeting,onamodifedstudyofcarcinogenicityconductedtoinves-
tigatetheeffectsoflong-termadministrationof2.5%,5%,or10%D-tagatose,20%
fructose or 10% D-tagatose plus 10% fructose on the liver of Wistar rats. In
response to the Committees request, the addendum included the results of
histopathologicalexaminationofthetestes,kidneysandadrenalsofallrats.The
incidence of nephrocalcinosis showed an apparent dose-related trend in both
sexes. This was statistically signifcant in all groups of males treated with D-
tagatose. There was a high (88%) incidence of nephrocalcinosis in the female
controlgroup,andasignifcanttreatment-relatedeffectwasobservedonlyinthe
groups given 10% D-tagatose. Mineralization occurred mainly in the pelvic and
medullary regions of the kidneys.The incidence of adrenomedullary proliferative
lesions was signifcantly increased in males at 5% and 10% D-tagatose, and
also at 5% D-tagatose in females. This was predominantly associated with
d-TAGATOSE 147
K2
pheochromocytomasinmalesandmedullaryhyperplasiainfemales.Therewere
no other reported treatment-related effects. The incidence of Leydig cell
hyperplasiaandadenomaswassimilarandwithintherangeforhistoricalcontrols
inallgroups(Lina&Br,2003).
3. INTAKE
Atitsffty-seventhmeeting,theCommitteeestimatedthatthemeanintakeof
D-tagatose was between 3 and 9g/day and the 95th percentile of consumption
was up to 18g/day. These estimates, based on data on food consumption from
Australia,MemberStatesoftheEuropeanUnionandtheUSA,wereconsidered
tobestillvalid.
4. COMMENTS
Additional histopathological examinations were conducted on the adrenals,
kidneysandtestesofWistarratsfeddietscontaining2.5,5or10%D-tagatose,or
10%D-tagatoseplus10%fructosefor2years.Theobservedchangesweresimilar
tothosereportedinstudieswithothercarbohydratesoflowdigestibility.TheCom-
mitteehaspreviouslynotedthatgrossdietaryimbalancecausedbyhighdosesof
polyols may result in metabolic and physiological disturbances in rats, and are
associatedwithchangesincalciumuptakeandexcretionaccompaniedbyneph-
rocalcinosis and adrenal medullary hyperplasia (Annex 1, reference 62). These
changes were not considered to be of relevance to the safety evaluation of D-
tagatose.Carbohydratesoflowdigestibilitydonotincreasetheintestinalabsorp-
tionofcalciuminhumanstothesameextentasinrats.Rats,especiallyfemales,
areparticularlypronetothedevelopmentofnephrocalcinosis.TheCommitteehas
previouslynotedtheuniquefeaturesoftheratadrenalmedullaandconcludedthat
theoccurrenceofproliferativelesionsoftheadrenalmedullainratsfedwithpolyols
and lactose is a species-specifc phenomenon (Annex 1, reference 122). An
increasedincidenceofLeydigcelltumourshasbeenreportedinmaleWistarrats
feddietscontaining10%lactitolor20%D-tagatose.Thisstudydemonstratedthat
there were no toxicologically signifcant fndings in rats fed D-tagatose at dietary
levels of up to 10% for 2 years (equal to approximately 4 and 5g/kgbw per day
formalesandfemales,respectively).
TheCommitteefurtherconsideredtherisktoindividualswithhereditaryfructose
intolerance,whichifuntreatedleadstometabolicdisturbances,liverdamage,renal
tubular disease and defective blood coagulation. Treatment requires almost
completeexclusionofsucrose,fructoseandsorbitol.Thereisnodirectevidence
establishingthatindividualswithhereditaryfructoseintolerancearealsointolerant
to D-tagatose, but in view of their common biochemical pathways it is probable
thatD-tagatosecouldproducethesameadverseeffectsasfructose.Atitsffty-ffth
meeting(Annex1,reference149),theCommitteenotedthattheabsorptionofD-
tagatose by humans is not expected to exceed 20% of the administered dose.
However, the rate of gluconeogenesis from D-tagatose is slower than that from
fructose.ThustheCommitteecouldnotdiscountthepossibilitythat,inindividuals
148 d-TAGATOSE
K2
withhereditaryfructoseintolerance,tissueconcentrationsofD-tagatosecouldbe
elevated or prolonged compared with those of fructose, leading to adverse
reactions.
The Committee has previously noted that gastrointestinal effects (nausea,
fatulence,diarrhoea)havebeenreportedinsomeindividualsaftertheconsump-
tionof30gofD-tagatoseinasingledose.
TheCommitteeatitsffty-seventhmeetingestimatedthatthemeandailyintake
ofD-tagatosewouldrangefrom3to9g/dayandthe95thpercentileofconsumption
would be up to 18g/day. These estimates, based on data on food consumption
fromAustralia,MemberStatesoftheEuropeanUnionandtheUSA,wereconsid-
eredtobestillvalid.
5. EVALUATION
At its sixty-frst meeting, the Committee concluded that, pending provision of
theresultsofhistopathologicalexaminationsfroma2-yearstudyinrats,thehuman
data provided the most relevant basis for assessing the acceptable intake of D-
tagatose. The histopathological data had now been provided and demonstrated
that there were no toxicologically signifcant fndings in rats given D-tagatose at
levelsofupto10%inthedietfor2years(equaltoapproximately4and5g/kgbw
per day for males and females, respectively). On the basis of the data reviewed
bytheCommitteeatitssixty-frstmeetingandatitspresentmeeting,andtaking
intoaccountthefactthatD-tagatosehasphysiologicalandtoxicologicalproperties
similartothoseofothercarbohydratesoflowdigestibility,theCommitteeremoved
thetemporaryADIandallocatedanADInotspecifedforD-tagatose.
Thefactthatingestionof30gormoreofD-tagatoseonasingleoccasionmay
causegastrointestinaleffectsinhumansshouldbetakenintoaccountwhencon-
sideringappropriatelevelsofuse.
TheADI not specifed does not apply to individuals with hereditary fructose
intolerance arising from 1-phosphofructoaldolase (aldolase B) defciency or fruc-
tose1,6-diphosphatasedefciency.
6. REFERENCE
Lina,B.A.R.&Br,A.(2003)ChronictoxicityandcarcinogenicitystudywithD-tagatoseand
fructoseinWistarrats.Addendum1tounpublishedreportNo.V4533fromTNONutrition
and Food Research, Zeist, Netherlands. Submitted to WHO by Bioresco Ltd., Basel,
Switzerland.
K2
149
XYLANASES FROM BACILLUSSUBTILIS
EXPRESSED IN B.SUBTILIS
First draft prepared by
M.E. van Apeldoorn
1
, M.E.J. Pronk
1
, C. Leclercq
2
, Z. Olempska-Beer
3

and Dr. D. Hattan
3
1
Centre for Substances and Integrated Risk Assessment, National
Institute for Public Health and the Environment, Bilthoven, Netherlands;
2
Research Group on Food Safety Exposure Analysis,
National Research Institute for Food and Nutrition, Rome, Italy; and
3
Center for Food Safety and Applied Nutrition, Food
and Drug Administration, College Park, MD, USA
Explanation............................................................................... 149
Geneticmodifcation........................................................... 150
Productcharacterization.................................................... 150
Biologicaldata.......................................................................... 151
Biochemicalaspects.......................................................... 151
Toxicologicalstudies.......................................................... 151
Acutetoxicity................................................................ 151
Short-termstudiesoftoxicity....................................... 151
Long-termstudiesoftoxicityandcarcinogenicity....... 153
Genotoxicity................................................................. 153
Reproductivetoxicity.................................................... 155
Observationsinhumans.................................................... 155
Intake ............................................................................... 155
Comments ............................................................................... 156
Evaluation ............................................................................... 156
References............................................................................... 156
1. EXPLANATION
Xylanases from Bacillus subtilis expressed in B. subtilis have not been
evaluatedpreviouslybytheCommittee.Xylanase(b-1,4-D-xylanxylanohydrolase,
b-1,4-D-xylanohydrolase, 1,4-xylanase, endo-1,4-xylanase) is an enzyme that
catalyses the hydrolysis of xylans and arabinoxylans to mono- and oligosaccha-
rides. The activity of the petitioned enzyme is measured relative to that of
thestandardenzymeandisexpressedintotalxylanaseunits(TXU)
1
.TheCom-
mitteereceivedinformationonthreexylanases,designatedBS1,BS2,andBS3.
These xylanases are derived from nonpathogenic and nontoxigenic, genetically
modifed strains of B. subtilis. B. subtilis has been a source of enzymes used in
1
Xylanase activity can also be expressed in Danisco xylanase units (DXU); 1TXU =
24.15DXU.
150 XYLANASE FROM BACILLUSSUBTILIS
K2
foodformanyyears.XylanasesBS1andBS2areidenticaltothenativexylanase
of B. subtilis. Xylanase BS3 differs from the native enzyme by two amino acids
andisresistanttothexylanaseinhibitorpresentinfour.XylanasesBS2andBS3
areusedasprocessingaidsinbakingapplicationstoincreasetolerancetowards
variationsinprocessparameters,improvethedough,andincreasethevolumeof
baked goods. Use levels range from 500 to 13300TXU/kg of four for xylanase
BS3,andfrom3000to40000TXU/kgoffourforxylanaseBS2.ThexylanaseBS2
preparation contains 0.3mg of total organic solids (TOS) per 1000TXU, and the
xylanaseBS3preparationcontains1.5mgofTOSper1000TXU.
1.1 Genetic modifcation
ThreeproductionstrainsforxylanasesBS1,BS2andBS3weredevelopedby
transformation of the B. subtilis host strain with an appropriate transformation
vector.Thehoststrainisderivedfromthewell-characterized,nonpathogenicand
nontoxigenic B. subtilis wild-type strain 168. Three transformation vectors were
constructed based on the commonly used plasmid pUB110.The vectors contain
the xylanase gene derived from B. subtilis strain 168. Two vectors encode xyla-
nases BS1 and BS2, both of which are identical to the native xylanase A from
strain168.ThevectorencodingxylanaseBS1alsocontainsgenesencodingpro-
teins that inactivate the antibiotics kanamycin/neomycin and phleomycin. These
proteins are intracellular and are not carried over into the xylanase preparation.
ThevectorencodingxylanaseBS2wasgeneticallymodifedtoremovethegenes
conferring resistance to the antibiotics. The third transformation vector encodes
xylanaseBS3,whichwasgeneticallymodifedbytwoaminoacidsubstitutionsto
beresistanttothexylanaseinhibitorpresentinfour.Thisvectordoesnotcontain
genesconferringresistancetotheantibiotics.Eachvectorwasintroducedintothe
host strain to obtain the corresponding xylanase production strain.All the intro-
ducedDNAiswell-characterizedandwouldnotresultintheproductionofanytoxic
or undesirable substances. The production strain is stable with respect to the
introducedDNA.
1.2 Product characterization
Eachxylanaseisproducedbypureculturefermentationoftherespectivepro-
duction strain. Xylanase is secreted into the fermentation medium, from which it
issubsequentlyrecovered,concentrated,andformulatedusingsubstancessuita-
bleforuseinfood,suchasstarchandsalt.Twoxylanasepreparations,onecon-
taining the native xylanase BS2 and the other containing the modifed xylanase
BS3, which is resistant to the xylanase inhibitor in four, have been marketed.
These xylanases would be denatured at temperatures >50C and would not be
enzymatically active in food as consumed. Both xylanase preparations
2
conform
2
Two specifcation monographs were prepared for xylanase preparations containing
xylanasesBS2andBS3,therespectivetitlesbeingXylanase from Bacillussubtilis expressed
in B.subtilis,andXylanase (resistant to xylanase inhibitor) from Bacillussubtilis containing
a modifed xylanase gene from B.subtilis.
XYLANASE FROM BACILLUSSUBTILIS 151
K2
to the General specifcations for enzyme preparations used in food processing
(Annex 1, reference 156). The xylanase preparation containing xylanase BS1 is
notintendedforcommercialization.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
Noinformationwasavailable.
2.2 Toxicological studies
Toxicological studies have been performed with different test batches of the
three enzyme preparations, each being brown, water-soluble liquid concentrates
fromafermentationwiththerecombinantstrain.Althoughfortheenzymeprepara-
tiondesignatedasxylanaseBS3theenzymecontentwasoriginallyexpressedin
DXU,thiswasconvertedtoTXUinthestudydescriptionsbelow,forcomparison
purposes.
2.2.1 Acute toxicity
Studiesofacutetoxicityhavebeenperformedwiththreeenzymetestprepara-
tions, designated as xylanases BS1 (or Bacillus xylanase), BS2 and BS3, with
enzyme contents of 100000TXU/ml, 106000TXU/g and 110000TXU/g, respec-
tively.TheTOScontentsofthesepreparationswere20.1%
3
,3.25%,and15.9%,
respectively.ThestudiesfollowedOECDtestguideline420(fxeddoseprocedure,
1992/2001),andwerecertifedforcompliancewithgoodlaboratorypractice(GLP)
andqualityassurance(QA).TheresultsaresummarizedinTable1.
2.2.2 Short-term studies of toxicity
Rats
GroupsoffvemaleandfvefemaleWistarrats(aged67weeks)weregiven
xylanaseBS3(batchTOX2;enzymecontent,41125TXU/g;TOScontent,6.25%)
at a dose equivalent to 0, 20000, 50000, or 200000TXU/kgbw by gavage (in
sterilewater),dailyfor4weeks.ThestudywasperformedaccordingtoOECDtest
guideline407(1995),andwascertifedforcompliancewithGLPandQA.Allvisible
signsofillhealthorbehaviouralchangeswererecordeddaily,asweremorbidity
andmortality.Onceperweek,bodyweightandfoodconsumptionwererecorded,
anddetailedclinicalobservationswereperformedoutsidethecage.Inweek4,all
animals were examined for sensory reactivity to different types of stimuli, grip
strength,andmotoractivity.Atterminationoftreatment,bloodandurinesamples
were collected from all animals for haematology, clinical chemistry, and urine
3
Thisisatheoreticalvalue,sincedrymatterandashcontentwerenotknown.
152 XYLANASE FROM BACILLUSSUBTILIS
K2
analysisdeterminations.Atnecropsy,amacroscopicexaminationwasperformed
on all animals, and absolute and relative (to body weight) weights of 11 organs
weredetermined.Microscopywascarriedoutonabout40organsandtissuesfrom
allanimalsinthecontrolgroupandinthegroupreceivingthehighestdose.
No treatment-related changes were observed in any of the parameters
examined.The no-observed-effect level (NOEL) was 200000TXU/kgbw per day
(equivalenttoanintakeofTOSof304mg/kgbwperday),thehighestdosetested
(Kaaber,2003).
Groups of ten male and ten female Wistar rats (aged 56 weeks) were
given xylanase BS1 (or Bacillus xylanase; batch 150699-1; enzyme content,
38900TXU/ml;andTOScontent,3.04%)atadoseequivalentto0,8000,20000,
or 80000TXU/kgbw by gavage (in sterile water), daily for 13 weeks. The study
wasperformedaccordingtoOECDtestguideline408(1998),andwascertifedfor
compliancewithGLPandQA.Allvisiblesignsofillhealthorbehaviouralchanges
were recorded daily, as were morbidity and mortality. Once weekly, body weight
and food consumption were recorded, and detailed clinical observations were
performedoutsidethecage.Ophthalmoscopywasperformedonallanimalsatthe
startoftheexperiment,andanimalsinthecontrolgroupandatthehighestdose
werere-examinedbeforetermination.Inweek12,allanimalswereexaminedfor
sensoryreactivitytodifferenttypesofstimuli,gripstrength,andmotoractivity.At
termination of treatment, blood samples were collected from all animals for hae-
matologyandclinicalchemistrydeterminations.Atnecropsy,amacroscopicexami-
nation was performed on all animals, and absolute and relative (to body weight)
weights of 11 organs were determined. Microscopy was carried out on about 40
organsandtissuesfromallanimalsinthecontrolgroupandatthehighestdose,
on all organs and tissues from animals dying or sacrifced during the study, and
onallgrosslesionsfromallanimals.
Table 1. Acute toxicity of xylanase administered orally
Enzymepreparation Species Sex LD
50
Reference
(mg/kgbw)
BS1,batch150699-3 Rat M,F >2000
a
Kaaber(1999);Harbak&
Thygesen(2002)
BS2,batchTOX1 Rat F >2000
b
Bollen(2003a)
BS3,batchTOX1 Rat F >2000
c
Bollen(2003b)
F,female;M,male.
a
Equivalentto200000TXU/kgbw.
b
Equivalentto212000TXU/kgbw.
c
Equivalentto220000TXU/kgbw.
XYLANASE FROM BACILLUSSUBTILIS 153
K2
Notreatment-relatedeffectswereseenonmortality,clinicalsigns,ophthalmos-
copy, sensory reactivity, body weights, and (in females) food consumption. In
males,statisticallysignifcantchangesinfoodconsumptionwereobservedduring
someweeksoftreatment.Thesechangesshowednodoseresponserelationship,
and there was no statistically signifcant change in food consumption over the
wholeperiodoftreatment.Theonlyeffectsseenonhaematologyweresmall,but
statisticallysignifcantlydecreasedrelativenumbersoflymphocytesinmalesinall
treatment groups (without a clear doseresponse relationship) and somewhat
greater, but not statistically signifcantly decreased absolute numbers of lym-
phocytes in males at the intermediate and highest doses. Females also showed
decreases in relative (at the highest dose only) and absolute numbers of lym-
phocytes (at the intermediate and highest dose), but these decreases were not
statisticallysignifcant.Examinationoftheindividualdatarevealedawidevariation
in relative and absolute numbers of lymphocytes in animals in the control group
and animals in the treated groups. In a 4-week study (Kaaber, 2003; described
above),higherdosesthanthoseusedinthepresentstudydidnotresultinsignif-
cant effects on lymphocytes.Taking the results as a whole, the fndings on lym-
phocytesinthisstudywerenotconsideredtobetoxicologicallyrelevant.Clinical
chemistryparameterswerenotaffectedbytreatment,norwereorganweights,or
fndingsonmacroscopyandmicroscopy.TheNOELwas80000TXU/kgbwperday
(equivalentto63mgofTOS/kgbwperday),thehighestdosetested(Glerup,1999;
Harbak&Thygesen,2002).
2.2.3 Long-term studies of toxicity and carcinogenicity
Noinformationwasavailable.
2.2.4 Genotoxicity
Theresultsoffourstudiesofgenotoxicitywithxylanaseinvitroaresummarized
inTable 2.The frst three studies followed OECD test guideline 471 (1997), and
were certifed for compliance with GLP and QA. In these studies, three enzyme
preparations designated as xylanases BS1 (or Bacillus xylanase; TOS content
4
,
7.8%;enzymecontent,38900TXU/ml),BS2(TOScontent,6.1%;enzymecontent,
156000TXU/g) and BS3 (TOS content, 8.2%; enzyme content, 39875TXU/g)
were tested. Xylanase BS1 was also tested in the fourth study, which followed
OECDtestguideline473(1997),andwasalsocertifedforcompliancewithGLP
andQA.
2.2.5 Reproductive toxicity
Noinformationwasavailable.
4
Values given for total organic solids contents are theoretical values. Since ash contents
werenotknown,itwasassumedthatalldrymatterwasorganicmaterial.
154 XYLANASE FROM BACILLUSSUBTILIS
K2
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XYLANASE FROM BACILLUSSUBTILIS 155
K2
2.3 Observations in humans
Noinformationwasavailable.
3. INTAKE
The petitioned enzyme preparations would be used in the milling and baking
industries, mainly to improve the dough. They may be used in yeast-raised or
chemically-leavened wheat and rye-based bakery products (including bread,
cakes,pastries,biscuits,crackers,pastaandnoodles)atuselevelsrangingfrom
12000to320000DXU/kgoffourforxylanaseBS3(equivalentto50013300TXU/
kg of four or 0.061.6mg of xylanase/kg of four, given the specifc activity of
202900DXU(or8400TXU)/mg).TheuselevelsforxylanaseBS2rangefrom3000
to40000TXU/kgoffour(equivalentto0.121.6mgofxylanase/kgoffour,given
thespecifcactivityof25000TXU/mg).Alowerminimumdosagemightbesuffcient
forxylanaseBS3,despitethefactthatithasalowerspecifcactivitythanthatof
xylanaseBS2,becauseitsamino-acidsequencehadbeenmodifedsuchthatthe
enzymedoesnotbindsoeasilytoaxylanaseinhibitorfoundnaturallyinfour.
As the enzyme is inactivated during baking (data provided suggest that it is
completelyinactivatedafter5minat70C),itisnotactiveinthefnalproductas
eaten.Itcanthusberegardedasaprocessingaid.
In order to estimate the daily intake resulting from consumption of bakery
productsinaworst-casesituation,thefollowingassumptionsweremade:
Allbakingproductsareproducedusingthexylanaseenzymepreparationsas
aprocessingaid;
Thefourcontentinbakeryproductsis66%;
The dosage is set at the maximum recommended level, that is: 1.6mg of
xylanase/kg of four for all preparations (equivalent to 13300TXU/kg of four
forxylanaseBS3,andto40000TXU/kgoffourforxylanaseBS2).
Theupperphysiologicalconsumptionoffoodis50g/kgbwperdayaccording
tothebudgetmethod(Hansen,1979).Aworst-casesituationisthatoftheinges-
tionofbakeryproductsat25g/kgbwperday.Thehypotheticalintakeoffourfrom
bakeryproductswouldthenbe16.5goffour/kgbwperday(250.66),resulting
in a daily enzyme intake of 26.4mg of xylanase/kgbw, equivalent to 219TXU (or
0.3mgofTOS)/kgbwperdayforxylanaseBS3and660TXU(or0.2mgofTOS)/
kgbwperdayforxylanaseBS2.
4. COMMENTS
Xylanases naturally present in food and xylanases used as enzymes in food
processing have not been reported to cause allergic reactions. By analogy, it is
notlikelythattheB. subtilisxylanasesunderevaluationwillcauseallergicreactions
afteringestionoffoodcontainingtheresiduesoftheseenzymes.
156 XYLANASE FROM BACILLUSSUBTILIS
K2
Toxicological studies were performed with test batches of the water-soluble
liquidenzymeconcentrates.Thesebacterialenzymepreparationswerenotacutely
toxicwhentestedinrats,norweretheymutagenicinassaysinbacteriainvitro or
clastogenicinanassayforchromosomalaberrationsinmammaliancellsinvitro.
Nosignifcanttreatment-relatedeffectswereseenina4-weekstudyinratstreated
bygavagewithxylanaseBS3atdosesuptoandincluding200000TXU/kgbwper
day(equivalentto304mgofTOS/kgbwperday),thehighestdosetested,orina
13-week study in rats treated by gavage with xylanase BS1 at doses up to and
including 80000TXU/kgbw per day (equivalent to 63mg of TOS/kgbw per day),
thehighestdosetested.Thesehighestdoseswerethereforeconsideredtobethe
NOELsinthesestudies.
Conservative estimates of daily intakes resulting from the use of xylanase in
bakingapplicationswere660TXU/kgbwperday(or0.2mgofTOS/kgbwperday)
for xylanase BS2, and 219TXU/kgbw per day (or 0.3mg of TOS/kgbw per day)
for xylanase BS3. When these intakes were compared with the NOEL of
200000TXU/kgbw per day (equivalent to 304mg of TOS/kgbw per day), the
highestdosetestedinthe4-weekstudyoforaltoxicity,themarginsofsafetywere
>1000forbothenzymepreparations.Whentheseintakeswerecomparedwiththe
NOEL of 80000TXU/kgbw per day (equivalent to 63mg of TOS/kgbw per day),
thehighestdosetestedinthe13-weekstudyoforaltoxicity,themarginsofsafety
were>200forbothenzymepreparations.
5. EVALUATION
The Committee allocated an acceptable daily intake (ADI) not specifed for
xylanasefromthisrecombinantstrainofB. subtilis,usedintheapplicationsspeci-
fedandinaccordancewithgoodmanufacturingpractice.
6. REFERENCES
Bollen,L.S.(2003a)XylanaseBS2acuteoraltoxicitystudyintherat.Unpublishedreport
No.51932fromScantox,LilleSkensved,Denmark.SubmittedtoWHObyDaniscoUSA
Inc.,Ardsley,NY,USA.
Bollen,L.S.(2003b)XylanaseBS3acuteoraltoxicitystudyintherat.Unpublishedreport
No.51228fromScantox,LilleSkensved,Denmark.SubmittedtoWHObyDaniscoUSA
Inc.,Ardsley,NY,USA.
Edwards,C.N.(1999a)BacillusxylanaseAmestest.UnpublishedreportNo.34923from
Scantox,LilleSkensved,Denmark.SubmittedtoWHObyDaniscoUSAInc.,Ardsley,NY,
USA.
Edwards,C.N.(1999b)Bacillusxylanasein vitromammalianchromosomeaberrationtest
performed with human lymphocytes. Unpublished report No. 34924 from Scantox, Lille
Skensved,Denmark.SubmittedtoWHObyDaniscoUSAInc.,Ardsley,NY,USA.
Edwards, C.N. (2003a) Xylanase BS2,TOX2 Ames test. Unpublished report No. 52910
fromScantox,LilleSkensved,Denmark.SubmittedtoWHObyDaniscoUSAInc.,Ardsley,
NY,USA.
XYLANASE FROM BACILLUSSUBTILIS 157
K2
Edwards, C.N. (2003b) Xylanase BS3,TOX3 Ames test. Unpublished report No. 52911
fromScantox,LilleSkensved,Denmark.SubmittedtoWHObyDaniscoUSAInc.,Ardsley,
NY,USA.
Glerup,P.(1999)Bacillusxylanasea13-weekoral(gavage)toxicitystudyinrats.Unpub-
lishedreportNo.34387fromScantox,LilleSkensved,Denmark.SubmittedtoWHOby
DaniscoUSAInc.,Ardsley,NY,USA.
Hansen,S.C.(1979)Conditionsforuseoffoodadditivesbasedonabudgetforanaccept-
abledailyintake.J. Food Protect.,42,42934.
Harbak, L. &Thygesen, H.V. (2002) Safety evaluation of a xylanase expressed in Bacillus
subtilis.Food Chem. Toxicol.,40,18.
Kaaber,K.(1999)Bacillusxylanaseacuteoraltoxicitystudyintherat.Unpublishedreport
No.34762fromScantox,LilleSkensved,Denmark.SubmittedtoWHObyDaniscoUSA
Inc.,Ardsley,NY,USA.
Kaaber,K.(2003)XylanaseBS3a4-weekoral(gavage)toxicitystudyinrats.Unpublished
reportNo.51173fromScantox,LilleSkensved,Denmark.SubmittedtoWHObyDanisco
USAInc.,Ardsley,NY,USA.
K2
K2
159
ZEAXANTHIN (synthetic)
First draft prepared by
Professor M.C. Archer,
1
Professor H. Ishiwata,
2
and Professor R. Walker
3
1
Department of Nutritional Sciences, University of Toronto, Toronto,
Ontario, Canada;
2
Seitoku University, Chiba, Japan; and
3
School of Biomedical and Molecular Sciences, University of Surrey,
Guildford, Surrey, England
Explanation............................................................................... 159
Biologicaldata.......................................................................... 160
Biochemicalaspects.......................................................... 160
Absorption,distribution,andexcretion........................ 160
Absorptionandavailability.................................... 160
Pharmacokineticstudies....................................... 162
Biotransformation......................................................... 166
Effectsonenzymesandotherbiochemical
parameters............................................................ 167
Toxicologicalstudies.......................................................... 167
Acutetoxicity................................................................ 167
Short-termstudiesoftoxicity....................................... 167
Long-termstudiesoftoxicityand
carcinogenicity....................................................... 170
Genotoxicity................................................................. 170
Reproductivetoxicity.................................................... 172
Multigenerationstudies......................................... 172
Developmentaltoxicity.......................................... 172
Specialstudies............................................................. 172
Immuneresponses................................................ 172
Oculartoxicity........................................................ 173
Ocularirritation...................................................... 174
Observationsinhumans.................................................... 174
Clinicalstudies............................................................. 174
Epidemiologicalstudies............................................... 175
Intake ..................................................................................... 176
Concentrationsinfoods..................................................... 176
Dietaryintake..................................................................... 176
Comments ............................................................................... 177
Evaluation ............................................................................... 179
References............................................................................... 179
1. EXPLANATION
Zeaxanthin (3R,3R-dihydroxy-b-carotene), a naturally occurring xanthophyll
pigment, is an oxygenated carotenoid that has no provitaminA activity. It occurs
together with the isomeric xanthophyll pigment, lutein (see monograph in this
160 ZEAXANTHIN
K2
volume,p49),inmanyfoods,particularlyvegetablesandfruit.Itisintendedtobe
usedasafoodcolourandasanutritionalsupplementinawiderangeofapplica-
tions at concentrations ranging from 0.5 to 70mg/kg. An extract from Tagetes
erecta L. containing primarily lutein with variable amounts of antheraxanthin and
other xanthophylls was considered by the Committee at its thirty-frst meeting
(Annex1,reference77).Atthattime,notoxicologicaldatawereavailableandno
evaluationwasmade.Forthepresentmeeting,informationwasreceivedfortwo
differentproducts:syntheticzeaxanthinandzeaxanthin-richextractfromTagetes
erectaL.However,theCommitteedidnotreceiveanytoxicologicaldatasupporting
thesafetyevaluationoftheextract.Anumberoftoxicologicalstudieshavebeen
carried out with respect to the safety of synthetic zeaxanthin for addition to food
andthesewereevaluatedbytheCommitteeatitspresentmeeting.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution, and excretion
(a) Absorption and availability
Xanthophylls may be ingested in either free or esterifed forms, although
unesterifedzeaxanthinisthesubjetofthepresentevaluation.Beforeabsorption,
the esters are hydrolysed by pancreatic esterases and lipases (Breithaupt et al.,
2002)suchthatonlythefreeformsarefoundinthecirculation(Wingerathetal.,
1995). Once released from their food matrix as a lipid emulsion, like other non-
polar lipids, these compounds must be solubilized within micelles in the gastro-
intestinal tract to permit absorption by mucosal cells (Erdman et al., 1993). The
transferofcarotenoidsfromlipidemulsiondropletstomixedmicellesdependson
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
HO
CH
3
CH
3
OH CH
3
Figure 1. Chemical structure of zeaxanthin
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
HO
CH
3
CH
3
CH
3
OH
Figure 2. Chemical structure of lutein
ZEAXANTHIN 161
K2
their hydrophobicity, as well as pH and concentration of bile acid (Tyssandier et
al., 2001). Other carotenoids such as lycopene and xanthophylls can impair the
transfer of b-carotene, but neither b-carotene nor other xanthophylls affect
the transfer of lutein (Tyssandier et al., 2001). The more polar carotenoids such
as the xanthophylls are preferentially solubilized in the surface of lipid emulsion
droplets and micelles, while the less polar carotenoids are incorporated into the
corearea(Boreletal.,1996).Thisfacilitatesthetransferofcompoundslikezea-
xanthinfromthelipiddropletstotheaqueousphase.Indeed,ithasbeendemon-
stratedthatthexanthophyllsaremorereadilyincorporatedintomicellesthanother
carotenoids(Garrettetal.,1999,Garrettetal.,2000).
Theabsorptionofcaroteneishigherwhenfatisaddedtothediet(Roodenburg
etal.,2000)andlowerindisease-inducedfatmalabsorption(Erdmanetal.,1993).
Thepresenceoffatinthesmallintestinestimulatesthesecretionofbileacidsfrom
thegallbladderandimprovestheabsorptionofcarotenoidsbyincreasingthesize
andstabilityofmicelles,thusallowingmorecarotenoidstobesolubilized.Absorp-
tion of carotenoids by mucosal cells is believed to occur by passive diffusion
(Hollander&Ruble,1978).
Afteruptakeintomucosalcells,carotenoidsareincorporatedintochylomicrons
and released into the lymphatics. When mucosal cells are sloughed off, carot-
enoidsthathavebeentakenupbythecells,butnotyetincorporatedintochylo-
microns, are lost into the lumen of the intestine. The carotenoids within the
chylomicrons are transported to the liver where they are distributed among the
lipoproteinfractions.Incontrasttothelesspolarcarotenoids,asignifcantfraction
ofthexanthophyllsiscarriedinthebloodstreambyhigh-densitylipoprotein(HDL)
(Romanchiketal.,1995).
The absorption of carotenoids including zeaxanthin is potentially affected by
thefoodmatrixinwhichthecarotenoidsareconsumed,dietaryfat,andthepres-
ence of other carotenoids in the diet (Castenmiller & West, 1998; Zaripheh &
Erdman,2002).
Whilenoinformationonzeaxanthinitselfwasprovided,therelativeavailability
of lutein from a mixed vegetable diet has been shown to be 67% relative to that
fromadietsupplementedwithpurelutein(vanhetHofetal.,1999a).Inanother
study, the relative bioavailability of lutein and b-carotene from various spinach
productswascomparedwiththatfromsupplements(6.6mgofluteinplus9.8mg
ofb-carotene).Thevaluesrangedfrom4554%forluteintoonly5.19.3%forb-
carotene(Castenmilleretal.,1999).Thepresenceofdietaryfbremayexplain,at
leastinpart,thelowavailabilityofcarotenoidsfromplantfoods.Ithasbeensug-
gestedthatfbreinterfereswiththeformationofmicellesbypartitioningbilesalts
andfatinthegelphaseofthefbre.Processing,suchasmechanicalhomogeniza-
tionorheattreatment,hasbeenshowntoincreasetheavailabilityof b-carotene
in vegetables from 18% to 600% (van het Hof et al., 2000). There is evidence,
however,thatdisruptionofthematrixaffectstheavailabilityofcarotenoidsdiffer-
entially,possiblybecauseofdifferencesintheirlipophiliccharacter.Forexample,
the plasma response of lutein was increased by about 14% when spinach was
consumedchoppedinsteadofwhole,whileb-carotenewasnotaffected(vanhet
Hofetal.,1999b).Thematricesofformulatednaturalorsyntheticcarotenoids(e.g.
162 ZEAXANTHIN
K2
water-dispersiblebeadlets,crystallinepowders,oilsuspensions)andwhetherthe
compoundsareesterifedornon-esterifedmayclearlyaffectavailability(Swanson
etal.,1996;Boileauetal.,1999).
Because the absorption of carotenoids occurs via incorporation into mixed
micelles,ingestionoffataffectstheiravailability.Theamountofdietaryfatrequired
toensuretheabsorptionofcarotenoidsseemstobelow(35g/meal),althoughit
depends on the physico-chemical characteristics of the carotenoids ingested. In
oneexperiment,lutein,addedasesters,gaveplasmaconcentrationsabout100%
higherwhenconsumedwith35goffatthanwith3goffat(vanhetHofetal.,2000).
Thesmallamountoffatmayhavelimitedthesolubilizationofluteinestersand/or
the release of esterases and lipases (Roodenburg et al., 2000). Availability of
carotenoids is also affected by the absorbability of the dietary fat (Borel et al.,
1998).
Eggyolkisanexampleofasourceofhighlyavailablezeaxanthinandlutein.
The lipid matrix of the egg yolk containing cholesterol, triacylglycerols and
phospholipids provides a vehicle for the effcient absorption of the xanthophylls
(Handelmanetal.,1999).
Interactions between carotenoids may decrease absorption. Competition for
absorptionmayoccuratthelevelofmicellarincorporation,intestinaluptake,lym-
phatictransportoratmorethanonelevel.Alternatively,simultaneousingestionof
variouscarotenoidsmayinduceanantioxidant-sparingeffectintheintestinaltract
resulting in increased levels of uptake of the protected carotenoids. It has been
demonstrated that in the presence of high amounts of b-carotene, the uptake of
xanthophylls from the intestinal lumen by chylomicrons is greater than that of b-
carotene (Grtner et al., 1996).A number of studies on the interaction of dietary
b-carotene and lutein have reported varying effects of one carotenoid on the
absorption or plasma concentrations of the other (Micozzi et al., 1992; Kostic et
al.,1995;vandenBerg,1998;Tyssandieretal.,2002).VandenBerg(1999)has
concludedthatingeneral,long-termsupplementationwithb-carotenehaslimited
or no effect on plasma concentrations of other carotenoids. However, in the a-
Tocopherol,b-CaroteneCancerPreventionStudy(ATBCStudy),atotalof29133
maleFinnishsmokersaged5069yearsweregivendailysupplementsof20mg
of b-carotene (0.3mg/kgbw per day) for an average of 6.7 years. Signifcantly
decreased serum concentrations of lutein (no changes in zeaxanthin) were
observedincomparisonwithgroupsthatdidnotreceiveb-carotenesupplements
(Albanesetal.,1997).
Anumberofnon-dietaryfactorsalsonegativelyaffecttheavailabilityofcaro-
tenoids, including exposure to tobacco smoke, alcohol consumption, intestinal
parasites, malabsorption diseases, liver and kidney diseases, hormone status,
pooriron,zincandproteinintake,gastricpHandhyperthyroidism(Albanesetal.,
1997;Williamsetal.,1998.,Patrick,2000;Alberg,2002).
(b) Pharmacokinetic studies
Pharmacokinetic studies with zeaxanthin have been performed in mice, rats
andhumans.
ZEAXANTHIN 163
K2
Mice
Inastudydesignedtoinvestigatetheuptakeoflutein/zeaxanthin,BALB/cmice
receiveddietscontaininganextractofmarigoldpetalsforupto28days(Parket
al.,1998).Basedondataonfoodintakeandbodyweight,dailyintakesoflutein
for each group of 36 mice corresponded to approximately 0, 75, 150, 300, and
600mg/kgbw,whileintakesofzeaxanthinwereapproximately0,1.0,2.0,4.0,and
8.0mg/kgbw,respectively.Sixmicepergroupwerekilledondays0,3,7,14,21
and 23. Body, liver and spleen weights did not differ throughout the experiment.
Plasmauptakeofluteinandzeaxanthin(inallcasesanalysedtogether)wasrapid
andreachedmaximumconcentrations(about3mmol/l)byday3ofdosing(thefrst
time-point examined after the start of dosing) and did not differ between groups
thereafteruntilday28.Therewasalsoarapidincreasetoday3inconcentrations
ofluteinandzeaxanthininliverandspleenwithcontinued,thoughsmall,increases
today28.Theliverwasconsideredtobethemajorstorageorganforluteinand
zeaxanthin.
Rats
Theabsorption,excretion,tissuedistributionandplasmakineticsofzeaxanthin
were investigated in groups of male and female Han Wistar rats given a single
oral (gavage) dose of [
14
C]R, R-all-E-zeaxanthin (99%) at 2 or 20mg/kgbw in a
beadlet formulation containing gelatin and vegetable oil (three to fve rats per
measurement). Before administration of the radiolabeled dose, rats had been
treated with nonradiolabelled synthetic zeaxanthin (99%) added to the diet as a
beadletformulationfor2weeksatthesamedailydose(2or20mg/kgbwperday)
to establish steady-state conditions. Radioactivity was determined in expired air,
urine,bile,faeces,plasma,bloodorgansandtissues.
Peak plasma concentrations of zeaxanthin were observed at 6 and 3h after
administrationofthelowerdose,andat8and6hafterthehigherdose,inmales
andfemalesrespectively.Plasmaconcentrationswerebelowthelimitofdetection
by3648hafterdosing.Tissueconcentrationsofzeaxanthinwerehighestinthe
gastrointestinal tract (stomach, and small and large intestines), liver, spleen,
kidney, pancreas, and lungs, with measurable amounts also in the thyroid and
adrenals. Radioactivity was almost completely cleared from tissues by 96h, the
lasttime-pointevaluated,andtherewasnoevidenceofaccumulation.Saturation
oftheabsorptionpathwayforzeaxanthinatthehigherdosewassuggestedboth
byanon-linearincreaseintheareaunderthecurveofconcentrationtime(AUC)
(anapproximatelytwofoldincreaseforatenfoldincreaseindose)andbydataon
urinaryexcretion.Chromatogramsobtainedforeachofthepooledplasmasamples
of each sex at both doses indicated the presence of up to seven components,
noneofwhichwasidentifed.
Most of the administered radiolabel was excreted in the faeces of both male
andfemalerats(about90%andabout100%atdosesof2mg/kgand20mg/kg,
respectively).Urinaryexcretionaccountedforameanofonly34%andabout1%
at the lower and higher doses, respectively, in both sexes. Excretion was rapid,
with most of the radioactivity being excreted in the frst 48h after dosing. Biliary
164 ZEAXANTHIN
K2
excretionwasminimalaftereitherdose(approximately0.5%oftheadministered
dose). No radiolabel was associated with the expired air in this study, indicating
that the site of labelling was stable. From the data on urinary excretion, the oral
absorptionoftotalradioactivitywasabout4%inbothsexesatthelowerdose,and
only about 1% in both sexes at the higher dose. There were reported to be no
relevantdifferencesbetweenmalesandfemales(Froescheisetal.,2001).
Adistributionstudywithzeaxanthinofahigherspecifcactivitythanthatused
in the study reported above was conducted in groups of three male albino Ibm:
ROROratspre-treatedwithzeaxanthin-poor(basicratdietcontainingamaximum
of0.0001%zeaxanthin,equivalenttoabout0.08mg/kgbwperday)orzeaxanthin-
enricheddiet(containing0.001%zeaxanthin,equivalenttoabout0.8mg/kgbwper
day)for5weeks.Asingledoseof(7,8,7,8[
14
C])-zeaxanthininaliposomalprepa-
rationwasadministeredorallybygavage.Asinthepreviousexperiment,zeaxan-
thinwasmainlyexcretedinthefaeces(5070%ofadministeredradiolabelin24h),
with 611% of the radioactivity in the urine after 24h. Approximately 1% of the
applieddose,orabout47%oftheabsorbeddose,wasmeasuredintheexpired
airduringthefrst24haftertheadministrationofradiolabeledzeaxanthin.Based
on the urine and tissue measurements, and assuming that there was no entero-
hepatic circulation, absorption ranged between 915% for the rats given the
zeaxanthin-poor diet to 1319% for the rats given the zeaxanthin-enriched diet
(biliary excretion was not considered). After 24h, about one-third of the
administeredradiolabelwasstillpresentinthebodyandintestinaltract,whileafter
1 week, <0.5% was present. It was concluded that the radioactivity from
[
14
C]zeaxanthinisrapidlydepletedfromthebodyandthegastrointestinaltractin
rats(Glatzleetal.,1999a,1999b).
Thedistributionofzeaxanthinwasinvestigatedingroupsof20maleFU-albino
(RORO) rats fed diets containing nonradiolabelled zeaxanthin at a concentration
of10mgor100mg/kgoffeed,addedasabeadletformulation(adoseofapproxi-
mately 0.8mg or 8mg/kgbw per day) (Bausch et al., 1999). Five rats per group
werekilledafter35daysofaccumulation(receivingadietcontainingzeaxanthin),
then after 7, 21 and 35 days of depletion (receiving a zeaxanthin-free diet). A
dose-dependentaccumulationofzeaxanthinwasfoundinseveraltissues,withthe
exceptionofthethyroidglandandtheeye,whereconcentrationswereundetect-
able.After35daysofaccumulation,thehighestconcentrationsofzeaxanthinwere
foundinthesmallintestine(about3.3mg/g)andspleen(about2.75mg/g),followed
byliver(about0.4mg/g),fat(about0.14mg/g)andadrenalglands(about0.12mg/g)
(values are for the higher dose). There was a pronounced decrease in tissue
concentrations of zeaxanthin during a subsequent 5-week period when rats
received a zeaxanthin-free diet. For many tissues, >50% of zeaxanthin was lost
inthefrstweek,followedbyaslowerrateofloss.
Humans
Concentrationsofluteinandzeaxanthininserumandtissueshavebeenshown
to be quite variable (Boileau et al., 1999), but to increase, as expected, with
increasedintakeeitherfromdietarysourcesorfromsupplements(e.g.Hammond
etal.,1997;Landrumetal.,1997a,1997b;Carrolletal.,1999;Tuckeretal.,1999;
Berendschot et al., 2000; Johnson et al., 2000; Curran-Celantano et al., 2001;
ZEAXANTHIN 165
K2
Olmedilla et al., 2001; Schalch et al., 2001; Bone et al., 2003). In a population-
basedstudy,Bradyetal.(1996)reportedthatlowerserumconcentrationsoflutein
andzeaxanthinaregenerallyassociatedwithmales,smoking,youngerage,lower
concentrationsofHDLcholesterol,greaterconsumptionofethanolandhigherbody
massindex.Carotenoidsarepresentinvariableamountsinmanytissuessuchas
kidneys, buccal mucosal cells and adrenal glands, but the main sites of storage
are adipose tissue and liver (Parker, 1996).As in serum, b-carotene, lutein and
lycopenearethemaincarotenoidsfoundintissues,althoughb-cryptoxanthinand
zeaxanthinarealsopresentinsignifcantamounts(Boileauetal.,1999).Theeye
ingeneral,andtheretinainparticular,containextraordinarilyhighconcentrations
of zeaxanthin and lutein (Bone et al., 1993). Other carotenoids are present in
onlytraceamountsintheretinaandlens(Khachiketal.,1997a,1997b,Yeumet
al., 1999, Bernstein et al., 2001). Zeaxanthin and lutein are the pigments
responsibleforthecolourationofthemaculalutea(yellowspot)(Landrum&Bone,
2001).
Therehavebeentwopharmacokineticstudieswithzeaxanthininhumans.The
plasma concentrations of lutein and zeaxanthin were measured in a small pilot
study in groups of eight volunteers (four men and four women) receiving daily
supplementscomprisingcapsulescontainingeither4.1mgoflutein(with0.34mg
ofzeaxanthin)or20.5mgoflutein(with1.7mgofzeaxanthin)for42days(Cohn
etal.,2001).Subjectsweremonitoredforafurther25daysafterthedosingphase.
Steady-stateconcentrationsofxanthophyllwerereachedbetweendays38to43
(0.06mmol/l and 0.13mmol/l for the lower and higher doses, respectively). Dose-
normalized incremental maximum and average steady state concentrations of
lutein and zeaxanthin were found to be comparable, indicating that they have
similarbioavailability.Theeliminationhalf-lifewascalculatedtobeapproximately
57daysforeithercompound.
Inasubsequentstudy,afterarun-inperiodof3daystodefnebase-linecon-
centrations,capsulesprovidingdosesofeither1mgor10mgofzeaxanthinwere
administereddailytogroupsoffvemenandfvewomenfor42days(Cohnetal.,
2002,Hartmannetal.,2004).Accumulationofplasmazeaxanthinwasmonitored
betweendays1and42,andthekineticsofdisappearancewerefollowedfromday
42 to day 76. Concentrations of zeaxanthin increased from 0.048 0.026mmol/l
at baseline to 0.20 0.07 and 0.92 0.28mmol/l at 1 and 10mg of zeaxanthin
respectively. The dose-normalized bioavailability of zeaxanthin after the 10mg
dosewas40%lowerthanafterthe1mgdose.After17daysofdosing,>90%of
the concentration at steady-state was reached, which was compatible with an
effectivehalf-lifeofaccumulationof5days.Theterminaleliminationhalf-lifewas
12 7 days. Multiple doses of 1 or 10mg of zeaxanthin did not affect plasma
concentrations of other carotenoids, retinol, a-tocopherol or lipids. 3-Dehydro-
lutein was shown to be derived from zeaxanthin and had the same plasma con-
centrationprofleaszeaxanthin.Itisreportedthatbothdosesofzeaxanthinwere
welltoleratedbyallsubjects.
2.1.2 Biotransformation
Anumberofcompoundsderivedfromluteinandzeaxanthinhavebeenidenti-
fed in human serum (Figure 3) (reviewed by Khachik et al., 1995a). These are
166 ZEAXANTHIN
K2
called metabolites, but they undoubtedly are formed by chemical rather than
enzymatic reactions. The metabolites result principally from three types of reac-
tions involving the end groups of these carotenoids oxidation, reduction and
double-bondmigration(Figure3).
Luteinandzeaxanthincanexistinequilibriuminvolvingtheintermediatecaro-
tenoid3-epilutein.AllylicoxidationofluteinatC3resultsintheformationofoxo-
luteinB,whichcanexistinequilibriumwithluteinand3-epiluteinthroughreduction
reactions.3-Epiluteinandzeaxanthincanalsoexistinequilibriumthroughrevers-
ible double-bond migration. Thus, presence of 3-epilutein in human serum may
be due to conversion of lutein and/or zeaxanthin. Acid-catalysed dehydration is
anotherreactionofcarotenoidswith3-hydroxy-eendgroups.Luteinisbelievedto
undergodehydrationinstomachacidtoform3-hydroxy-3,4-didehydro-b,g-caro-
tene and 3-hydroxy-2,3-didehydro-b,e-carotene (anhydroluteins) that have been
isolatedfromserum.Inadditiontotheirpresenceinhumanserum,thesemetabo-
liteshavealsobeendetectedinbreastmilkaswellasretinalextracts(Khachiket
al., 1995b; Khachik et al., 1997a, 1997b). The toxicological importance of these
compoundsisnotknown.
Figure 3. Proposed reactions of lutein and zeaxanthin in humans
AdaptedfromKhachiketal.(1995a,1997b)
HO
(P )
OH
(3R,3'R,6'R)-Lutein
Natural Lutei n
HO
(P)
O
(3R,6'R)-3-Hydroxy-
b,e- caroten-3'-one
Oxolutein B
OH
HO
(P)
(3R,3'S,6'R)-Lutein
3'-Epilutein
HO
(P)
OH
(3R,3'R)-Zeaxanthi n
HO
(P)
OH
(3R,3'S,meso)-Zeaxanthin
P=
OH
HO
(P)
(3S,6S,3'S,6'S) - e,e -carotene-
3,3'-diol ( Lactucaxanthin)
O
(P)
O
(6S,6'S)- e,e -carotene-3,3'-dione
O
HO
(P)
(6S,3'S,6'S)-3'-Hydroxy-
e,e -carotene-3-one
HO
(P)
OH
(3R,6S,3'S,6'S)- e,e -carotene-
3,3'-diol
Double Bond
Migratio n
Reduction Oxidatio n
Reductio n
Oxidation
Reductio n
Oxidatio n
Oxidation
Reductio n
Oxidatio n
Reductio n
Double Bond
Migratio n
Double Bond
Migratio n
Double Bond
Migratio n
ZEAXANTHIN 167
K2
2.1.3 Effects on enzymes and other biochemical parameters
Thexanthophyllsarenotconsideredtobeprecursorsofretinol.Indeed,they
have been shown to have little or no activity as substrates of b-carotene-15,15-
dioxygenase, although they are able to inhibit the conversion of b-carotene to
retinol(Ershovetal.,1993;vanVlietetal.,1996;Grolieretal.,1997).However,
in a model in rats, Weiser & Korman (1993) showed that the xanthophylls have
smallbutsignifcantprovitaminAactivity(45%oftheactivityofb-carotene),prob-
ably via a vitaminA-sparing effect. Furthermore, Weiser & Korman reported that
dietaryzeaxanthinisabletoinduceduodenal15,15-dioxygenaseactivityinchicks
aged1day.
Althoughtherearenostudiesontheeffectsofzeaxanthinontheenzymesof
drugmetabolism,studieshaveshownthatlutein(extractedfrommarigoldpetals
andlikelytocontainsmallamountsofzeaxanthin)hasnoeffectonanumberof
phaseIandphaseIIenzymesinratliver,lungandkidney(Gradeletetal.,1996;
Jewell&OBrien,1999).
2.2 Toxicological studies
2.2.1 Acute toxicity
Studies of acute toxicity with zeaxanthin were performed in rats and mice
(Baechtold, 1977a, 1977b).All mice and rats survived for 10 days after a single
oraldoseofzeaxanthinofupto4000mg/kgbwinratsand8000mg/kgbwinmice.
The LD
50
values in rats and mice are therefore >4000 and 8000mg/kgbw,
respectively.
2.2.2 Short-term studies of toxicity
Mice
In a study of oral toxicity, which did not comply with good laboratory practice
(GLP), albino-SPF mice were diets containing all-trans-3R,3R-zeaxanthin incor-
porated into gelatin-coated beadlets containing a fne suspension of 9.3% of the
purecompound(purity,97.6%)for13weeks.Somebatchesofbeadletscontained
upto0.15%chloroform,aresiduefromthesyntheticprocedure.Groupsof10male
and10femalemicereceivedzeaxanthinatnominaldosesof250,500,or1000mg/
kgbwperday.Usingplacebobeadlets,adjustmentsweremadesuchthatallfour
groups received the same amount of beadlets (about 10% of feed). In addition,
therewasacontrolgroupthatreceivedbeadletsthatdidnotcontainzeaxanthin.
No treatment-related effects were observed throughout the study. Haematology,
bloodchemistryandurineanalysismeasurementsshowednoevidenceoftoxicity
causedby.Therewerenotreatment-relatedfndingsbyophthalmoscopicexamina-
tion. In contrast with later studies in rats and dogs, no discolouration of adipose
tissue was reported. Findings at necropsy and histopathological examination of
tissuesrevealednosignifcanttreatment-relatedchanges.Theno-observed-effect
level (NOEL) was 1000mg/kgbw per day, the highest dose tested (Ettlin et al.,
1980a).
168 ZEAXANTHIN
K2
Rats
In a 13-week study of oral tolerance study, which did not comply with GLP,
albino-SPF rats were given diets containing a beadlet formulation of zeaxanthin
exactlyasdescribedaboveforthestudyinmice.Groupsof16maleand16female
ratsweregivenzeaxanthinatadoseof250,500,or1000mg/kgbwperday.The
additionofplacebobeadletsensuredthatallfourgroupsreceivedsimilaramounts
ofbeadlets(about20%offeed),andabeadlet-onlycontrolgroupwasincluded.
Towardstheendofthestudy,ratsatthehighestdosetendedtoavoideatingthe
beadlets, so the dose was reduced by about 40% in females and about 65% in
males.Onemaleratatthelowestdosediedatweek8;therehadbeennoprevi-
ousclinicalsignsoftoxicityandtherewerenoobvioushistopathologicalfndings.
There was a slight reduction in body-weight gain in all groups consuming the
beadlets,whichwasunrelatedtointakeofzeaxanthin.Asforthemice,therewere
no treatment-related effects throughout the study. Haematology, blood chemistry
andurineanalysismeasurementsshowednoevidenceoftoxicitycausedbyzea-
xanthin. There were no treatment-related fndings by ophthalmoscopic examina-
tion. In contrast with later 13-week studies of toxicity in rats and dogs, no
discolouration of adipose tissue was reported in the current study. Findings at
necropsy and histopathological examination of tissues revealed no signifcant
treatment-related changes.The NOEL was 500mg/kgbw per day (in view of the
reducedintakeatthehighestdoseduringthelatterpartofthestudy,thedatafrom
thisgroupcouldnotbeused)(Ettlinetal.,1980b).
Inasecond13-weekstudyoforaltoleranceinrats,adifferentbatchofbeadlets
wasusedinwhichmethylenechloridewasemployedinsteadofchloroforminthe
synthesisofthezeaxanthin.Theresidueofmethylenechlorideafterevaporation
wasestimatedtobeapproximately150250mg/kg.Inthisstudy,whichcomplied
withGLP,theratswereunabletoexcludethebeadletsfromtheirdiets.Groupsof
12 male and 12 female rats were given zeaxanthin at the same doses as those
usedpreviously(0,250,500,and1000mg/kgbwperday).Asbefore,therewere
nobiologicallysignifcantchangesresultingfromexposuretozeaxanthin,butthere
were a number of statistically signifcant changes: decreased numbers of leuko-
cytes (in males at the highest dose), decreased concentrations of bilirubin (in
malesandfemalesatthehighestdose),andincreasedconcentrationsofsodium,
decreasedconcentrationsoftotalserumproteinanddecreasedconcentrationsof
a-1-globulin(infemalesattheintermediateandhighestdoses).Allthesechanges,
however, were considered by the investigators to be within normal limits for the
rat. There were no treatment-related changes in organ weights, and no macro-
scopicormicroscopicfndingsthatcouldbeattributedtotoxiceffectsofzeaxan-
thin.Overall,theresultsweresimilartothoseoftheearlierstudydescribedabove,
withtheexceptionthattherewasayellow-orangediscolourationofthefaecesand
a slight orange discolouration of the adipose tissue, particularly at the higher
doses.Thiswasattributedtocolourimpartedbythezeaxanthinbeadlet.TheNOEL
was1000mg/kgbwperday,thehighestdosetested(thediscolourationnotbeing
consideredtobeanadverseeffect)(Buser,1985).
ZEAXANTHIN 169
K2
Dogs
Ina13-weekstudyoforaltoxicity,whichcompliedwithGLP,groupsofthree
maleandthreefemalebeagledogsaged10monthsweregivendietscontaining
abeadletformulationofzeaxanthin,exactlyasdescribedaboveformiceandrats.
Methylene chloride was used instead of chloroform in the formulation of the
beadlets.Beadletswereincorporatedintofeedpelletsatconcentrationsof0,4,8,
or16%,toachievedosesofzeaxanthinof0,123,204,or422mg/kgbwperday
inmalesand0,104,238,and442mg/kgbwperdayinfemales,respectively.The
percentage of beadlets present in the feed was kept constant (16% w/w) for all
treatment groups.Actual concentrations of zeaxanthin measured throughout the
studywerewithin90106%ofthenominalconcentrations.Therewerenodiffer-
encesinbodyweightsbetweenthegroupsthroughoutthestudy.Furthermore,no
treatment-related toxicity was observed in any of the dogs. Haematology, blood
chemistryandurineanalysismeasurementsshowednoevidenceoftoxicitycaused
byzeaxanthin.Thetestcompoundwasfoundtodiscolourstronglyandtoslightly
softenthefaeces,particularlyatthehighestdose.Therewasalsoaslightorange
discolouration of the adipose tissue, particularly in males at the highest dose.
There were no treatment-related fndings by ophthalmoscopic examination. At
necropsy, male dogs in the groups receiving the intermediate or highest doses
showedslighttomoderatediscolouration(yellowtoreddish)oftheadiposetissue
(attributed to colour imparted by the zeaxanthin beadlet). Histolopathological
examination of the tissues showed splenic adhesions (subacute haemorrhagic
perisplenitis) in one male at the intermediate dose, which was considered not to
be treatment-related (possibly arising owing to trauma). Slight changes in organ
weights(increasedweightsofthethyroidinfemalesatthelowestdoseandmales
at the highest dose compared with controls, decreased weights of the heart in
females at the lowest dose, and decreased weights of the kidney in dogs at the
intermediate dose) were not accompanied by histological changes and were not
dose-dependent. Overall, it was concluded that there were no treatment-related
microscopic fndings. The NOEL was 420mg/kgbw per day, the highest dose
tested (the discolouration not being considered to be an adverse effect) (Ettlin,
1985).
Monkeys
InastudythatcompliedwithGLPandthatwasdesignedprimarilytoinvesti-
gate the effects on the eye of long-term exposure to zeaxanthin, cynomologus
monkeysaged47yearsweregivenzeaxanthinatadoseof0,0.2,or20mg/kgbw
perdaybygavagefor52weeks.Thesolutionsforgavagewerepreparedbydis-
solvingbeadletsinwater.Thecontrolwaspreparedusingbeadletsthatcontained
no zeaxanthin. There were two males and two females in each group, with an
additionalmaleandfemaleincludedinthegroupreceivingthehigherdose,des-
ignatedforexaminationat6months.Allanimalssurvivedthetreatmentperiod.All
animals at 20mg/kg per day showed orange/yellow discolouration of the faeces
fromday2ofthestudyonwards(attributedtotreatmentwithzeaxanthin).There
wasnoeffectonoverallmeanbody-weightgainandonoverallgroupmeanfeed
intakeineitherofthetreatmentgroups.Therewerenotreatment-relatedchanges
170 ZEAXANTHIN
K2
inhaematology,bloodchemistry,orurineanalysismeasurements.Therewereno
changesinelectrocardiogramwaveformordataonbloodpressurethatcouldbe
regarded as being related to the administration of zeaxanthin. There were no
treatment-relatedorganweightchanges.Mostoftheanimalsshoweddarkyellow-
colouredmesentericfatatinterimsacrifceandgold-yellowmesentericfatatter-
minal sacrifce (attributed to treatment with zeaxanthin). Histopathological
examinations revealed no treatment-related fndings (a single adenoma of the
thyroid observed in a single female at the higher dose was considered to be an
isolatedincidentandnotoftoxicologicalsignifcance).Comprehensiveophthalmic
examinationsshowednoevidenceoftreatment-relatedadversechanges.Animals
showedadose-relatedincreaseinplasmaandliverconcentrationsofzeaxanthin.
Thus,zeaxanthinwasconsideredtobewelltolerated.TheNOELincynomologus
monkeys was 20mg/kgbw per day, the highest dose tested (Pfannkuch et al.,
2000a,2000b;Pfannkuch,2001).
2.2.3 Long-term studies of toxicity and carcinogenicity
Therehavebeennostudiesoftoxicitywithzeaxanthinwithadurationofgreater
than1year.
2.2.4 Genotoxicity
Thereissomeconcern,inthefrstthreestudieslistedinTable1,thatzeaxan-
thin precipitated out of solution, despite the use of dimethylsulfoxide (DMSO), at
thehighestconcentrationstested(Strobel,1986,1987)andatallconcentrations
tested(Strobel&Bonhoff,1987).Itisclear,however,thatzeaxanthininabeadlet
formulationgavenegativeresultsinanassayformicronucleusformationinbone
marrow of mice. In these studies, there was no evidence for genotoxicity. The
concentrationsanddosesusedinsomeofthestudieswereconsideredtobelow,
butwerethemaximumfeasibledoses.
InadditiontothestudiesreportedinTable1,abeadletformulationofzeaxan-
thinwasevaluatedformutagenicactivityinanAmesassay,whichcompliedwith
GLP, using both the plate incorporation and the pre-incubation methods (Gocke,
1987). Seven Salmonella typhimurium standard tester strains were employed
(TA1535,TA1537,TA1538,TA97,TA98,TA100,andTA102),withandwithoutan
exogenousmicrosomalfraction(S9)derivedfromliversofmalealbinoratstreated
withphenobarbital/b-naphthofavone.Owingtoprecipitationofthetestcompound
in the aqueous medium, concentrations of 2.4 to 1500mg/plate and 5 to 500mg/
plateweretestedintheplateincorporationandpre-incubationmethods,respec-
tively.Therewasnoincreaseofthenumbersofmutantsinanyofthetesterstrains,
whilethepositivecontrolsverifedthesensitivityofthestrainsandtheactivityof
theS9mix.
ZEAXANTHIN 171
K2 T
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172 ZEAXANTHIN
K2
2.2.5 Reproductive toxicity
(a) Multigeneration studies
Nostudiesofthistypewereavailable.
(b) Developmental toxicity
Rats
In a segment II study of teratology, which complied with GLP, groups of 36
mated female FU-albino outbred rats (aged 23 months at the beginning of the
experiment) were given diets containing zeaxanthin at a dose of 0, 250, 500, or
1000mg/kgbwperdayorallyasadietaryadmixtureina10%beadletformulation
from days 7 to 16 of gestation (Kistler, 1984).Actual doses of zeaxanthin were
closetothenominallevels,basedonfoodintake.Damswerenecropsiedonday
21anduteriwereexaminedfornumbersandlocationsofimplantationsandresorp-
tions.Fetusesfrom15litterspergroupunderwentskeletalorsofttissueexamina-
tions. Litters from each group were raised until weaning and examined for
abnormalities.There were no treatment-related maternal deaths and no signs of
maternal toxicity. There were no reproductive effects (resorption rates, average
littersizes,meanbodyweightsoflivefetuses)andnoteratagenicordevelopmental
effects(noskeletal,softtissue,orexternalabnormalitiesrelatedtotreatment).One
fetus in the group receiving the highest dose exhibited severe malformations
(representing 1 out of 422 fetuses at this dose). The NOEL was 1000mg/kgbw
perday,thehighestdosetested.
Rabbits
In a segment II study of teratology, which complied with GLP, groups of 20
matedfemaleFU-albinorabbits(aged23monthsatthebeginningoftheexperi-
ment)werefedzeaxanthinatadoseof0,100,200,or400mg/kgorallyinrapeseed
oil from days 7 to 19 of gestation (Kistler, 1983). Dams were necropsied on day
30ofgestationanduteriwereexaminedfornumbersandlocationsofimplantations
and resorptions. Fetal viability in incubators was tested and gross and skeletal
examinationswereconducted.Therewerenotreatment-relatedmaternaldeaths
and no signs of maternal toxicity.There were no reproductive effects (resorption
rates, average litter sizes, mean body weights of live fetuses, survival rates of
foetuses)andnoteratogeniceffects(noskeletal,softtissue,orexternalabnormali-
ties). Some malformed fetuses were observed, but there was no consistency in
thepatternandnodose-dependencewithdistributioninallgroupsincludingcon-
trols.Thus,theincidenceofmalformedfetuseswasconsideredtobeunrelatedto
treatment.TheNOELwas400mg/kgbwperday,thehighestdosetested.
2.2.6 Special studies
(a) Immune responses
In order to assess the allergenic potential of zeaxanthin, a maximization test
forskinsensitization,whichcompliedwithGLP,wasperformedin15(10testand
ZEAXANTHIN 173
K2
5 control) female Himalayan spotted guinea-pigs, aged 46 weeks (Csato &
Arcelin, 2000a). The intradermal induction of sensitization in animals in the test
groupwasperformedusinga3%solutionofzeaxanthin(purity,98.3%)inpolyeth-
ylene glycol 300 (PEG 300) and in a 1:1 mixture of Freund complete adjuvant
(FCA)andphysiologicalsaline,appliedinthenuchalregion.Theepidermalinduc-
tionofsensitizationwasconductedfor48hunderanocclusivedressingwith25%
zeaxanthininPEG300,1weekaftertheintradermalinductionandafterpretreat-
ment of the test areas with 10% sodium dodecyl sulfate (SDS). Animals in the
controlgroupweretreatedidenticallyexceptfortheabsenceofzeaxanthininthe
vehiclesolutions.Twoweeksafterepidermalinduction,thecontrolandtestanimals
werechallengedbyepidermalapplicationof25%zeaxanthininPEG300andPEG
300 alone under the occlusive dressing. Cutaneous reactions were evaluated at
24 and 48h after removal of the dressing. None of the control or test animals
showed skin reactions after the challenge treatment and it was concluded that
zeaxanthinisnotaskinsensitizer,andthattherisk,ifany,tohumansislow.
(b) Ocular toxicity
The long-term ingestion of canthaxanthin at high doses has been shown to
leadtoaccumulationandcrystallizationintheretinaofhumans(Arden&Barker,
1991)andmonkeys(Goralczyketal.,1997),andthequestionhasthereforearisen
astowhetherzeaxanthinbehavessimilarly.Theeffectsofzeaxanthinontheeye
wereinvestigatedinaGLP-compliantstudyincynomolgusmonkeystreatedorally
withzeaxanthinina10%beadletformulation(describedabove)bygavagefor52
weeks.Thecynomolgusmonkeywaschosensinceitwasshowntobeanexcellent
modeltoinvestigatetheinductionanddose-dependencyofcarotenoidcrystalfor-
mation in the retina (Goralczyk et al., 1997; Goralczyk, 2000; Goralczyk et al.,
2002).Groupsoftwomaleandtwofemalemonkeysweregivenzeaxanthinata
doseof0(placebobeadlet),0.2,or20mg/kgbwperday,withoneadditionalmale
and female included in the group receiving the higher dose. One male and one
femaleinthegroupreceivingthehigherdosewerealsokilledat6months.Occa-
sionalretinalchanges,suchasinclusionsinthemacula,wereobservedinsome
groupsofanimals,includingthecontrols,andwereconsideredtobeunrelatedto
treatment.Overall,comprehensiveophthalmicexaminations(ophthalmoscopyand
biomicroscopyexaminations,fundusphotography,electroretinography(considered
to be a very sensitive procedure for the detection of early signs of generalized
retinal degeneration), and post-mortem examinations of the right retina including
macroscopicinspection,microscopicpathologyunderpolarizedandbrightlightfor
peripheralretinaandmacula,confocalmicroscopyofthemaculaandhistopatho-
logical examination of the peripheral retinal) showed no evidence of treatment-
related adverse changes, including no evidence for crystal formation in the eyes
duringorafter52weeksoftreatmentwithzeaxanthin.Dose-dependentincreases
inconcentrationsofzeaxanthinwerereportedintheperipheralretina.Inthecentral
retina and lens, zeaxanthin content was markedly increased in animals at the
higherdose,buttherewasnoevidenceforcrystallinedeposits.Itwasconcluded
thatadministrationofzeaxanthinfor52weeksincynomolgusmonkeysatdoses
of0.2and20mg/kgbwperdayresultedinnotoxiceffectstotheeye(Pfannkuch
etal.,2000a,2000b;Pfannkuch,2001).
174 ZEAXANTHIN
K2
(c) Ocular irritation
Zeaxanthin, as described above, was also tested in a study of primary eye
irritationinthreeadultNewZealandwhiterabbits.Theprimaryirritationscorefor
zeaxanthin was 0.78 (maximum potential score is 13.0) and it was classifed as
not irritating to the rabbit eye in this study, which complied with GLP (Csato &
Arcelin,2000b).
2.3 Observations in humans
2.3.1 Clinical studies
Therehavebeenanumberofstudiesdesignedtoinvestigatethepharmaco-
kineticsofluteinandzeaxanthinthatdidnotnecessarilyincludesafetyend-points,
butalsodidnotreportanyadverseeffectscausedbythexanthophylls(seesec-
tions2.1.1and2.1.2).Arelativelylargenumberofstudiesinhumanshasexamined
correlations between dietary intake of lutein and/or zeaxanthin, the effects of
dietarysupplements,orserumconcentrationsofluteinand/orzeaxanthinandthe
incidence of age-related macular degeneration (AMD), macular pigment density
orcataractogenesiswithvaryingresults(EyeDiseaseCaseControlStudyGroup,
1993; Seddon et al., 1994; Mares-Perlman et al., 1995a, 1995b; Khachik et al.,
1997c; Lyle et al., 1997b; Beatty et al., 1999; Chasan-Taber et al., 1999; Lyle et
al.,1999a;Pratt,1999;Richer,1999;Boneetal.,2000;Johnsonetal.,2000;Gale
et al., 2001; Schalch et al., 2001; Bone et al., 2003; Gale et al., 2003). These
studieswillnotbereviewedhere,butinmanycases,weakinverseassociations
havebeenfound,althoughitisapparentthattheprotectiveeffectofthexantho-
phyllsagainstAMDorcataractformationremainsunproven.Ofimportancehere,
however, is that none of these studies reported adverse effects, including ocular
toxicity, caused by the xanthophylls, even though in some cases, high dietary
levelsorsupplementswereconsumed.
In a pharmacokinetic study described earlier (section 2.1.1), which complied
withGLP,andinwhichgroupsoffvemenandfvewomenweregivencapsules
containing zeaxanthin at either 1mg or 10mg of zeaxanthin per day for 42 days
(correspondingtodosesofapproximately0.014and0.14mg/kgbwperdayfora
70kgadult),clinicalchemistrymeasures(haematology,bloodchemistryandurine
analysis)andadverseeventswererecorded.Severalclinicallaboratoryresultsat
differentassessmenttimesfelloutsideofthenormalranges,butallweredeemed
to be without clinical relevance. There were no relevant changes in blood pres-
sures, heart rate, or body temperature; one subject in the group receiving the
higher dose showed an electrocardiogram (ECG) abnormality that was deemed
nottobeofclinicalrelevance.Inthegroupsreceivingtheloweedoseandhigher
doses, there was one adverse event (an infection of the upper respiratory tract)
deemedtohaveonlyaremotepossibilityofbeingrelatedtodosing.Inthegroup
receiving the higher dose there were three adverse events (one case of bilirubi-
naemia,onecaseofabnormalvision,andonecaseofabnormalaccommodation)
that were deemed to be remotely or possibly related to treatment. The case of
abnormal accommodation was accompanied in the same subject by reports of
dyspnoeaandsleepdisorder,bothofwhichweredeemedtohaveonlyaremote
ZEAXANTHIN 175
K2
possibilityofbeingrelatedtotreatment.Therewasalsoonereportofsyncopein
the group receiving the lower dose, which was deemed to be unrelated to treat-
ment.Alltheadverseeventswereratedasmildtomoderateinseverity(Cohnet
al.,2002).
2.3.2 Epidemiological studies
Mostepidemiologicalstudiesonthexanthophyllshaveaddressedthehypoth-
esisthatintakeofthesecompoundsisinverselyrelatedtodevelopmentofcancer.
A number of such studies have suggested that dietary xanthophylls may protect
againstthedevelopmentofavarietyofcancersincludingthoseoftheoesophagus,
colon, breast, prostate and lung (e.g. Le Marchand et al., 1995; Freudenheim et
al.,1996;Zhangetal.,1997;Franceschietal.,2000;Levietal.,2000;Luetal.,
2001;Nkondjock&Ghadirian,2004),althoughrecentstudiesonbreastandlung
cancerhaveindicatedthatthesecompoundsarenotprotective(Terryetal.,2002;
Mannistoetal.,2004).
A recent large prospective study in a region of China with epidemic rates of
oesophageal and gastric cancer examined the relationship between serum con-
centrations of carotenoids and subsequent risk of developing cancers of the
stomachandupperdigestivetract(Abnetetal.,2003).Therewasanassociation
betweentheincidenceofgastricnon-cardiacancerandserumconcentrationsof
luteinand/orzeaxanthinderivedfromnormaldietarysources.Theseobservations,
however,areonlycorrelativeand,indeed,inaDutchcohortstudy,dietaryintake
of lutein/zeaxanthin was not associated with risk of gastric cancer (pathological
type not specifed), although intakes of retinal and b-carotene were positively
associatedwithriskofthiscancer(Botterwecketal.,2000).
Inviewofthestructuralsimilaritiesbetweenxanthophyllsandb-carotene,the
Committeeconsideredtheoutcomeoftwotrialsthatshowedthatsupplementation
withb-caroteneincreasesriskoflungcancerinheavysmokers;onestudyinvolved
theadministrationofb-caroteneat30mg/dayplus25000IUofretinylpalmitatein
18314smokers,formersmokersandworkersexposedtoasbestos(Omennetal.,
1996), while in the second study, b-carotene at 20mg/day with or without 50mg
of a-tocopherol was given to 29133 male smokers (The a-Tocopherol and b-
Carotene Cancer Prevention Study Group, 1994). However, in the light of the
negative results in studies of genotoxicity and the absence of tumour-promoting
activity of lutein, it was considered that these intervention studies on b-carotene
werenotappropriatefortheriskassessmentofzeaxanthin.
Theresultsofanumberofepidemiologicalstudies,includingdescriptive,cohort
andcasecontrolstudies,suggestthatdietsrichincarotenoidsorb-caroteneare
associatedwithareducedriskofcardiovasculardisease(reviewedinInstituteof
Medicine,2000).Furthermore,noadverseoutcomeshavebeenreportedbetween
increased serum concentrations of lutein and zeaxanthin and risk of subsequent
myocardialinfarction(Streetetal.,1994).Recentepidemiologicalfndings,aswell
as those from studies in vitro and in mouse models, support the hypothesis that
increased dietary intake of zeaxanthin protects against the development of early
atherosclerosis(Dwyeretal.,2001).
176 ZEAXANTHIN
K2
3. INTAKE
3.1 Concentrations in foods
Adatabaseofconcentrationsofcarotenoids,includingluteinandzeaxanthin,
in120foodswasassembledbyMangelsetal.(1993),andwasupdatedbyHolden
etal.(1999).Itshouldbenotedthatthecarotenoidcontentoffoodishighlyvariable
and depends on a number of factors, including geographical area and growing
conditions,cultivarorvariety,processingtechniques,preparationandlengthand
conditionsofstorage(Holdenetal.,1999andreferencestherein).Majorsources
oflutein/zeaxanthinareleafygreenvegetables(e.g.rawspinach,11.9mg/100g),
corn (boiled, 1.8mg/100g) and green vegetables such as broccoli (raw,
2.4mg/100g), brussel sprouts (boiled, 1.3mg/100g), green beans (boiled,
0.7mg/100g),andpeas(canned,1.3mg/100g).Althoughitisnotamajorpartof
the diet in western Europe and NorthAmerica, kale has the highest lutein/zeax-
anthincontentofallfoodsanalysed(raw,39.5mg/100g).Anumberoffoodshave
been analysed specifcally for zeaxanthin. Major sources are corn (canned,
0.5mg/100g),cornmeal(0.5mg/100g),Japanesepersimmons(0.5mg/100g)and
leafygreens(e.g.rawspinach,0.3mg/100g).
3.2 Dietary intake
Dietaryrecalldatafrom1102adultwomenparticipatinginthe1986Continuing
SurveyofFoodIntakebyIndividualsindicatemeanintakesoflutein/zeaxanthinof
1.3mg/day,withatotalintakeofcarotenoidsof6mg/day(Chug-Ahujaetal.,1993).
Food frequency data from 8341 adults participating in the 1992 National Health
InterviewSurveyindicatethatmeanintakesofxanthophyllsformenwere2.2mg/
dayandforwomen1.9mg/day(Nebelingetal.,1997).TheNutritionalFactorsin
Eye Disease Study reported mean dietary intakes of lutein/zeaxanthin of 0.7
0.8mg/day(VandenLangenbergetal.,1996).Inapooledanalysisofsevencohort
studiesdesignedtoassesstheeffectofdietarycarotenoidsonriskoflungcancer,
intakesoflutein/zeaxanthinwereenergy-adjustedbyusingthepredictedintakeof
2100kcal/dayformenand1600kcal/dayforwomen(Mannistoetal.,2004).Food
consumptionwasassessedatbaselineusingavalidateddietaryquestionnairefor
eachstudypopulation.Forthesesevenpopulations,themeanintakeoflutein/zea-
xanthinformenandwomencombinedwas3.7mg/day(range,16mg/day).
Themeanand90thpercentileconsumptionofzeaxanthinintheUnitedStates
ofAmerica(USA)estimatedbysurveyedfoodsampleswas1.42and2.68mg/day
respectively (Table 2). Simulations considering proposed food use levels in the
totalpopulationoftheUSAresultedinestimatedmeanand90thpercentileintakes
forallusersofzeaxanthinof1.46and2.68mg/dayrespectively(DSMNutritional
Products,2004)(Table2).ThesamemethodwasappliedtotheUnitedKingdom
(UK)usingdataonfoodconsumptionfromtheUK.Theestimatedmeanand90th
percentileconsumptionoftotalzeaxanthininthesamplefoodssurveyedwas1.02
and 1.81mg/day respectively for males, and 0.95 and 1.63mg/day for females
(DSMNutritionalProducts,2004)(Table2).
Lutein/zeaxanthin formuations are also available as dietary supplements, but
therearenoreliableestimatesofintakefromthesesources.
ZEAXANTHIN 177
K2
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178 ZEAXANTHIN
K2
4. COMMENTS
Inratsgivendietscontainingzeaxanthinfor5weeks,thehighesttissuecon-
centrations were present in the small intestine, spleen, liver and adipose tissue.
Sevendaysaftercessationofadministration,concentrationsinplasmaandtissues
haddecreasedbybetweentwo-andfourfold,indicatingthattheeliminationhalf-life
wasabout45days.
In humans treated with daily administration of zeaxanthin at a dose of 1 or
10mgfor42days,thetimetosteady-stateplasmaconcentrationswasabout30
days.Thisisconsistentwithaneliminationhalf-lifeofabout5days.Theplasma
concentrations indicated that uptake and availability were not proportional to
dose.
The food matrix, including its fbre and lipid contents, and the concentrations
ofothercarotenoidsinthedietmayinfuencetheextentofabsorptionofcarotenoid
compounds. Studies have shown that zeaxanthin/lutein does not infuence the
absorptionofb-carotene.
ZeaxanthinhasoralLD
50
valuesof>4000mg/kgbwinratsand>8000mg/kgbw
inmice.Ninety-daystudiesoftoxicitywithzeaxanthininratsgivendosesofupto
1000mg/kgbw per day, and in dogs given doses of up to 442mg/kgbw per day,
produced no treatment-related effects even at the highest doses. In a 52-week
study in monkeys, which was designed primarily to investigate possible adverse
effectsontheeye,zeaxanthinwasadministeredatadoseof0.2or20mg/kgbw
perdaybygavage.Thisstudywasperformedbecauseadverseoculareffectshad
beenseenwithcanthaxanthin(Annex1,references78,89,117).Therewereno
treatment-relatedeffectsonawiderangeoftoxicologicalend-points.Furthermore,
comprehensive ophthalmic examinations, including electroretinography, showed
noevidenceoftreatment-relatedadversechanges.
Nolong-termstudiesoftoxicityorcarcinogenicitywereavailable.
Zeaxanthingavenegativeresultsinseveralstudiesofgenotoxicityinvitroand
in vivo.Although the Committee noted that the doses in these tests were low, it
recognizedthatmaximumfeasibledoseswereused.
Inastudyofdevelopmentaltoxicitywithzeaxanthininrats,therewasnoevi-
dence for toxicity at doses of up to 1000mg/kgbw per day, the highest dose
tested.
Inthepharmacokineticstudyinhumansdescribedabove,avarietyofclinical
chemistrymeasurementsaswellasanyadverseeventswererecordedduringthe
study. In the groups of fve men and fve women receiving zeaxanthin at a dose
of1or10mgperdayfor42days,therewerenoreportedtreatment-relatedadverse
effects. There has been a relatively large number of human studies that have
examinedcorrelationsbetweenmaculardegenerationandexposuretolutein/zeax-
anthinviaintakefromtraditionalfoodorfromdietarysupplements,orviameasure-
mentsofserumconcentrations.Althoughthesestudiesweredesignedtolookfor
ocular effects, where clinical or biochemical parameters were also examined, no
adverseeffectsofthexanthophyllswerereported.
ZEAXANTHIN 179
K2
Dietary intake
Dietary intake data from a number of studies in North America and the UK
indicatethatintakeofzeaxanthinfromnaturalsourcesisintherangeof12mg/
day (about 0.010.03mg/kgbw per day). Simulations considering proposed use
levels as a food ingredient resulted in an estimated mean and 90th percentile
intakeofluteinpluszeaxanthinofapproximately7andapproximately13mg/day,
respectively.Formulationscontainingluteinandzeaxanthinarealsoavailableas
dietary supplements, but there were no reliable estimates of intakes from these
sources.
5. EVALUATION
In several studies of toxicity, including developmental toxicity, no adverse
effects were documented in animals, including monkeys, or humans.Taking into
accountdatashowingthatzeaxanthinwasnotgenotoxic,hadnostructuralalert,
thattheisomericxanthophyllluteindidnotexhibittumourpromotingactivity,and
thatzeaxanthinisanaturalcomponentofthebody(theeye),theCommitteecon-
cludedthattherewasnoneedforastudyofcarcinogenicity.
Zeaxanthin has some structural similarities to b-carotene, which has been
reported to enhance the development of lung cancer when given in supplement
formtoheavysmokers.Theavailabledataindicatedthatzeaxanthininfoodwould
notbeexpectedtohavethiseffect.TheCommitteewasunabletoassesswhether
zeaxanthin in the form of supplements would have the reported effect in heavy
smokers.
In view of the toxicological data and structural and physiological similarities
betweenthexanthophyllsluteinandzeaxanthin,theCommitteedecidedtoinclude
zeaxanthinintheacceptabledailyintake(ADI),02mg/kgbw,forlutein,whichhad
a stronger toxicological database, and to make this a group ADI for these two
substances.ThisgroupADIdoesnotapplytootherzeaxanthinpreparationsthat
donotcomplywithestablishedspecifcations.
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K2
K2
SAFETY EVALUATIONS OF GROUPS OF
RELATED FLAVOURING AGENTS
K2
INTRODUCTION
K2
Eight groups of favouring agents were evaluated using the Procedure for the
Safety Evaluation of Flavouring Agents as outlined in Figure 1 (Annex 1, references
116, 122, 131, 137, 143, 149, 154 and 160). In applying the Procedure, the chemi-
cal is frst assigned to a structural class as identifed by the Committee at its forty-
sixth meeting (Annex 1, reference 122). The structural classes are as follows:
Class I. Flavouring agents that have simple chemical structures and effcient
modes of metabolism, which would suggest a low order of toxicity by the oral
route.
Class II. Flavouring agents that have structural features that are less innocuous
than those of substances in Class I but are not suggestive of toxicity. Sub-
stances in this class may contain reactive functional groups.
Class III. Flavouring agents that have structural features that permit no strong
initial presumption of safety, or may even suggest signifcant toxicity.
A key element of the Procedure involves determining whether a favouring
agent and the product(s) of its metabolism are innocuous and/or endogenous
substances. For the purpose of the evaluations, the Committee used the following
defnitions, adapted from the report of its forty-sixth meeting:
Innocuous metabolic products are defned as products that are known or readily
predicted to be harmless to humans at the estimated intake of the favouring
agent.
Endogenous substances are intermediary metabolites normally present in
human tissues and fuids, whether free or conjugated; hormones and other sub-
stances with biochemical or physiological regulatory functions are not included.
The estimated intake of a favouring agent that is, or is metabolized to, an endog-
enous substance should be judged not to give rise to perturbations outside the
physiological range.
Intake
Estimates of the intake of favouring agents by populations typically involve the
acquisition of data on the amounts used in food. These data were derived from
surveys in Europe and the USA. In Europe, a survey was conducted in 1995 by
the International Organization of the Flavour Industry, in which favour manufactur-
ers reported the total amount of each favouring agent incorporated into food sold
in the European Union during the previous year. Manufacturers were requested
to exclude use of favouring agents in pharmaceutical, tobacco or cosmetic
products.
In the USA, a series of surveys was conducted between 1970 and 1987 by the
National Academy of Sciences National Research Council (under contract to the
Food and Drug Administration) in which information was obtained from ingredient
manufacturers and food processors on the amount of each substance destined for
191
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addition to the food supply and on the usual and maximal levels at which each
substance was added in a number of broad food categories.
In using the data from these surveys to estimate intakes of favouring agents,
it was assumed that only 60% of the total amount used is reported in Europe and
80% of the amount used is reported in the USA and that the total amount used in
food is consumed by only 10% of the population.
Intake
annual volume of production kg g
=
( ) ( / 10
9
kkg
population of consumers or day
)
. ( . ) 0 6 0 8 365 ss g per person per day ( )
The population of consumers was assumed to be 32 10
6
in Europe and 26
10
6
in the USA.
Several of the favouring agents that were evaluated at the present meeting
were not included in the above surveys or were placed on the market after the
surveys were conducted. Intakes of these favouring agents were estimated on the
basis of anticipated use by the manufacturer in the USA, and the standard formula
was applied.
INTRODUCTION 193
K2
K2
PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES
First draft prepared by
Dr P.J. Abbott
1
and Dr D.G. Hattan
2
1
Food Standards Australia New Zealand, Canberra, Australian Capital
Territory, Australia; and
2
Offce of Food Additives Safety, Center for Food Safety and Applied
Nutrition, Food and Drug Administration, College Park, MD, USA
Evaluation ............................................................................... 195
Introduction......................................................................... 195
Estimated daily intake ........................................................ 196
Absorption, distribution, metabolism,
and elimination ............................................................. 196
Application of the Procedure for the Safety
Evaluation of Flavouring Substances .......................... 197
Consideration of secondary components .......................... 198
Consideration of combined intakes from use
as favouring agents .................................................... 198
Conclusions ........................................................................ 198
Relevant background information............................................. 198
Explanation......................................................................... 198
Additional considerations on intake ................................... 198
Biological data .................................................................... 208
Biochemical data ......................................................... 208
Absorption, distribution, and excretion .................. 208
Metabolism ............................................................ 209
Toxicological studies .................................................... 215
Acute toxicity ......................................................... 215
Short-term studies of toxicity ................................. 216
Long-term studies of toxicity
and carcinogenicity ......................................... 221
Genotoxicity ........................................................... 221
Other relevant studies ........................................... 227
References ............................................................................... 228
1. EVALUATION
1.1 Introduction
The Committee evaluated a group of 22 favouring agents (Table 1) by the
Procedure for the Safety Evaluation of Flavouring Agents (see Figure 1, p 192).
This group included:
six pyrroles (Nos 1314, 13051307, 1310 and 1319);
two indoles (Nos 1301 and 1304);
195
K2
196 PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES
K2
12 pyridine derivatives (Nos 1308, 1309, 13111313, 13151318 and 1320
1322); and
a quinoline derivative and an isoquinoline derivative (Nos 1302 and 1303).
The Committee has not previously evaluated any member of the group.
Nineteen of the 22 substances (Nos 13011307, 1309, 1310, 13121320 and
1322) have been reported to occur naturally in foods. They have been detected
in fresh and cooked vegetables, uncured meats, a variety of whole grains, green
and black teas, coffee, alcoholic beverages, whiskeys, shellfsh, and a wide variety
of fresh fruits (Nijssen et al., 2003).
1.2 Estimated daily intake
The total annual volume of production of the 22 favouring agents in this group
is approximately 1000 kg in Europe (International Organization of the Flavor Indus-
try, 1995) and 650 kg in the USA (National Academy of Sciences, 1982; Lucas et
al., 1999). More than 41% of the total annual volume of production in Europe and
>79% in the USA is accounted for by a single substance in this group, namely 2-
acetylpyridine (No. 1309). The estimated daily intakes of 2-acetylpyridine in Europe
and the USA are 59 and 68 mg/person, respectively. The daily intakes of all other
favouring agents in the group ranged from 0.001 to 30 mg/person (National
Academy of Sciences, 1982; International Organization of the Flavor Industry,
1995; Lucas et al., 1999), most values being at the lower end of this range. The
estimated daily per capita intake of each agent is reported in Table 1.
1.3 Absorption, distribution, metabolism, and elimination
Pyridine, pyrrole and quinoline derivatives are expected to be rapidly absorbed
from the gastrointestinal tract, oxidized to polar metabolites, and eliminated primar-
ily in the urine and, to a minor extent, in the faeces.
Alkyl-substituted pyrroles and indoles may undergo cytochrome P450 (CYP)-
mediated side-chain oxidation to yield the corresponding alcohol, which may be
excreted as the glucuronic acid or sulfate conjugate (Ruangyuttikarn et al., 1992;
Thornton-Manning et al., 1993; Gillam et al., 2000). To a lesser extent, the double
bond of the indole ring may undergo epoxidation (Skiles et al., 1991; Smith et al.,
1993).
Alkyl-substituted pyridines and quinolines are principally subject to side-chain
oxidation, primarily at the C1 position. Minor pathways include ring hydroxylation
and epoxidation for substituted quinolines. N-Oxide formation has also been
reported (Cowan et al., 1978; Schwartz et al., 1978; Damani et al., 1980; Nguyen
et al., 1988).
Methyl nicotinate (No. 1320), the only ester in the group, is rapidly hydrolysed
by carboxyesterase to yield nicotinic acid and methanol (Heymann, 1980; White
et al., 1990; Durrer et al., 1992).
PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES 197
K2
1.4 Application of the Procedure for the Safety Evaluation of
Flavouring Substances
Step 1. In applying the Procedure, the Committee assigned three (Nos 1301,
1304 and 1314) of the 22 agents to structural class I. Thirteen agents
(Nos 13051307, 1309, 1312, 1313, 13151320 and 1322) were assigned
to structural class II and the remaining six (Nos 1302, 1303, 1308, 1310,
1311, and 1321) were assigned to structural class III (Cramer et al.,
1978).
Step 2. Twenty favouring agents in this group are predicted to be metabolized
to innocuous products (Nos 13011307, 1309 and 13111322). The
evaluation of these favouring agents therefore proceeded via the A-side
of the decision-tree. Two favouring agents (Nos 1308 and 1310) cannot
be predicted to be metabolized to innocuous products. The evaluation
of these two favouring agents therefore proceeded via the B-side of the
decision-tree.
Step A3. The estimated daily intakes of all three of the favouring agents in struc-
tural class I (Nos 1301, 1304 and 1314), all thirteen of the favouring
agents in structural class II (Nos 13051307, 1309, 1312, 1313, 1315
1320 and 1322), and of the four favouring agents in structural class III
(Nos 1302, 1303, 1311 and 1321) are below the respective thresholds
of concern (i.e. 1800 mg/person for class I, 540 mg/person for class II, and
90 mg/person for class III). According to the Procedure, the use of these
20 favouring agents raises no safety concern at estimated current
intakes.
Step B3. The estimated daily intakes in Europe and the USA of the remaining two
favouring agents in this group (Nos 1308 and 1310), which cannot be
predicted to be metabolized to innocuous products, are also below the
threshold of concern for structural class III (i.e. 90 mg/person). Accord-
ingly, the evaluation of both favouring agents in the group proceeded to
step B4.
Step B4. For N-furfurylpyrrole (No. 1310), the no-observed-effect level (NOEL) of
12 mg/kg bw per day from a 90-day feeding study in rats (Morgareidge,
1971) is >1 000 000 greater than the estimated current intake of this
substance as a favouring agent. For 2-pyridinemethanethiol (No. 1308),
the NOEL of 3.4 mg/kg bw per day from a 90-day feeding study in rats
(Posternak et al., 1969) is >20 000 000 times greater than the estimated
current intake of this substance as a favouring agent.
The intake considerations and other information used to evaluate the 22
favouring agents in this group according to the Procedure are summarized in
Table 3.
198 PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES
K2
1.5 Consideration of secondary components
No favouring agents in this group have minimum assay values of <95%.
1.6 Consideration of combined intakes from use as favouring agents
In the event that all three agents in structural class I were consumed concur-
rently on a daily basis, the estimated combined intake would not exceed the human
intake threshold for class I (1800 mg/person per day). In the unlikely event that all
13 agents in structural class II were consumed concurrently on a daily basis, the
estimated combined intake would not exceed the human intake threshold for class
II (540 mg/person per day). In the unlikely event that all six agents in structural
class III were consumed concurrently on a daily basis, the estimated combined
intake would not exceed the human intake threshold for class III (90 mg/person per
day). Overall evaluation of the data indicated that combined intake would not raise
safety concerns at estimated current intakes.
1.7 Conclusions
The Committee concluded that none of the favouring agents in this group of
pyridine, pyrrole and quinoline derivatives would present safety concerns at esti-
mated current intakes. Other available data on the toxicity and metabolism of these
pyridine, pyrrole and quinoline derivatives were consistent with the results of the
safety evaluation.
2. RELEVANT BACKGROUND INFORMATION
2.1 Explanation
The background information summarizes key data relevant to the safety evalu-
ation of the 22 pyridine, pyrrole and quinoline derivatives used as favouring
agents.
2.2 Additional considerations on intake
Quantitative data on natural occurrence and consumption ratios reported for
indole (No. 1301), skatole (No. 1304), 1-methyl-2-acetylpyrrole (No. 1306), 2-
acetylpyridine (No. 1309), N-furfurylpyrrole (No. 1310), and pyrrole (No. 1314)
indicate that exposure occurs predominantly from consumption of traditional
foods (i.e. consumption ratio >1) (Stofberg & Kirschman, 1985; Stofberg &
Grundschober, 1987). Volumes of production and intake values for each favouring
agent in this group are shown in Table 2.
PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES 199
K2
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200 PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES
K2
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PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES 201
K2
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202 PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES
K2
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t
y


H N
O


E
u
r
o
p
e
:

0
.
0
1



1
,

4


c
o
n
c
e
r
n




U
S
A
:

2
M
e
t
h
y
l

n
i
c
o
t
i
n
a
t
e

1
3
2
0

9
3
-
6
0
-
7

Y
e
s

N
o


S
e
e

N
o

s
a
f
e
t
y


N
O O


E
u
r
o
p
e
:

0
.
6



n
o
t
e

6


c
o
n
c
e
r
n




U
S
A
:

0
.
2
2
-
P
r
o
p
y
l
p
y
r
i
d
i
n
e

1
3
2
2

6
2
2
-
3
9
-
9

Y
e
s

N
o


S
e
e

N
o

s
a
f
e
t
y


N


E
u
r
o
p
e
:

N
D



n
o
t
e

3


c
o
n
c
e
r
n




U
S
A
:

0
.
9
S
t
r
u
c
t
u
r
a
l

c
l
a
s
s

I
I
I
6
-
M
e
t
h
y
l
q
u
i
n
o
l
i
n
e

1
3
0
2

9
1
-
6
2
-
3

Y
e
s

N
o


S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y


N


E
u
r
o
p
e
:

4



2
,

5


c
o
n
c
e
r
n




U
S
A
:

0
.
0
1
PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES 203
K2 I
s
o
q
u
i
n
o
l
i
n
e

1
3
0
3

1
1
9
-
6
5
-
3

Y
e
s

N
o


S
e
e

N
o

s
a
f
e
t
y


N


E
u
r
o
p
e
:

0
.
0
1



n
o
t
e

2


c
o
n
c
e
r
n




U
S
A
:

0
.
0
7
2
-
(
2
-
M
e
t
h
y
l
p
r
o
p
y
l
)

1
3
1
1

6
3
0
4
-
2
4
-
1

Y
e
s

N
o


S
e
e

N
o

s
a
f
e
t
y

p
y
r
i
d
i
n
e


N


E
u
r
o
p
e
:

N
D



n
o
t
e

3


c
o
n
c
e
r
n




U
S
A
:

0
.
9
2
-
(
3
-
P
h
e
n
y
l
p
r
o
p
y
l
)

1
3
2
1

2
1
1
0
-
1
8
-
1

Y
e
s

N
o


S
e
e

N
o

s
a
f
e
t
y

p
y
r
i
d
i
n
e


N


E
u
r
o
p
e
:

2


n
o
t
e

3


c
o
n
c
e
r
n




U
S
A
:

0
.
7
F
l
a
v
o
u
r
i
n
g

N
o
.

C
A
S

N
o
.

a
n
d

S
t
e
p

2

S
t
e
p

B
3

D
o
e
s

S
t
e
p

B
4

A
d
e
q
u
a
t
e

C
o
m
m
e
n
t
s

C
o
n
c
l
u
s
i
o
n
a
g
e
n
t


s
t
r
u
c
t
u
r
e

P
r
e
d
i
c
t
e
d

i
n
t
a
k
e

e
x
c
e
e
d

m
a
r
g
i
n

o
f

s
a
f
e
t
y

f
o
r


b
a
s
e
d

o
n



t
o

b
e

t
h
e

t
h
r
e
s
h
o
l
d

t
h
e

f
a
v
o
u
r
i
n
g

a
g
e
n
t


c
u
r
r
e
n
t

i
n
t
a
k
e



m
e
t
a
b
o
l
i
z
e
d

f
o
r

h
u
m
a
n

o
r

r
e
l
a
t
e
d



t
o

i
n
n
o
c
u
o
u
s

i
n
t
a
k
e
?
a

c
h
e
m
i
c
a
l
?



m
e
t
a
b
o
l
i
t
e
s
?
S
t
r
u
c
t
u
r
a
l

c
l
a
s
s

I
I
I
2
-
P
y
r
i
d
i
n
e
m
e
t
h
a
n
e
t
h
i
o
l

1
3
0
8

2
0
4
4
-
7
3
-
7

N
o

N
o

Y
e
s
.

T
h
e

N
O
E
L

S
e
e

N
o

s
a
f
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t
y


N
S
H


E
u
r
o
p
e
:

0
.
0
0
1


o
f

3
.
4
2

m
g
/
k
g

b
w


n
o
t
e

3


c
o
n
c
e
r
n




U
S
A
:

0
.
0
0
7


p
e
r

d
a
y

i
n

r
a
t
s






(
P
o
s
t
e
r
n
a
k






e
t

a
l
.
,

1
9
6
9
)

i
s


>
2
0

m
i
l
l
i
o
n


t
i
m
e
s

t
h
e


e
s
t
i
m
a
t
e
d

d
a
i
l
y


i
n
t
a
k
e

o
f







2
-
p
y
r
i
d
i
n
e
m
e
t
h
a
n
e
t
h
i
o
l
.
204 PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES
K2
T
a
b
l
e

1
.

(
c
o
n
t
d
)
F
l
a
v
o
u
r
i
n
g

N
o
.

C
A
S

N
o
.

a
n
d

S
t
e
p

2

S
t
e
p

B
3

D
o
e
s

S
t
e
p

B
4

A
d
e
q
u
a
t
e

C
o
m
m
e
n
t
s

C
o
n
c
l
u
s
i
o
n
a
g
e
n
t


s
t
r
u
c
t
u
r
e

P
r
e
d
i
c
t
e
d

i
n
t
a
k
e

e
x
c
e
e
d

m
a
r
g
i
n

o
f

s
a
f
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t
y

f
o
r


b
a
s
e
d

o
n



t
o

b
e

t
h
e

t
h
r
e
s
h
o
l
d

t
h
e

f
a
v
o
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r
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n
g

a
g
e
n
t


c
u
r
r
e
n
t

i
n
t
a
k
e



m
e
t
a
b
o
l
i
z
e
d

f
o
r

h
u
m
a
n

o
r

r
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l
a
t
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d



t
o

i
n
n
o
c
u
o
u
s

i
n
t
a
k
e
?
a

c
h
e
m
i
c
a
l
?



m
e
t
a
b
o
l
i
t
e
s
?
r
f
u
r
y
l
p
y
r
r
o
l
e

1
3
1
0

1
4
3
8
-
9
4
-
4

N
o

N
o

Y
e
s
.

T
h
e

N
O
E
L

o
f

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y


N
O


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u
r
o
p
e
:

0
.
1


1
2
.
2

m
g
/
k
g

b
w

p
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r


1
,

4

c
o
n
c
e
r
n




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S
A
:

0
.
0
7


d
a
y

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n

r
a
t
s






(
M
o
r
g
a
r
e
i
d
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e
,






1
9
7
1
)

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s

>
1

m
i
l
l
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n






t
i
m
e
s

t
h
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s
t
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m
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d






d
a
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n
t
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f






N
-
f
u
r
f
u
r
y
l
p
y
r
r
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e
.
C
A
S
:

C
h
e
m
i
c
a
l

A
b
s
t
r
a
c
t
s

S
e
r
v
i
c
e
;

N
D
:

N
o

i
n
t
a
k
e

d
a
t
a

r
e
p
o
r
t
e
d
;

N
R
:

N
o
t

r
e
q
u
i
r
e
d

f
o
r

e
v
a
l
u
a
t
i
o
n

b
e
c
a
u
s
e

c
o
n
s
u
m
p
t
i
o
n

o
f

t
h
e

s
u
b
s
t
a
n
c
e

w
a
s

d
e
t
e
r
m
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d

t
o

b
e

o
f

n
o

s
a
f
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t
y

c
o
n
c
e
r
n

a
t

s
t
e
p

A
3

o
f

t
h
e

P
r
o
c
e
d
u
r
e
.
a

T
h
e

t
h
r
e
s
h
o
l
d
s

f
o
r

h
u
m
a
n

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n
t
a
k
e

f
o
r

s
t
r
u
c
t
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r
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l

c
l
a
s
s
e
s

I
,

I
I
,

a
n
d

I
I
I

a
r
e

1
8
0
0
,

5
4
0

a
n
d

9
0

m
g
/
p
e
r
s
o
n

p
e
r

d
a
y
,

r
e
s
p
e
c
t
i
v
e
l
y
.

A
l
l

i
n
t
a
k
e

v
a
l
u
e
s

a
r
e

e
x
p
r
e
s
s
e
d

i
n

m
g
/
p
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r
s
o
n

p
e
r

d
a
y
.

T
h
e

c
o
m
b
i
n
e
d

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n
t
a
k
e

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f

t
h
e

f
a
v
o
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r
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n
g

a
g
e
n
t
s

i
n

s
t
r
u
c
t
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r
a
l

c
l
a
s
s

I

i
s

3
3

m
g
/
p
e
r
s
o
n

p
e
r

d
a
y

i
n

E
u
r
o
p
e

a
n
d

1
1

m
g
/
p
e
r
s
o
n

p
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r

d
a
y

i
n

t
h
e

U
S
A
.

T
h
e

c
o
m
b
i
n
e
d

i
n
t
a
k
e

o
f

t
h
e

f
a
v
o
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r
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n
g

a
g
e
n
t
s

i
n

s
t
r
u
c
t
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r
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l

c
l
a
s
s

I
I

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s

1
0
3

m
g
/
p
e
r
s
o
n

p
e
r

d
a
y

i
n

E
u
r
o
p
e

a
n
d

7
6

m
g
/
p
e
r
s
o
n

p
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r

d
a
y

i
n

t
h
e

U
S
A
.

T
h
e

c
o
m
b
i
n
e
d

i
n
t
a
k
e

o
f

t
h
e

f
a
v
o
u
r
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n
g

a
g
e
n
t
s

i
n

s
t
r
u
c
t
u
r
a
l

c
l
a
s
s

I
I
I

i
s

6

m
g
/
p
e
r
s
o
n

p
e
r

d
a
y

i
n

E
u
r
o
p
e

a
n
d

1

m
g
/
p
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r
s
o
n

p
e
r

d
a
y

i
n

t
h
e

U
S
A
.
N
o
t
e
s
:
1

T
h
e

p
y
r
r
o
l
e

r
i
n
g

u
n
d
e
r
g
o
e
s

h
y
d
r
o
x
y
l
a
t
i
o
n

a
t

t
h
e

C
2

p
o
s
i
t
i
o
n

a
n
d

i
s

e
x
c
r
e
t
e
d

i
n

t
h
e

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r
i
n
e

a
s

t
h
e

c
o
r
r
e
s
p
o
n
d
i
n
g

g
l
u
c
u
r
o
n
i
c

a
c
i
d

c
o
n
j
u
g
a
t
e
.
2

T
h
e

r
i
n
g

s
y
s
t
e
m

u
n
d
e
r
g
o
e
s

h
y
d
r
o
x
y
l
a
t
i
o
n

a
t

t
h
e

C
3

p
o
s
i
t
i
o
n

a
n
d

i
s

e
x
c
r
e
t
e
d

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n

t
h
e

u
r
i
n
e

a
s

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h
e

c
o
r
r
e
s
p
o
n
d
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n
g

g
l
u
c
u
r
o
n
i
c

a
c
i
d

c
o
n
j
u
g
a
t
e
.
3

A
l
k
y
l

s
i
d
e
-
c
h
a
i
n

o
x
i
d
a
t
i
o
n

f
o
l
l
o
w
e
d

b
y

g
l
u
c
u
r
o
n
i
c

a
c
i
d

c
o
n
j
u
g
a
t
i
o
n

a
n
d

e
x
c
r
e
t
i
o
n

o
r

o
x
i
d
a
t
i
o
n

t
o

n
i
c
o
t
i
n
i
c

a
c
i
d
.
4

T
h
e

a
c
e
t
y
l

g
r
o
u
p

i
s

r
e
d
u
c
e
d

a
n
d

c
o
n
j
u
g
a
t
e
d

w
i
t
h

g
l
u
c
u
r
o
n
i
c

a
c
i
d
.
5

F
o
r
m
s

a

r
e
a
c
t
i
v
e

e
p
o
x
i
d
e

m
e
t
a
b
o
l
i
t
e

t
h
a
t

i
s

d
e
t
o
x
i
f
e
d

t
h
r
o
u
g
h

g
l
u
t
a
t
h
i
o
n
e

c
o
n
j
u
g
a
t
i
o
n
.
6

E
s
t
e
r

r
e
a
d
i
l
y

u
n
d
e
r
g
o
e
s

h
y
d
r
o
l
y
s
i
s

a
n
d

r
e
s
u
l
t
i
n
g

n
i
c
o
t
i
n
i
c

a
c
i
d

i
s

e
i
t
h
e
r

u
s
e
d

i
n

n
u
m
e
r
o
u
s

m
e
t
a
b
o
l
i
c

p
r
o
c
e
s
s
e
s

o
r

e
x
c
r
e
t
e
d

a
s

t
h
e

m
e
r
c
a
p
t
u
r
i
c

a
c
i
d

c
o
n
j
u
g
a
t
e
.
PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES 205
K2
T
a
b
l
e

2
.

A
n
n
u
a
l

v
o
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206 PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES
K2
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PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES 207
K2
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208 PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES
K2
2.3 Biological data
2.3.1 Biochemical data
(a) Absorption, distribution, and excretion
(i) Pyrroles (Nos 1314, 13051307, 1310 and 1319) and
indoles (Nos 1301 and 1304)
Weak bases with pK
a
values of >3, such as pyrroles and indoles, are readily
absorbed from the intestine by passive diffusion because the base will not be
ionized and pass through intestinal membranes with ease (Hogben et al., 1959).
Pyrrole (No. 1314) has a pK
a
of 3.8, while indole (No. 1301) has a pK
a
of 3.2.
Female albino Wistar rats given [2
14
C]indole as a single oral dose at 64 to
80 mg/kg bw eliminated >80% of the radiolabel in the urine within 48 h. Urine,
faeces and expired air contained 80.6, 11.1, and 2.4% of the administered dose,
respectively (King et al., 1966).
Groups of 24 male Holtzman rats were maintained on diets supplemented with
indole at 0 (control), 0.25, 0.50, or 0.75% for 3 weeks. During week 3, all groups
(including controls) received a single dose of
14
C-labelled indole via stomach tube.
Urine collected for 48 h before and for an additional 24 h after administration of the
single dose revealed that 32.1, 46.8, 49.4, 50.9, and 61.5% of indole and indole
metabolites were recovered from the groups receiving indole at 0 (control), 0.25,
0.50, 0.75%, respectively, demonstrating a rapid elimination of indole (Martinez &
Roe, 1972).
Mice and rats given a single intraperitoneal injections of [
14
C-3]methylindole at
400 mg/kg bw excreted 69.4 and 66.2% respectively in the urine within 48 h (Skiles
et al., 1991).
(ii) Pyridines (Nos 1308, 1309, 13111313, 13151318, 1320
1322) and quinolines (Nos 1302 and 1303)
Pyridines (pK
a
= 5.2), quinolines (pK
a
= 4.85), and isoquinolines (pK
a
= 5.14)
are weak tertiary bases and undergo rapid absorption in the gastrointestinal tract
(Hogben et al., 1959).
2-Methylpyridine, at a dose of 500 mg/kg bw administered orally to rats, was
distributed to the liver, heart spleen, lungs, and muscles within 1020 min and an
unidentifed amount was excreted in the urine 48 h after dosing (Kupor, 1972).
In dogs treated orally with 3-acetylpyridine at a dose of 40 mg/kg bw per day
for 2 days, metabolites were detected in the urine within 48 h (McKennis et al.,
1964).
PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES 209
K2
(b) Metabolism
(i) Pyrroles (Nos 1314, 13051307, 1310, and 1319) and
indoles (Nos 1301 and 1304)
Unsubstituted pyrrole and indole are metabolized primarily by ring hydroxyl-
ation at the C2 position in pyrrole (Figure 1) (Town et al., 1992) and the C3 position
in indole (Figure 2); Posner et al., 1961; King et al., 1966; Ruangyuttikarn et al.,
1992; Thornton-Manning et al., 1993; Gillam et al., 2000). The resulting hydroxyl
derivative is subsequently conjugated with glucuronic acid or sulfuric acid and
excreted in the urine. A minor pathway involves epoxidation of the pyrrole ring
double bond to yield an epoxide that is readily conjugated with glutathione (York
et al., 1993).
Alkyl-substituted pyrroles and indoles may also undergo CYP-induced side-
chain oxidation to yield the corresponding alcohol, which may be excreted as the
glucuronic acid or sulfuric acid conjugate (Ruangyuttikarn et al., 1992; Thornton-
Manning et al., 1993; Gillam et al., 2000). To some extent, epoxidation of the indole
ring double bond has been considered as another metabolic pathway for metabo-
lism of alkyl-substituted pyrroles and indole derivatives (Skiles & Yost, 1989; Smith
et al., 1993).
Experiments in vitro have demonstrated that pyrroles and indoles also undergo
ring hydroxylation. Hydroxylation at the C2 position of the pyrrole ring occurs when
human liver microsomes are incubated with a pyrrole-substituted heterocyclic
derivative (HIV tat inhibitor Ro 5-3335) (see Figure 1) (Town et al., 1992).
The pyrrole ring or its metabolites also react with glutathione. A novel class of a-
glutathione-S-transferase (GST) isozymes is expressed in rat liver fractions after
treatment with pyrroles (York et al., 1993; Primiano & Novack, 1989).
Ring hydroxylation of indole (No. 1301) has been well documented (see Figure
2). Approximately 63% of total 3-hydroxyindole was present as 3-hydroxyindole
sulfate (49.6%) and 3-hydroxyindole glucuronide (13.2%) in pooled samples of
urine at 48 h after oral treatment of three albino Wistar rats with [
14
C]2-indole as a
N
H
N
H
OH
N
H
OR
R = sulfate or glucuronide
N
H
N
H
N
H
R = sulfate or glucuronide
OH OR
Figure 1. Metabolic options for pyrrole and alkyl-substituted pyrroles
210 PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES
K2
single dose at 64 to 74 mg/kg bw. In two of the rats, other metabolites identifed in
the urine included 5-hydroxyindole (3.5%), 2H-indole-2-one (1.4%), and indole-2,3-
dione (5.8%). Analysis of the faecal excretions showed that 0.14, 0.40, and 0.64%
of the radiolabel was present as indole, 3-hydroxyindole sulfate and total 3-
hydroxyindole metabolites, respectively (King et al., 1966). In a parallel study, bile
samples collected at 48 h via cannulation of the common bile duct of two female
albino Wistar rats treated orally with
14
C-labelled 2-indole at a dose of 49 to 63 mg/
kg bw showed that 0.56, 0.80, and 0.82% of the radiolabel was present as 5-
hydroxyindole, 3-hydroxyindole sulfate, and total 3-hydroxyindole metabolites,
respectively (King et al., 1966).
3-Hydroxyindole was the primary metabolite isolated when indole was incu-
bated with freshly prepared rabbit liver microsomes (Posner et al., 1961).
3-Hydroxyindole may further oxidize to indigo (2-(1,3-dihydro-3-oxo-2H-indol-
2-ylidene)-1,2-dihydro-3H-indol-3-one) (Posner et al., 1961). Aerobic incubation of
indole with rat liver microsomes in the presence of glucose-6-phosphate, nicotin-
amide, and nicotinamide adenine dinucleotide phosphate, reduced (NADPH) for
1 h demonstrated the formation of ring-oxidized metabolites including indigo, indi-
rubin (3-(1,3-dihydro-3-oxo-2H-indol-2-ylidene)-1,3-dihydro-2H-indol-2-one), and
oxindole (1,3-dihydro-2H-indol-2-one). Under anaerobic conditions, oxindole was
detected (King et al., 1966). The presence of metabolites of indole observed under
aerobic conditions were also reported when indole was incubated with recombinant
human CYP enzymes, 2A6, 2C19, and 2E1 coexpressed with CYP reductase in
Escherichia coli (Gillam et al., 2000). These studies support the role of CYP
enzymes in the oxidation of the indole ring.
Alkyl-substituted pyrroles and indoles undergo mainly side-chain oxidation,
although there is some evidence that epoxidation of the indole ring alkene also
occurs. Mice and rats given
14
C-labelled 3-methylindole (No. 1304) as a single
intraperitoneal injection at 400 mg/kg bw excreted 69.4 and 66.2%, respectively, of
the administered dose as indole-3-carbinol (i.e. 3-hydroxymethylindole) and 2.6
and 7.3%, respectively, as the mercapturic acid conjugate of 3-methylindole, 3-[(N-
acetylcystein-S-yl)methyl]indole (see Figure 2) (Skiles et al., 1991). The mercap-
turic acid conjugate is likely to be formed via a reactive 3-methylene iminium ion
(Figure 1) that may be generated either directly via CYP mediated oxidation of the
methyl substituent or indirectly via ready dehydration of indole-3-carbinol (Skiles
& Yost, 1992).
At least six metabolites were isolated from the urine of male Swiss-Webster
mice at 36 h after administration of ring-labelled [
14
C]3-methylindole at a dose of
400 mg/kg bw by intraperitoneal injection. Two primary pathways were character-
ized. In one, side-chain oxidation yields indole-3-carbinol that is dehydrated to 3-
methyleneindolenine which subsequently is conjugated with glutathione to yield
3-[(N-acetylcystein-S-yl)methyl]indole. Indole-3-carbinol is then oxidized to the cor-
responding carboxylic acid. In the other pathway, the 2,3-alkene is epoxidized to
yield 3-methyloxindole or 3-hydroxy-3-methylindolenine intermediates. These
intermediary metabolites are conjugated with glucuronic acid or sulfuric aicd, fol-
lowed by excretion in the urine, or are further oxidized to yield a series of dihy-
droxy-3-methyloxindole metabolites that are also conjugated and excreted (Smith
PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES 211
K2
et al., 1993). When (
2
H-2-)-3-methylindole was incubated with microsomal CYP in
the presence of
18
O
2
, 3-methyloxindole was formed with an
18
O label incorporated
at position 2. Also, an intramolecular shift (NIH shift) of
2
H from position 2 to posi-
tion 3 was observed. These experiments provide evidence for the formation of the
epoxide followed by a well recognized NIH shift to yield 3-methyloxindole (Skordos
et al., 1998).
Evidence for the presence of the 3-methyleneindolenine intermediate has been
demonstrated by numerous experiments in vitro. Incubation of 3-methylindole
(0.5 mmol/l) with rabbit Clara cells and alveolar macrophages yielded four metabo-
lites: the two metabolites derived from side-chain oxidation were indole-3-carbinol
and the glutathione conjugate, 3-(N-acetylcysteine-S-yl)-3-methylindole. Two other
metabolites, presumably derived from epoxidation, were 3-methyloxindole, and 2-
(N-acetylcystein-S-yl)-3-hydroxy-3-methylindoline, a mercapturic acid (Thornton-
Manning et al., 1993). Incubation with microsomal CYP, NADPH and excess
glutathione (4 mmol/l) produced the corresponding glutathione conjugates in place
N
H
N
H
OH
N
H
OR
N
H
O
O
N
H
O
H
N
O
N
H
O
N
H
O
HO
N
H
O
RO
H
N
O
N
H
O
Indole
Indigo
Indirubin
Oxindole
R = sulfate or glucuronide
3-Hydroxyindole
5-Hydroxyoxindole
Isatin
Figure 2. Metabolism of indole under aerobic conditions
212 PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES
K2
of the mercapturic acid conjugates (Thornton-Manning et al., 1993). Human liver
microsomes were incubated with [
14
C]3-methylindole in the presence of NADPH.
Hydrolysis of the isolated protein fraction indicated the presence of a cysteine
conjugate at position 3 of 3-methylindole. The authors suggested that a reactive
3-metyhyleneindolenine intermediate reacts with the cysteine thiol groups of target
proteins (Ruangyuttikarn et al., 1992).
Experiments have been performed in vitro to better characterize the enzyme-
catalysed formation of the reactive intermediate 3-methyleneindolenine and the
intermediates produced in subsequent reaction with glutathione and cellular pro-
teins. When liver homogenates isolated from rats treated with b-naphthofavone
or phenobarbitone, known inducers of CYP, were incubated with 3-methylindole
(skatole, No. 1304) at 1 mmol/l, rates of glutathione depletion were signifcantly
higher than rates of depletion for untreated liver homogenates (i.e. 32.1 (p < 0.05)
and 48 (p < 0.001) nmol/mg protein per 30 min, respectively, versus 20 nmol/mg
protein per 30 min for untreated controls) (Garle & Fry, 1989). Incubation of 3-
methylindole with HepG2 cell lysates containing vaccinia-expressed CYP2A6 or
CYP2F1 in vitro produced an intermediate that binds covalently to cellular proteins,
presumably through a similar mechanism (Thornton-Manning et al., 1992).
Swiss-Webster mice given L-buthionine-(S,R)-sulfoximine, a specifc inhibitor
of GSH synthesis, at a dose of 06 mmol/kg bw, 3 h before treatment with [
14
C-
methyl]3-methylindole at a dose of 75 mg/kg bw, showed that the covalent binding
of 3-methylindole-derived radiolabel to cellular proteins increased with increasing
concentration of L-buthionine-(S,R)-sulfoximine. The binding was greater in the
renal tissue (3.4-fold increase) than in pulmonary (2.1-fold increase) or hepatic
(1.5-fold increase) tissues. Increased binding to cellular proteins correlated with
lower concentrations of glutathione (Yost et al., 1990).
H
N
H
N
H
N
H
N
HO
RS
Skatole
(3 -m ethylin dol e)
In do le-3-car bi no l
(m aj or meta bo li te )
3- Me th yleneindolenine
(p r opo se d inte rm edi at e)
3-[(glutath ion-S-yl ) -meth yl]indole
or
3-[(N-acetylcystei n- S-yl )- methyl ]indol e
(minor excretion product)
Ma jor
Pa thwa y
Mi no r
Pa th wa y
Figure 3. Metabolic pathway of skatole in rats and human microsomal
preparations
PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES 213
K2
(ii) Pyridines (Nos 1308, 1309, 13111313, 13151318,
13201322) and quinolines (Nos 1302 and 1303)
Alkyl-substituted pyridines and quinolines are subject to side-chain oxidation,
usually at the C1 position (Hawksworth & Scheline, 1975). The polar hydroxy- or
acyl-substituted metabolites are readily excreted in conjugated form (see Figure
4). Ring hydroxylation and epoxidation have also been observed for substituted
quinolines. In addition, pyridine derivatives may form polar N-oxides (Cowan et al.,
1978) (Figure 4). Pyridine itself has been found to undergo N-oxidation and N-
methylation in vivo in most species (Damani & Crooks, 1982). Instances of com-
bined side-chain oxidation and N-oxide formation have been observed (Cowan et
al., 1978; Schwartz et al., 1978; Damani et al., 1980; Nguyen et al., 1988).
Methyl nicotinate is the only ester in the group and is rapidly hydrolysed to
yield pyridinecarboxylic acid (nicotinic acid) and methyl alcohol by carboxylester-
ases (Heymann, 1980; White et al., 1990; Durrer et al., 1992).
In a study of the effect of substrate on the rate of carboxylesterase-catalysed
hydrolysis, the steady state kinetic constants for a series of nicotinate esters were
determined. Purifed hog liver carboxylesterase and human plasma containing
carboxylesterase were incubated with methyl nicotinate. The maximal velocity
(V
max
) for ester hydrolysis in homogeneous hog liver carboxyesterase and human
plasma is 24.4 and 46.4 mmol/min per mg protein, respectively, indicating rapid
hydrolysis of ester (Durrer et al., 1992).
Nicotinic acid, or niacin, is ubiquitous in living cells and is essential for the
production of nicotinamide adenine dinucleotide (NAD) and its derivatives. As a
result of normal turnover of NAD or excess dietary intake, nicotinic acid is excreted
as the glycine conjugate, nicotinuric acid (Miller et al., 1960). In humans, approxi-
mately 88% of an oral dose of 3000 mg of nicotinic acid was recovered in the urine
within 1 h (Miller et al., 1960).
There is substantial experimental evidence that alkyl-substituted pyridines
undergo extensive side-chain oxidation to yield the corresponding carboxylic acid
derivatives. Ninety per cent of a dose of 2-methylpyridine of 100 mg/kg bw or 96%
of a dose of 2,6-dimethylpyridine (No. 1317) of 100 mg/kg bw administered by
gavage to male albino Wistar rats was excreted as the glycine conjugate of the
corresponding pyridine-2-carboxylic acid derivative (Hawksworth & Scheline,
1975).
In groups of three male and three female Wistar rats given 4-methylpyridine at
a dose of 300 mg/kg bw by gavage, >50% was excreted in the urine principally as
pyridine-4-carboxylic acid (50%) or its glycine conjugate (5%) after 24 h. Minor
metabolites included unchanged 4-methylpyridine (approximately 2.5%) and
4-methylpyridine-N-oxide (1.5%), demonstrating that N-oxidation is also a
minor metabolic pathway (Nguyen et al., 1988). In fact, in mice, hamsters, rats,
guinea-pigs, or rabbits given the related substance 3-methylpyridine at a dose of
40 mg/kg bw by intraperitoneal administration, 6.4, 0.3, 4.0, 0.7, and 0.1%, respec-
tively, of the administered dose was excreted in the urine as the corresponding
N-oxide within 24 h (Gorrod & Damani, 1980).
214 PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES
K2
When incubated with hepatic and pulmonary microsomal preparations isolated
from male Wistar rats, albino Dunkin-Hartley guinea-pigs, albino New Zealand
rabbits, and LACA albino mice, 3-methylpyridine, 3-ethylpyridine (No. 1315), and
3-acetylpyridine (No. 1316) were converted to N-oxides by the hepatic microsomal
fractions and not the pulmonary microsomal fractions (Cowan et al., 1978).
Although no metabolic data are available on 2-acetylpyridine, data are available
on the structurally related compound, 3-acetylpyridine. 3-Acetylpyridine is metabo-
lized by both N-oxidation and side-chain reduction. In rats, 3-acetylpyridine is
reported to be metabolized to 1-(3-pyridyl)ethanol and 1-(3-pyridyl-N-oxide)ethanol
(Schwartz et al., 1978). Both 1-(3-pyridyl)ethanol and (3-pyridyl)-1,2-ethandiol
were isolated as N-oxide derivatives from the urine of a dog given daily oral doses
of 3-acetylpyridine for 8 consecutive days (McKennis et al., 1964). Similar results
were obtained in vitro when 3-acetylpyridine was incubated successively with rat
microsomal and cytoplasmic preparations. In both experiments, metabolites
included 1-(3-pyridyl-N-oxide)ethanol and 1-(3-pyridyl)ethanol (Damani et al.,
1980) (Figure 2).
2-Pyridinemethanethiol (No. 1308) is oxidized to the polar sulfonic acid metabo-
lite and subsequently excreted in the urine. The oxidation of thiol is catalysed by
two enzyme systems, CYP and the favin-containing monooxygenases (Renwick,
1989). Aromatic and aliphatic sulfdes are primarily oxidized by favin-containing
monooxygenases and, to a lesser extent, CYP to form sulfonic acids. Based on
the numerous examples of successive oxidation of thiols to sulfonic acids by favin-
containing monooxygenases and CYP enzymes in a variety of test systems
(Cashman & Williams, 1990; Cashman et al., 1990; Rettie et al., 1990; Yoshihara
& Tatsumi, 1990; Sadeque et al., 1992, 1995; Cashman et al., 1995a, 1995b;
Elfarra et al., 1995; Nnane & Damani, 1995), it is concluded that the S-oxidation
pathway is the major route of detoxication of 2-pyridinemethanethiol in humans
(Ziegler, 1980; Nickson & Mitchell, 1994).
N
O
N
O
N
OH
O
N
3-Ethylpyridine
3-Acetylpyridine
3-Acetylpyridine-N-oxide
1-(3-Pyridyl)ethanol
(major metabolite)
Figure 4. Metabolic pathway for 3-alkyl- and 3-acetylpyridine
PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES 215
K2
Alkyl-substituted quinolines undergo side-chain oxidation, ring epoxidation and
ring hydroxylation. When 6-methylquinoline (No. 1302) was incubated with rat liver
microsomes, 6-hydroxylmethylquinoline, 5-hydroxyl-6-methylquinoline, and 6-
methyl-7,8-oxoquinoline were the major metabolites. Other minor metabolites
include 6-methyl-5,6-oxoquinoline, quinoline-6-carboxaldehyde, and quinoline-6-
carboxylic acid (Scharping et al., 1993).
Isoquinoline (No. 1303), lacking ring substituents, is subject to N-oxide forma-
tion, epoxidation and ring hydroxylation. When groups of fve male Wistar rats were
given isoquinoline at a dose of 75 mg/kg bw via intragastric tube for three consecu-
tive days, induction of UDP-glucuronosyltransferase, microsomal epoxide hydro-
lase, and GST activities were observed. Isoquinoline showed a 1.4 to 1.8-fold
increase in UDP-glucuronosyltransferase activity and a 20% increase in GST activ-
ity (p < 0.05) (Le & Franklin, 1997).
On the basis of the evidence above, pyrrole and indole undergo ring hydroxyl-
ation. Alkyl-substituted pyrroles and indoles mainly undergo CYP-mediated oxida-
tion of the side-chain to yield the corresponding side-chain alcohol that may be
excreted as the glucuronic acid or sulfuric acid conjugate or further oxidized. To
some extent, epoxidation of the pyrrole ring may occur leading to hydroxylated
polar metabolites excreted mainly in the urine. In a similar manner, alkyl-
substituted pyridines and quinolines are subject to side-chain oxidation and in the
case of substituted quinolines, ring epoxidation and ring hydroxylation. The
polar hydroxy- or acyl-substituted metabolites are readily excreted in conjugated
form. In addition, pyridine and quinoline derivatives may form polar N-oxides.
Instances of combined side-chain oxidation and N-oxide formation have been
observed.
2.3.2 Toxicological studies
The available data on the toxicity of pyridine, pyrrole and quinoline derivatives
in this group of 22 favouring agents are presented below. Although the studies of
acute toxicity and and short-term studies of toxicity were of limited use for evaluat-
ing the safety of these substances, because of their short duration, they are
included for completeness.
(a) Acute toxicity
Oral median lethal dose (LD
50
) values have been reported for 10 of the 22
agents in this group (see Table 3). In rats, LD
50
values are in the range from 51 to
3450 mg/kg bw; however most LD
50
values range from approximately 300 to
1500 mg/kg bw (Smyth et al., 1951, 1962; Spanjers & Til, 1968; McGee, 1974;
Posternak et al., 1975; Moreno, 1976; Izamerov et al., 1982; Costello et al., 1992;
Myers & Ballantyne, 1997), demonstrating that the acute oral toxicity of pyridine,
pyrrole and quinoline derivatives is low. In mice, LD
50
values are in the range of
282 to 2800 mg/kg bw (Shellenberger, 1971; Pellmont, 1977; Moran et al., 1980;
Izamerov et al., 1982).
216 PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES
K2
(b) Short-term studies of toxicity
The results of short-term studies with representative pyridine, pyrrole and qui-
noline derivatives are summarized in Table 4 and are described below.
(i) Indole (No. 1301)
Rats
Groups of six male Holtzman rats were fed a diet containing indole at a con-
centration of 0 (control), 0.25, 0.50, or 0.75% for 3 weeks (equivalent to a dose of
approximately 0, 125, 250, or 375 mg/kg bw per day. An additional group received
a diet containing indole at 0.75%, supplemented with methionine at 0.25%. The
animals were monitored for food consumption and food effciency, uptake, body-
weight gain, and haematological effects. At termination of the study, the animals
were necropsied and the livers were weighed. A statistically signifcant reduction
in food intake was reported at 0.50 and 0.75%. All groups fed indole, including the
group receiving indole at 0.25% in the diet, exhibited a statistically signifcant
reduction in body-weight gain. However, animals in the group given the diet sup-
Table 3. Studies of the acute toxicity of pyridine, pyrrole and quinoline
derivatives administered orally
No. Flavouring agent Species Sex LD
50
Reference
(mg/kg bw)
1301 Indole Rat M 1000 Smyth et al. (1962)
1302 6-Methylquinoline Rat NR 1260 Moreno (1976)
1303 Isoquinoline Rat NR 360 Smyth et al. (1951)
1304 Skatole Rat NR 3450 McGee (1974)
1309 2-Acetylpyridine Rat NR 2280 Posternak et al. (1975)
1309 2-Acetylpyridine Rat M, F 2160
a
Spanjers & Til (1968)
1310 N-Furfurylpyrrole Mice F 335 Shellenberger (1971)
1310 N-Furfurylpyrrole Mice M 580 Shellenberger (1971)
1310 N-Furfurylpyrrole Mice M, F 380 Moran & Easterday (1980)
1316 3-Acetylpyridine Rat M 57
b
Costello et al. (1992)
1316 3-Acetylpyridine Rat F 51
b
Costello et al. (1992)
1318 5-Ethyl-2-methylpyridine Rat NR 1540 Smyth et al. (1951)
1318 5-Ethyl-2-methylpyridine Rat NR 368 Izamerov et al. (1982)
1318 5-Ethyl-2-methylpyridine Mice NR 282 Izamerov et al. (1982)
1318 5-Ethyl-2-methylpyridine Rat M 1195
c
Myers & Ballantyne (1997)
1319 2-Propionylpyrrole Mice M, F 1620 Moran & Easterday (1980)
1320 Methyl nicotinate Mice NR 2800 Pellmont (1977)
F, female; M, male; NR, not reported.
a
Calculated using density = 1.08 g/ml (Sigma-Aldrich, 2003; available from
http://www.sigmaaldrich.com).
b
Calculated using density = 1.102 g/ml (Sigma-Aldrich, 2003; available from
http://www.sigmaaldrich.com).
c
Calculated using density = 0.919 g/ml (Sigma-Aldrich, 2003; available from
http://www.sigmaaldrich.com).
PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES 217
K2 T
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218 PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES
K2
plemented with methionine at 0.25% had body-weight gains similar to those of the
controls. There were no signifcant differences between test and control animals
with respect to concentration of haemoglobin or erythrocyte volume fraction, and
relative weights of the liver (Martinez & Roe, 1972).
(ii) Indole-3-carbinol, a metabolite of skatole (No. 1304)
Indole-3-carbinol is a major urinary metabolite formed by side-chain oxidation
of skatole (3-methylindole, No. 1304) and is therefore available data on its toxicity
is included in this evaluation
Rats
In a study designed to evaluate the ability of indole-3-carbinol to prevent
cancer, groups of 20 female Sprague-Dawley rats were given a tricapylin vehicle
with or without different tumour-inducing agents on days 1, 8, 15 to 26, and 29.
Animals were maintained on a basal diet with access to food and water ad libitum.
From weeks 5 to 30, control animals continued to be given a basal diet or a diet
supplemented with indole-3-carbinol at a concentration of 2000 mg/kg of diet
(approximately equivalent to an intake of 100 mg/kg bw per day). Animals were
weighed weekly and the size and location of palpable mammary gland tumours
were recorded. After 30 weeks, animals were killed and subjected to macroscopic
and histopathological examination. Total foci and aberrant crypts per focus were
evaluated by light microscopy. Mean body weights in the groups treated with
indole-3-carbinol were signifcantly (p < 0.05) lower than those of the control group.
There was no signifcant difference in survival in the control group and in the
groups treated with indole-3-carbinol. A higher incidence of mammary gland
tumours (10%) was detected in the control group that was treated with vehicle
only, when compared with that (0%) in the group treated with vehicle and main-
tained on a diet supplemented with indole-3-carbinol at a concentration of 2000 mg/
kg of diet from weeks 5 to 30. Treatment with indole-3-carbinol resulted in a 100-
fold increase in liver GST-P (placental form of glutathione-S-transferase) foci, but
a decreased number of aberrant crypts of the colon compared with the control
group (Stoner et al., 2002).
In a 6-week study, groups of 10 female Sprague-Dawley rats were given indole-
3-carbinol at a dose of 0, 5, 25, 50, 100, or 200 mg/kg bw per day by gavage 5
days per week. Animals underwent daily observation for clinical signs of toxicity
and weekly measurements of body weight. In addition, at the conclusion of the
study, gross necropsy, organ weight measurements, and histopathological exami-
nation were conducted. At the highest dose, a 10% reduction in body-weight gain
and a 20% increase in absolute weight of the liver was reported. No other adverse
effects were reported (Grubbs et al., 1995).
A group of male inbred ACI/N rats was maintained on a basal diet or a diet
containing indole-3-carbinol at a concentration of 1000 mg/kg of diet (approxi-
mately equivalent to a dose of 50 mg/kg bw per day) for 37 weeks. Animals were
observed daily for general health, and at 37 weeks, body weights were measured
and animals were killed. At necropsy, liver weights were recorded and organs were
evaluated for gross lesions. Histological examination of major organs was per-
PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES 219
K2
formed and the oral cavity was evaluated for pre-neoplastic and neoplastic lesions.
There were no treatment-related effects on body and liver weights, or on gross
pathology. Histopathological examinations did not reveal any differences between
treated and control animals, and there was no treatment-related increase in the
incidence of neoplasms of the oral cavity (Tanaka et al., 1992)
(iii) 6-Methylquinoline (No. 1302), methyl- 2-pyrrolyl ketone
(No. 1307), 2-pyridinemethanethiol (No. 1308), and
2-acetylpyridine (No. 1309)
Rats
In a series of 90-day single-dose studies, groups of 1016 Charles River CD
rats were fed diets containing either 6-methylquinoline, methyl 2-pyrrolyl ketone,
2-pyridinemethanethiol, or 2-acetylpyridine. The rats were housed in same-sex
pairs and given access to water and food ad libitum. The concentration of the test
material in the diet was adjusted during the study to maintain constant levels of
dietary intake. The doses were calculated to be >100 times greater than the pos-
sible average daily intake, which was determined by multiplying usual levels of use
of the favouring agent in each of 33 food categories (e.g. baked goods and meat
products) by the average amount of that food category consumed daily and
summing the intake over all 33 food categories. Body weights of the individual rats
and the food consumption of pairs of rats were measured weekly and the effciency
of food utilization was calculated. Haematological examinations were carried out
on half the animals at 7 weeks and on all animals at 13 weeks. After 13 weeks,
all animals were killed, liver and kidney weights were measured, and gross and
histological examinations were carried out on a wide range of organs. Tissues
samples from various organs from each animal were preserved for histopathologi-
cal evaluation.
Based on measurements of growth, food intake, haematological and clinical
chemistry parameters, organ weights, and gross and histopathological examina-
tion, no differences were observed between groups of animals receiving 2-pyrid-
inemethanethiol, 6-methylquinoline, or 2-acetylpyridine and control animals.
Similarly, with the exception of a signifcant decrease in body-weight gain of <10%
accompanied by a decrease in food utilization in males only, no other variations
were reported in groups of rats receiving methyl 2-pyrrolyl ketone, compared with
controls. Therefore, the NOELs were: 2-acetylpyridine, 3.13 and 3.06 mg/kg bw per
day (Posternak et al., 1975); 2-pyridinemethanethiol, 3.42 mg/kg bw per day
(Posternak et al., 1969); 6-methylquinoline, 2.2 and 2.7 mg/kg per day (Posternak
et al., 1969); and methyl-2-pyrrolyl ketone, 87.5 and 86.3 mg/kg bw per day, for
males and females, respectively (Posternak et al., 1975).
(iv) 2-Acetylpyridine (No. 1309)
Rats
Groups of 10 male and 10 female albino rats were given 2-acetylpyridine at a
dose of 0, 37, 110, 330, or 1000 mg/kg bw per day by gavage in a propylene glycol
220 PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES
K2
vehicle, 6 days a week for 13 weeks. Clinical signs of toxicity, body-weight gain,
and food intake were recorded and haematological parameters were assessed.
Serum enzyme activities of serum glutamic pyruvic transaminase, serum glutamic
oxaloacetic transaminase, and serum alkaline phosphatase were monitored. In
addition, urine analysis was performed at the end of the study. At week 14, all
surviving animals were killed and examined macroscopically. Selected organs
were weighed and microscopic examinations were performed. 2-Acetylpyridine
had no effect on general appearance, behaviour, mortality, growth, water intake,
serum enzyme activity, or urine composition. All early deaths were attributed to
dosing errors. Slight anaemia was indicated by a decrease in erythrocytes in males
at the two higher doses (330 and 1000 mg/kg bw per day) and in females at 110
and 330 mg/kg bw per day. While the decrease in erythrocyte count was less
distinct at 1000 mg/kg bw per day, females at the highest dose (1000 mg/kg bw
per day) exhibited decreased lymphocyte counts and increased neutrophil counts.
Relative weights of the kidney, liver, and spleen were increased at the highest dose
in both males and females. Slightly elevated absolute weights of the liver
were also observed in both sexes at 330 mg/kg bw per day; however, the increase
was not statistically signifcant. Microscopic examination revealed changes in
centrolobular hepatocytes (slightly swollen cells with homogeneous, eosinophilic
cytoplasm, individual cell necrosis, and increased number of mitotic fgures),
slight proliferation of bile duct epithelium, and increased haematopoietic activity in
the spleen at the two higher doses (330 and 1000 mg/kg bw per day) in both sexes,
with the exception of the hepatic changes, which were observed only at the
highest dose (1000 mg/kg bw per day) in females. The NOEL for 2-acetylpyridine
was 37 mg/kg bw per day in albino rats (Til & van der Meulen, 1971).
(v) N-Furfurylpyrrole (No. 1310)
Rats
Groups of 15 male and 15 female albino FDRL rats were fed a diet containing
N-furfurylpyrrole, which was adjusted at 2-week intervals to maintain an intake of
11.2 mg/kg bw per day for 90 days. The average actual intake of N-furfurylpyrrole
was calculated to be 12.21 mg/kg bw per day. Rats were given access to drinking-
water ad libitum. Daily observation for survival, behaviour and physical appearance
produced no indications of changes in behaviour or signs of toxicity. All animals
survived to the end of the study. Weekly measurements of body weight, food
consumption, and calculation of effciency of food utilization, revealed no differ-
ences between test and control animals. Haematological examination, clinical
chemistry (blood urea), and urine analysis conducted during weeks 6 and 12 on
eight males and eight females from each group showed normal values. After 90
days, all animals were killed and subjected to a detailed gross examination, and
liver and kidney weights were recorded. Histological examination of a wide range
of tissues and organs from each animal revealed no treatment-related changes
(Morgareidge, 1971).
PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES 221
K2
(c) Long-term studies of toxicity and carcinogenicity
The results of a study of carcinogenicity with indole are summarized in Table
4 and are described below.
(i) Indole (No. 1301)
Rats
Groups of fve male and twenty female strain W rats were maintained on a diet
providing indole at a dose of 0 or 100 mg/kg bw per day for 460 days, followed by
a 30-day period of no treatment to examine the possible reversibility of any
treatment-related effects. Following the reversibility period, treatment with a diet
providing indole at a dose of 200 mg/kg bw per day was continued for 100 additional
days. A concurrent control group was maintained, but was not described.
Animals were monitored every 2 weeks for food consumption, body weight,
survival and haematological effects. At necropsy, liver, kidney, and spleen were
examined microscopically. Rats maintained on a diet supplemented with indole
showed a 20% reduction in body-weight gain compared with controls. The
haematological profle obtained for the frst 460 days revealed slightly reduced
concentrations of haemoglobin and erythrocyte counts compared with thoseof the
controls (i.e. haemoglobin, 15.2 versus 13.8 g Hb/100 ml blood; and erythrocyte
count, 8 versus 6.4 million cells/ml blood, respectively), which were reversible upon
cessation of treatment. By day 460, the leukocyte counts reached twice their
original values, which the authors stated were still within the normal range of values
for rats. No leukaemia or other tumours attributable to administration of indole were
observed in the animals. The average life expectancy of the rats was not altered
by the inclusion of indole in the diet. Except for indications of moderate reversible
anaemia, no other adverse effects were reported (Kaiser, 1953).
(d) Genotoxicity
Studies of mutagenicity and genotoxicity were available for nine pyridine,
pyrrole and quinoline derivatives. The results of these tests are summarized in
Table 5 and described below.
1
(i) In vitro
There was no evidence of mutagenicity in the assay for reverse mutation in
bacteria when various strains of Salmonella typhimurium (TA97, TA98, TA100,
TA102, TA104, TA1535, TA1537, TA1538, and TM677) were incubated with indole
1
For conversions used, see Table 5
222 PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES
K2
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a
n
d

T
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8
PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES 223
K2
1
3
0
2

6
-
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(
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(
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9
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(
1
9
8
9
)




T
A
9
8

a
n
d

T
A
1
0
0
224 PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES
K2
T
a
b
l
e

5
.

(
c
o
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t
d
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o
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9
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(
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9
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(
1
9
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6
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PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES 225
K2
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.
226 PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES
K2
(No. 1301) at a concentration of up to 30 mmol/plate (3515 mg/plate) (Anderson &
Styles, 1978; Kaden et al., 1979; Florin et al., 1980; Ochiai et al., 1986; Vance et
al., 1986; Sasagawa & Matsushima, 1991; Fujita et al., 1994), isoquinoline (No.
1303) at a concentration of up to 20 000 mg/ml (Sugimura et al., 1976; Nagao et
al., 1977; Epler et al., 1979; Kaden et al., 1979; Sideropoulos & Specht, 1984; ),
skatole (No. 1304) at a concentration of up to 3 mmol/plate (394 mg/plate) (Florin
et al., 1980; Ochiai et al., 1986; Kim et al., 1989; Sasagawa & Matsushima, 1991),
pyrrole (No. 1314) at a concentration of up to 1.4 mmol/plate (93 926 mg/plate)
(Florin et al., 1980; Aeschbacher et al., 1989; Lee et al., 1994), and 3-ethylpyridine
(No. 1315) at a concentration of up to 3 mmol/plate (321 mg/plate) (Florin et al.,
1980) with and without metabolic activation. Methyl 2-pyrrolyl ketone (No. 1307)
at concentrations of 4 to 100 mmol/plate induced a > 2-fold increase in the number
of revertants per plate compared with the control when tested in S. typhimurium
TA98 in the absence of metabolic activation (Lee et al., 1994). However, negative
results were obtained with metabolic activation as well as in S. typhimurium TA100
(both with and without metabolic activation). Furthermore, no mutagenic activity
was reported in either strain when incubated with methyl 2-pyrrolyl ketone at a
concentration of up to 200 mg/plate with and without metabolic activation (Wang et
al., 1994). 6-Methylquinoline (No. 1302) at a concentration of 3.3 to 3600 mg/plate)
gave uniformly positive results in the presence of metabolic activation (Sugimura
et al., 1976; Nagao et al., 1977; Dong et al., 1978; Wild et al., 1983; Takahashi et
al., 1988; Debnath et al., 1992; Zeiger et al., 1992). Methylquinolines, tested at a
concentration of 400 mg/plate, showed a potent bactericidal or bacteriostatic effect,
with only 6% survival of S. typhimurium TA100 treated with 6-methylquinoline
(Dong et al., 1978).
There was no evidence of mutagenicity when Escherichia coli (strains WP2
uvr4A/pKM101, SD-4-73, or B/r HCR+) were incubated with indole (No. 1301) at
a concentration of up to 0.4 mmol/plate (47 mg/plate) (Sasagawa & Matsushima,
1991), isoquinoline (No. 1303) at a concentration of up to 50 mg/ml, skatole (No.
1304) at a concentration of up to 0.4 mmol/plate (52 mg/plate) (Szybalski, 1958;
Sasagawa & Matsushima, 1991), or 3-acetylpyridine (No. 1316) at a concentration
of up to 10 000 mg/plate of (Pai et al., 1978).
In non-standardized assays, 2-acetylpyridine (No. 1309) at 0.50 to 0.87%
(54 000 to 939 600 mg/ml) and 3-acetylpyridine (No. 1316) at 0.5 to 1.11% (55 100
to 122 322 mg/ml) caused a dose-dependent increase in mitotic aneuploidy in strain
D61.M of Saccharomyces ceverisiae (Zimmermann et al., 1986). At the higher test
concentrations, the growth of D61.M was strongly or completely inhibited. The
authors noted that it is generally recognized that there is a threshold dose for
induction of aneuploidy in yeast (Zimmermann et al., 1985a, 1985b, 1985c).
Assays in mammalian cell lines have been performed for isoquinoline
(No. 1303) (Williams, 1984), skatole (No. 1304) (Kim et al., 1989), and pyrrole
(No. 1314) (Williams, 1984). There was no evidence of increased unscheduled
DNA synthesis when freshly isolated rat liver cells were incubated with pyrrole or
isoquinoline (concentrations not specifed) (Williams, 1984). Single-strand DNA
breaks and inhibition of growth were reported when undeuterated or deuterated
(at C2 or C3 positions) 3-methylindole (skatole) at 10 mmol/l to 1 mmol/l (1.31 to
131.18 mg/ml) was incubated with isolated cultured bovine kidney cells. However,
PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES 227
K2
there was no evidence of DNA interstrand crosslinks (Kim et al., 1989). These
observations are consistent with reports that, at high concentrations, indoles
deplete glutathione, leading to increased formation of DNA adducts (Nichols et al.,
2000; Regal et al., 2001).
(ii) In vivo
There was no evidence for mutation in a standard assay for sex-linked
recessive lethal mutation when adult Drosophila melanogaster were fed 6-
methylquinoline (No. 1302) at a concentration of 10 mmol/l (1432 mg/ml) in a
5% sucrose solution for 3 days (Wild et al., 1983). Furthermore, 6-methylquinoline
did not induce micronucleus formation in bone marrow cells obtained from male
and female NMRI mice 30 h after treatment with the test compound as a
single intraperitoneal dose at 0, 286, 429, or 572 mg/kg bw (Wild et al., 1983).
(iii) Conclusions
Overall, negative results were reported in assays for reverse mutation in bac-
teria for six representative pyridine, pyrrole and quinoline derivatives (i.e. indole,
No. 1301; isoquinoline, No. 1303; skatole, No. 1304; methyl 2-pyrrolyl ketone,
No. 1307; pyrrole, No. 1314; and 3-ethylpyridine, No. 1315). Although
6-methylquinoline gave positive results with metabolic activation, it gave negative
results in studies in vivo, indicating that there are adequate detoxication
mechanisms for the rapid absorption, distribution, biotransformation, and
elimination of the N-containing heteroaromatic derivatives. 2-Acetylpyridine and
3-acetylpyridine produced positive results in yeast, but this is unlikely to occur at
low doses because yeast is generally believed to have a threshold for the induction
of aneuploidy. The positive results reported in bacteria for skatole are consistent
with observations that, at high concentrations, indoles depletes glutathione, leading
to reduced detoxifcation.
On the basis of the available evidence, the 22 pyridine, pyrrole and quinoline
derivatives in this group do not demonstrate genotoxic potential.
(e) Other relevant studies
(i) DNA adducts
Numerous studies have been undertaken to investigate the alkylating potential
of 3-methylindole and its principal metabolite indole-3-carbinol. A recent study has
investigated the formation of DNA adducts with metabolites of 3-methylindole
(skatole) in vitro. When 3-methylindole at a concentration of 200 mmol/l is incubated
with calf thymus DNA in the presence of CYP obtained from goat lung, rat liver,
or human liver microsomes, DNA adducts are formed. In all three microsomal
preparations, the 3-methylindole-deoxyguanosine adduct was the primary adduct.
3-Methylindole-deoxyadenosine and 3-methylindole-deoxycytosine adducts were
formed in smaller amounts. No adducts were reported in untreated hepatocytes.
Analysis of adducts formed when 3-[
2
H
3
]-methylindole was incubated with micro-
somes revealed that the 3-methyleneindolenine intermediate undergoes nucle-
228 PYRIDINE, PYRROLE AND QUINOLINE DERIVATIVES
K2
ophilic attack with the nucleotide amine function. When mammalian cell lines were
exposed to 3-methylindole at 200 mmol/l, or more likely its activated metabolite(s),
concentrations of GSH were depleted, leading to increased formation of protein
and DNA adducts (Regal et al., 2001).
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ALIPHATIC AND ALICYCLIC HYDROCARBONS
First draft prepared by
Mrs M.E.J. Pronk
1
and Professor J.R. Bend,
2
1
Centre for Substances and Integrated Risk Assessment, National Institute
for Public Health the Environment, Bilthoven, Netherlands; and
2
Department of Pharmacology & Toxicology, Faculty of Medicine and
Dentistry, University of Western Ontario, London, Ontario, Canada;
Evaluation ............................................................................... 235
Introduction....................................................................... 235
Estimateddailyintake........................................................ 242
Absorption,distribution,metabolismandelimination........ 242
ApplicationoftheProcedurefortheSafety
EvaluationofFlavouringAgents.................................. 246
Considerationofsecondarycomponents.......................... 247
Considerationofcombinedintakesfromuseas
favouringagents......................................................... 247
Conclusions........................................................................ 247
Relevantbackgroundinformation............................................. 248
Explanation......................................................................... 248
Additionalconsiderationsonintake................................... 248
Biologicaldata.................................................................... 248
Biochemicaldata......................................................... 248
Absorption,distribution,andexcretion.................. 251
Metabolism............................................................ 257
Toxicologicalstudies.................................................... 257
Acutetoxicity......................................................... 257
Short-termstudiesoftoxicity................................. 257
Long-termstudiesoftoxicityand
carcinogenicity................................................ 266
Genotoxicity........................................................... 269
Reproductivetoxicity............................................. 279
References............................................................................... 283
1. EVALUATION
1.1 Introduction
The Committee evaluated a group of 20 aliphatic and alicyclic hydrocarbons
(Table 1) by the Procedure for the Safety Evaluation of FlavouringAgents (see
Figure1,p192).Onememberofthisgroup,d-limonene(No.1326),wasevaluated
by the Committee at its thirty-ninth meeting (Annex 1, reference 101) and was
assigned an acceptable daily intake (ADI) of 01.5mg/kg bw. The Committee
at that meeting recommended, however, that intake of this substance as a food
additive be restricted to 0.075mg/kg bw per day, or 5% of theADI.At its forty-
frst meeting (Annex 1, reference 107), the Committee re-evaluated the ADI for
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u
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.
242 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
d-limoneneandrecommendedthatitbewithdrawnandreplacedwithanADInot
specifed.
Nineteenofthe20favouringagentsinthisgroup(Nos1323,1324,13261331,
13361343and13451347)havebeenreportedtooccurnaturallyinfoods.They
havebeendetectedin,forexample,coffee,alcoholicbeverages,bakedandfried
potato,heatedbeans,tea,breadandcheese(Nijssenetal.,2003).Thesubstance
withthehighestnaturaloccurrenceisd-limonene(No.1326).
1.2 Estimated daily intake
Thetotalannualvolumeofproductionofthe20favouringagentsinthisgroupis
approximately380000kginEurope(InternationalOrganizationoftheFlavorIndus-
try,1995)and140000kgintheUSA(NationalAcademyofSciences,1989;Lucas
etal.,1999).d-Limonene(No.1326)accountsforapproximately73%ofthetotal
annualvolumeofproductioninEuropeand71%intheUSA.Theestimateddaily
intakes of d-limonene in Europe and the USA are approximately 40000mg and
13000mg/person, respectively. Myrcene (No. 1327), a- and b-pinene (Nos 1329
and 1330, respectively), terpinolene (No. 1331), b-caryophyllene (No. 1324), a-
phellandrene(No.1328),andp-mentha-1,4-diene(No.1340)accountformostof
theremaining(approximately2627%)totalannualvolumeofproduction.Theesti-
mateddailyintakesofthesefavouringagentsareintherangeof928300mg/person
in Europe and 702400mg/person in the USA. The reported annual volumes of
productionoftheremainderofthefavouringagentsinthisgroupareextremelylow,
accountingfor<1and3%ofthetotalannualvolumeofproductioninEuropeandthe
USA,respectively.Theestimateddailyintakesoftheseagentsrangefrom<0.1to
93mg/personinEuropeandtheUSA,exceptfor1-methyl-1,3-cyclohexadiene(No.
1344)whichhasanestimateddailyintakeofapproximately300mg/personinthe
USA.TheestimateddailypercapitaintakeofeachagentisreportedinTable2.
1.3 Absorption, distribution, metabolism and elimination
Beinglipophilic,thealiphaticandalicyclichydrocarbonsinthisgrouparelikely
tocrossbiologicalmembranesbypassivediffusion.Afteroralandinhalationexpo-
sure, they are rapidly absorbed and distributed to body tissues, elimination from
bloodbeingtriphasic,withaslowterminalphase.
Onthebasisoftheavailabledata,itisanticipatedthatallthealiphaticandali-
cyclichydrocarbonsinthisgroupwillparticipateinsimilarpathwaysofmetabolic
detoxifcation in mammals, including humans. After absorption, these hydrocar-
bons are oxidized to polar oxygenated metabolites via cytochrome P450 (CYP)
enzymes and alcohol and aldehyde dehydrogenases. The aliphatic and alicyclic
substances are oxidized either by side-chain oxidation or by epoxidation of an
exocyclic or endocyclic double bond. Alkyl oxidation initially yields hydroxylated
metabolitesthatmaybeexcretedinconjugatedformorundergofurtheroxidation,
yielding more polar metabolites that are also excreted in conjugated form in the
urine. If a double bond is present, epoxide metabolites may form and these
metabolitesaredetoxifedeitherbyhydrolysistoyielddiols,orbyconjugationwith
glutathione.
ALIPHATIC AND ALICYCLIC HYDROCARBONS 243
L1
T
a
b
l
e

2
.

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7
244 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
B
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ALIPHATIC AND ALICYCLIC HYDROCARBONS 245
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246 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
1.4 Application of the Procedure for the Safety Evaluation of
Flavouring Agents
Step1. InapplyingtheProcedure,theCommitteeassignedallthe20favouring
agentsinthisgrouptostructuralclassI(Crameretal.,1978).
Step2. All the favouring agents in this group are expected to be metabolized
toinnocuousproducts.Theevaluationofallagentsinthisgroupthere-
foreproceededviatheA-sideofthedecision-tree.
StepA3. Theestimateddailyintakesof17ofthe20favouringagents(Nos1323,
1324, 1328, 1330, 1331 and 13361347) are below the threshold of
concern(i.e.1800mg/personperdayforclassI).AccordingtothePro-
cedure,theuseofthese17favouringagentsraisesnosafetyconcern
at estimated current intakes.The estimated daily per capita intakes of
theremainingthreeagentsinthisgroup,d-limonene(No.1326),myrcene
(No. 1327) and a-pinene (No. 1329), exceed the threshold of concern
forclassI.Accordingly,theevaluationofthesethreeagentsproceeded
tostepA4.
StepA4. d-Limonene, myrcene and a-pinene are not endogenous in humans.
Therefore,theevaluationoftheseagentsproceededtostepA5.
StepA5. For myrcene (No. 1327) a lowest-observed-effect level (LOEL) of
250mg/kgbwperdaywasreportedformalemiceandmaleandfemale
rats treated by gavage for 13 weeks (National Toxicology Program,
2004a, 2004b), while the same dose was the no-observed-effect level
(NOEL)infemalemice.Thisdoseisapproximately1800timesgreater
thantheestimatedintakeofmyrcenefromitsuseasafavouringagent
in Europe (140mg/kg bw per day) and 83000 times greater than the
estimatedintakeofmyrceneintheUSA(3mg/kgbwperday).TheCom-
mittee concluded that myrcene would not pose a safety concern at
estimatedcurrentintake.
Atitsforty-frstmeeting,theCommitteeestablishedanADInotspecifed
ford-limonene(No.1326)onthebasisofshort-andlong-termstudiesof
toxicity in female rats and male and female mice, and studies of
developmentaltoxicityinmice,ratsandrabbits.Inthesestudies, d-limo-
nenewastestedatdosesrangingfrom250to2800mg/kgbwperday.
BasedontheADInotspecifed,theCommitteeconcludedthatd-limo-
nenewouldnotposeasafetyconcernattheestimatedcurrentintakes
(660mg/kgbwperdayinEuropeand210mg/kgbwperdayintheUSA).
Notoxicologicaldataona-pinene(No.1329)wereavailable.d-Limonene
shares structural characteristics with a-pinene in that both contain a
methyl-substituted cyclohexene ring, which contains a second alkyl
substituent.Ind-limonene,thisisanisopropenylgroup,whileina-pinene
thesecondsubstituentisadimethyl-substitutedmethylenebridge.Based
onthesechemicalstructures,itwouldbepredictedthatthetoxicityofa-
pinenewouldbeunlikelytoexceedthatofd-limonene.Bothcompounds
arepredictedtobemetabolizedtoinnocuousproducts.Metabolismof
ALIPHATIC AND ALICYCLIC HYDROCARBONS 247
L1
bothcompoundsisbyhydroxylationofthecyclohexeneringandoxidation
of its methyl substituent. d-Limonene undergoes epoxidation of the
endocyclic and allylic double bonds, leading to dihydroxy products. a-
Pineneisconvertedtoseveralmetabolites,includingd-limonene,byrat
liver microsomes in vitro. The Committee concluded that d-limonene
sharedsuffcientchemicalandmetabolicsimilaritieswitha-pinenetobe
usedasastructuralanaloguefora-pineneatthisstepoftheProcedure.
Theestimatedcurrentpercapitaintakesofa-pineneinEurope(36mg/kg
bwperday)andintheUSA(41mg/kgbwperday)areapproximately5%
and20%,respectively,ofthoseofd-limonene,andarealmostfourorders
ofmagnitudelowerthanthelowestdosesofd-limoneneconsideredin
the establishment of its ADI not specifed. On the basis of these
considerations,theCommitteeconcludedthata-pinenewouldnotpose
asafetyconcernatestimatedcurrentintakes.
The intake considerations and other information used to evaluate the 20 ali-
phatic and alicyclic hydrocarbons in this group according to the Procedure are
summarizedinTable1.
1.5 Consideration of secondary components
Nine members (Nos 1323, 1324, 1327, 13371339 and 13411343) of this
groupoffavouringagentshaveassayvaluesof<95%.TheCommitteeevaluated
the secondary components in No. 1339 (1,4- and 1,8-cineole) at a previous
meetingandconsideredthattheydidnotpresentasafetyconcern.Thesecondary
components in Nos 1323, 1324, 1337 and 1343 (C
15
H
24
terpene hydrocarbons)
andinNo.1342(b-pinene,d-limonene,myrceneandp-cymene)wereallevaluated
according to the Procedure by the Committee at its present meeting. The Com-
mittee did not consider any of these secondary components to present a safety
concern. The secondary components in No. 1327 (dihydromyrcene), No. 1338
(cis-b-ocimene), No. 1341 (2,4,6-undecatriene), and the remaining secondary
components in No. 1343 (other isomers of farnesene) are all structurally related
to the primary favouring agents and are expected to share the same metabolic
fate.Thereforenoneofthesesecondarycomponentswasconsideredtopresent
asafetyconcern.
1.6 Consideration of combined intakes from use as favouring agents
In the unlikely event that all 20 agents in this group were consumed concur-
rently on a daily basis, the estimated combined intake would exceed the human
intakethresholdof1800mg/personperdayforclassI.However,these20agents
are all expected to be effciently metabolized and would not saturate metabolic
pathways.Overallevaluationofthedataindicatedthatcombinedintakeofthese
agentswouldnotraiseasafetyconcern.
1.7 Conclusions
The Committee maintained the previously establishedADI not specifed for
d-limonene (Annex 1, reference 107).The Committee concluded that use of the
248 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
favouring agents in this group of aliphatic and alicyclic hydrocarbons would not
presentasafetyconcernatestimatedcurrentintakes.TheCommitteealsonoted
thattheavailabledataonthetoxicityandmetabolismofthesefavouringagents
wereconsistentwiththeresultsofthesafetyevaluation.
2. RELEVANT BACKGROUND INFORMATION
2.1 Explanation
The relevant background information summarizes the key scientifc data ap-
plicable to the safety evaluation of 20 aliphatic and alicyclic hydrocarbons used
as favouring agents (see Table 1).All substances in this group are unsaturated
hydrocarbonsthatareacyclic,monocyclic,orbicyclic.Membersofthegroupthat
exhibitthehighestannualvolumesofuseasfavouringagentsincludeaseriesof
naturallyoccurringC10terpenehydrocarbons(e.g.d-limonene,myrcene,anda-
andb-pinene).
2.2 Additional considerations on intake
Volumesofproductionandintakevaluesforeachfavouringagentarereported
in Table 2. The majority of favouring agents in this group are products of plant
biosynthesis.
Nineteenofthe20favouringagentsinthegrouphavebeenreportedtooccur
naturally in traditional foods (Nijssen et al., 2003; Table 2). Quantitative data on
naturaloccurrencedatahavebeenreportedfor14favouringagentsinthegroup
(Stofberg & Grundschober, 1987). The consumption of all of these 14 agents is
derived predominantly from their presence in traditional foods (i.e. they have a
consumptionratioof1;Table2).
Owing to their volatility, several C
10
terpene hydrocarbons in this group have
alsobeenreportedtoemitfromvegetationtotheatmosphere.InNorthAmerica,
the annual emissions for these C
10
terpene hydrocarbons approach 18 million
tonnes,withmajorcontributionscomingfroma-pinene(No.1329),b-pinene(No.
1330),andd-3-carene(No.1342)(Guentheretal.,2000).
2.3 Biological data
2.3.1 Biochemical data
(a) Absorption, distribution, and excretion
(i) Acyclic hydrocarbons
InmaleJapanesewhiterabbitsgivenmyrcene(No.1327)atadoseof670mg/
kgbwperdaybygavagefor2days,approximately25%ofthetotaladministered
amount (19g to six rabbits) could be recovered from the urine excreted over a
period of 3 days after administration. More than 80% of the metabolites in urine
wereneutralmetabolites,therestwereacidicmetabolites(Ishidaetal.,1981).
ALIPHATIC AND ALICYCLIC HYDROCARBONS 249
L1
(ii) Monocyclic hydrocarbons
MaleWistarratsweregiven[
14
C
9
]d-limonene(No.1326)bystomachtubeata
doseof800mg/kgbw,andtheconcentrationofradiolabelwasdeterminedinblood,
tissues(fatnotincluded),excreta,bile,andexpiredair.Theanimalsweresacrifced
48h after dosing. Radiolabel reached a peak plasma concentration at 2h after
dosing, and after maintaining high levels for 10h, declined to negligible levels at
48h. In most tissues, peak concentrations of radiolabel were reached within 2h
afterdosing,indicatingrapiddistribution.Liver,kidneyandadrenalscontainedthe
highest concentrations of radiolabel (higher than blood or serum); other tissues
(includingbrain)contained<0.2%oftheadministeredradiolabel.Hardlyanyradio-
label could be detected at 48h after dosing. Whole body autoradiography con-
frmedthesefndings.At48hafterdosing,about60%oftheadministeredradiolabel
wasrecoveredfromtheurine,5%fromfaecesand2%fromexhaledairascarbon
dioxide(CO
2
).Approximately25%oftheadministeredradiolabelwasexcretedin
thebileduringthefrst24hafteradministration.Totalrecoveryofradiolabelwas
<100%andastherewashardlyanyradiolabelpresentinthetissuesat48h,this
couldpointtolossofvolatile
14
Cfromtheexcretaortotheeliminationofvolatile
14
C-labelledcompoundsotherthanCO
2
(Igimietal.,1974).Whenasimilardose
of[
14
C
9
]d-limonenewasgiventomalerabbits,72%and7%oftheradiolabelwas
excretedintheurineandfaecesduringthefrst72h,respectively(Kodamaetal.,
1974).
Inanadditionalstudywithseveralspecies(rats,hamsters,guinea-pigs,rabbits,
dogs,andhumans)dosedorallywith[
14
C
9
]-d-limonene,urinaryexcretionofradio-
labelinrodentsandrabbitscomprised8296%ofthedosewithin72handfaecal
excretionwas29%.Thetotalexcretionrateindogswassomewhatlower(77%
via urine and 9% via faeces within 72h), while two human volunteers excreted
5583% of the administered dose in the urine. Faecal excretion in humans was
notmeasured,butmayhavebeenconsiderableinthepersonwithlowerurinary
excretion as this person developed diarrhoea shortly after administration. In all
species,mostexcretionoccurredwithinthefrst24h(Kodamaetal.,1976).
Invitro,thesolubilityofd-limoneneinbloodandoliveoilwashigh,butlowin
water(partitioncoeffcientswere42,5700,1.8,and140forblood/air,oil/air,water/
air,andoil/blood,respectively).Thissuggestsahighrespiratoryuptakeandaccu-
mulationinadiposetissues(Falketal.,1990a).Indeed,uptakewasrapidandhigh
(68%) in an experiment in which human volunteers were exposed to d-limonene
in air at 0.225 and 0.450mg/l for 2h while doing light physical exercise. The
absorbed d-limonene was metabolized rapidly. Elimination followed a triphasic
pattern, with a short half-life in blood immediately after exposure (2.6min) but a
longhalf-lifeduringthelateeliminationphase(12.5h),whichindicatesslowelimi-
nationfromadiposetissues.Approximately1%ofthetotaluptakewaseliminated
unchangedinexpiredair,whileapproximately0.003%waseliminatedunchanged
inurine(Falk-Filipssonetal.,1993).
(iii) Bicyclic hydrocarbons
When male Japanese white rabbits were given (+)-a-pinene (No. 1329) in a
single dose at 560mg/kg bw by gavage, only 3% of the total administered dose
250 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
(10g to six rabbits) could be recovered from the urine collected for 3 days after
administration.Allthemetabolitesinurinewereneutralmetabolites(Ishidaetal.,
1981).Thesameauthorsalsoinvestigatedtheurinaryexcretionofd-3-carene(No.
1342) and (-)-b-pinene (isomer of (+)-b-pinene, No. 1330) in rabbits, and found
thatapproximately18%ofthetotaladministeredamount(26gtosixrabbits)ofd-
3-carenecouldberecoveredfromthe3-dayurine,withapproximately55%ofthe
metabolitesinurinebeingacidicmetabolites,andtheremaining45%beingneutral
metabolites. For (-)-b-pinene, approximately 15% of the total administered dose
(12gtosixrabbits)couldberecoveredfromtheurine,ofwhich43%wereneutral
and57%wereacidicmetabolites(Ishidaetal.,1981).
Invitrodataonthesolubilityofa-pinene,b-pinene(No.1330)andd-3-carene
inblood,oliveoilandwatersuggestahighrespiratoryuptakeandaccumulation
inadiposetissues(partitioncoeffcientswere1532,29005000,0.120.41,and
160190 for blood/air, oil/air, water/air, and oil/blood, respectively). For a-pinene
this is supported by a high estimated brain/blood partition coeffcient of 18 (Falk
etal.,1990a).Experimentsinwhichhumanvolunteerswereexposedto(+)-and
(-)-a-pineneord-3-careneat0.225and0.450mg/linairfor2hwhiledoinglight
physicalexerciseconfrmedthatuptakewasrapidandhighfortheseagents(58
60%for(+)-and(-)-a-pineneand70%ford-3-carene),andthattheyweremetabo-
lized rapidly. Elimination followed a triphasic pattern, with (+)- and (-)-a-pinene
exhibitingarapidinitial(distribution)phase(4.8and5.6min,respectively),arapid
second distribution phase (38 and 40min, respectively), and a slow elimination
phase (695 and 555min, respectively). Triphasic elimination was also observed
ford-3-carenewithhalf-livesof4.5,35,and1800minfortheinitial,rapid,andslow
phases,respectively.Itwasestimatedthatitwouldrequiremorethan2or6days
toeliminatea-pineneord-3-carene,respectively,fromthebody.Thelonghalf-lives
indicate slow elimination from adipose tissues. Less than 0.001% of the total
uptake of a-pinene or d-3-carene was eliminated unchanged in the urine, while
7.57.8% and 3% of the inhaled amount of the a-pinenes and d-3-carene was
exhaled(Falketal.,1990b,1991).
Inanotherstudy,humanswereexposedfor4or6htoatmospherescontaining
amixtureofvolatileorganicsubstances,whichincludeda-pinene,attotalconcen-
trationsof0.012or0.024mg/l.At0.024mg/l,theairconcentrationofa-pinenewas
0.775mg/l.Themeanpre-exposurebloodconcentrationof a-pineneof0.035mg/l
increasedtoanaverageconcentrationof1.9mg/lduringthe4hofexposure(50
240min). Thereafter (330450min), the mean blood concentration decreased to
0.15mg/l.Changesproportionaltothoseobservedat0.024mg/lwererecordedat
0.012mg/l.Similarresultswererecordedfor6hofexposure.Plasmaelimination
for a-pinene was best described with a three-exponential curve, with half-lives
rangingfrom0.227.8min,1958minand>150minfortheinitial,intermediateand
terminalphases,respectively(Ashley&Prah,1997).
In summary
Beinglipophilic,thealiphaticandalicyclichydrocarbonsinthisgrouparelikely
tocrossbiologicalmembranesbypassivediffusion.Afteroralandinhalationexpo-
sure, they are rapidly absorbed and distributed to body tissues, with elimination
frombloodbeingtriphasicwithasloweliminationphase.
ALIPHATIC AND ALICYCLIC HYDROCARBONS 251
L1
(b) Metabolism
(i) Acyclic hydrocarbons
In the urine of rabbits given myrcene (No. 1327) via oral gavage, the main
metabolites identifed were myrcene-3,10-glycol, myrcene-1,2-glycol, and uroter-
penol(asacetate)(40.7,20.8and11.8%,respectively,oftheneutralmetabolites).
Additionally,theglycolsunderwentfurtheroxidationtoyield2-hydroxymyrcene-1-
carboxylic acid and 3-hydroxymyrcene-10-carboxylic acid (no quantitative data
weregivenfortheseacidicmetabolites).Theauthorssuggestedthaturoterpenol
(orlimonene-8,9-diol)mayhavebeenformedfromlimonene,whichisderivedfrom
cyclizationofmyrceneintheacidicconditionsoftherabbitstomach(Ishidaetal.,
1981).
When rats were given myrcene at a dose of 800mg/kg bw per day orally by
gavage for 20 days, the principal metabolites isolated from the urine were 10-
hydroxylinalool(ormyrcene-3,10-glycol)and,toalesserextent,7-methyl-3-methy-
lene-oct-6-ene-1,2-diol(ormyrcene-1,2-glycol).Otherminormetabolitesincluded
the hydroxy acids of both the 3,10- and 1,2-glycols (10-carboxylinalool (or 3-
hydroxymyrcene-10-carboxylic acid) and 2-hydroxy-7-methyl-3-methylene-oct-6-
enoicacid(or2-hydroxymyrcene-1-carboxylicacid),respectively)andacyclicdiol,
1-hydroxymethyl-4-isopropenylcyclohexanol (or p-menth-8-ene-1,7-diol), formed
byintramolecularcyclizationofanopenchainmetabolite(Madyastha&Srivatsan,
1987). It was demonstrated that the biotransformation of myrcene was CYP-
mediatedandthatitcouldbeenhancedbypre-treatmentofanimalswithpheno-
barbital(Madyastha&Srivatsan,1987).AsidefrombeingasubstrateforCYPen-
zymes,myrcenehasalsobeenshowntoinducetheseenzymes,especiallythose
fromtheCYP2B(phenobarbital-inducible)subfamily(De-Oliveiraetal.,1997).
Theseresultsindicatethattheprincipalurinarymetabolitesinratsandrabbits
afteradministrationofmyrcenebygavagearemyrcene-3,10-glycolandmyrcene-
1,2-glycol formed from the hydration of the respective epoxide intermediates. In
bothspecies,epoxidationofthe3,10-doublebondwasfavouredoverepoxidation
ofthe1,2-doublebond,whileepoxidationofthe6,7-doublebonddidnotseemto
occur.Furtheroxidizedbiotransformationproducts,notablycarboxylicacids,and
cyclizationproductswereobservedinbothratsandrabbits.Theproposedmetabo-
lismschemeisgiveninFigure1.
(ii) Monocyclic hydrocarbons
Morethan10metaboliteswerefoundintheurineofratsgivend-limonene(or
p-mentha-1,8-diene; No. 1326) at a dose of 800mg/kg bw by oral gavage. Four
ofthemetaboliteswereidentifedasperillicacid,p-menth-1-ene-8,9-diol(oruro-
terpenol,orlimonene-8,9-diol),perillicacid-8,9-diol,and8-hydroxy-p-menth-1-en-
9-yl-b-D-glucopyranosiduronicacid(theglucuronicacidconjugateofuroterpenol).
The bile of these rats contained three metabolites, of which the most important
was 8-hydroxy-p-menth-1-en-9-yl-b-D-glucopyranosiduronic acid (Igimi et al.,
1974).Sixmetaboliteswereidentifedintheurineofrabbitsgiventhesameoral
dose. In addition to the four metabolites identifed in rat urine, the rabbit urine
containedp-mentha-1,8-dien-10-ol(orlimonene-10-ol)andp-mentha-1,8-dien-10-
252 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
Figure 1. Metabolism of myrcene in rats and rabbits
O
OH
OH
COOH
OH
O
OH
OH
COOH
OH
O H
O H
OH
OH
OH
OH
C
+
b-Myrcene myrcene-1,2-glycol
myrcene-3,10-glycol
2-hydroxymyrcene-1-carboxylic acid
3-hydroxymyrcene-10-carboxylic acid
uroterpenol
[cyclic diol]
rat
rabbit
rabbit
rabbit
rat
rat
yl-b-D-glucopyranosiduronicacid(theglucuronicacidconjugateofp-mentha-1,8-
dien-10-ol).Althoughnotdeterminedquantitatively,perillicacid,perillicacid-8,9-diol
and both glucuronic acid conjugates were the major metabolites in rabbit urine,
and no unchanged d-limonene was detected (Kodama et al., 1974). The same
authorsidentifedanadditionalfvemetabolitesintheurineofratsanddogstreated
orallywithd-limonene.Thesewerecharacterizedas2-hydroxy-p-menth-8-en-7-oic
acid, perillylglycine, perillyl-b-D-glucopyranosiduronic acid (the glucuronic acid of
perillicacid),p-mentha-1,8-dien-6-ol(orlimonene-6-ol),andp-menth-1-ene-6,8,9-
triol.Theyalsofoundsomespeciesdifferencesinthenatureofthemajormetabo-
lites in urine. Perillic acid-8,9-diol was the main metabolite in rats and rabbits,
perillyl-b-D-glucopyranosiduronic acid in hamsters, uroterpenol in dogs, and the
ALIPHATIC AND ALICYCLIC HYDROCARBONS 253
L1
glucuronicacidconjugateofuroterpenolinguinea-pigsandhumans.Itshouldbe
notedthatthefateofonly4065%ofthed-limonenedoseadministeredorallyto
theseanimalsandhumanswasaccountedfor(Kodamaetal.,1976).
Perillicacid,dihydroperillicacid,andlimonene-1,2-diolwerethemajormetabo-
lites identifed in the plasma of humans given an oral dose of d-limonene. Minor
metaboliteswerethemethylestersofperillicacidanddihydroperillicacid,andd-
limoneneitself(Crowelletal.,1994).Apartfromtheparentcompound,Poonetal.
(1996) and Vigushin et al. (1998) also identifed perillic acid, dihydroperillic acid,
andlimonene-1,2-diolasmajormetabolitesinhumanplasma.However,theyalso
foundtwoothermetabolites,i.e.p-mentha-1,8-diene-10-carboxylicacidandlimo-
nene-8,9-diol(oruroterpenol),whiletheydidnotdetectthemethylestersofperillic
acidanddihydroperillicacid.Peakplasmaconcentrationsofallmetaboliteswere
achieved46hafteradministration,withtheexceptionoflimonene-8,9-diolwhich
reacheditspeak1hafteradministration(Poonetal.,1996).Metabolitesinhuman
urinecomprisedtheglucuronicacidconjugatesofperillicacid,dihydroperillicacid,
p-mentha-1,8-diene-10-carboxylic acid, limonene-8,9-diol, and limonene-10-ol
(Poonetal.,1996).
Theseresultsindicatethatmetabolismofd-limonene(seeFigure2)proceeds
eitherbyallylicoxidationoftheexocyclicmethylgrouptoyieldperillicacidderiva-
tives (e.g. perillic acid and dihydroperillic acid), by epoxidation and hydrolysis to
yield diols (e.g. limonene-1,2-diol and limonene-8,9-diol), or by hydroxylation to
yieldmonohydroxycompounds(e.g.limonene-6-ol,limonene-10-ol).Experiments
invitrohaveshownthatepoxidationoftheC8doublebondisfavouredoverepoxi-
dationoftheC1

doublebond,duetosterichindranceofthe1-methylgroup:upon
incubationwithratlivermicrosomes,themajorityofd-limonenewasconvertedto
the8,9-epoxideandthe8,9-diol,andtoamuchlesserextent,tothe1,2-epoxide
andthe1,2-diol(Watabeetal.,1981).Otherexperimentsinvitrohaveshownthat
male rats can convert d-limonene into perillyl alcohol (by hydroxylation of the
methyl group at C7) and carveol (or limonene-6-ol; by ring C6-hydroxylation).
ThesereactionsarecatalysedbyCYP2C11and,whenpre-treatedwithphenobar-
bital,CYP2B1.Infemalerats,theactivityforconversiontoeitheralcoholismuch
lower.Apparently,thefemale-specifcCYP2C12hasnoactivitywithrespecttod-
limonenehydroxylation.Inmales,thehydroxylationactivitieswerenotdetectable
with fetal liver microsomes, but they increased after birth, closely related to the
developmentalincreaseinCYP2C11(Miyazawaetal.,2002).Limonenehasalso
beenshowntoinduceCYPenzymesoftheCYP2BandCYP2Csubfamilies(Austin
etal.,1988;Maltzmanetal.,1991).
Themetabolicproductsconstitutethemajorplasmametabolites,butunchanged
d-limonenewasalsopresent,aswereperillicacidartefacts(methylesters)inone
study.Perillicacidcanbeexcretedunchanged,orastheglycineorglucuronicacid
conjugate in the urine, or it can be further oxidized to perillic acid-8,9-diol or 2-
hydroxy-p-menth-8-en-7-oic acid. Glucuronic acid conjugates in urine have also
beenidentifedfordihydroperillicacid,forp-mentha-1,8-diene-10-carboxylicacid,
for limonene-8,9-diol, and for limonene-10-ol. Although most metabolites have
been identifed in several species, there are species differences as to the im-
portanceofthedifferentpathways.Intherat,themajorpathwayisviaperillicacid
254 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
andmetabolitesthereof(especiallyperillicacid-8,9-diol).Minorpathwaysintherat
include epoxidation of the 8,9-double bond, with subsequent hydrolysis to the
correspondingdiolandglucuronidation,oroxidationatC10orC6positions,with
subsequentglucuronidation.Inthehamster,theperillicacidderivativesweremore
important (especially the conjugate of perillic acid) than the 8,9-epoxide-deriva-
tives,whileinguinea-pigsandinrabbitstheywereequallyimportant.Indogsand
humans,however,the8,9-epoxidederivativesweremoreimportantthantheperil-
licacidderivativesinthestudybyKodamaetal.(1976),whileotherinvestigators
(Crowelletal.,1994;Poonetal.,1996;Vigushinetal.,1998)identifedperillicacid
anditsderivativesasmajormetabolitesinhumans,nextto8,9-and1,2-epoxide
derivatives.
6
5
1
4
2
3
CH
3
7
8
C H
2
9
CH
3
10
CH
3
C H
2
CH
3
OH
CH
3
C H
2
CH
2
OH
CH
3
C H
2
CH
3
O H
O H
CH
3
HOH
2
C CH
3
O H
CH
3
HOH
2
C CH
3
O H
OH
COOH
C H
2
CH
3
COOH
C H
2
CH
3
O H
CH
3
C H
2
COOH
COOH
HOH
2
C CH
3
O H
COOH
C H
2
CH
3
glucuronide
glucuronide
glucuronide
glucuronide
d-Limonene
M-I
M-XIV
M-V
M-VIII
M-IX
M-II
M-XVI
M-X
M-XI
CONHCH
2
COOH
C H
2
CH
3
M-XII
M-XIII
M-III
M-VII
M-XV
glucuronide
M-VI
M-IV
Figure 2. Metabolism of d-limonene
M-I limonene-10-ol M-IX glucuronicacidofM-III
M-II limonene-8,9-diol M-X limonene-6-ol
M-III perillicacid M-XI p-menth-1-ene-6,8,9-triol
M-IV perillicacid-8,9-diol M-XII dihydroperillicacid
M-V glucuronicacidofM-I M-XIII limonene-1,2-diol
M-VI glucuronicacidofM-II M-XIV p-mentha-1,8-diene-10-carboxylic
acid
M-VII 2-hydroxy-p-menth-8- M-XV glucuronicacidofM-XII
en-7-oicacid
M-VIII perillylglycine M-XVI glucuronicacidofM-XIV
ALIPHATIC AND ALICYCLIC HYDROCARBONS 255
L1
(iii) Bicyclic hydrocarbons
Intheurineofsawmillworkersexposedtoanatmospherecontaininga-pinene
(No.1329)at0.0310.210mg/l,b-pinene(No.1330)at0.0020.017mg/l,andd-
3-carene(No.1342)at0.0060.090mg/lfor3days,cisandtrans-verbenolwere
identifedasmetabolites.Theywereexcretedasconjugates,probablywithgluc-
uronicacid.Theauthorssuggestedthatthesemetaboliteswereformedbyhydrox-
ylation of a-pinene (Eriksson & Levin, 1990). Analysis of urinary metabolites
eliminated by human volunteers within 4h after exposure to a-pinene at 0.010
0.450mg/l

for 2h revealed that a-pinene is indeed eliminated as cis- and trans-

verbenol, at a ratio of 1:10, within 20h after exposure (Levin et al., 1992). In a
moreextensivemetabolicstudy,urinewascollectedfromsawmillworkersatthe
endofan89hworkshiftorfromchamber-exposedindividuals.Afterhydrolysis
ofglucuronicacidconjugates,cis-andtrans-verbenolwereidentifedintheurine,
togetherwithtwodiols,cis-andtrans-4-hydroxymyrtenol,formedbymethylgroup
hydroxylation of cis- and trans-verbenol. Trans-4-hydroxymyrtenal was also
detected(Eriksson&Levin,1996).
In the urine of rabbits given (+)-a-pinene, (-)-a-pinene, ()-a-pinene, (-)-b-
pinene,ord-3-careneorally viagavage,bicyclicterpenehydrocarbonmetabolites
wererecoveredas(glucuronicacid)conjugatesorasfurtheroxidizedmetabolites,
notably carboxylic acids. The principal neutral metabolite formed by oxidation at
the C4 position in the alicyclic ring of each of the three stereochemical forms of
a-pinenewastrans-verbenol.Asaminorpathway,allylicoxidationoftheexocyclic
methylgrouptoyieldmyrtenolwasobservedforallthreea-pinenestereoisomers,
withalsomyrtenicacidasminormetabolite(Ishidaetal.,1981).Thepresenceof
anexocyclicalkenefunctionin(-)-b-pineneprovidedadditionalmetabolicoptions,
andfourneutralandoneacidicmetaboliteswereidentifed.Allylicoxidationofthe
C2positionyields(+)-trans-pinocarveol,whileepoxidationoftheexocyclicalkene
followed by hydration or rearrangement yields (-)-trans-10-pinanol and (-)-1-p-
menthene-7,8-diol, respectively. Ring cleavage yields (-)-a-terpineol. These
metabolites comprised 11, 39, 30 or 5% of the total urinary neutral metabolite
fraction, respectively. The acidic metabolite identifed was identical to the one
identifedforthea-pinenes(Ishidaetal.,1981).Themetabolismofa- andb-pinene
isdepictedinFigure3.Astudywithratlivermicrosomesinvitrodemonstratedthe
involvementofCYPenzymesinthemetabolismofa-pinene.Metabolitespresent
were b-pinene and limonene together with smaller amounts of trans-verbenol,
myrtenol,verbenone,andpineneoxide(White&Agosin,1980).a-Pinene,aswell
asanotherbicyclichydrocarbon,i.e.cadinene,havebeenshowntoinduceCYP
enzymes,especiallythosefromtheCYP2Bsubfamily,andtoalesserextentalso
CYP3A2 (cadinene) and CYP4A2 (a-pinene) (Austin et al., 1988; Hiroi et al.,
1995).
d-3-Careneundergoesstereoselectivehydroxylationatthegem-methylgroup
(yielding3-caren-9-ol),followedbycarboxylation,allylicoxidationoftheC10methyl
group followed by carboxylation or, as the main route, allylic ring opening and
hydroxylation at a secondary carbon atom, yielding (-)-m-mentha-4,6-dien-8-ol
(72% of the total urinary neutral metabolite fraction) and m-cymen-8-ol (Ishida
etal.,1981).
256 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
Inrabbits,b-caryophyllene(No.1324)undergoesepoxidationoftheendocylic
5,6-double bond to yield a stable epoxide metabolite and hydroxylation at the
gem-dimethyl group. Epoxidation of the exocyclic 2,12-double bond, ultimately
resultingin2,12-diolformation,wasalsoreported(Asakawaetal.,1981,1986).
In summary
Onthebasisoftheavailabledata,itisanticipatedthatallthealiphaticandali-
cyclichydrocarbonsinthisgroupwillparticipateinsimilarpathwaysofmetabolic
detoxifcation in mammals, including humans. After absorption, these hydrocar-
bonsareoxidizedtopolaroxygenatedmetabolitesviaCYPenzymesandalcohol
andaldehydedehydrogenases.Thealiphaticandalicyclicsubstancesareoxidized
OH
OH
OH
OH
O H
OH COOH a-Pinene
cis/trans-verbenol cis/trans-4-hydroxymyrtenol 4-hydroxymyrtenal
myrtenol
myrtenic acid
OH
O
OH
OH
OH
OH
b-Pinene trans-pinocarveol
a-terpineol
trans-10-pinanol
p-menthen-7,8-diol
Figure 3. Metabolism of a-pinene and b-pinene
ALIPHATIC AND ALICYCLIC HYDROCARBONS 257
L1
either by side-chain oxidation or by epoxidation of an exocyclic or endocyclic
double bond.Alkyl oxidation initially yields hydroxylated metabolites that may be
excreted in conjugated form or undergo further oxidation, yielding more polar
metabolitesthatarealsoexcretedinconjugatedformintheurine.Ifadoublebond
is present, epoxide metabolites may form and these metabolites are detoxifed
eitherbyhydrolysistoyielddiols,orbyconjugationwithglutathione.
2.3.2 Toxicological studies
Alltoxicologicalstudieswithd-limonene(No.1326)describedinthefollowing
sections(a)to(e)havebeenevaluatedpreviouslybytheCommitteeandformed
the basis for setting theADI not specifed for d-limonene (Annex 1, references
101 and 107). Since d-limonene is now being evaluated as part of the group of
aliphaticandalicyclichydrocarbons,thestudiesarereportedagaininthismono-
graph, in somewhat more detail, in order to have present all toxicological in-
formationonthisgroupoffavouringagents.
(a) Acute toxicity
Oral median lethal dose (LD
50
) values have been reported for 16 of the 20
substances in this group, three of them being tested in both mice and rats, and
theother13onlyinrats(seeTable3).Inmice,oralLD
50
valueswereall>2000mg/
kgbw(Hoffmann-LaRoche,1967;Pellmont,1973;Tsujietal.,1975a).Inrats,oral
LD
50
valuesrangedfrom1590to>8000mg/kgbw(Brownlee,1940;Wong&Hart,
1971; Keating, 1972; Moreno, 1972a, 1972b, 1972c, 1972d, 1973a, 1973b;
Pellmont, 1973; Moreno, 1974a, 1974b; Levenstein, 1975; Moreno, 1975; Tsuji
etal.,1975a;Moreno,1976a,1976b,1980).TheseLD
50
valuesindicatethatthe
acuteoraltoxicityofaliphaticandalicyclichydrocarbonsislow.
(b) Short-term studies of toxicity
Short-term studies of toxicity were available for four of the 20 substances in
thisgroup(Tsujietal.,1975a,1975b;Kanervaetal.,1987;Shapiro,1988;Webb
etal.,1989;NationalToxicologyProgram,1990,Webbetal.,1990;NationalToxi-
cology Program, 2004a, 2004b).The results of these studies are summarized in
Table4(exceptforthesubstanced-limonene,forwhichalreadyanADInotspeci-
fedwasestablished;see1.1)anddescribedbelow.
(i) d-Limonene (No. 1326)
Mice
GroupsoffvemaleandfvefemaleB6C3F
1
miceweregivend-limoneneata
doseof0,413,825,1650,3300,or6600mg/kgbwperdayincornoilbygavage
for12days,overa16-dayperiod.Theanimalswereobservedtwiceperdayand
weighedonceperweek.Necropsieswereperformedonallanimals.Histopathol-
ogywasperformedonthesurvivorsinthegroupsreceivingthehighestdose.The
studycompliedwithgoodlaboratorypractice(GLP).
258 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
Allexceptoneanimalat3300or6600mg/kgbwperdaydiedwithin3daysof
studyinitiation.At1650mg/kgbwperday,twoanimalsdied(oneowingtogavage
error). No treatment-related clinical signs were observed in mice that survived
dosesof1650mg/kgbwperdayorlower,norweretreatment-relatedhistopatho-
logicallesionsobserved(NationalToxicologyProgram,1990).
Groups of 10 male and 10 female B6C3F
1
mice were given d-limonene at a
doseof0,125,250,500,1000or2000mg/kgbwperdayincornoilbygavage,
5 days per week for 13 weeks. The animals were observed twice per day and
weighedonceperweek.Necropsieswereperformedonallanimals.Histological
examinationswereperformedonallcontrolanimalsandonanimalsatthehighest
dose.Tissuesexaminedincludedawholerangeoforgansandtissues.Thestudy
compliedwithGLP.
Table 3. Studies of the acute toxicity of aliphatic and alicyclic hydrocarbons
administered orally
No. Flavouringagent Species Sex LD
50
Reference
(mg/kgbw)
1323 Camphene Rat NR >5000 Moreno(1974a)
1324 b-Caryophyllene Rat M,F >5000 Wong&Hart
(1971)
1326 d-Limonene Mouse M,F 5600(M) Tsujietal.(1975a)
6600(F)
1326 d-Limonene Rat M >5000 Moreno(1972a)
1326 d-Limonene Rat M,F 4400(M) Tsujietal.(1975a)
5200(F)
1327 Myrcene Rat M >5000 Moreno(1972b)
1328 a-Phellandrene Rat M 5700 Moreno(1972c)
1328 a-Phellandrene Rat M,F 1.87ml/kg Brownlee(1940)
(1590
a
)
1330 b-Pinene Rat NR >5000 Moreno(1975)
1331 Terpinolene Rat NR 4.39ml/kg Levenstein(1975)
(3753
b
)
1336 Bisabolene Rat NR >5000 Moreno(1974b)
1336 Bisabolene Mouse M,F >13360 Hoffmann-LaRoche
(1967)
1337 Valencene Rat M >5000 Moreno(1980)
1338 3,7-Dimethyl-1,3, Rat M,F 5000 Moreno(1976a)
6-octatriene
1339 p-Mentha-1,3-diene Rat NR 1680 Moreno(1973a)
1340 p-Mentha-1,4-diene Rat NR 3650 Moreno(1973b)
1341 1,3,5-Undecatriene Mouse M,F >2000 Pellmont(1973)
1341 1,3,5-Undecatriene Rat M,F >8000 Pellmont(1973)
1342 d-3-Carene Rat M 4800 Moreno(1972d)
1346 Cadinene Rat NR >5000 Keating(1972)
1347 Guaiene Rat NR >5000 Moreno(1976b)
F,female;M,male;NR,notreported.
a
Calculatedusingadensityofa-phellandreneof0.85(0.8350.865)g/ml(Lewis,1999).
b
Calculatedusingadensityofterpinoleneof0.855g/ml(Lewis,1999).
ALIPHATIC AND ALICYCLIC HYDROCARBONS 259
L1
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260 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
Onemaleandtwofemalesat2000mg/kgbwperday,aswellasonefemale
at500mg/kgbwperdaydiedbeforetheendofthestudy.Severalotheranimals
diedasaresultofgavageerror.At1000and2000mg/kgbwperday,malemice
gainedlessweightthanthecontrolanimals,withfnalbodyweightsbeing8990%
ofthoseofthecontrols.Clinicalsignsofroughhaircoatsanddecreasedactivity
were observed for animals at the two highest doses. An alveolar cell adenoma
wasreportedinthelungofonefemaleat2000mg/kgbwperday(NationalToxicol-
ogyProgram,1990).
Rats
Groups of fve male and fve female F344/N rats were given d-limonene at a
doseof0,413,825,1650,3300,or6600mg/kgbwperdayincornoilbygavage
for12days,overa16-dayperiod.Theanimalswereobservedtwiceperdayand
weighedonceperweek.Necropsieswereperformedonallanimals.Histopathol-
ogywasperformedonthesurvivorsinthegroupreceivingthehighestdose.The
studycompliedwithGLP.
All except two animals at 3300 or 6600mg/kg bw per day died within 2 days
ofstudyinitiation.At1650mg/kgbwperday,theanimalsgainedlessweightthan
thecontrolanimals,withfnalbodyweightsbeing9092%ofthoseofthecontrols.
No treatment-related clinical signs or histopathological lesions were observed in
rats receiving doses of 1650mg/kg bw per day or lower (National Toxicology
Program,1990).
Groupsof910maleand910femaleSpragueDawleyJCLratsweregiven
d-limonene (in 1%Tween 80) at oral doses of 0, 277, 554, 1385, or 2770mg/kg
bwperdayfor1month.
Final body weights and body-weight gains were reduced in all treated males
(in a dose-dependent manner), and in females at 1385mg/kg bw per day. Food
consumptionwasalsoreducedintreatedmales.Bloodandurineanalysisrevealed
increases in haemoglobin, erythrocyte volume fraction, erythrocyte counts and
total protein, and decreases in leukocyte count, alanine aminotransferase, total
cholesterol, bilirubin and blood urea nitrogen. Changes were observed in both
sexes,buttoagreaterextentinmalesthaninfemales,andsomechangeswere
alreadyobservedatthelowestdose,277mg/kgbwperday.Absoluteweightsof
thethymus,lungs,heart(malesonly),spleen,andovariesweredecreased,while
absolute weights of the kidneys (males only), liver (females only), and adrenals
wereincreased.Relativeweightsoftheseorgansweresometimesalsoaffected.
Someorganweightchangeswerealreadyobservedatthelowestdosetested.No
signifcanttreatment-relatedchangescomparedwithcontrolswereobservedupon
histologicalexamination,withtheexceptionofgranularcastformationinkidneys
inmales,startingat277mg/kgbwperday(Tsujietal.,1975a).
Groupsof10maleand10femaleF344/Nratsweregivend-limoneneatadose
of0,150,300,600,1200,or2400mg/kgbwperdayincornoilbygavage,5days
per week for 13 weeks.The animals were observed twice per day and weighed
onceperweek.Necropsieswereperformedonallanimals.Histologicalexamina-
tionswereperformedonallanimalsinthevehiclecontrolgroupandatthehighest
dose,andallfemaleratsat1200mg/kgbwperday.Arangeoforgansandtissues
ALIPHATIC AND ALICYCLIC HYDROCARBONS 261
L1
wereexamined.Kidneyswereexaminedforallmalerats.Thestudycompliedwith
GLP.
Ninefemaleandfvemaleratsat2400mg/kgbwperdaydiedwithinthefrst
week of the study. Male rats gained less weight than the control animals at the
threehighestdoses,andfnalmeanbodyweightsat600,1200,or2400mg/kgbw
perdaywere94%,88%,and77%ofthoseofthecontrols,respectively.Theone
surviving female at 2400mg/kg bw per day also gained less weight, with a fnal
bodyweightthatwas11%lowerthanthatofthecontrols.Roughhaircoats,leth-
argy, and excessive lacrimation were observed for animals at the two highest
doses.Nephropathywithadose-dependentincreaseinseveritywasnotedinall
treatedmalerats.Thenephropathywascharacterizedbydegenerationofepithe-
liumintheconvolutedtubules,granularcastswithintubularlumens,andregenera-
tionofthetubularepithelium.Hyalinedropletswereobservedintheepitheliumof
theproximalconvolutedtubulesinallgroupsofmalerats,includingcontrols.Re-
evaluationbytwopathologistsconfrmedthattherewasnoincreaseinthenumber
of these droplets in treated males compared with controls (National Toxicology
Program,1990).
Special studies on kidney toxicity of d-limonene
Inashort-termstudytoinvestigaterenaleffects,groupsoffvemaleF344rats
(aged10weeks)weregivend-limoneneatadoseof0,75,150,or300mg/kgbw
per day in corn oil via gavage, 5 days per week, for a total of 5 or 20 doses.
Observations included daily and fnal body weights, weekly food intake, relative
and absolute liver and kidney weights, and light microscopy of the liver and
kidneys.The kidneys were examined for hyaline droplet formation, granular cast
formation, and nephrosis. Examinations were conducted on animals that were
killed on day 6 (after fve doses) and day 27 (after 20 doses). In addition, two-
dimensional gel electrophoresis evaluation of protein profles was conducted on
samplesfromthekidneysofanimalsinthecontrolgroupandinthegroupreceiving
theintermediatedosekilledonday6.
Noindicationsoftoxicitywereseenupondailyin-lifeobservationsandgross
necropsy, and weight gain and food consumption were comparable to those of
controls.Ondays6and27ofthestudy,increasesinrelativeweightsoftheliver
(notdose-dependent)andthekidney(dose-dependent)wereobservedinalltreated
rats, reaching statistical signifcance only at the highest dose. Light microscopy
revealed no effects of d-limonene on the liver, but the kidney showed a dose-
dependentexacerbationoftheminimalhyalinedropletformationobservedincon-
trols,ofapproximatelysimilarseverityondays6and27.Onday27,hyalinedroplet
formation was accompanied by granular cast formation (dose-dependent) and
nephrosis.Comparedwithcontrols,ratsattheintermediatedosehadsignifcantly
greater concentrations of a
2u
-globulin in renal cortical tissues (Kanerva et al.,
1987).
Afurtherstudyinvestigatingtherenaleffectsofd-limonenewascarriedoutby
Webbetal.(1989).Inthefrstexperiment,groupsofthreemaleandthreefemale
Fischer 344 rats (aged 12 weeks) received radiolabelled d-limonene in a single
262 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
doseat0or200mg/kgbwincornoilbygavage.Theanimalsweresacrifced24h
afterdosingandkidneyswereremovedandexaminedhistopathologically.Inthe
secondexperiment,groupsofmaleFischer344rats(aged6weeks)receivedd-
limoneneatadoseof0,2,5,10,30,or75mg/kgbwperdayincornoilbygavage,
5daysperweekfor13weeks.Interimnecropsieswereconductedonfveratsper
groupondays8,15,22,and29inthecontrolgroupandthegroupsreceivinga
doseof10(days8and15only)and75mg/kgbwperday.Attheendofthe90-day
study,10ratsateachdoseweresacrifced.Atnecropsy,liverandkidneyweights
were recorded, and a range of tissues were processed for histopathology. On a
dailybasis,ratswereobservedforsignsoftoxicityandbodyweightswererecorded.
Foodconsumptionwasrecordedweekly.
Inthefrstexperiment,treatmentresultedinanincreaseintheincidenceand
severityofhyalinedropletsinthekidneysofmalesonly.Thiswasassociatedwith
an increase in a
2u
-globulin and a greater accumulation of radiolabel in the renal
cortex.Femaleshadnohyalinedropletsora
2u
-globulinaccumulation.
Inthesecondexperiment,relativeweightsoftheliverandkidneywereincreased
inadose-dependentmannerat30and75mg/kgbwperday,withstatisticalsig-
nifcance reached only at the highest dose. No histopathological changes were
noted in the livers of treated rats, while examination of the kidneys revealed
changes characterized as hyaline droplet formation, granular casts and multiple
cortical changes, all of which were classifed as chronic nephrosis. Exacerba-
tion of hyaline droplet formation and chronic nephrosis were time- and dose-
dependent,andalreadyobservedattheearliestnecropsyonday8oftreatment
(Webbetal.,1989).
After evaluation of a study of carcinogenicity with d-limonene in rats (see
section (c) below), a supplemental short-term study was initiated to investigate
effectsontheratkidney.Groupsof12maleand12femaleF344/Nrats(aged18
weeks) were given d-limonene at a dose of 0, 75, 150, 300, 600, or 1200mg/kg
bwperdayincornoilbygavagefor14days,overa21-dayperiod.Theanimals
wereobservedtwiceperdayandweighedonceperweek.Microscopicexamina-
tion of kidney sections from these rats indicated a treatment-related increase in
intracytoplasmicgranulesintheproximalconvolutedtubulesofdosedmalerats,
butnotoffemalerats.Thegranuleswereshowntocontaina
2u
-globulinbyimmu-
nohistochemical staining for protein. a
2u
-Globulin was shown to be increased in
kidney homogenates from dosed male rats by enzyme-linked immunosorbent
assay(ELISA)(NationalToxicologyProgram,1990).
Dogs
Groupsofthreemaleandthreefemalebeagledogsweregivend-limoneneat
adoseof0,0.4,1.2,or3.6ml/kgbwperday(equivalenttoapproximately0,340,
1000, or 3000mg/kg bw per day, respectively) by oral administration for 6
months.
Frequentvomitingandnauseawerereportedatthetwohigherdoses.Atthe
endoftreatment,femalesatthetwohigherdosesandmalesatthehighestdose
had lost weight. Food consumption was not affected. Several changes were
ALIPHATIC AND ALICYCLIC HYDROCARBONS 263
L1
observeduponanalysisofblood(after1,3and6monthsoftreatment)andurine
(after1and6months),anddeterminationofabsoluteandrelativeorganweights,
with some of them already present at the lowest dose tested. However, as most
changeswerewithoutadoseresponserelationship,someweremorepronounced
inonesexthantheother,statisticswerenotperformed,andinsomecasesstarting
valuesalreadydifferedconsiderablybetweengroups,itisdiffculttointerpretthe
fndings. The authors only reported decreased total cholesterol and blood sugar
concentrationsinbothmalesandfemalesatthehighestdose.Histologicalexami-
nations revealed an increase in protein cast formation in the renal tubules of
femalesandmalesatdosesof340and1000mg/kgbwperday,respectively,but
nootherremarkablefndingsinotherorgansexamined(Tsujietal.,1975b).
The fndings in the kidney reported in the previous study were not confrmed
in a later study, in which groups of fve male and fve female beagle dogs (aged
1015 months) were given d-limonene at a dose of 0 (tap water only), 0.12, or
1.2ml/kg bw per day (equivalent to approximately 0, 100, or 1000mg/kg bw per
day) by gavage for 6 months.The highest dose was determined in a pilot study
to be close to the maximum tolerated dose for emesis.To minimize emesis, the
total daily doses were divided into two equal amounts and administered in the
morningandtheafternoon.
Treatmentwithd-limonenewasassociatedwithexcretionofsoftfaeces.Diar-
rhoeaandemesisoccurredperiodically,withthesamefrequencyinanimalsatthe
lowest and highest doses. Food consumption and fnal body weights were unaf-
fected by treatment.Absolute and relative weights of the kidney were increased
withadose-dependenttrend,reachingstatisticalsignifcanceatthehighestdose
only in females (absolute and relative weight) and males (relative weight only).
Haematologyandclinicalchemistrydeterminationsat1,3and6months,andurine
analysisat6monthsrevealednotreatment-relateddifferencesotherthana35%
increaseinserumcholesterolandatwofoldincreaseinserumalkalinephospha-
tase activities in animals at the highest dose. Neither the gross post-mortem
examinations nor the histological examinations revealed any evidence of treat-
ment-related alterations in a whole range of tissues and organs. Specifcally, no
hyalinedropletnephropathywasobserved(Webbetal.,1990).
(ii) Myrcene (No. 1327)
Mice
Ina13-weekstudyoftoxicity,groupsof20maleand20femaleB6C3F
1
mice
(10 of each sex for the core study, 10 of each sex for the clinical chemistry part
ofthestudy)weregivenmyrceneatadoseof0,250,500,1000,2000,or4000mg/
kg bw per day by gavage, on weekdays only.Animals were observed twice per
day for moribundity and death, and clinical observations were recorded weekly.
Weightmeasurementsweretakeninitiallyandthenweeklyuntilterminationofthe
study.Onday23,bloodwasdrawnfromtheclinicalchemistrystudymiceforclini-
calchemistryanalysis,andatweek13fromallsurvivinganimalsinthecorestudy
forhaematologyandclinicalchemistryanalysis.Attermination,allmiceinthecore
study were necropsied and weights of heart, right kidney, liver, lung, right testis,
264 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
andthymuswererecorded.Afullhistopathologicalexaminationwasconductedon
animalsinthecontrolgroupandinthegroupsreceivingadoseof1000,2000,or
4000mg/kg bw per day. In addition, the liver, forestomach, and kidneys were
examinedhistopathologicallytoano-effectlevel.
Allanimalsat4000mg/kgbwperdaydiedorwerekilledinamoribundcondi-
tion within the frst 3 days of treatment, while nine out of ten male and eight out
of ten female mice at 2000mg/kg bw per day died or were killed in a moribund
condition before week 5 of treatment. Clinical signs of toxicity in these animals
included lethargy, abnormal breathing, or thin appearance. All other animals
appearednormalandsurviveduntilstudytermination.Comparedwiththecontrols,
thesurvivinganimalsinthegroupreceivingadoseof2000mg/kgbwperdayhad
decreased body weights. In all other treatment groups, body weights were not
affected.Effectsonorganweightsweremainlyobservedinthesurvivinganimals
at2000mg/kgbwperday:intheonemale,therelativeweightsofliver,lungand
kidneywereincreased,asweretheabsoluteandrelativeweightsofthethymus;
the two females had increased absolute and relative weights of the liver and
kidney.Intheothertreatmentgroups,organweightchangesdidnotexceed20%
inmales,whileinfemalestherelativekidneyweightandtheabsoluteandrelative
liver weights were increased by more than 20% at 1000mg/kg bw per day only.
Haematologyanalysisrevealedverysmalldecreases(1.56.2%)inerythrocytes,
haemoglobin,anderythrocytevolumefractionandverysmallincreases(<3%)in
meancorpuscularvolumeandmeancorpuscularhaemoglobinat1000mg/kgbw
per day in both sexes. In the few surviving animals at 2000mg/kg bw per day,
these changes were somewhat more pronounced (5.121%). Treatment also
affected the number of reticulocytes, platelets and leukocytes, but the changes
were not consistent between doses and/or between sexes. Clinical chemistry
analysisrevealeddecreasedbloodureanitrogenconcentrationsat1000mg/kgbw
per day and decreased alanine aminotransferase and sorbitol dehydrogenase
activityat500and1000mg/kgbwperdayinbothsexes.Intheanimalsthatdied
beforeterminationofthestudy,minimalcentrilobularhypertrophyandminimalto
markednecrosisoftheliver,irritationofthenoseandforestomach,atrophyand/or
necrosisofthebonemarrow,spleenandthymus,andnecrosisoftherenaltubules
were observed. Minimal centrilobular hypertrophy of the liver was also observed
in females at 1000 and 2000mg/kg bw per day (8 out of 10, and 2 survivors,
respectively),andinmalesat250,500,1000and2000mg/kgbwperday(1out
of10,4outof10,10outof10and1survivor,respectively).Minimaltomoderate
forestomach epithelial hyperplasia was found in females at 500 and 1000mg/kg
bwperday(2outof10inbothcases)andin2outof10malesat1000mg/kgbw
perday.Somefemalesalsoshowedirritationofthenoseat1000and2000mg/kg
bw per day (2 out of 10 and 1 out of 2 survivors, respectively).Thymus atrophy
wasobservedinsurvivinganimalsat2000mg/kgbwperday.Minimalnephropathy
andadrenalcortexhyperplasiawereobservedinanimalsofalltreatmentgroups,
including controls. In males, minimal renal cytoplasmic vacuolation was also
observed,withtheincidencedecreasingupontreatment(10outof10,8outof10,
and 1 out of 10 at 0, 250, and 500mg/kg bw per day, respectively). The NOEL
was<250mg/kgbwperdayformalemice,onthebasisofliverhypertrophy.The
ALIPHATIC AND ALICYCLIC HYDROCARBONS 265
L1
NOEL for female mice was 250mg/kg bw per day, on the basis of forestomach
lesions(NationalToxicologyProgram,2004a;draftresults).
Rats
Ina13-weekstudyoftoxicity,groupsof20maleand20femaleF344rats(10
ofeachsexforthecorestudy,10ofeachsexfortheclinicalpathologypartofthe
study)weregivenmyrceneatadoseof0,250,500,1000,2000,or4000mg/kg
bwperdaybygavage,onweekdaysonly.Animalswereobservedtwicedailyfor
moribundity and death, and clinical observations were recorded weekly. Weight
measurementsweretakeninitiallyandthenweeklyuntilterminationofthestudy.
On day 23, blood was drawn from the rats in the clinical pathology part of the
study,andatweek13fromallsurvivinganimalsinthecorestudyforhaematology
andclinicalchemistryanalysis.Attermination,allratsinthecorestudywerenec-
ropsiedandweightsofheart,rightkidney,liver,lung,righttestis,andthymuswere
recorded. A full histopathological examination was conducted on animals in the
controlgroupandinthegroupsreceivingadoseof2000,and4000mg/kgbwper
day.Inaddition,thenose,lymphnodes,liver,Harderiangland,andkidneyswere
histopathologicallyexaminedtoano-effectlevel.
Allanimalsat4000mg/kgbwperdaydiedorwerekilledinamoribundcondi-
tion within the frst 12 days of treatment. Treatment-related mortality was also
observedat2000mg/kgbwperday,withtwomaleandfourfemaleratsdyingor
killed a moribund condition. Clinical signs of toxicity in these animals included
lethargy, ruffed fur, abnormal breathing, or thin appearance. Three accidental
deathsoccurredatlowerdoses(onemaleandonefemaleratat1000mg/kgbw
per day, and one male rat at 500mg/kg bw per day).A decrease in mean body-
weightgainofmorethan10%wasobservedinmales(at500onlyduringthe
lastweekoftreatment1000,and2000mg/kgbwperday),butnotinfemales.
Absoluteandrelativekidneyweightswereincreasedinadose-dependentmanner
bymorethan20%inmaleandfemaleratsatalldoses.Inmales,dose-dependent
changes of more than 20% were also observed in relative weight of the liver
(increased at 1000 and 2000mg/kg bw per day), relative weight of the testis
(increased at 2000mg/kg bw per day), and absolute and relative weights of the
thymus (decreased at 1000 absolute weight only and 2000mg/kg bw per
day). In females, absolute and relative weights of the liver were increased in a
dose-dependent manner by more than 20% at 500 (relative weight only), 1000,
and2000mg/kgbwperday.Haematologyanalysisrevealeddecreasesof2535%
inleukocytesandlymphocytesatday23,butnotatday93,inmalesandfemales
at2000mg/kgbwperday.Increasesofmorethan30%(notdose-dependent)in
reticulocytes were reported in males and females at 500, 1000, and 2000mg/kg
bw per day.Treatment affected clinical chemistry parameters, but most changes
werenotconsistentbetweenday23andweek13,betweendoses,and/orbetween
sexes. At week 13, both males and females at 2000mg/kg bw per day had
decreasedconcentrationsofcreatinine,aswellasslightlydecreasedbloodurea
nitrogenconcentrations.Males,butnotfemales,alsohaddecreasedalanineami-
notransferaseandsorbitoldehydrogenaseactivitiesat500,1000and2000mg/kg
bwperday.Intheanimalsthatdiedbeforeterminationofthestudy,mildtomoder-
ateirritationofthenoseandforestomach,minimaltomarkedatrophyofthespleen,
266 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
necrosis of the thymus, degeneration of the renal tubules (except in males at
4000mg/kgbwperday),andporphyrinpigmentationoftheHarderianglandwere
observed.Inthesurvivinganimals,minimalforestomachepithelialhyperplasiawas
alsofoundinonefemaleat2000mg/kgbwperday,andminimaltomarkednose
irritationinmalesat500,1000and2000mg/kgbwperday(oneoutofnine,fve
out of nine and eight out of eight, respectively) and in females at 1000 and
2000mg/kg bw per day (nine out of nine and six out of six, respectively). At
2000mg/kgbwperday,minimaltomildatrophyofthespleenwasobservedinall
survivinganimals,andinoneoutoftwofemalesminimalnecrosisofthethymus
wasalsoobserved.Nephropathywasobservedinanimalsinalltreatmentgroups,
including controls, but with a somewhat higher incidence and severity in males
than in females. All surviving males in all treatment groups, including controls,
showedminimaltomoderateaccumulationofhyalinedropletsintherenaltubules.
Allsurvivinganimalsinalltreatmentgroups,butnotincontrols,showeddegenera-
tionoftherenaltubulesofincreasingseverity,whichwasaccompaniedbyminimal
mineralizationinsomefemales.MinimalporphyrinpigmentationoftheHarderian
glandwasobservedinanimalsofalltreatmentgroups,includingcontrols,witha
dose-dependent increase in incidence in males but not in females. On the basis
ofthekidneyfndings,whichwereobservedatalldoses,theNOELwas<250mg/
kgbwperday(NationalToxicologyProgram,2004b;draftresults).
(iii) b-Pinene (No. 1330) and 1,3,5-undecatriene (No. 1341)
Rats
In a 14-day feeding study, groups of fve male and fve female young adult
Sprague-DawleyratswerefeddietscalculatedtoprovideGalbelicaatadoseof
10mg/kgbwperday.Galbelicaisaformulationcomposedof80%b-pineneand
20% 1,3,5-undecatriene. It was administered in a vehicle called Pinene Beta
Supra,inaratioof1(Galbelica):4(vehicle).Twoadditionalgroupsservedascon-
trols and were maintained on a basal diet, one group with and the other without
thePineneBetaSupravehicle.Dailyobservationsforgrosssignsoftoxicityand
mortality were made. Body weight and food consumption were recorded weekly.
Onday14,allanimalsweresacrifcedandsubjectedtogrossnecropsy.Liverand
kidneyweightswererecorded.Histopathologywasperformedonlywhenfndings
were remarkable upon gross necropsy. The study complied with GLP, and was
certifedforqualityassurance.
Allratssurvivedandnoclinicalsignsoftoxicitywereobserved.Foodconsump-
tion, body weights, and liver and kidney weights were not signifcantly different
betweengroups.Atgrossnecropsy,allfndingswerereportedtobeunremarkable.
The NOEL for Galbelica was 10mg/kg bw per day (equivalent to 8 and 2mg/kg
bw per day for b-pinene and 1,3,5-undecatriene respectively), the highest dose
tested(Shapiro,1988).
(c) Long-term studies of toxicity and carcinogenicity
Long-term studies of toxicity and carcinogenicity were only available for d-
limonene (No. 1326) (National Toxicology Program, 1990). The results of these
ALIPHATIC AND ALICYCLIC HYDROCARBONS 267
L1
studiesaredescribedbelow(theywerenotsummarizedintabularformbecause
an ADI not specifed for d-limonene was established by the Committee at its
forty-frstmeeting(Annex1,reference107)).
Mice
Inastudyofcarcinogenicity,whichcompliedwithGLP,groupsof50maleand
50femaleB6C3F
1
miceweregivend-limoneneatadoseof0,250,or500mg/kg
bwperdayformales,or0,500,or1000mg/kgbwperdayforfemales,incornoil
bygavage,5daysperweekfor103weeks.Theanimalswereobservedtwiceper
day and weighed once per week for 12 weeks and once per month thereafter.
Necropsies were performed on all animals. Histopathological examinations were
performed on all animals in the control group and at the higher dose, and on
animalsatthelowerdosethatdiedbeforetheendofthestudy.Inaddition,histo-
pathologicalexaminationswereperformedonallgrosslyvisiblelesionsandtarget
organsortissuesatalldoses.Tissuesexaminedincludedarangeoforgansand
tissues,includingtheliverinfemalesatthelowerdose.
No treatment-related clinical signs were observed. Treatment did not affect
survivalinmalesandfemalesatthehigherdose,orinfemalesatthelowerdose.
In males at the lower dose, survival was signifcantly lower than that of controls
(24outof50comparedwith33outof50,respectively).Noeffectsonbodyweight
were observed in male mice in both treatment groups or in female mice at the
lowerdose.Femalesatthehigherdosehadmeanbodyweightsthatwere515%
lowerthanthoseofcontrolsfromweek28tostudytermination,resultingina25%
lowermeanbody-weightgainoverthewholetreatmentperiod.Theonlyfndings
inmalemiceweresignifcantlyincreasedincidencesofmultinucleatedhepatocytes
andcentrilobularlivercytomegalyatthehigherdose.However,theincidencesof
hepatocellularadenomasorcarcinomas(combined)inthisgroupwerenotdifferent
fromthoseincontrols.Infact,notreatment-relatedincreasesinneoplasmswere
observedinthisstudy.Asignifcantdecreasewasobservedinincidenceofneo-
plasms of the anterior pituitary gland in females at the higher dose. Overall, the
National Toxicology Program concluded that under the conditions of this 2-year
gavage study, there was no evidence of carcinogenic activity of d-limonene for
maleB6C3F
1
micethatreceived250or500mg/kgorforfemalemicethatreceived
500or1000mg/kg (NationalToxicologyProgram,1990).
Rats
Inastudyofcarcinogenicity,whichcompliedwithGLP,groupsof50maleand
50femaleF344/Nratsweregivend-limoneneatadoseof0,75,or150mg/kgbw
perdayformales,and0,300,or600mg/kgbwperdayforfemales,incornoilby
gavage, 5 days per week for 103 weeks. The animals were observed twice per
day and weighed once per week for 12 weeks and once per month thereafter.
Necropsies were performed on all animals. Histopathological examinations were
performedonallanimalsinthecontrolgroupandatthehigherdose,onanimals
atthelowerdoseanimalsthatdiedbeforetheendofthestudy,andonallfemales
atthelowerdose.Inaddition,histopathologicalexaminationswereperformedon
allgrosslyvisiblelesionsandtargetorgansortissuesatalldoses.Tissuesexam-
268 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
inedincludedarangeoforgansandtissues,includingtheadrenalglands,kidney,
liver,spleen,andtestisinmaleratsatthelowerdose.
No treatment-related clinical signs were observed. Treatment did not affect
survivalinmalesandfemalesatthelowerdose.Atthehigherdose,thesurvival
ofmaleswassignifcantlyhigherthanthatofcontrols(40outof50comparedwith
29outof50,respectively),whileatthehigherdose,thesurvivaloffemaleswas
signifcantlylowerthanthatofcontrols(24outof50comparedwith42outof50,
respectively).Animalsatthehigherdosehadmeanbodyweightsthatwere47%
lowerthanthoseofcontrols(inmalesfromweek2tostudytermination,infemales
from week 28 to study termination), but this did not result in lower mean body-
weightgainsoverthewholetreatmentperiod.Histopathologyshowedthekidney
to be the primary target organ in male rats. Spontaneous nephropathy occurred
in all males, including controls, but in treated males the incidence and severity
wereincreasedinadose-dependentmanner.Intreatedmales,theincidencesof
mineralizationoftherenalpapillaandfocalhyperplasiaofthetransitionalepithe-
lium overlying the papilla were also increased in a dose-dependent manner, as
wastheincidenceoftubularcellhyperplasia.Tubularcelladenomasandtubular
celladenomasoradenocarcinomas(combined)occurredwithsignifcantpositive
trends in treated males. These neoplasms were not observed in controls or in
treated females. Some statistically signifcant effects were found in the uterus of
female rats and in the testis, haematopoietic system, and skin of male rats.
However,thesefndingswerenotconsideredtobetreatment-related,owingtothe
absenceofadoseresponserelationship,lowincidenceincontrolscomparedwith
historicalcontrols,lowsurvivalofcontrols,and/orbecauseincidenceswerewithin
therangeforhistoricalcontrols.Thefndingofincreasedincidencesofcataracts
inmalesandfemalesatthehigherdosewasattributedtothepositionofthecages,
andthereforenotconsideredtobetreatment-related.Overall,theNationalToxicol-
ogy Program concluded that under the conditions of this 2-year gavage study,
there was clear evidence of carcinogenic activity of d-limonene for male F344/N
rats,asshownbyincreasedincidencesoftubularcellhyperplasia,adenomas,and
adenocarcinomas of the kidney. There was no evidence of carcinogenic activity
of d-limonene for female F344/N rats that received 300 or 600mg/kg (National
ToxicologyProgram,1990).
Relevance of d-limonene-induced kidney tumours to
humans
During the frst evaluation of d-limonene by the Committtee at its thirty-ninth
meeting, there was an extensive discussion of the mechanism involved in the
exacerbation of the spontaneously occurring nephropathy in male rats, with the
subsequent occurrence of renal tumours.A number of mechanistic studies were
evaluated(Annex1,reference102),alsotakingintoaccountanextensivereview
onthistopicbytheUnitedStatesEnvironmentalProtectionAgency(Environmental
Protection Agency, 1991). Based on these data, the Committee concluded that
the postulated mechanism behind the male rat-specifc renal tubule cell carcino-
genesis, being a
2u
-globulin-associated nephropathy following a specifc, time-
dependent, sequence of pathological changes, was probably not relevant to
ALIPHATIC AND ALICYCLIC HYDROCARBONS 269
L1
humans,andthattoxicend-pointsassociatedwiththiseffectwerenotanappropri-
atebasisforthederivationofanADIford-limonene(Annex1,reference101).
Afewyearsafterthisevaluation,IARCexaminedthescientifcbasisforpossi-
blespeciesdifferencesinmechanismsbywhich,amongothers,renaltubulecell
tumoursinmaleratsmaybeproduced,andaddressedalsothepredictivevalue
of this type of tumour for the identifcation of carcinogenic hazards to humans
(IARC,1999a).Intheresultingconcensusdocument,asetofcriteriawasformu-
latedwhichmustbemetbeforeitcanbeconcludedthatanagentcauseskidney
tumoursthroughana
2u
-globulin-associatedresponse.Thecriteriaare:
Lackofgenotoxicactivity(agentand/ormetabolite)basedonanoverallevalu-
ationofinvitroandinvivodata;
Maleratspecifcityfornephropathyandrenaltumorigenicity;
Inductionofthecharacteristicsequenceofhistopathologicalchangesinshorter-
termstudies,ofwhichproteindropletaccumulationisobligatory;
Identifcationoftheproteinaccumulatingintubulecellsasa
2u
-globulin;
Reversiblebindingofthechemicalormetabolitetoa
2u
-globulin;
Inductionofsustainedincreasedcellproliferationintherenalcortex;
Similarities in doseresponse relationship of the tumour outcome with the
histopathological end-points (protein droplets, a
2u
-globulin accumulation, cell
proliferation).
Ifaparticularagentmeetsallthesecriteria,accordingtotheconsensusdocu-
ment it can subsequently be concluded that the production of renal cell tumours
in male rats by an a
2u
-globulin-associated response is not predictive of carcino-
genic hazard to humans. a
2u
-Globulin is not present in humans, and although
closely related proteins may be present, differences in ligand-binding of these
proteinscomparedwiththeuniquebindingpropertiesofa
2u
-globulinprecludetheir
involvementinthecharacteristicproteindropletnephropathy(IARC,1999a).
WhenIARCevaluatedthecarcinogenichazardbyd-limonene(IARC,1999b),
d-limonene fulflled all the criteria, hence it was concluded that ...d-limonene
produces renal tubular tumours in male rats by a non-DNA-reactive mechanism,
throughana
2u
-globulin-associatedresponse.Therefore,themechanismbywhich
d-limonene increases the incidence of renal tubular tumours in male rats is not
relevanttohumans.
TheCommitteeatitspresentmeetingthusconfrmeditsearlierpositionthatthe
mechanismbehindtheinductionofrenaltubulecellcarcinogenesisbyd-limonene
inmaleratsisa
2u
-globulin-associatednephropathy,andthatneitherthesetumours,
northeassociatednon-tumorigeniclesionsarerelevanttohumans.
(d) Genotoxicity
Testingforgenotoxicityhasbeenperformedon10ofthe20favouringagents
inthisgroup(Nos1323,1324,13261330,1340,1342,1346).Theresultsofthese
testsaresummarizedinTable5anddescribedbelow.
270 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
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i
n
e
s
e

1
0

1
0
0
0

m
m
o
l
/
l

N
e
g
a
t
i
v
e
f

S
a
s
a
k
i

e
t

a
l
.

e
x
c
h
a
n
g
e

h
a
m
s
t
e
r

(
2
.
0

2
0
4
.
4

m
g
/
m
l
)
i
,
e

(
1
9
8
9
)

o
v
a
r
y

K
-
1

c
e
l
l
s
1
3
2
4

b
-
C
a
r
y
o
p
h
y
l
l
e
n
e

U
n
s
c
h
e
d
u
l
e
d

R
a
t

h
e
p
a
t
o
c
y
t
e
s

U
p

t
o

1
0

0
0
0

m
g
/
m
l

N
e
g
a
t
i
v
e

H
e
c
k

e
t

a
l
.

D
N
A

s
y
n
t
h
e
s
i
s

(
1
9
8
9
)
ALIPHATIC AND ALICYCLIC HYDROCARBONS 271
L1
1
3
2
6

d
-
L
i
m
o
n
e
n
e
j

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

0
.
0
3

3
0

m
m
o
l
/
p
l
a
t
e

N
e
g
a
t
i
v
e
c

F
l
o
r
i
n

e
t

a
l
.

T
A
9
8
,

T
A
1
0
0
,

(
4
.
1

4
0
8
7

m
g
/
p
l
a
t
e
)
k
,
l

(
1
9
8
0
)

T
A
1
5
3
5
,

T
A
1
5
3
7
1
3
2
6

d
-
L
i
m
o
n
e
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

0
.
3

3
3

m
g
/
p
l
a
t
e

-
S
9
;

N
e
g
a
t
i
v
e
c

H
a
w
o
r
t
h

e
t

a
l
.

T
A
9
8
,

T
A
1
0
0
,

1
0

3
3
3
3

m
g
/
p
l
a
t
e

+
S
9

(
1
9
8
3
)
;

T
A
1
5
3
5
,

N
a
t
i
o
n
a
l

T
A
1
5
3
7

T
o
x
i
c
o
l
o
g
y

P
r
o
g
r
a
m

(
1
9
9
0
)
1
3
2
6

d
-
L
i
m
o
n
e
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

1
0

5
0
0

m
g
/
p
l
a
t
e

N
e
g
a
t
i
v
e
c

C
o
n
n
o
r

e
t

a
l
.

T
A
9
8
,

T
A
1
0
0
,

(
1
9
8
5
)

U
T
H
8
4
1
3
,

U
T
H
8
4
1
4
1
3
2
6

d
-
L
i
m
o
n
e
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

U
p

t
o

1
5
0

0
0
0

m
g
/
p
l
a
t
e

N
e
g
a
t
i
v
e
c

H
e
c
k

e
t

a
l
.

T
A
9
8
,

T
A
1
0
0
,

(
1
9
8
9
)

T
A
1
5
3
5
,

T
A
1
5
3
7
,

T
A
1
5
3
8
1
3
2
6

d
-
L
i
m
o
n
e
n
e
j

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

U
p

t
o

0
0
0

m
g
/
p
l
a
t
e

N
e
g
a
t
i
v
e
b

l
l
e
r

e
t

a
l
.

T
A
1
0
2

(
1
9
9
3
)
1
3
2
6

d
-
L
i
m
o
n
e
n
e

F
o
r
w
a
r
d

m
u
t
a
t
i
o
n
,

S
a
c
c
h
a
r
o
m
y
c
e
s

U
p

t
o

2
3
0

m
m
o
l
/
l

N
e
g
a
t
i
v
e
f

F
a
h
r
i
g

(
1
9
8
4
)

(
n
o
n
-
)
r
e
c
i
p
r
o
c
a
l

c
e
r
e
v
i
s
i
a
e

M
P
1

(
3
1

3
3
5

m
g
/
m
l
)
k

r
e
c
o
m
b
i
n
a
t
i
o
n
1
3
2
6

d
-
L
i
m
o
n
e
n
e

F
o
r
w
a
r
d

m
u
t
a
t
i
o
n

M
o
u
s
e

l
y
m
p
h
o
m
a

U
p

t
o

1
0
0

m
g
/
m
l

N
e
g
a
t
i
v
e
c

H
e
c
k

e
t

a
l
.

L
5
1
7
8
Y

T
k
+
/
-

(
1
9
8
9
)

c
e
l
l
s
1
3
2
6

d
-
L
i
m
o
n
e
n
e

F
o
r
w
a
r
d

m
u
t
a
t
i
o
n

M
o
u
s
e

l
y
m
p
h
o
m
a

U
p

t
o

1
0
0

m
g
/
m
l
m

N
e
g
a
t
i
v
e
c

M
y
h
r

e
t

a
l
.

L
5
1
7
8
Y

T
k
+
/
-

(
1
9
9
0
)
;

c
e
l
l
s

N
a
t
i
o
n
a
l

T
o
x
i
c
o
l
o
g
y

P
r
o
g
r
a
m

(
1
9
9
0
)
272 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
T
a
b
l
e

5
.

(
c
o
n
t
d
)
N
o
.

F
l
a
v
o
u
r
i
n
g

a
g
e
n
t

E
n
d
-
p
o
i
n
t

T
e
s
t

o
b
j
e
c
t

D
o
s
e

o
r

c
o
n
c
e
n
t
r
a
t
i
o
n

R
e
s
u
l
t

R
e
f
e
r
e
n
c
e
1
3
2
6

d
-
L
i
m
o
n
e
n
e

S
i
s
t
e
r

c
h
r
o
m
a
t
i
d

C
h
i
n
e
s
e

h
a
m
s
t
e
r

1
0

3
3
3

m
m
o
l
/
l

N
e
g
a
t
i
v
e
f

S
a
s
a
k
i

e
t

a
l
.

e
x
c
h
a
n
g
e

o
v
a
r
y

K
-
1

c
e
l
l
s

(
1
.
4

4
5
.
4

m
g
/
m
l
)
k
,
e

(
1
9
8
9
)
1
3
2
6

d
-
L
i
m
o
n
e
n
e

S
i
s
t
e
r

c
h
r
o
m
a
t
i
d

C
h
i
n
e
s
e

h
a
m
s
t
e
r

1
5

1
6
2

m
g
/
m
l

-
S
9
;

N
e
g
a
t
i
v
e
c

A
n
d
e
r
s
o
n

e
x
c
h
a
n
g
e

o
v
a
r
y

c
e
l
l
s

1
6
.
2

1
6
2

m
g
/
m
l

+
S
9

e
t

a
l
.







(
1
9
9
0
)
;

N
a
t
i
o
n
a
l

T
o
x
i
c
o
l
o
g
y

P
r
o
g
r
a
m

(
1
9
9
0
)
1
3
2
6

d
-
L
i
m
o
n
e
n
e

C
h
r
o
m
o
s
o
m
a
l

C
h
i
n
e
s
e

h
a
m
s
t
e
r

1
0

1
0
0

m
g
/
m
l

-
S
9
;

N
e
g
a
t
i
v
e
c

A
n
d
e
r
s
o
n

a
b
e
r
r
a
t
i
o
n

o
v
a
r
y

c
e
l
l
s

5
0

5
0
0

m
g
/
m
l

+
S
9

e
t

a
l
.







(
1
9
9
0
)
;

N
a
t
i
o
n
a
l

T
o
x
i
c
o
l
o
g
y

P
r
o
g
r
a
m

(
1
9
9
0
)
1
3
2
6

d
-
L
i
m
o
n
e
n
e
j

C
e
l
l

S
y
r
i
a
n

h
a
m
s
t
e
r

0
.
1

1
0
0

m
g
/
m
l

N
e
g
a
t
i
v
e

P
i
e
n
t
a

(
1
9
8
0
)

t
r
a
n
s
f
o
r
m
a
t
i
o
n

e
m
b
r
y
o

c
e
l
l
s
1
3
2
6

d
-
L
i
m
o
n
e
n
e
j

C
e
l
l

S
y
r
i
a
n

h
a
m
s
t
e
r

0
.
1

m
m
o
l
/
l

P
o
s
i
t
i
v
e
n

R
i
v
e
d
a
l

e
t

a
l
.

t
r
a
n
s
f
o
r
m
a
t
i
o
n

e
m
b
r
y
o

c
e
l
l
s

(
1
3
.
6

4
0
8
.
7

m
g
/
m
l
)
k

(
2
0
0
0
)
1
3
2
7

M
y
r
c
e
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

3
3

3
3
3

m
g
/
p
l
a
t
e

-
S
9
o
;

N
e
g
a
t
i
v
e
c

N
a
t
i
o
n
a
l

T
A
9
7
,

T
A
9
8
,

3
3

1
0

0
0
0

m
g
/
p
l
a
t
e

+
S
9
o

T
o
x
i
c
o
l
o
g
y

T
A
1
0
0
,

T
A
1
5
3
5

P
r
o
g
r
a
m

(
2
0
0
4
d
)
1
3
2
7

M
y
r
c
e
n
e

G
e
n
e

m
u
t
a
t
i
o
n

C
h
i
n
e
s
e

h
a
m
s
t
e
r

1
0
0

0
0
0

m
g
/
m
l

N
e
g
a
t
i
v
e
c

K
a
u
d
e
r
e
r

V
7
9

H
p
r
t

c
e
l
l
s

e
t

a
l
.

(
1
9
9
1
)
1
3
2
7

M
y
r
c
e
n
e

S
i
s
t
e
r

c
h
r
o
m
a
t
i
d

H
u
m
a
n

1
0
0

0
0
0

m
g
/
m
l

N
e
g
a
t
i
v
e
c

K
a
u
d
e
r
e
r

e
x
c
h
a
n
g
e

l
y
m
p
h
o
c
y
t
e
s

e
t

a
l
.

(
1
9
9
1
)
1
3
2
7

M
y
r
c
e
n
e

S
i
s
t
e
r

c
h
r
o
m
a
t
i
d

C
h
i
n
e
s
e

h
a
m
s
t
e
r

1
0
0

5
0
0

m
g
/
m
l

-
S
9
;

N
e
g
a
t
i
v
e
c

s
c
h
e
i
s
e
n

e
x
c
h
a
n
g
e

V
7
9

c
e
l
l
s

5
0
0

m
g
/
m
l

+
S
9

e
t

a
l
.

(
1
9
9
1
)
1
3
2
7

M
y
r
c
e
n
e

S
i
s
t
e
r

c
h
r
o
m
a
t
i
d

H
e
p
a
t
i
c

t
u
m
o
u
r

1
0
0

5
0
0

m
g
/
m
l

N
e
g
a
t
i
v
e
p

s
c
h
e
i
s
e
n

e
x
c
h
a
n
g
e

c
e
l
l
s

e
t

a
l
.

(
1
9
9
1
)
ALIPHATIC AND ALICYCLIC HYDROCARBONS 273
L1
1
3
2
7

M
y
r
c
e
n
e

C
h
r
o
m
o
s
o
m
a
l

H
u
m
a
n

1
0
0

0
0
0

m
g
/
m
l

N
e
g
a
t
i
v
e
c

K
a
u
d
e
r
e
r

a
b
e
r
r
a
t
i
o
n

l
y
m
p
h
o
c
y
t
e
s

e
t

a
l
.

(
1
9
9
1
)
1
3
2
8

a
-
P
h
e
l
l
a
n
d
r
e
n
e

S
i
s
t
e
r

c
h
r
o
m
a
t
i
d

C
h
i
n
e
s
e

h
a
m
s
t
e
r

3
3
.
3

0
0
0

m
m
o
l
/
l

N
e
g
a
t
i
v
e
f

S
a
s
a
k
i

e
t

a
l
.

e
x
c
h
a
n
g
e

o
v
a
r
y

K
-
1

c
e
l
l
s

(
4
.
5

1
3
6
.
2

m
g
/
m
l
)
q

(
1
9
8
9
)
1
3
2
9

a
-
P
i
n
e
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

0
.
0
5

1
0
0

m
l
/
p
l
a
t
e

N
e
g
a
t
i
v
e
b

R
o
c
k
w
e
l
l

&

T
A
9
8
,

T
A
1
0
0

(
4
3

8
5

9
2
0

m
g
/
p
l
a
t
e
)
r

R
a
w

(
1
9
7
9
)
1
3
2
9

a
-
P
i
n
e
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

0
.
0
3

3
0

m
m
o
l
/
p
l
a
t
e

N
e
g
a
t
i
v
e
c

F
l
o
r
i
n

e
t

a
l
.

T
A
9
8
,

T
A
1
0
0
,

(
4
.
1

0
8
7

m
g
/
p
l
a
t
e
)
s
,
l

(
1
9
8
0
)

T
A
1
5
3
5
,

T
A
1
5
3
7
1
3
2
9

a
-
P
i
n
e
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

0
.
1

2
5

m
l
/
p
l
a
t
e

N
e
g
a
t
i
v
e
c

J
a
g
a
n
n
a
t
h

T
A
9
8
,

T
A
1
0
0
,

(
8
5
.
9

2
1

4
8
0

m
g
/
p
l
a
t
e
)
r
,
t

(
1
9
8
4
b
)

T
A
1
5
3
5
,

T
A
1
5
3
7
,

T
A
1
5
3
8
1
3
2
9

a
-
P
i
n
e
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

1
0

5
0
0

m
g
/
p
l
a
t
e

N
e
g
a
t
i
v
e
c

C
o
n
n
o
r

e
t

a
l
.

T
A
9
8
,

T
A
1
0
0
,

(
1
9
8
5
)

U
T
H
8
4
1
3
,

U
T
H
8
4
1
4
1
3
2
9

a
-
P
i
n
e
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

U
p

t
o

2
5

0
0
0

m
g
/
p
l
a
t
e

N
e
g
a
t
i
v
e
c

H
e
c
k

e
t

a
l
.

T
A
9
8
,

T
A
1
0
0
,

(
1
9
8
9
)

T
A
1
5
3
5
,

T
A
1
5
3
7
,

T
A
1
5
3
8
1
3
2
9

a
-
P
i
n
e
n
e

U
n
s
c
h
e
d
u
l
e
d

R
a
t

h
e
p
a
t
o
c
y
t
e
s

U
p

t
o

1
0

0
0
0

m
g
/
m
l

N
e
g
a
t
i
v
e

H
e
c
k

e
t

a
l
.

D
N
A

s
y
n
t
h
e
s
i
s

(
1
9
8
9
)
1
3
3
0

b
-
P
i
n
e
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

0
.
0
3

3
0

m
m
o
l
/
p
l
a
t
e

N
e
g
a
t
i
v
e
c

F
l
o
r
i
n

e
t

a
l
.

T
A
9
8
,

T
A
1
0
0
,

(
4
.
1

0
8
7

m
g
/
p
l
a
t
e
)
u
,
v

(
1
9
8
0
)

T
A
1
5
3
5
,

T
A
1
5
3
7
1
3
3
0

b
-
P
i
n
e
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

0
.
0
1

m
l
/
p
l
a
t
e

N
e
g
a
t
i
v
e
c

D
e
G
r
a
f
f

T
A
9
8
,

T
A
1
0
0
,

(
8
.
6

3
2
0

m
g
/
p
l
a
t
e
)
w
,
x

(
1
9
8
3
)

T
A
1
5
3
5
,

T
A
1
5
3
7
,

T
A
1
5
3
8
274 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
T
a
b
l
e

5
.

(
c
o
n
t
d
)
N
o
.

F
l
a
v
o
u
r
i
n
g

a
g
e
n
t

E
n
d
-
p
o
i
n
t

T
e
s
t

o
b
j
e
c
t

D
o
s
e

o
r

c
o
n
c
e
n
t
r
a
t
i
o
n

R
e
s
u
l
t

R
e
f
e
r
e
n
c
e
1
3
3
0

b
-
P
i
n
e
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

U
p

t
o

0
0
0

m
g
/
p
l
a
t
e

N
e
g
a
t
i
v
e
c

H
e
c
k

e
t

a
l
.

T
A
9
8
,

T
A
1
0
0
,

(
1
9
8
9
)

T
A
1
5
3
5
,

T
A
1
5
3
7
,

T
A
1
5
3
8
1
3
3
0

b
-
P
i
n
e
n
e

S
i
s
t
e
r

c
h
r
o
m
a
t
i
d

C
h
i
n
e
s
e

3
3
.
3

0
0
0

m
m
o
l
/
l

N
e
g
a
t
i
v
e
f

S
a
s
a
k
i

e
t

a
l
.

e
x
c
h
a
n
g
e

h
a
m
s
t
e
r

(
4
.
5

1
3
6
.
2

m
g
/
m
l
)
u

(
1
9
8
9
)

o
v
a
r
y

K
-
1

c
e
l
l
s
1
3
4
0

p
-
M
e
n
t
h
a
-
1
,
4
-
d
i
e
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

U
p

t
o

5
0

0
0
0

m
g
/
p
l
a
t
e

N
e
g
a
t
i
v
e
c

H
e
c
k

e
t

a
l
.

T
A
9
8
,

T
A
1
0
0
,

(
1
9
8
9
)

T
A
1
5
3
5
,

T
A
1
5
3
7
,

T
A
1
5
3
8
1
3
4
0

p
-
M
e
n
t
h
a
-
1
,
4
-
d
i
e
n
e

U
n
s
c
h
e
d
u
l
e
d

R
a
t

h
e
p
a
t
o
c
y
t
e
s

U
p

t
o

3
0

m
g
/
m
l

N
e
g
a
t
i
v
e

H
e
c
k

e
t

a
l
.

D
N
A

s
y
n
t
h
e
s
i
s

(
1
9
8
9
)
1
3
4
2

d
-
3
-
C
a
r
e
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

1
.
2
5

m
l
/
p
l
a
t
e

P
o
s
i
t
i
v
e
z

K
u
r
t
t
i
o

e
t

a
l
.

T
A
9
8
,

T
A
1
0
0
,

(
1

0
7
8

3
1
4

m
g
/
p
l
a
t
e
)
y

(
1
9
9
0
)

T
A
1
0
2
1
3
4
6

C
a
d
i
n
e
n
e
j

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

1
0
0

m
g
/
p
l
a
t
e

-
S
9
1
;

N
e
g
a
t
i
v
e
;

N
a
t
i
o
n
a
l

T
A
9
7
,

T
A
9
8
,

1
0
0

1
0

0
0
0

m
g
/
p
l
a
t
e

+
S
9
1

P
o
s
i
t
i
v
e
2

T
o
x
i
c
o
l
o
g
y

T
A
1
0
0
,

P
r
o
g
r
a
m

T
A
1
5
3
5
,

(
2
0
0
4
e
)

T
A
1
5
3
7
1
3
4
6

C
a
d
i
n
e
n
e
3

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

1
0

3
3
3

m
g
/
p
l
a
t
e

-
S
9
;

N
e
g
a
t
i
v
e
c

H
a
w
o
r
t
h

e
t

a
l
.

T
A
9
8
,

T
A
1
0
0
,

1
0
0

1
0

0
0
0

m
g
/
p
l
a
t
e

+
S
9
o

(
1
9
8
3
)
;

T
A
1
5
3
5
,

N
a
t
i
o
n
a
l

T
A
1
5
3
7

T
o
x
i
c
o
l
o
g
y

P
r
o
g
r
a
m

(
2
0
0
4
f
)
ALIPHATIC AND ALICYCLIC HYDROCARBONS 275
L1
1
3
4
6

C
a
d
i
n
e
n
e
3

F
o
r
w
a
r
d

m
u
t
a
t
i
o
n

M
o
u
s
e

l
y
m
p
h
o
m
a

0
.
0
0
5

0
.
0
5

m
g
/
m
l

-
S
9

N
e
g
a
t
i
v
e
c

N
a
t
i
o
n
a
l

L
5
1
7
8
Y

(
4
.
6

4
6
.
2

m
g
/
m
l
)
4
,
5
;

T
o
x
i
c
o
l
o
g
y

T
k
+
/
-

c
e
l
l
s

0
.
0
1

0
.
0
8

m
l
/
m
l

+
S
9

P
r
o
g
r
a
m

(
9
.
2

7
3
.
9

m
g
/
m
l
)
4
,
6

(
2
0
0
4
g
)
1
3
4
6

C
a
d
i
n
e
n
e
3

S
i
s
t
e
r

c
h
r
o
m
a
t
i
d

C
h
i
n
e
s
e

8
.
9

2
6
.
6

m
g
/
m
l

-
S
9
;

E
q
u
i
v
o
c
a
l
;

N
a
t
i
o
n
a
l

e
x
c
h
a
n
g
e

h
a
m
s
t
e
r

o
v
a
r
y

2
2
.
2

3
1
.
1

m
g
/
m
l

+
S
9

N
e
g
a
t
i
v
e

T
o
x
i
c
o
l
o
g
y

c
e
l
l
s

P
r
o
g
r
a
m

(
2
0
0
4
h
)
1
3
4
6

C
a
d
i
n
e
n
e
3

C
h
r
o
m
o
s
o
m
a
l

C
h
i
n
e
s
e

h
a
m
s
t
e
r

2
4
.
9

3
5
.
5

m
g
/
m
l

-
S
9
;

N
e
g
a
t
i
v
e
c

N
a
t
i
o
n
a
l

a
b
e
r
r
a
t
i
o
n

o
v
a
r
y

c
e
l
l
s

3
0
.
2

4
0

m
g
/
m
l

+
S
9

T
o
x
i
c
o
l
o
g
y

P
r
o
g
r
a
m

(
2
0
0
4
h
)
I
n

v
i
v
o
1
3
2
6

d
-
L
i
m
o
n
e
n
e

M
a
m
m
a
l
i
a
n

s
p
o
t

M
o
u
s
e

(
C
5
7
B
L
x
T
)

2
1
5

m
g
/
k
g

b
w
7

N
e
g
a
t
i
v
e

F
a
h
r
i
g

(
1
9
8
4
)

t
e
s
t

e
m
b
r
y
o
s
1
3
2
7

M
y
r
c
e
n
e

C
h
r
o
m
o
s
o
m
a
l

R
a
t

b
o
n
e

1
0
0

0
0
0

m
g
/
k
g

b
w
8

N
e
g
a
t
i
v
e
9

Z
a
m
i
t
h

e
t

a
l
.

a
b
e
r
r
a
t
i
o
n

m
a
r
r
o
w

c
e
l
l
s

(
1
9
9
3
)
1
3
2
7

M
y
r
c
e
n
e

M
i
c
r
o
n
u
c
l
e
u
s

M
o
u
s
e

2
5
0

0
0
0

m
g
/
k
g

b
w
1
0

N
e
g
a
t
i
v
e

N
T
P

(
2
0
0
4
i
)

f
o
r
m
a
t
i
o
n

p
e
r
i
p
h
e
r
a
l

b
l
o
o
d
a

C
a
l
c
u
l
a
t
e
d

u
s
i
n
g

d
e
n
s
i
t
y

o
f

c
a
m
p
h
e
n
e

o
f

0
.
8
4
2

g
/
m
l

(
L
e
w
i
s
,

1
9
9
9
)
.
b

W
i
t
h

m
e
t
a
b
o
l
i
c

a
c
t
i
v
a
t
i
o
n
.
c

W
i
t
h

a
n
d

w
i
t
h
o
u
t

m
e
t
a
b
o
l
i
c

a
c
t
i
v
a
t
i
o
n
.
d

C
a
l
c
u
l
a
t
e
d

u
s
i
n
g

r
e
l
a
t
i
v
e

m
o
l
e
c
u
l
a
r

m
a
s
s

o
f

c
a
m
p
h
e
n
e

o
f

1
3
6
.
2
4
.
e

C
y
t
o
t
o
x
i
c
i
t
y

o
b
s
e
r
v
e
d

a
t

t
h
e

h
i
g
h
e
s
t

d
o
s
e
/
c
o
n
c
e
n
t
r
a
t
i
o
n

t
e
s
t
e
d
.
f

W
i
t
h
o
u
t

m
e
t
a
b
o
l
i
c

a
c
t
i
v
a
t
i
o
n
.
g

C
a
l
c
u
l
a
t
e
d

u
s
i
n
g

d
e
n
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276 ALIPHATIC AND ALICYCLIC HYDROCARBONS
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.
ALIPHATIC AND ALICYCLIC HYDROCARBONS 277
L1
(i) In vitro
NoevidenceofmutagenicitywasobservedinAmesassayswhencamphene
(No. 1323; up to 84200mg/plate), b-caryophyllene (No. 1324; up to 150000mg/
plate), d-limonene (No. 1326; up to 150000mg/plate), myrcene (No. 1327; up to
10000mg/plate),a-pinene(No.1329;upto25000mg/plate),b-pinene(No.1330;
upto5000mg/plate),orp-mentha-1,4-diene(No.1340;upto50000mg/plate)were
incubated with Salmonella typhimurium strains TA97, TA98, TA100, TA1535,
TA1537,TA1538,and/orUTH8413,UTH8414,orTA102withandwithoutmetabolic
activation (Rockwell & Raw, 1979; Florin et al., 1980; DeGraff, 1983; Haworth
et al., 1983; Jagannath, 1984a, 1984b; Connor et al., 1985; Heck et al., 1989;
National Toxicology Program, 1990; Mller et al., 1993; National Toxicology
Program, 2004c, 2004d). Without metabolic activation, d-3-carene (No. 1342) at
dosesofbetween2157and4314mg/plategavepositiveresultsintheAmesassay
in S. typhimurium strains TA100 and TA102, but gave negative results in both
strainswithmetabolicactivation(Kurttioetal.,1990). d-3-Careneatdosesofup
to4314mg/platealsogavenegativeresultsinS. typhimuriumstrainTA98withand
without metabolic activation (Kurttio et al., 1990). In one Ames assay with S.
typhimuriumstrainsTA98,TA100,TA1535andTA1537,thebisomerofcadinene
(No. 1346) gave negative results at doses of up to 10000 and 3333mg/plate,
respectively,withandwithoutmetabolicactivation(Haworthetal.,1983;National
ToxicologyProgram,2004f).InanotherAmesassay,cadinene(isomernotspeci-
fed)gaveequivocal/weakpositiveresultsatdosesofupto10000mg/plateinS.
typhimuriumstrainsTA97andTA100withmetabolicactivation,butgavenegative
resultsatdosesofupto100mg/plateinbothstrainswithoutmetabolicactivation,
aswellasinstrainsTA98,TA1535andTA1537withandwithoutmetabolicactiva-
tion(NationalToxicologyProgram,2004e).
Camphene (No. 1323; 1.4136.2mg/ml), b-caryophyllene (No. 1324; 2.0
204.4mg/ml),a-phellandrene(No.1328;4.5136.2mg/ml),andb-pinene(No.1330;
4.5136.2mg/ml) did not induce sister chromatid exchanges (SCE) in Chinese
hamsterovarycellswithoutmetabolicactivation(Sasakietal.,1989).
b-Caryophyllene (No. 1324; up to 10000mg/ml), a-pinene (No. 1329; up to
10000mg/ml), and p-mentha-1,4-diene (No. 1340; up to 30mg/ml) did not induce
unscheduledDNAsynthesisinrathepatocytes(Hecketal.,1989).
d-Limonene(No.1326)didnotinducegeneticeffectswhentestedinSaccha-
romyces cerevisiaestrainMP1,withoutmetabolicactivation,atconcentrationsof
up to 230mmol/l (Fahrig, 1984). In Chinese hamster ovary cells, d-limonene did
not induce chromosomal aberrations at concentrations of 10500mg/ml, or SCE
atconcentrationsof1.4162mg/ml,withandwithoutmetabolicactivation(Sasaki
et al., 1989; Anderson et al., 1990; National Toxicology Program, 1990). In an
assayforforwardmutationinmouselymphomacells,d-limoneneproducednega-
tiveresultsinL5178Ycellswithandwithoutmetabolicactivation,ataconcentration
of up to 100mg/ml (Heck et al., 1989; Myhr et al., 1990; National Toxicology
Program, 1990). When incubated with Syrian hamster embryo cells, limonene
(isomernotspecifed)didnotinducecelltransformationinoneassayataconcen-
trationofupto100mg/ml(Pienta,1980),whileinanotherassayconcentrationsup
278 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
to 408.7mg/ml increased the transformation frequency, albeit not in a statistically
signifcant manner (Rivedal et al., 2000).The latter study also tested the effects
oflimonene(isomernotspecifed)ongapjunctionintercellularcommunicationin
Syrianhamsterembryocells.Limoneneatconcentrationsof1.4136.2mg/mldid
notshoweffects(Rivedaletal.,2000).
Withandwithoutmetabolicactivation,myrcene(No.1327)didnotinducegene
mutationsatthehypoxanthineguaninephosphoribosyltransferase(Hprt)locusin
ChinesehamsterV79cells,atconcentrationsofupto1000mg/ml(Kaudereretal.,
1991), nor did it induce SCE in these cells at concentrations up to 500mg/ml
(Rscheisenetal.,1991).MyrcenealsodidnotinduceSCEorchromosomalaber-
rationsinhumanlymphocytes,withandwithoutmetabolicactivation,atconcentra-
tionsofupto1000mg/ml(Kaudereretal.,1991),nordiditinduceSCEinhepatic
tumourcells,atconcentrationsofupto500mg/ml,althoughaslight,reproducible
butnotdose-dependentincreasewasnoted(Rscheisenetal.,1991).
The b isomer of cadinene (No. 1346) gave negative results in an assay for
forwardmutationinmouselymphomacells,atconcentrationsofupto46.2mg/ml
without metabolic activation, and at up to 73.9mg/ml with metabolic activation
(National Toxicology Program, 2004g). In Chinese hamster ovary cells, this b
isomerdidnotinducechromosomalaberrationwithorwithoutmetabolicactivation
atconcentrationsof24.940mg/ml,orSCEwithmetabolicactivationatconcentra-
tionsofupto31.1mg/ml(NationalToxicologyProgram,2004h).Withoutmetabolic
activation,anequivocalresultwasobtainedforinductionofSCE,atconcentrations
upto26.6mg/ml(NationalToxicologyProgram,2004g).
Inastudyconductedinvivo-invitro,designedtoinvestigatethemutagenicity
of urinary metabolites of a number of food additives, Sprague-Dawley rats were
givenasingledoseof0.5mlofcamphene(No.1323;approximately1684mg/kg
bw) anda-pinene (No. 1329; approximately 1718mg/kg bw) via gavage and the
urinewascollectedfor24h.ThreetypesofurinesampleweretestedintheAmes
assay with S. typhimurium strains TA98 and TA100 with metabolic activation: a
direct urine sample, a urine-ether extract, and the aqueous fraction of the urine-
ether extract. The urine samples of rats treated with a-pinene did not show any
evidence of mutagenicity, either in the presence or absence of b-glucuronidase.
Oftheurinesamplesofcamphene-treatedratsonlytheurine-etherextractshowed
a weak mutagenic response, and only in TA100, not in TA98 (Rockwell & Raw,
1979).
(ii) In vivo
Inamammalianspottest,noevidenceofmutagenicitywasobservedinmouse
C57BLxT embryos in utero after intraperitoneal injection of the dam with d-
limonene(No.1326)atadoseof215mg/kgbwperdayondays9and10ofges-
tation(Fahrig,1984).
Inanassayforcytogeneticchangesinbonemarrow,groupsofWistarrats(two
orfourofeachsexpergroup)weregivenmyrcene(No.1327)atadoseof100,
500,or1000mg/kgbwviagavage.Anegativecontrolgroup(tworatsofeachsex)
receivedonlythevehicle(cornoil)viagavage,whileapositivecontrolgroup(two
ALIPHATIC AND ALICYCLIC HYDROCARBONS 279
L1
ratsofeachsex)receivedcyclophosphamideatadoseof30mg/kgbwviaintra-
peritonealinjection.Amitoticinhibitor(colchicine,administeredatadoseof5mg/
kg bw via intraperioneal injection) was administered 1h before sacrifce at 24 or
48h after treatment, at which time the bone-marrow cells were harvested. Com-
paredwiththenegativecontrolgroup,treatmentwithmyrcenedidnotresultinan
increase of metaphase cells with chromosomal aberrations upon examination at
24or48h.Incontrast,inthepositivecontrolgroupchromosomalaberrationswere
found in 19% of the bone-marrow metaphase cells examined. Although not
clastogenic, myrcene caused a dose-dependent increase in the mitotic index in
bone-marrowcells,indicatingthatitwaspresentatasuffcientdoseinthetarget
tissue(Zamithetal.,1993).
An assay for micronucleus formation in mouse peripheral blood erythrocytes
wasperformed,withsamplesofperipheralbloodobtainedwithin24hofthefnal
exposure in a 13-week study of toxicity in which male and female B6C3F
1
mice
were given myrcene (No. 1327) at a dose of up to 2000mg/kg bw per day via
gavage. Scoring of 1000 normochromatic erythrocytes (NCEs) for micronuclei
revealed no increase in micronucleated NCEs at any dose (National Toxicology
Program,2004i).
(iii) Conclusion
Seven substances in this group of favouring agents have been tested in the
Ames assay and found not to be mutagenic in bacteria in vitro. One favouring
agent,d-3-carene,producedapositiveresultinthisassay,onlywithoutmetabolic
activation,inS. typhimuriumstrainsTA100andTA102butnotTA98.Anotherfa-
vouringagent,cadinene(isomernotspecifed),gaveweaklypositiveresultsinthe
Ames assay, only with metabolic activation, in S. typhimurium strains TA97 and
TA100butnotTA98,TA1535,andTA1537.
Inmammaliancellsystems,predominantlynegativeresultswereobtainedfor
representative members of this group with respect to induction of SCE, chromo-
somal aberrations, unscheduled DNA synthesis, and gene mutations. In assays
forcelltransformationinSyrianhamsterembryocells,limonene(isomernotspeci-
fed)gavenegativeresultsinoneassay,butweakpositiveresultsinanother,the
increaseintransformationfrequencybeingnotstatisticallysignifcant.
Myrcene and d-limonene showed no signs of genetic toxicity in cytogenetic
assays for micronucleus formation in bone marrow and peripheral erythrocytes
(myrcene)andamammalianspottest(d-limonene)performedinvivo.
Onthebasisoftheresultsofavailablestudiesofgenotoxicity,theCommittee
concludedthatthefavouringagentsinthisgroupofaliphaticandalicyclichydro-
carbonsarenotgenotoxic.
(e) Reproductive toxicity
(i) d-Limonene (No. 1326)
Three groups of 20 female Wistar rats were given d-limonene (in 1% gum
arabicsolution)atadoseof0,591,or2869mg/kgbwperdaybyoraladministra-
280 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
tionondays915ofgestation.Fifteenanimalspergroupweresacrifcedatday
20ofgestation.Theremainingfveanimalspergroupwereallowedtogivebirth
totheiroffspring,andtheoffspringwerefolloweduntilpostnatalweek7.
Maternaltoxicitywasnotedatthehigherdoseonly,evidencedbymortality(8
outof20animalsdiedcomparedwithnoneofthe20animalsinthecontrolgroup
andinthegroupreceivingthelowerdose)anddecreasedbody-weightgain.The
fetusesofdamsatthehigherdoseexhibiteddelayedossifcationofthemetacarpal
boneandtheproximalphalanx.Thiswasreportedtoberestoredtonormalwithin
severalweeksafterbirth.Atthehigherdose,offspringshoweddecreasesinpost-
natal weight gain in males, in absolute and relative weights of the thymus and
spleeninmalesandfemales,andinabsoluteandrelativeweightsoftheovaryin
females.Nostatisticallysignifcantdifferenceswereobservedinphysicalsignsof
postnataldevelopment(openingoftheear-shell,eyelidandvaginalorifce,odon-
tiasis, descending of the testis, and coating with hair) at the doses tested (Tsuji
etal.,1975c).
In a similar experiment, groups of 20 pregnant SLC-ICR mice were given d-
limoneneatadoseof0,591,or2363mg/kgbwperdaybyoraladministrationon
days 712 of gestation. Fifteen animals per group were sacrifced at day 18 of
gestation.Theremainingfveanimalspergroupwereallowedtogivebirthtotheir
offspring,andtheoffspringwasfolloweduntilpostnatalweek7.
Compared with controls, dams at the higher dose gained less weight during
administration,butweightgainwasnormalaftertreatmentstopped.Asimilareffect
wasseenindamsatthelowerdose,buttoasmallerdegree.Atthehigherdose,
also the numbers of implantations and live fetuses were slightly decreased, and
the fetal weights were slightly decreased at both doses. External and visceral
examination of the fetuses did not reveal treatment-related changes. However,
increasedincidencesoflumbarandfusedribs,aswellasdelayedossifcationof
some bones were observed in fetuses of the group receiving the higher dose.A
delay in ossifcation of the middle phalanx was also observed in fetuses of the
groupreceivingthelowerdose.Allretardedossifcationswererestoredtonormal
during postnatal development. Males born to dams in the group receiving the
higherdoseshowedasignifcantdecreaseinpostnatalbody-weightgain.Several
organweightswereaffectedbytreatment(e.g.testes,ovaries,liver),mostlyatthe
higher dose, but also incidently at the lower dose. Histology of the testes and
ovaries did not reveal any abnormalities. No statistically signifcant differences
were observed in physical signs of postnatal development at the doses tested
(Kodamaetal.,1977a).
Thesameresearchgroupalsostudiedthedevelopmentaleffectsofd-limonene
inrabbitfetusesandoffspring.Groupsof1321pregnantJapaneseWhiterabbits
weregivend-limoneneatadoseof0,250,500,or1000mg/kgbwperdaybyoral
administration from days 618 of gestation. Ten to 18 animals per group were
sacrifced on day 28 of gestation. The remaining three animals per group were
allowedtogivebirthtotheiroffspring,andtheoffspringwerefolloweduntilpost-
natalweek7.
Signs of maternal toxicity included increased mortality at the highest dose (6
out of 21 animals died as compared with none of the 13 animals in the control
ALIPHATIC AND ALICYCLIC HYDROCARBONS 281
L1
group and in other treatment groups) and a temporary decrease in body-weight
gainandfoodconsumptionattheintermediateandhighestdose.Noabnormalities
werenotedinanyofthefetusesuponexternalexamination.Visceralandskeletal
examinations revealed some anomalies (such as incomplete lobulation of the
lungs, lumbar rib variations, and retarded ossifcation of the middle phalanx and
ffth sternebrae) in all treatment groups, including controls, but without a dose
responserelationship.Moreover,theseanomalieswererestoredtonormalduring
postnataldevelopment.Postnatalweightgainwasdecreasedinfemaleoffspring
attheintermediatedosebutnotatthelowestorhighestdoses.Treatmentdidnot
affectopeningoftheear-shellandeyelids,odontiasisorcoatingwithhair(Kodama
etal.,1977b).
(ii) Myrcene (No. 1327)
Groupsof1629femaleWistarratsweregivenmyrcene(dissolvedincornoil)
atadoseof250,500,or1200mg/kgbwperdaybygavageondays615ofges-
tation.Controlrats(16intotal)receivedeitherthevehicleonly,ornotreatmentat
all. All animals were sacrifced on day 20 of gestation. The gravid uterus was
weighed, and the number of implantation sites, living and dead fetuses, resorp-
tions,andcorporaluteawererecorded.Thefetuseswereweighed,andexamined
forexternal,visceral,andskeletalabnormalities.
Signsofmaternaltoxicitywereobservedonlyatthehighestdoseandconsisted
ofdecreasedmaternalbody-weightgain(especiallyduringthefrstdaysoftreat-
ment)andgraviduterusweight,andmortalityinonedam.Embryo-andfetotoxicity
were also only observed at the highest dose, evidenced by reduced numbers of
visibleimplantationsitesandlivefetuses,andincreasedincidencesoffetuseswith
delayedossifcationandotherskeletalmalformations.TheNOELforbothmaternal
andembryo-fetotoxicitywas500mg/kgbwperday(Delgadoetal.,1993a).
In a follow-up study designed to test for peri- and postnatal developmental
toxicityinrats,groupsof1218pregnantWistarratsweregivenmyrcene(incorn
oil) at a dose of 250, 500, 1000, or 1500mg/kg bw per day by gavage from day
15ofgestationuntilweaningoftheoffspringonpostnatalday21.Acontrolgroup
of20animalsreceivedthevehicleonly,bygavage.Theprogenywereexamined
formortality,weightgain,physicalsignsofpostnataldevelopment(earunfolding,
incisoreruption,furdevelopmentandeyeopening),andwhentheyreachedmatu-
rity,forreproductivecapacity.
Maternaltoxicitywasmainlyevidentatthehighestdose;5ofthe15damsdied
within 4 days of treatment, and all dams showed a weight defcit at term which
persistedafterdelivery(postnatalday1).At1000mg/kgbwtherewasalsoaweight
defcit observed at term, but this was no longer detectable on postnatal day 1.
Necropsyofthedamsrevealedhyperkeratosisintheforestomachinmostratsat
thetwohigherdoses.Whilethedurationofgestation,littersize,andpost-weaning
mortality did not differ signifcantly between groups, the duration of labour was
increasedat500and1000mg/kgbwperday,aswasthenumberofstillbirthsat
these doses. There was a dose-dependent decrease in birth weight at doses of
500mg/kgbwandgreater,whichgraduallyreturnedtocontrolvaluesatweaning.
Postnatalmortalitywasincreased,especiallyduringthefrstweekoflactation,and
282 ALIPHATIC AND ALICYCLIC HYDROCARBONS
L1
developmentallandmarksweredelayedat500,1000and1500mg/kgbwperday.
In addition, female offspring at the two higher doses displayed impaired fertility.
The male sex organs were not affected by treatment, nor was the sperm count.
TheNOELforperi-andpostnataltoxicitywas250mg/kgbwperday(Delgadoet
al.,1993b).
Inaone-generationstudy,groupsof60Wistarrats(15male,45female)were
givenmyrcene(inpeanutoil)atadoseof0,100,300,or500mg/kgbwperday
bygavage.Malesweretreatedfor91daysbeforematingandduringthemating
period,whilefemalesweretreatedcontinuouslyfrom21daysbeforematinguntil
theoffspringwereweanedat21daysafterbirth.Malesweresacrifcedafterthe
matingperiod,andone-thirdofthefemalesineachgroupweresacrifcedonday
21ofgestation.Theweightofthegraviduteruswasrecorded,resorptions,living
anddeadfetuses,andimplantationsiteswerecounted,andalllivingfetuseswere
weighed and examined for external and skeletal abnormalities. The remaining
pregnantfemaleswereallowedtogivebirthtotheiroffspring,andweresacrifced
afterweaning(postnatalday21).Thenumbersofviableanddeadnewbornswere
counted, and the pups were sexed, weighed on days 1, 6, 11, 16 and 21 and
examinedforsignsofphysicaldevelopment.
The only effects observed in male rats were slight but statistically signifcant
increasesintherelativeandabsoluteweightsoftheliverandkidneyinthegroup
receiving the highest dose. Body weight and body-weight gain were reduced at
thisdose,althoughnotstatisticallysignifcantly.Treatmentdidnotaffectthemating
or pregnancy index, and maternal toxicity was not observed, except for slightly
increased liver and kidney weights. However, at the highest dose a signifcant
increase in the number of resorptions and a parallel decrease in the number of
livefetusesperimplantationsitewereobserved,aswellasasignifcantincrease
inthenumberoffetuseswithskeletalmalformations.Theauthorsnotedthatmost
of the malformations that were observed (e.g. fused os zygomatic, dislocated
sternum,andextralumbarribs)alsooccurredwithhighspontaneousfrequencyin
thecontrolsandhistoricalcontrolsofthestrainofratsused.Noeffectswereseen
onmaternalweightchangesduringgestationandlactation,andonoffspringweight
changes during lactation. During postnatal days 221, pup mortality was slightly
increasedat500mg/kgbwperday,buttheincreasewasnotstatisticallysignifcant.
Slightdelayswerenotedineyeopening,incisoreruption,andprimarycoatappear-
ance of offspring of dams treated with myrcene, but without a doseresponse
relationship.TheNOELwas300mg/kgbwperday(Paumgarttenetal.,1998).
(iii) Rowachol
Groupsof1217JCL-SpragueDawleyratsweregivenRowacholatadose
of0.16,0.8,or1.6ml/kgbwperdaybyoraladministrationondays914ofgesta-
tion.Rowacholisaliquidterpenemixturecontainingl-menthol(32%),a,b-pinene
(17%),menthone(6%),borneol(5%),d-camphene(5%),cineol(2%),rheochrysin
(0.1%),andoliveoil(32.9%).Acontrolgroupof21ratsreceivedoliveoilataoral
dose of 0.80ml/kg bw per day. Autopsies were performed on day 20 of
gestation.
ALIPHATIC AND ALICYCLIC HYDROCARBONS 283
L1
No adverse effects were observed at 0.16 and 0.8ml/kg bw per day. In the
groupreceivingadoseof1.6ml/kgbwperday,thedamslostweightduringtreat-
ment,butgainedweightagainaftertreatmentstopped.Placentalweightandthe
numbersofimplantationsandlivefetuseswerereduced,aswerefetalweightand
body weight at birth. Within 1 week after birth, however, weight gain of offspring
wascomparabletothatofcontroloffspring.Thefetusesofthegroupreceivingthe
highestdosedidnotshowanyretardedossifcationorgrossorvisceralanomalies,
nor were there signifcant increases in the incidences of skeletal malformations.
TheNOELforRowacholformaternalandembryo-fetotoxicitywas0.8ml/kgbw
perday(Hasegawa&Toda,1978).
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L1
L1
291
AROMATIC HYDROCARBONS
First draft prepared by
Mrs M.E.J. Pronk
1
and Professor J.R. Bend
2
1
Centre for Substances and Integrated Risk Assessment, National Institute
for Public Health and the Environment, Bilthoven, Netherlands; and
2
Department of Pharmacology & Toxicology, Faculty of Medicine and
Dentistry, University of Western Ontario, London, Ontario, Canada;
Evaluation ............................................................................... 291
Introduction......................................................................... 291
Estimateddailyintake........................................................ 292
Absorption,distribution,metabolismandelimination........ 292
ApplicationoftheProcedurefortheSafetyEvaluation
ofFlavouringAgents................................................... 292
Considerationofsecondarycomponents.......................... 296
Considerationofcombinedintakesfromuseas
favouringagents......................................................... 296
Conclusions........................................................................ 296
Relevantbackgroundinformation............................................. 296
Explanation......................................................................... 296
Additionalconsiderationsonintake................................... 296
Biologicaldata.................................................................... 297
Biochemicaldata......................................................... 297
Absorption,distribution,andexcretion........... 297
Metabolism...................................................... 298
Toxicologicalstudies.................................................... 301
Acutetoxicity................................................... 301
Short-termstudiesoftoxicity.......................... 301
Long-termstudiesoftoxicityand
carcinogenicity.......................................... 304
Genotoxicity.................................................... 308
Reproductivetoxicity....................................... 312
References............................................................................... 313
1. EVALUATION
1.1 Introduction
TheCommitteeevaluatedagroupoffvearomatichydrocarbons(Table1)by
theProcedurefortheSafetyEvaluationofFlavouringAgents(seeFigure1,p192).
Onememberofthisgroup,biphenyl(No.1332),wasevaluatedbytheCommittee
atitseighthmeeting(Annex1,reference8)andwasassignedanacceptabledaily
intake(ADI)foritsuseasafungistaticagent.Thefungistaticuseofbiphenylwas
alsoevaluatedbytheJointFAO/WHOMeetingonPesticideResidues(JMPR)in
1966and1967(WHO,1967;WHO,1968),whenanADIof00.125mg/kgbwwas
established.
292 AROMATIC HYDROCARBONS
L1
Four (Nos 1325, 1332, 1333 and 1335) of the fve favouring agents in this
group have been reported to occur naturally in foods. They have been detected
in,forexample,coffee,alcoholicbeverages,bakedandfriedpotato,heatedbeans,
tea, bread and cheese (Nijssen et al., 2003). The substance with the highest
naturaloccurrenceisp-cymene(No.1325).
1.2 Estimated daily intake
Thetotalannualvolumeofproductionofthefvefavouringagentsinthisgroup
isapproximately7800kginEurope(InternationalOrganizationoftheFlavorIndus-
try,1995)and3600kgintheUnitedStatesofAmerica(USA)(NationalAcademy
ofSciences,1989;Lucasetal.,1999)(seeTable2).Morethan98%ofthetotal
annualvolumeofproductioninEuropeandtheUSAisaccountedforbythemono-
aromaticterpenehydrocarbonp-cymene.Theestimateddailyintakesofp-cymene
in Europe and the USA are approximately 1100mg/person and 470mg/person,
respectively.The reported annual volumes of production of the remainder of the
favouringagentsinthisgrouparelowtoverylow.Theestimateddailyintakesof
theseagentsrangefrom0.001to21mg/personinEuropeandtheUSA.Theesti-
mateddailypercapitaintakeofeachagentisreportedinTable1.
1.3 Absorption, distribution, metabolism and elimination
Being lipophilic, the aromatic hydrocarbons in this group are likely to cross
biologicalmembranesbypassivediffusion.Availabledataonp-cymeneandbiphe-
nyl indicated that these materials are readily absorbed from the gastrointestinal
tract, widely distributed in the body, metabolized and excreted mainly in the
urine.
Onthebasisoftheavailabledata,itisanticipatedthatthearomatichydrocar-
bonsinthisgroupwillparticipateinsimilarpathwaysofmetabolicdetoxifcationin
mammals, including humans.After absorption, these hydrocarbons are oxidized
topolaroxygenatedmetabolitesviacytochromeP450(CYP)enzymesandalcohol
andaldehydedehydrogenases.Themajormetabolicpathwayofaromaticterpene
hydrocarbons involves hepatic microsomal CYP-mediated oxidation of ring side-
chains, yielding alcohols, aldehydes, and acids.The metabolites are then conju-
gatedwithglycine,glucuronicacid,orglutathione,andexcretedintheurineand/or
bile.Thebiotransformationofbiphenylproceedsviaringhydroxylation,preferen-
tiallyattheC4position,yieldingphenolicderivativesthataresubsequentlymetab-
olizedtoglucuronideandsulfateconjugates,whichareexcretedintheurine.
1.4 Application of the procedure for the safety evaluation of
favouring agents
Step 1. InapplyingtheProcedure,theCommitteeassignedtwo(Nos1325and
1333)ofthefvefavouringagentsinthisgrouptostructuralclassI.The
remaining three favouring agents (Nos 1332, 1334 and 1335) were
assignedtostructuralclassIII(Crameretal.,1978).
AROMATIC HYDROCARBONS 293
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AROMATIC HYDROCARBONS 295
L1
Table 2. Annual volumes of use of aromatic hydrocarbons used as favouring
agents in Europe and the USA
Flavouringagent(No.) Mostrecent Intake
b
Annual Consumption

annual
mg/day mg/kgbw
volumein ratio
d

volume(kg)
a
perday
naturally

occurring
foods(kg)
c
p-Cymene(1325)
Europe 7602 1085 18
USA 3583 472 8 29302 8
Biphenyl(1332)
Europe 0.01 0.001 0.00002
USA
e
4 0.7 0.01 401 100
p,a-Dimethylstyrene
(1333)
Europe 144 21 0.3
USA 2 0.3 0.004 8 4
4-Methylbiphenyl
(1334)
Europe 0.1 0.01 0.0002
USA
e
0.45 0.08 0.001 NA
1-Methylnaphthalene
(1335)
Europe 6 0.9 0.01
USA 0.45 0.06 0.001 218 484
Total
Europe 7752
USA 3590
NA,notapplicable;+,reportedtooccurnaturallyinfoods(Nijssenetal.,2003),butno
quantitativedataavailable;-,notreportedtooccurnaturallyinfoods.
a
FromInternationalOrganizationoftheFlavorIndustry(1995)andLucas etal.(1999)or
NationalAcademyofSciences(1989).
b
Intakeexpressedasmg/personperdaywascalculatedasfollows:[(annualvolume,kg)
(110
9
mg/kg)/(populationsurveycorrectionfactor365days)],wherepopulation
(10%,eatersonly)=3210
6
forEuropeand2610
6
fortheUSA.Thecorrection
factor=0.6forEurope,and0.8fortheUSA,representingtheassumptionthatonly60%
and80%oftheannualvolumeofthefavour,respectively,wasreportedinthepoundage
surveys(InternationalOrganizationoftheFlavorIndustry,1995;Lucas etal.,1999;
NationalAcademyofSciences,1989).
Intakeexpressedasmg/kgbwperdaywascalculatedasfollows:[(mg/personperday)/
bodyweight],wherebodyweight=60kg.Slightvariationsmayoccurfromrounding.
c
QuantitativedatafortheUSAreportedbyStofberg&Grundschober(1987).
d
Consumptionratiowascalculatedasfollows:(annualconsumptioninfood,kg)/(most
recentreportedvolumeasafavouringagent,kg).
e
Thevolumecitedistheanticipatedannualvolume,whichwasthemaximumamountof
favourestimatedtobeusedannuallybythemanufactureratthetimethematerialwas
proposedforfavouruse.Nationalsurveys(NationalAcademyofSciences1970,1975,
1976,1982or1987,asreportedinNationalAcademyofSciences,1989;Lucasetal.,
1999)revealednoreporteduseasafavouringagentatthattime.
296 AROMATIC HYDROCARBONS
L1
Step 2. Allthefavouringagentsinthisgroupareexpectedtobemetabolizedto
innocuousproducts.Theevaluationofallagentsinthisgrouptherefore
proceededviatheA-sideofthedecision-tree.
Step A3. The estimated daily per capita intakes of the two favouring agents in
structuralclassIandthethreefavouringagentsinstructuralclassIIIare
allbelowthethresholdsofconcern(i.e.1800mgforclassIand90mgfor
class III). According to the Procedure, the use of these fve favouring
agentsraisesnosafetyconcernatestimatedcurrentintakes.
Theintakeconsiderationsandotherinformationusedtoevaluatethefvearo-
matic hydrocarbons in this group according to the Procedure are summarized in
Table1.
1.5 Consideration of secondary components
All fve favouring agents in this group have minimum assay values of>95%.
Hence,itisnotnecessarytoconsidersecondarycomponents.
1.6 Consideration of combined intakes from use as favouring agents
In the event that the two agents in structural class I were consumed concur-
rentlyonadailybasis,theestimatedcombinedintakewouldnotexceedthehuman
intake threshold of 1800mg/person per day for class I. In the event that all three
agents in structural class III were consumed concurrently on a daily basis, the
estimatedcombinedintakewouldnotexceedthehumanintakethresholdof90mg/
personperdayforclassIII.Overallevaluationofthedataindicatedthatcombined
intakewouldnotraiseasafetyconcern.
1.7 Conclusions
The Committee concluded that none of the favouring agents in this group of
aromatichydrocarbonswouldpresentsafetyconcernsatcurrentestimatedintakes.
The Committee noted that all the available data on toxicity and metabolism of
the favouring agents in the group were consistent with the results of the safety
evaluation.
2. RELEVANT BACKGROUND INFORMATION
2.1 Explanation
Therelevantbackgroundinformationsummarizesthekeyscientifcdataappli-
cable to the safety evaluation of fve aromatic hydrocarbons used as favouring
agents(seeTable1).
2.2 Additional considerations on intake
ProductionVolumesofproductionandintakevaluesforeachfavouringagent
arereportedinTable2.
AROMATIC HYDROCARBONS 297
L1
Fourofthefvefavouringagentsinthegrouphavebeenreportedtooccurnatu-
rallyintraditionalfoods(Nijssenetal.,2003;Table2).Quantitativedataonnatural
occurrence have been reported for all four agents (Stofberg & Grundschober,
1987). The consumption of all these agents is derived predominantly from their
presenceintraditionalfoods(i.e.theyhaveaconsumptionratioof1;Table2).
2.3 Biological data
2.3.1 Biochemical data
(a) Absorption, distribution, and excretion
InmaleWistarratsorDunkinHartleyguinea-pigsgivenp-cymene(No.1325)
orally at a dose of 100mg/kgbw, 80% or 71%, respectively, of the administered
dose was excreted in the form of extractable metabolites in the urine within the
following48h.Itwasspeculatedthattherestofthedosewaseitherexcretedvia
thefaecesorasunextractablemetabolitesintheurine(Waldeetal.,1983).Ina
studyinasinglemaleJapanesewhiterabbitgivenp-cymeneasasingleoraldose
at670mg/kgbw,Ishidaetal.(1981)observedatotalurinaryexcretionof20%of
theadministereddoseasneutraloracidicmetaboliteswithin72h.
Inmalealbinoratsgivenbiphenyl(No.1332)asasingleoraldoseat100mg/
kgbw, nearly 30% of the administered dose was excreted in the urine within 4
days.Approximatelyequalamounts(5%oftheadministereddose)wererecovered
from the faeces and bile within 24h. The metabolites found in excreta and bile
mainly consisted of several mono-, di- and trihydroxy-metabolites of biphenyl.
Biphenylitselfwaspresentonlyinverysmallamountsintheurine,andwasnot
detectableinfaecesorbile.Mostofthebiotransformationproductswereexcreted
within2448hafterdosing(Meyer&Scheline,1976).Inasubsequentexperiment
followingthesameprocedures,maleguinea-pigsandrabbitsweregivenbiphenyl
atthesameoraldose.After4days,approximately33%and49%oftheadminis-
tereddosewasexcretedintheurineofguinea-pigsandrabbits,respectively,with
most being excreted within the frst 2448h, and only as biphenyl metabolites,
sincenounchangedbiphenylwasdetected.Intheguinea-pig,approximately20%
ofthedosewasrecoveredinthefaeces,nearly75%ofwhichwasbiphenylitself.
Therabbitexcretedonly1.6%oftheadministereddoseinthefaeces,almost90%
of which was unmetabolized biphenyl. Small amounts of the administered dose
were excreted via bile (3.3% and 0.3% in guinea-pigs and rabbits, respectively).
Nobiphenylwasdetectedinbileofeitherspecies(Meyer,1977).Theguinea-pig
thusexhibitedamuchhigherleveloffaecalexcretionofunchangedbiphenylthan
either the rat or the rabbit, suggesting decreased absorption compared with the
otherspecies.
Ratsfedbiphenylat1%inthediet(equivalenttoadoseof500mg/kgbwper
day),untilatotalof15gofdiphenylhadbeenconsumed,excretednearly60%of
thetotaldoseintheurineduringthefeedingperiodandthe48hthereafter(West
etal.,1956).
Sixmalerabbitsgivenbiphenylasasingleoraldoseof1000mgincornoilby
gavageexcretednearly65%oftheadministereddoseintheurineasfreephenolic
298 AROMATIC HYDROCARBONS
L1
metabolites or glucuronic acid conjugates within 4 days after dosing (Block &
Cornish,1959).
In summary
Being lipophilic, the aromatic hydrocarbons in this group are likely to cross
biologicalmembranesbypassivediffusion.Availabledataonp-cymeneandbiphe-
nyl indicate that these materials are readily absorbed from the gastrointestinal
tract, widely distributed in the body, metabolized and excreted mainly in the
urine.
(b) Metabolism
Themainmetabolitesintheurineofrabbitsgivenp-cymene(No.1325)orally
were p-cymen-9-ol and p-cymen-8-ol (50% and 28%, respectively, of the neutral
metabolites).Acidic metabolites identifed were a-p-tolylpropionic acid, a-tolyl-a-
hydroxylpropionicacid,p-isopropylbenzoicacidandp-1-hydroxyisopropylbenzoic
acid.Ringhydroxylationdidnotoccur(Ishidaetal.,1981).
In male rats given p-cymene orally at a dose of 100mg/kgbw, the principal
urinarymetaboliteswerep-isopropylbenzoicacid(19%oftheadministereddose)
and2-p-carboxyphenylpropionicacid(16%).Otherlessimportanturinarymetabo-
lites included 2-p-tolylpropan-1-ol (8%), 2-p-olylpropan-2-ol (9%), 2-p-carboxy-
phenylpropan-2-ol (9%), 2-p-(hydroxymethyl)phenylpropionic acid (4%),
2-p-carboxyphenylpropan-1-ol(11%),p-isopropylbenzoylglycine(2%),p-isopropyl-
benzylalcohol(1%),and2-p-tolylpropionicacid(1%)(Waldeetal.,1983).When
the same dose was given to male guinea-pigs, similar urinary metabolites were
identifed,howeverindifferentquantities.Theprimaryurinarymetaboliteinguinea-
pigswasp-isopropylbenzoylglycine(31%),indicatingthatconjugationwithglycine
wasmoreprevalentinguinea-pigsthaninrats.Anothermajormetaboliteinguinea-
pigs was 2-p-tolylpropan-2-ol (14%). In addition, while ring hydroxylation of p-
cymenewasnotreportedinrats(Bakke&Scheline,1970;Waldeetal.,1983)and
rabbits (Ishida et al., 1981), trace amounts of the ring hydroxylation metabolites
carvacrol and hydroxycarvacrol were detected in the urine in guinea-pigs. Ring
hydroxylationinguinea-pigsonlyoccurredorthotothemethylgroup(Waldeetal.,
1983).
Boyleetal.(1999)studiedthemetabolitepatternofp-cymeneinratsafteroral
administrationofdosesequivalentto50and200mg/kgbw.Themajormetabolites
intheurine048hafteradministrationofadoseof50mg/kgbwwere2-p-tolypro-
pan-2-ol(39%oftherecovereddose)and2-p-carboxyphenylpropan-2-ol(19%of
therecovereddose).Theformermetaboliteistheproductofallylichydroxylation
of the isopropyl substituent, while the latter metabolite is the product of allylic
hydroxylation of both the isopropyl substituent and the methyl substituent. Minor
metabolites in rat urine were 2-p-carboxyphenylpropan-1-ol (10%), 2-p-carboxy-
phenylpropionicacid(14%),andp-isopropylbenzoicacid(17%).Alargepercent-
ageoftheurinarymetabolitesatthisdosewasconjugated(66%conjugatedversus
34%free).Thesamemetaboliteswereobservedafterthehighestdose,butcon-
jugationwasconsiderablyreduced(18%conjugatedversus82%free),suggesting
saturationoftheconjugationpathway(Boyleetal.,1999).
AROMATIC HYDROCARBONS 299
L1
In a study designed to identify the stereochemistry of p-cymene metabolites,
fourrabbitsweregiven2.5gofp-cymene,afterwhichurinewascollectedfor72h.
Sevendifferenthydroxylatedandcarboxylatedmetaboliteswererecoveredinthe
urine.Fourwereopticallyactiveandidentifedas2-(p-tolyl)-1-propanol,2-(p-tolyl)-
propanoic acid, p-(2-hydroxy-1-methylethyl)benzoic acid, and p-(1-carboxyethyl)
benzoicacid.Threewereopticallyinactiveandidentifedas2-(p-tolyl)-2-propanol,
p-isopropylbenzoic acid, and p-(1-hydroxy-1-methylethyl)benzoic acid. Oxidation
ofthemethylgroupoftheisopropylsubstituentyielded2-(p-tolyl)-1-propanolinan
R/S ratio of 65:35. The (R)-alcohol is then further oxidized to the corresponding
acid (R)-2-(p-tolyl)propanoic acid, which undergoes complete stereochemical
inversionto(S)-2-(p-tolyl)propanoicacid.Subsequently,thealcoholoracidmetab-
olite may undergo oxidation of the tolyl methyl group to yield the corresponding
hydroxy acid and diacid, respectively. If the tolyl methyl is oxidized before the
isopropyl group, no stereochemical inversion is observed upon oxidation of the
isopropyl side-chain. On the basis of the observed stereochemical changes, it
wasconcludedthatomega-hydroxylationofp-cymeneorp-isopropylbenzoicacid
occurs preferentially at the pro-S-methyl group of the isopropyl substituent
(Matsumotoetal.,1992).
According to these studies, p-cymene undergoes extensive oxidation of the
methylsubstituentandisopropylside-chaintoyieldpolaroxygenatedmetabolites
(Figure 1). These metabolites are either excreted unchanged in the urine, or
undergoconjugationwithglucuronicacidand/orglycine,followedbyexcretionin
theurine.
Studiesinvitrowithsupernatantsofhomogenizedratandmouseliver(Creaven
& Parke, 1966) and rat hepatocytes (Wiebkin et al., 1976) have shown that
OH
2-(p-tolyl)-2-propanol p-Cymene
HO
2-(p-tolyl)-1-propanol
2-(p-tolyl)-2-propanoic
acid
HO
O
OH
O
OH
p-(1-hydroxy-1-methyl
ethyl)benzoic acid
O
OH
p-isopropylbenzoic
acid
O
OH
HO
p-(2-hydroxy-1-methyl
ethyl)benzoicacid
O
OH
HO
O
p-(1-carboxyethyl)
benzoicacid
Figure 1. Metabolism of p-cymene
300 AROMATIC HYDROCARBONS
L1
biphenyl (No. 1332) is largely metabolized by hydroxylation at C4, followed by
conjugationand/orfurtherhydroxylation.HydroxylationatC2inbiphenyloccurred
toamuchsmallerextent.Studiesinratsinvivo(oralandintraperitonealadminis-
trationofbiphenyl)andmice(intraperitonealadministrationonly)confrmedthese
fndings,i.e.more4-hydroxybiphenylthan2-hydroxybiphenyl(bothasconjugates)
waspresentintheurine(Creaven&Parke,1966).
In another study in rats, several hydroxylated metabolites of biphenyl were
present in the urine after oral administration of biphenyl. The major metabolites
were4,4-dihydroxybiphenyland4-hydroxybiphenyl,butseveralothermono-,di-,
and trihydroxy compounds were reported, including 2- and 3-hydroxybiphenyl,
3,4- and 3,4-dihydroxybiphenyl, and 3,4,4-trihydroxybiphenyl.Trace amounts of
the3-and4-methylethersof3,4-dihydroxybiphenyland3,4,4-trihydroxybiphenyl
were also detected. Only small amounts were present as free phenolic
compounds; nearly all the metabolites in urine were detected after enzymatic
hydrolysis. The most prominent metabolites present in the bile of these rats
were the conjugates of 4-hydroxybiphenyl, 4,4-dihydroxybiphenyl and 3,4,4-
trihydroxybiphenyl(Meyer&Scheline,1976).
Westetal.(1956)identifeddiphenylmercapturicacidasanadditional(minor)
metaboliteintheurineofbiphenyl-treatedrats,aswellas4-hydroxybiphenyl(plus
conjugate),4,4-dihydroxybiphenyland3,4-dihydroxybiphenyl,demonstratingthat
formationofareneoxideistheinitialstepinthemetabolismofbiphenyl.
Studiesinrabbitsandguinea-pigsshowedthatmostofanoraldoseofbiphenyl
is excreted in the urine as 4-hydroxybiphenyl conjugated with either sulfate or
glucuronicacid.Di-and/ortrihydroxymetaboliteswerealsodetectedintheurine
in rabbits, but in much smaller amounts than previously reported for the rat. In
guinea-pigs,onlysmallamountsofdihydroxymetabolitesandnotrihydroxymetab-
oliteswereseen,(Meyer,1977).
Itcanbeconcludedthatthemetabolismofbiphenylproceedsviaringhydrox-
ylation, followed by conjugation and/or further hydroxylation and subsequent
conjugation.
In summary
Onthebasisoftheavailabledata,itisanticipatedthatthearomatichydrocar-
bonsinthisgroupwillparticipateinsimilarpathwaysofmetabolicdetoxicationin
mammals, including humans.After absorption, these hydrocarbons are oxidized
to polar oxygenated metabolites via CYP enzymes and alcohol and aldehyde
dehydrogenases.
The major metabolic pathway of aromatic terpene hydrocarbons involves
hepaticmicrosomalCYP-mediatedoxidationofringside-chains,yieldingalcohols,
aldehydes,andacids.Themetabolitesarethenconjugatedwithglycine,glucuronic
acid, or glutathione, and excreted in the urine and/or bile.The biotransformation
ofbiphenylproceedsviaringhydroxylation,preferentiallyattheC4position,yield-
ing phenolic derivatives that are subsequently metabolized to glucuronide and
sulfateconjugates,whichareexcretedintheurine.
AROMATIC HYDROCARBONS 301
L1
2.3.2 Toxicological studies
(a) Acute toxicity
Oralmedianlethaldose(LD
50
)valueshavebeenreportedforthreeofthefve
substancesinthisgroup,onebeingtestedinbothratsandrabbits,andtheother
two only in rats (see Table 3). The only reported oral LD
50
value in rabbits was
2410mg/kgbw(Deichmannetal.,1947).Inrats,oralLD
50
valuesrangedfrom2570
to 5040mg/kgbw (Deichmann et al., 1947; Pecchiai & Saffotti, 1957; Jenner et
al.,1964;Posternaketal.,1975;Clarketal.,1979;Hasegawaetal.,1989).These
LD
50
valuesindicatethattheacuteoraltoxicityofaromatichydrocarbonsislow.
(b) Short-term studies of toxicity
Short-termstudiesoftoxicitywereavailableforthreeofthefvesubstancesin
thisgroup(Boothetal.,1961;Posternaketal.,1969,1975).Theresultsofthese
studiesaresummarizedinTable4anddescribedbelow.
(i) Biphenyl (No. 1332)
Inordertofurtherinvestigatethenephrotoxiceffectsofbiphenylasobserved
inalong-termstudyoftoxicityinrats(seesection2.3.2(c)),astudywascarried
out to determine the extent of potential kidney damage in relation to dose and
exposuretimeandtoassessthereversibilityoftheinjuries.Groupsof42maleand
42 female rats (strain not specifed) were given diets containing 0, 0.1, 0.25, or
0.5%biphenyl,calculated(FoodandDrugAdministration,1993)toprovideaverage
dailyintakesof0,50,125,or250mg/kgbw.Urinewascollectedperiodicallyfrom
fveratsofeachsexpergroup.After30,60,and120daysoftreatment,fverats
ofeachsexpergroupweresacrifcedandsectionsofthekidneyweretakenand
preparedforhistopathologicalexamination.After165daysoftreatment,10ratsof
eachsexatthehighestdosewerereturnedtothecontroldiet;fveratsweresub-
sequentlykilled30dayslater,whiletheotherfvewerekilled60dayslater.
Table 3. Studies of the acute toxicity of aromatic hydrocarbons
administered orally
No. Flavouringagent Species Sex LD
50
Reference
(mg/kgbw)
1325 p-Cymene Rat M,F 4750 Jenneretal.(1964)
1332 Biphenyl Rat M,F 3150(M) Hasegawaetal.(1989)
3550(F)
1332 Biphenyl Rat NR 3280 Deichmannetal.(1947)
1332 Biphenyl
a
Rat F 4100 Clarketal.(1979)
1332 Biphenyl Rat F 5040 Pecchiai&Saffotti(1957)
1332 Biphenyl Rabbit NR 2410 Deichmannetal.(1947)
1334 4-Methylbiphenyl Rat M,F 2570 Posternaketal.(1975)
F,female;M,male;NR,notreported.
a
DowthermA,amixtureofdiphenyl(26%)anddiphenyloxide(72%),wastested.
302 AROMATIC HYDROCARBONS
L1
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AROMATIC HYDROCARBONS 303
L1
Comparedwiththecontrols,ratsatthehighestdosedisplayedanincreasein
urinaryvolumeaswellasurineturbidityandprecipitatebeginningondays27and
28, and continuing progressively until day 165. The same effect, but to a lesser
degree,wasseeninthegroupreceivingtheintermediatedose,butnotinthegroup
receiving the lowest dose. Within 10 days of returning to the control diet, the
volumes of urine, turbidity and precipitate in rats at the highest dose decreased
markedly and returned to normal within 30 days. Histopathological examinations
ofthekidneypreparationsrevealedabnormalitiesthatwereonlyobservedatthe
highestdose,withtheexceptionofasingleprominentlesionfoundinonefemale
ratattheintermediatedose.After30daysoftreatment,thekidneylesionsfound
atthehighestdoseincludedseveralsmallcystsanddilatedtubulesinthemedulla
and inner cortex of one male, and mild local tubular dilation with some epithelial
fattening in two females.The frequency and severity of histopathological effects
on the kidney increased as the study continued.After 60 days of treatment, the
kidneysofthreemalesandallfemalesshoweddistinctlesions.After120daysof
treatment,allratsexhibiteddistinctfocaltubulardilationofthekidneys,involving
only a few tubules in females but several in males. Some small atrophic tubules
andsomecellularfbroustissuewereobservednexttothedilatedtubules.Upon
returning to the control diet after 165 days of exposure to biphenyl, rats showed
aregressionoftubulardilationwithscarformation,buttheregressionwasmore
pronounced in males than in females.The no-observed-effect level (NOEL) was
50mg/kgbwperday,sinceatthisdosetherewasnoevidenceofeitherpolyuria
orhistopathologicaldamagetothekidney(Boothetal.,1961).
(ii) p-a-dimethylstyrene (No. 1333)
Inastudydesignedtotestthetoxicityof42favouringagentsatalevelequiva-
lent to 100 times the maximum estimated daily dietary intake of a human with a
bodyweightof50kg,groupsof1016maleand1016femaleCharlesRiverCD
rats were given diets containing p-a-dimethylstyrene for 90 days, resulting in a
meanintakeof0.625mg/kgbwperday.Bodyweightandfoodconsumptionwere
measuredweeklyandtheeffciencyoffoodutilizationwascalculated.Haematol-
ogyexaminations(onhaemoglobin,erythrocytevolumefraction,erythrocytecount,
and total and differential leukocyte counts) and blood urea determinations were
carriedoutonhalftheanimalsatweek7andonallanimalsatweek13.Atautopsy,
liverandkidneyweightsweremeasuredandgrossandhistologicalexaminations
werecarriedoutonawiderangeoforgans(notspecifed).
No effects of p-a-dimethylstyrene on any of the tested parameters were
reported. The NOEL was 0.625mg/kgbw per day, the highest dose tested
(Posternaketal.,1969).
(iii) 4-methylbiphenyl (No. 1334)
In a study that was similar to that described above, in which 12 favouring
agents were tested, groups of 16 male and 16 female Charles River Sprague
DawleyCDratsweregivendietscontaining4-methylbiphenylfor90days,resulting
inameanintakeof8.73mg/kgbwperday.
304 AROMATIC HYDROCARBONS
L1
Statisticallysignifcant,butveryslight(<10%)decreaseswereobservedinthe
meanterminalbodyweightofmaleratsandinfoodeffciencyforfemalerats.No
effects of 4-methylbiphenyl were reported on haematology parameters, on blood
urealevel,oronweightsoftheliverandkidney.Grossandhistologicalexamina-
tionsonarangeofspecifedorgans/tissuesdidnotrevealanyeffects.TheNOEL
was8.73mg/kgbwperday,thehighestdosetested(Posternaketal.,1975).
(c) Long-term studies of toxicity and carcinogenicity
Long-termstudiesoftoxicityandcarcinogenicitywereonlyavailableforbiphe-
nyl(Ambroseetal.,1960;Innesetal.,1969;Umedaetal.,2002).Theresultsof
thesestudiesaresummarizedinTable4anddescribedbelow.
(i) Biphenyl (No. 1332)
Mice
In a study designed to evaluate the tumorigenicity of a series of substances
used in industrial and agricultural applications, mice were given biphenyl for 18
months.Twostrainsofmicewereused,obtainedbymatingC57BL/6femaleswith
eitherC3H/AnfmalesorAKRmales.Miceweregiventhemaximaltolerateddose,
which was defned as the highest dose that caused zero mortality in a series of
preliminarystudiesinwhichbiphenylwasadministeredfor1,6,andthen19con-
secutivedays.Themaximaltolerateddoseofbiphenylwas2.5mg/kgbwperday.
Thisdosewasgivento18micepersexperstrainbygavageuntiltheanimalswere
aged4weeks,andtheninthedietfortheremainderofthestudy.Theconcentration
ofbiphenylinthedietwas517mg/kg,whichwascalculatedaccordingtotheweight
andfoodconsumptionofthemiceaged4weekstoprovidethemaximaltolerated
dose. Groups of untreated negative controls and treated positive controls were
maintainedunderthesameconditions.Postmortemexaminationsattheendofthe
studyincludedanexternalexaminationandathoroughexaminationofthethoracic
andabdominalcavities,withhistologicalexaminationsofallgrosslyvisiblelesions
andallmajororgans,exceptthecranium.Bloodsmearsweretakenfromallanimals
in the event that either splenomegaly or lymphadenopathy was observed. The
authorsreportednosignifcantincreaseintumoursinmiceofeitherstraintreated
withbiphenylwhencomparedwithnegativecontrols.TheNOELwas2.5mg/kgbw
perday,thehighestdosetested(Innesetal.,1969).
Rats
Groupsof15maleand15femaleweanlingalbinoratsweregivendietscon-
taining0,0.001,0.005,0.01,0.05,0.1,0.5,or1.0%biphenylfor750days,calcu-
lated (Food and Drug Administration, 1993) to provide average daily intakes of
biphenyl of 0, 0.5, 2.5, 5, 25, 50, 250, or 500mg/kgbw. Measurements of body
weightandfoodconsumptionwererecordedperiodicallyforallratsthroughoutthe
study. Haemoglobin determinations were made at regular intervals for male and
female rats assigned to the control group and groups receiving the three higher
doses.Atstudytermination,weightsofliver,kidneys,heartandtestesweredeter-
AROMATIC HYDROCARBONS 305
L1
minedandmajororgans/tissuesweresectionedandstainedformicroscopicexam-
inations.Bone-marrowsmearswerepreparedfromrepresentativeanimalsonly.
Effectsweremainlyobservedinanimalsinthegroupsreceivingthetwohigher
doses (0.5 and 1%). They included dose-dependent reductions in body-weight
gain,foodconsumptionandconcentrationsofhaemoglobin.Mortalitywasincreased
atthesedosesinmales(13outof15diedatboth0.5%and1%,comparedwith
6 out of 15 controls) and females (10 out of 15 at 0.5% and 13 out of 15 at 1%
died, compared with 6 out of 15 controls).Additional paired feeding experiments
conductedatthesedosesfor98daysshowedthattheeffectsonbody-weightgain
wereattributabletoadecreaseinfoodintake,possiblybecauseofreducedpalat-
ability.Organsweightswerenotaffectedatdosesof0.1%andbelow.Inthetwo
survivingmalesat0.5%,absoluteandrelativeweightsofkidneyswereincreased
andoftestesweredecreased.Femalesat0.5%alsohadincreasedabsoluteand
relative kidney weights and, owing to reduced body weights, increased relative
liverweights.Theonlyhistopathologicalchangethatcouldbeattributedtoadmin-
istrationofbiphenyloccurredinthekidneysofbothsexesofratsatthetwohigher
doses. Prominent scarring, lymphocytic infltration, tubular atrophy and dilation,
andhaemorrhageinthedilatedtubulesandrenalpelvisweresomeoftheeffects
observed.Hydronephrosiswascommon,aswassquamouscellmetaplasiaofthe
renal pelvis epithelium, although this did not appear to be neoplastic. The fre-
quency of scars and dilated tubules, as well as the severity of hydronephrosis,
was greater in males at all doses (including controls) than in females. At lower
doses (0.1%), histopathological fndings in the kidneys were not different from
thoseinthecontrols.Treatmentwithbiphenyldidnotresultinincreasedincidences
of tumours. The NOEL was 50mg/kgbw per day on the basis of mortality and
kidneyfndings(Ambroseetal.,1960).
In a long-term study of toxicity and carcinogenicity (reported to comply with
OECDtestguidelineNo.453andGLP),groupsof50maleand50femaleF344/
DuCrjrats(aged6weeks)weregivendietscontainingbiphenylatadietarycon-
centrationof0,500,1500,or4500mg/kgfor105weeks.Thesedietarylevelshave
been calculated (Food and DrugAdministration, 1993) to provide average daily
intakesofbiphenylof0,25,75,or225mg/kgbw.Theanimalswereobserveddaily
for clinical signs, behavioural changes and mortality. Body weight and food con-
sumption were recorded once a week for the frst 14 weeks, and every 4 weeks
thereafter.Urinaryparameters,includingpHandoccultblood,weremeasuredin
allsurvivingratsinthefnalweekofthestudy.Necropsies,organweightdetermi-
nations and histological examinations of a whole range of organs/tissues were
performedonallanimals.
Comparedwiththoseofthecontrols,themeanbodyweightsoftheanimalsat
thehighestdoseweresignifcantlydecreased.Survivalratesinallgroupstreated
withbiphenyl,withtheexceptionofmalesatthehighestdose,werecomparable
tothoseofthecontrolanimals.Nineteenoutof50malesatthehighestdosedied
before the end of the study, and their death was attributed primarily to bladder
tumours and haematuria.Although three females at the highest dose died from
markedmineralizationofthekidneysandtheheartafter1326weeksoftreatment
with biphenyl, the survival rate did not differ from controls thereafter. Clinical
306 AROMATIC HYDROCARBONS
L1
haematuriawasobservedin32malesatthehighestdosefromweeks40105of
exposure;14oftheseanimalshadaskinoreyecolourthatsuggestedanaemia.
No clinical haematuria was observed in the other groups of treated males or in
anyofthegroupsoftreatedfemales.UrineanalysisrevealedincreasedpHofthe
urineinmalesatthehighestdose,whiletheincidenceofpositiveoccultbloodwas
signifcantlyincreasedinmalesand,toalesserdegree,infemalesatthehighest
dose.Relativeweightsofthekidneyweresignifcantlyincreasedinbothsexesat
theintermediateandhighestdoses,whileabsoluteweightofthekidneywassig-
nifcantly increased only in males at the highest dose. Gross fndings included
bladdercalculiinbothsexesat4500mg/kg(in43outof50malesand8outof50
females). No bladder calculi were found in animals at 500 or 1500mg/kg. The
bladder calculi frst appeared at around week 40 of the study, together with the
occurrenceofhaematuria.Inaddition,thickeningofthebladderwallwasreported
infouroftheeightcalculi-bearingfemales.Forty-oneofthe50malesatthehighest
dosehadpolyp-likeorpapillarynodulesprotrudingfromthebladderwallintothe
lumen;38ofthese41werebearingcalculi.Notumourortumour-relatedlesions
were observed in organs other than the urinary tract. In the bladder, tumour or
tumour-related lesions were almost exclusively found in animals at the highest
dose, and included increased incidences of transitional cell hyperplasia in the
epithelium of both males (45 out of 50) and females (10 out of 50), squamous
metaplasia(in19outof50malesand4outof50females),squamouscellhyper-
plasia(in13outof50malesand1outof50females),infammatorypolyps(in10
out of 50 males, not in females), and calculi (in 43 out of 50 males and 8 out of
50females).Thetransitionalcellhyperplasiawerecharacterizedassimple,nodular
and papillary and developed focally on the bladder epithelium. Bladder tumours
wereonlyfoundinmalesatthehighestdose,andincludedtransitionalcellpapil-
lomas(10outof50)andcarcinomas(24outof50),andsquamouscellpapilloma
and carcinoma (1 out of 50).The authors noted that all 24 of the male rats with
transitionalcellcarcinomasand8ofthe10maleratswithtransitionalcellpapillo-
mas were found to have bladder calculi. In other parts of the urinary tract, only
non-neoplastic lesions were observed, no tumours. In the ureter, fndings were
limitedtothehighestdoseandincludedincreasedincidences(statisticallysignif-
cantlyonlyinmales)ofsimpletransitionalcellhyperplasia(8outof50malesand
2outof50females)anddilation(14outof50malesand6outof50females).In
the kidneys, incidences of simple and nodular transitional cell hyperplasia of the
renal pelvis were signifcantly increased in males at the highest dose and in
females at the intermediate and highest doses. Mineralization of the renal pelvis
wasincreasedinmalesatthehighestdoseandinfemalesattheintermediateand
highestdoses,butstatisticalsignifcancewasonlyreachedforthelatter.Theinci-
dencesofcalculianddesquamationoftherenalpelviswereincreasedinanimals
atthehighestdose(statisticallysignifcantlyonlyinmales).Othersignifcantfnd-
ingsinthekidneyincludedincreasedincidencesofpapillarymineralization,papil-
lary necrosis and infarct in females at the highest dose, and of haemosiderin
deposits in females at the intermediate and highest doses. In males, increased
incidencesofmineralizationofthecortico-medullaryjunctionandpapillarynecrosis
(not statistically signifcantly) were observed at the highest dose, and increased
incidences of papillary mineralization at the intermediate (not statistically signif-
cantly) and highest doses. Chronic nephropathy was reported for both sexes of
AROMATIC HYDROCARBONS 307
L1
thecontrolanimalsandthetreatedanimalsatalldoses(in45,45,43,and34out
of 50 males, and in 33, 35, 30, and 26 out of 50 females, at 0, 500, 1500, and
4500mg/kg, respectively). The NOEL in this study was 25mg/kgbw per day
(Umedaetal.,2002).
Mechanism of induction of bladder tumours by biphenyl
in rats and relevance to humans
Studieshavebeenconductedonthemechanismunderlyingthepredominant
formationofbladdercalculiinmaleratscomparedwithfemaleratsafteradminis-
tration of biphenyl.Analysis of the urinary calculi formed in the 105-week study
described above revealed that the calculi consisted principally of potassium 4-
hydroxybiphenyl O-sulfate (4-HBPOSK) in males and of 4-hydroxybiphenyl (4-
HBP) and KHSO
4
in females. The shape and colour of the calculi were also
differentbetweensexes,aswerethestructureanddistributionofcomponentele-
ments. The calculi in males had a multilayer structure with alternating layers of
calcium phosphate and 4-HBPOSK, with the latter being the centre component
andtheinsideareaofeachlayer.Infemales,thecalculiweredescribedasbeing
single layered with open holes where needle shaped crystals were present.The
authors attributed the differences in the principal constituents and the structural
formation of the calculi to the increased hydrolysis of 4-HBPOSK to 4-HBP and
KHSO
4
inthefemaleratascomparedwiththemalerat.Theyalsonotedthatthis
wasconsistentwiththeobservedlowerpHofthefemaleurinecomparedwiththe
male urine, as the lower the pH, the greater the extent of hydrolysis (Ohnishi et
al., 2000). Ohnishi et al. (2001) observed that co-adminstration of biphenyl and
potassiumbicarbonate(KHCO
3
)inthedietofmaleratsfor13weeksresultedin
theformationofurinecrystalsconsistingof4-HBPOSK.Thiswasinducedbythe
higher concentration of potassium in the urine and the higher urinary pH as a
consequenceoffeedingwithKHCO
3
.Theseurinecrystalsproducedhyperplasia
of the transitional epithelium of the ureter, uretal obstruction and hydronephrosis
intheurinarytract(Ohnishietal.,2001).
Themodeofactionbywhichlong-termadministrationofbiphenylathighdoses
inducesmale-specifcbladdertumoursinratsisnotfullyunderstood.However,in
all probability, the induction of tumours is secondary to the formation of bladder
calculi, which in males results from the precipitation of the potassium salt of
4-hydroxybiphenyl-O-sulfate. These calculi then induce sustained mechanical
damage, thereby evoking haematuria and a regenerative response. This is sup-
ported by the fndings that bladder tumours occurred in close association with
calculusformationandhaematuria.Itisalsoconsistentwiththeobservedsexdif-
ferencesinstructureandcompositionofcalculiandinoccurrenceofhaematuria,
which was absent in females. The postulated mechanism appears to be dose-
dependent,giventheverysteepdoseresponserelationshipsfoundfortheneo-
plastic and associated preneoplastic lesions, i.e. complete lack of these fndings
at500and1500mg/kg,butpresenceatthenexthigherdoseof4500mg/kg.
Whenaddressingthepredictivevalueofcertaintypesoftumoursfortheiden-
tifcationofcarcinogenichazardstohumans,theInternationalAgencyforResearch
on Cancer (IARC) (IARC, 1999) concluded the following with respect to the
308 AROMATIC HYDROCARBONS
L1
relevance of calculi- and microcrystalluria-associated urinary bladder neoplasms
inrodents:
Forchemicalsproducingbladderneoplasmsinratsandmiceasaresultofcalculusformation
intheurinarybladder,theresponsecannotbeconsideredtobespecies-specifc;thus,the
tumourresponseisrelevanttoanevaluationofcarcinogenicitytohumans.Therearequan-
titativedifferencesinresponsebetweenspeciesandsexes.Calculusformationisdependent
ontheattainmentintheurineofcriticalconcentrationsofconstituentchemicalswhichform
thecalculus;therefore,thebiologicaleffectsaredependentonreachingthresholdconcentra-
tionsforcalculusformation.Microcrystalluriaisoftenassociatedwithcalculusformation,but
itsrelevancetospecies-specifcmechanismscannotbeassessed.
TheCommittee,however,concludedthatthebladdertumoursinducedbylong-
term administration of biphenyl at high doses are not relevant for human risk
assessment.Owingtothedifferenceinanatomicalpositionofthebladder(vertical
in humans versus horizontal in rodents), humans will more easily lose calculi
formed,ifany.Itisalsoveryunlikelythathumanswillbeexposedtobiphenylat
thehighdosesneededtoinducetheformationofcalculi,owingtoprecipitationof
4-hydroxybiphenylO-sulfate.
(d) Genotoxicity
Testingforgenotoxicityhasbeenperformedonfourofthefvefavouringagents
in this group (Nos 1325, 1332, 1334, 1335).The results of these tests are sum-
marizedinTable5anddescribedbelow.
(i) In vitro
No evidence of mutagenicity was observed in standard or modifed Ames
assayswhenp-cymene(No.1325;upto85300mg/plate),biphenyl(No.1332;up
to10000mg/plate),4-methylbiphenyl(No.1334;upto1000mg/plate),or1-methyl-
naphthalene (No. 1335; up to 4266mg/plate) were incubated with Salmonella
typhimuriumstrainsTA97,TA98,TA100,TA1535,TA1537,TA1538,and/orTA1532,
TA2636, TA2637, G46, C3076, or D3052 with and without metabolic activation
(Clarketal.,1977,1979;Anderson&Styles,1978;Rockwell&Raw,1979;Florin
et al., 1980; Hirayama et al., 1981; Probst et al., 1981; Haworth et al., 1983;
Pagano et al., 1983; Nohmi et al., 1985; Brams et al., 1987; Houk et al., 1989;
NationalToxicologyProgram,2004a,2004b,2004c;Zeigeretal.,1992).Biphenyl
alsogavenegativeresultswhenincubatedwithEscherichia colistrainsWP2and
WP2uvrA
-
inthemodifedAmestest(Probstetal.,1981),strainPQ37intheSOS
chromotest (up to 154mg/ml) (Brams et al., 1987), and with strains WP2, WP2
uvrA,WP100,andCM571inatestforDNArepair(upto4000mg/disk)(Hirayama
etal.,1981).
IncontrasttothenegativeresultsobtainedforbiphenylinS. typhimuriumand
E. coli systems, biphenyl (No. 1332) produced genetic effects in an assay with
Saccharomyces cerevisiae strain D7, with and without metabolic activation, at
concentrations of up to 1mmol/l (Pagano et al., 1983). In an assay for forward
mutation in mouse lymphoma cells, biphenyl produced signifcant increases in
mutation frequency in L5178Y cells at concentrations of 45.660.9mg/ml without
AROMATIC HYDROCARBONS 309
L1
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/
p
l
a
t
e
d

N
e
g
a
t
i
v
e
e

C
l
a
r
k

e
t

a
l
.

(
1
9
7
7
)

T
A
1
5
3
5
,

T
A
1
5
3
7
,

T
A
1
5
3
8
1
3
3
2

B
i
p
h
e
n
y
l

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
A
9
8
,

2
5
0
0

m
g
/
p
l
a
t
e

N
e
g
a
t
i
v
e
b

A
n
d
e
r
s
o
n

&

S
t
y
l
e
s

T
A
1
0
0
,

T
A
1
5
3
5
,

(
1
9
7
8
)

T
A
1
5
3
8
1
3
3
2

B
i
p
h
e
n
y
l
c

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
A
9
8
,

1
0

0
0
0

m
g
/
p
l
a
t
e

N
e
g
a
t
i
v
e
e

C
l
a
r
k

e
t

a
l
.

(
1
9
7
9
)

T
A
1
0
0
,

T
A
1
5
3
5
,

T
A
1
5
3
7
,

T
A
1
5
3
8
1
3
3
2

B
i
p
h
e
n
y
l

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
A
9
8
,

1
0
0

m
g
/
p
l
a
t
e

N
e
g
a
t
i
v
e
e

H
i
r
a
y
a
m
a

e
t

a
l
.

T
A
1
0
0

(
1
9
8
1
)
1
3
3
2

B
i
p
h
e
n
y
l

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n
f

S
.

t
y
p
h
i
m
u
r
i
u
m

G
4
6
,

N
R
g

N
e
g
a
t
i
v
e
e

P
r
o
b
s
t

e
t

a
l
.

(
1
9
8
1
)

T
A
9
8
,

T
A
1
0
0
,

T
A
1
5
3
5
,

T
A
1
5
3
7
,

T
A
1
5
3
8
,

C
3
0
7
6
,

D
3
0
5
2
;

E
s
c
h
e
r
i
c
h
i
a

c
o
l
i

W
P
2

a
n
d

W
P
2
u
v
r
A
-
1
3
3
2

B
i
p
h
e
n
y
l

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
A
9
8
,

1
0
0

m
g
/
p
l
a
t
e
h

N
e
g
a
t
i
v
e
e

H
a
w
o
r
t
h

e
t

a
l
.

T
A
1
0
0
,

T
A
1
5
3
5
,

T
A
1
5
3
7

(
1
9
8
3
)
;

N
T
P

(
2
0
0
4
a
/
b
)
1
3
3
2

B
i
p
h
e
n
y
l

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
A
9
8
,

0
.
1

5
0
0

m
g
/
p
l
a
t
e
i

N
e
g
a
t
i
v
e
e

P
a
g
a
n
o

e
t

a
l
.

T
A
1
0
0
,

T
A
1
5
3
2
,

T
A
1
5
3
5
,

(
1
9
8
3
)

T
A
1
5
3
7
,

T
A
1
5
3
8
,

T
A
2
6
3
6
310 AROMATIC HYDROCARBONS
L1
T
a
b
l
e

5
.

(
c
o
n
t
d
)
N
o
.

F
l
a
v
o
u
r
i
n
g

a
g
e
n
t

E
n
d
-
p
o
i
n
t

T
e
s
t

s
y
s
t
e
m

D
o
s
e

o
r

c
o
n
c
e
n
t
r
a
t
i
o
n

R
e
s
u
l
t

R
e
f
e
r
e
n
c
e
1
3
3
2

B
i
p
h
e
n
y
l

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
A
9
8
,

1
0

5
0
0
0

m
g
/
p
l
a
t
e
j

N
e
g
a
t
i
v
e
e

N
o
h
m
i

e
t

a
l
.

T
A
1
0
0
,

T
A
2
6
3
7

(
1
9
8
5
)
1
3
3
2

B
i
p
h
e
n
y
l

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

1
0
0

m
g
/
p
l
a
t
e

N
e
g
a
t
i
v
e
e

B
r
a
m
s

e
t

a
l
.

(
1
9
8
7
)

T
A
9
7
,

T
A
9
8
,

T
A
1
0
0
1
3
3
2

B
i
p
h
e
n
y
l

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
A
9
8
,

2
0
0
0

m
g
/
p
l
a
t
e

N
e
g
a
t
i
v
e
e

H
o
u
k

e
t

a
l
.

(
1
9
8
9
)

T
A
1
0
0
1
3
3
2

B
i
p
h
e
n
y
l

S
O
S

i
n
d
u
c
t
i
o
n

E
s
c
h
e
r
i
c
h
i
a

c
o
l
i

P
Q
3
7

2
.
4

1
5
4

m
g
/
m
l

N
e
g
a
t
i
v
e
e

B
r
a
m
s

e
t

a
l
.

(
1
9
8
7
)
1
3
3
2

B
i
p
h
e
n
y
l

D
N
A

r
e
p
a
i
r

E
.

c
o
l
i

4
0
0
0

m
g
/
d
i
s
c

N
e
g
a
t
i
v
e

H
i
r
a
y
a
m
a

e
t

a
l
.

W
P
2
,

W
P
2
u
v
r
A
,

C
M
5
7
1
,

(
1
9
8
1
)

W
P
1
0
0
1
3
3
2

B
i
p
h
e
n
y
l

M
i
t
o
t
i
c

r
e
c
o
m
b
i
n
a
t
i
o
n
,

S
a
c
c
h
a
r
o
m
y
c
e
s

c
e
r
e
v
i
s
i
a
e

m
m
o
l
/
l

P
o
s
i
t
i
v
e
e

P
a
g
a
n
o

e
t

a
l
.

g
e
n
e

c
o
n
v
e
r
s
i
o
n
,

D
7

p
l
a
t
e
)
a

(
1
5
4

m
g
/
m
l
)
k

(
1
9
8
3
)

r
e
v
e
r
s
i
o
n
1
3
3
2

B
i
p
h
e
n
y
l

F
o
r
w
a
r
d

m
u
t
a
t
i
o
n

M
o
u
s
e

l
y
m
p
h
o
m
a

L
5
1
7
8
Y

9
8
.
7

3
9
5

m
m
o
l
/
l

-
S
9

P
o
s
i
t
i
v
e
n
;

W
a
n
g
e
n
h
e
i
m

&

T
k
+
/
-

c
e
l
l
s

(
1
5
.
2

6
0
.
9

m
g
/
m
l
)
k
,
l
;

B
o
l
c
s
f
o
l
d
i

(
1
9
8
8
)

5
.
0
1

6
0

m
m
o
l
/
l

+
S
9

P
o
s
i
t
i
v
e
o

(
0
.
8

9
.
3

m
g
/
m
l
)
k
,
m
1
3
3
2

B
i
p
h
e
n
y
l

S
i
s
t
e
r

c
h
r
o
m
a
t
i
d

C
h
i
n
e
s
e

h
a
m
s
t
e
r

D
o
n

c
e
l
l
s

0
.
1

m
m
o
l
/
l

N
e
g
a
t
i
v
e
p
,
q

A
b
e

&

S
a
s
a
k
i

e
x
c
h
a
n
g
e

(
1
5
.
4

1
5
4

m
g
/
m
l
)
k

(
1
9
7
7
)
1
3
3
2

B
i
p
h
e
n
y
l

C
h
r
o
m
o
s
o
m
a
l

a
b
e
r
r
a
t
i
o
n

C
h
i
n
e
s
e

h
a
m
s
t
e
r

D
o
n

c
e
l
l
s

0
.
1

m
m
o
l
/
l

N
e
g
a
t
i
v
e
p

A
b
e

&

S
a
s
a
k
i

(
1
5
.
4

1
5
4

m
g
/
m
l
)
k

(
1
9
7
7
)
1
3
3
2

B
i
p
h
e
n
y
l

U
n
s
c
h
e
d
u
l
e
d

D
N
A

R
a
t

h
e
p
a
t
o
c
y
t
e
s

0
.
0
1

1
0
0
0

m
m
o
l
/
l

N
e
g
a
t
i
v
e

B
r
o
u
n
s

e
t

a
l
.

s
y
n
t
h
e
s
i
s

(
0
.
0
0
2

1
5
4

m
g
/
m
l
)
k

(
1
9
7
9
)
1
3
3
2

B
i
p
h
e
n
y
l

U
n
s
c
h
e
d
u
l
e
d

D
N
A

R
a
t

h
e
p
a
t
o
c
y
t
e
s

0
.
5

1
0
0
0

n
m
o
l
/
m
l

N
e
g
a
t
i
v
e

P
r
o
b
s
t

e
t

a
l
.

(
1
9
8
1
)

s
y
n
t
h
e
s
i
s

(
0
.
0
8

1
5
4

m
g
/
m
l
)
k
,
r
1
3
3
2

B
i
p
h
e
n
y
l

U
n
s
c
h
e
d
u
l
e
d

D
N
A

R
a
t

h
e
p
a
t
o
c
y
t
e
s

0
.
1

1
0
0

m
m
o
l
/
l

N
e
g
a
t
i
v
e

H
s
i
a

e
t

a
l
.

(
1
9
8
3
)

s
y
n
t
h
e
s
i
s

(
0
.
0
2

1
5
.
4

m
g
/
m
l
)
k
AROMATIC HYDROCARBONS 311
L1 1
3
3
4

4
-
M
e
t
h
y
l
b
i
p
h
e
n
y
l
s

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
A
9
7
,

0
.
1

1
0

m
g
/
p
l
a
t
e

-
S
9
h
;

N
e
g
a
t
i
v
e
e

Z
e
i
g
e
r

e
t

a
l
.

(
1
9
9
2
)

T
A
9
8
,

T
A
1
0
0
,

T
A
1
5
3
5

1
0

1
0
0
0

m
g
/
p
l
a
t
e

+
S
9
h
1
3
3
5

1
-
M
e
t
h
y
l
n
a
p
h
t
h
a
l
e
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
A
9
8
,

0
.
0
3

3
0

m
m
o
l
/
p
l
a
t
e

N
e
g
a
t
i
v
e
e

F
l
o
r
i
n

e
t

a
l
.

(
1
9
8
0
)

T
A
1
0
0
,

T
A
1
5
3
5
,

T
A
1
5
3
7

(
4
.
3

4
2
6
6

m
g
/
p
l
a
t
e
)
t
,
u
1
3
3
5

1
-
M
e
t
h
y
l
n
a
p
h
t
h
a
l
e
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
A
9
7
,

0
.
3

3
3

m
g
/
p
l
a
t
e

-
S
9
h
;

N
e
g
a
t
i
v
e
e

N
a
t
i
o
n
a
l

T
A
9
8
,

T
A
1
0
0
,

T
A
1
5
3
5

1
0
0

m
g
/
p
l
a
t
e

+
S
9

T
o
x
i
c
o
l
o
g
y

P
r
o
g
r
a
m

(
2
0
0
4
c
)
a

C
a
l
c
u
l
a
t
e
d

u
s
i
n
g

d
e
n
s
i
t
y

o
f

p
-
c
y
m
e
n
e

o
f

0
.
8
5
3

g
/
m
l

(
L
e
w
i
s
,

1
9
9
9
)
.
b

W
i
t
h

m
e
t
a
b
o
l
i
c

a
c
t
i
v
a
t
i
o
n
.
c

D
o
w
t
h
e
r
m

A
,

m
i
x
t
u
r
e

o
f

d
i
p
h
e
n
y
l

(
2
6
%
)

a
n
d

d
i
p
h
e
n
y
l

o
x
i
d
e

(
7
2
%
)
,

w
a
s

t
e
s
t
e
d
.
d

C
y
t
o
t
o
x
i
c
i
t
y

o
b
s
e
r
v
e
d

a
t

t
h
e

h
i
g
h
e
s
t

d
o
s
e
/
c
o
n
c
e
n
t
r
a
t
i
o
n

t
e
s
t
e
d
.
e

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312 AROMATIC HYDROCARBONS
L1
metabolicactivation,and3.19.3mg/mlwithactivation(Wangenheim&Bolcsfoldi,
1988).However,theincreaseswere2-foldonlyat60.9mg/mlwithoutactivation,
and6.29.3mg/mlwithactivation.Attheseconcentrations,cellviabilitywas15%.
At lower concentrations of 15.230.4mg/ml without metabolic activation, and
0.81.5mg/ml with activation, biphenyl gave negative results (Wangenheim &
Bolcsfoldi,1988).Cellviabilitywasmuchhigherattheselowerconcentrations(at
least49%).Biphenyldidnotinducesisterchromatidexchanges(SCE)orchromo-
somalaberrationsinChinesehamsterDoncellsatconcentrationsofupto154mg/
mlwithoutmetabolicactivation(Abe&Sasaki,1977),nordiditinduceunscheduled
DNA synthesis in rat hepatocytes at concentrations of 0.002154mg/ml (Brouns
etal.,1979;Probstetal.,1981;Hsiaetal.,1983).
In a study designed to investigate the mutagenicity in vivo-in vitro of urinary
metabolitesofanumberoffoodadditives,Sprague-Dawleyratsweregiven0.5ml
ofp-cymene(No.1325;approximately1706mg/kgbw)bygavageandurinewas
collected for 24h. Three types of urine samples were tested in theAmes assay
with S. typhimurium strains TA98 and TA100 with metabolic activation: a direct
urine sample, a urine-ether extract, and the aqueous fraction of the urineether
extract. The urine samples of rats treated with p-cymene did not show any evi-
dence of mutagenicity, either in the presence or absence of b-glucuronidase
(Rockwell&Raw,1979).
(ii) Conclusion
Four substances in this group of favouring agents have been tested in the
Ames assay and found not to be mutagenic in vitro in bacteria. In addition to
showing no mutagenic potential in theAmes assay, biphenyl produced negative
resultsinE. coliintheSOSchromotestandDNArepairtest.Ontheotherhand,
biphenylproducedgeneticeffectsinyeast(S. cerevisiae).
In mammalian cell systems, negative results were obtained for biphenyl with
respect to induction of SCE, chromosomal aberration, and unscheduled DNA
synthesis. The positive fnding for biphenyl in an assay for forward mutation in
mouselymphomacellswasobtainedatnearlethalconcentrations.
Onthebasisoftheresultsofavailablestudiesofgenotoxicity,theCommittee
concluded that the favouring agents in this group of aromatic hydrocarbons are
notgenotoxic.
(e) Reproductive toxicity
(i) Biphenyl (No. 1332)
The potential effects of biphenyl on reproduction and survival of pups were
examined in ten female and fve male weanling albino rats (strain not specifed)
that were mated (one male to two female rats) after 60 days of being fed a diet
containing biphenyl at 0 or 0.1%. Another nine females and three males were
mated(onemaletothreefemales)after60daysofdietaryexposuretobiphenyl
at 0.5%. All the rats were kept on their respective diets until all the pups were
AROMATIC HYDROCARBONS 313
L1
weaned. In a second experiment with rats aged 90 days, eight to nine females
andthreetofourmaleswereplacedondietscontainingbiphenylat0,0.1,or0.5%
for11daysbeforebeingmated.
Biphenylwasreportedtohavenosignifcanteffectonreproduction,although
the number of litters and pups born was slightly lower in the second experiment
atthehighestdoseof0.5%(Ambroseetal.,1960).
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Arch. Biochem. Biophys.,60,1420.
WHO (1967) Evaluations of some pesticide residues in food (WHO Food Add./67.32).
Geneva:WorldHealthOrganization
WHO (1968) Evaluations of some pesticide residues in food (WHO Food Add./68/30).
Geneva:WorldHealthOrganization.
Wiebkin, P., Fry, J.R., Jones, C.A., Lowing, R. & Bridges, J.W. (1976) The metabolism of
biphenylbyisolatedviablerathepatocytes.Xenobiotica,6,725743.
Zeiger,E.,Anderson,B.,Haworth,S.,Lawlor,T.&Mortelmans,K.(1992)Salmonellamuta-
genicitytests:V.Resultsfromthetestingof311chemicals.Environ. Mol. Mutag.,19(Suppl.
21),2141.
L1
317
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES, ACIDS AND
RELATED ALCOHOLS, ACETALS AND ESTERS
First draft prepared by
Professor G.M. Williams
1
and Professor J.R. Bend
2
1
Department of Pathology, New York Medical College, NY, USA; and
2
Department of Pharmacology & Toxicology, Faculty of Medicine and
Dentistry, University of Western Ontario, London, Ontario, Canada
Evaluation ............................................................................... 317
Introduction......................................................................... 317
Estimateddailyintake........................................................ 326
Absorption,distribution,metabolismandelimination........ 326
ApplicationoftheProcedurefortheSafetyEvaluation
ofFlavouringAgents.................................................... 332
Considerationofsecondarycomponents.......................... 332
Considerationofcombinedintakesfromuseas
favouringagents......................................................... 332
Conclusions........................................................................ 333
Relevantbackgroundinformation............................................. 333
Additionalconsiderationsonintake................................... 333
Biologicaldata.................................................................... 333
Biochemicaldata......................................................... 333
Hydrolysis.............................................................. 333
Absorption,distribution,andexcretion.................. 336
Metabolism............................................................ 338
Toxicologicalstudies.................................................... 347
Acutetoxicity......................................................... 347
Short-termstudiesoftoxicity................................. 348
Long-termstudiesoftoxicityandcarcinogenicity. 354
Genotoxicity........................................................... 360
References............................................................................... 375
1. EVALUATION
1.1 Introduction
TheCommitteeevaluatedagroupof37aliphatic,lineara,b-unsaturatedalde-
hydes,acidsandrelatedalcohols,acetalsandestersfavouringagents(Table1)
by the Procedure for the Safety Evaluation of FlavouringAgents (see Figure 1,
p 192). The Committee has not previously evaluated any member of the group.
The group included nine 2-alkenals (Nos 1349, 1350, 1353, 1359, 1360, 1362
1364and1366),six2-alken-1-ols(Nos1354,1365,1369,1370,1374and1384),
fve 2-alkenoic acids (Nos 1361, 13711373 and 1380), 16 related alkenoic and
alkynoicacidesters(Nos1348,1351,1352,13551358,1367,1368,13751379,
1381,1382),andoneacetal(No.1383).
318 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
T
a
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e

1
.

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.
7
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 319
L1
E
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3
8
320 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
T
a
b
l
e

1
.

(
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)
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4
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 321
L1
2
-
O
c
t
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a
l

1
3
6
3

2
3
6
3
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9
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5

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9
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1
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7
6
4
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6

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3
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c
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c
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1
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4
1
4
5
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0
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0
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2
322 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
T
a
b
l
e

1
.

(
c
o
n
t
d
)
F
l
a
v
o
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g

a
g
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o
.

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A
S

N
o
.

a
n
d

s
t
r
u
c
t
u
r
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S
t
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p

A
3
b

C
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m
m
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C
o
n
c
l
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s
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b
a
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D
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1
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l

1
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0

1
8
4
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9
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1
7
-
1

N
o

S
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e

n
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N
o

s
a
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t
y

c
o
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0
.
2
(
E
)
-
2
-
B
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t
e
n
o
i
c

a
c
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d

1
3
7
1

1
0
7
-
9
3
-
7

N
o

S
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n
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N
o

s
a
f
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t
y

c
o
n
c
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r
n

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7
(
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)
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a
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1
3
7
2

3
3
4
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4
9
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6

N
o

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a
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4
(
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)
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n
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a
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1
3
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3

1
0
3
5
2
-
8
8
-
2

N
o

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e

n
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t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

O
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H

E
u
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4
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 323
L1
(
E
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3
324 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
T
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1
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ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 325
L1 2
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.
326 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
Twenty-eightofthe37favouringagents(Nos13491351,13531355,1359
1366,13691378,13801382and1384)inthisgrouphavebeenreportedtooccur
naturallyinfoodsandhavebeendetectedinbeef,chicken,fsh,freshfruit,cheese,
tea, coffee and beer (Nijssen et al., 2003). Exposure to an important favouring
agent in the group, 2-hexenal (No. 1353), occurs primarily through consumption
oftraditionalfoods(Stofberg&Grundschober,1987).
1.2 Estimated daily intake
Thetotalannualvolumeofproductionofthe37favouringagentsinthisgroup
is approximately 10000kg in Europe (International Organization of the Flavour
Industry, 1995) and 7100kg in the USA (National Academy of Sciences, 1970,
1982, 1987; Lucas et al., 1999) (see Table 2). Approximately 95% of the total
annual volume of production in Europe and 81% in the USA is accounted for by
2-hexenal(No.1353),thecorrespondingalcohol2-hexen-1-ol(No.1354),andthe
correspondingacetateester(E)-2-hexen-ylacetate(No.1355).Ofthese,2-hexenal
accountsforapproximately54%ofthetotalannualvolumeofproductioninEurope
and44%intheUSA.Theestimateddailyintakesof2-hexenalinEuropeandthe
USAwere791and409mg/person,respectively.Thedailyintakesofalltheother
favouringagentsinthegroupwereintherangeof0.01395mg/person(National
AcademyofSciences,1970,1982,1987;InternationalOrganizationoftheFlavour
Industry, 1995; Lucas et al., 1999), with most values being at the lower end of
this range. The estimated daily per capita intake of each agent is reported in
Table1.
1.3 Absorption, distribution, metabolism and elimination
In general, aliphatic esters formed from 2-alkenols and carboxylic acids are
more rapidly hydrolysed than their saturated alcohol counterparts (Heymann,
1980). Hydrolysis of esters or acetals has been shown to occur in simulated
stomach juice, simulated intestinal fuid, plasma, and liver microsomes (Knoefel,
1934;Morgareidge,1962,Longlandetal.,1977).Ifhydrolysedbeforeabsorption,
the resulting aliphatic alcohols and carboxylic acids are rapidly absorbed in the
gastrointestinal tract. The unsaturated alcohols are successively oxidized to the
corresponding aldehydes and carboxylic acids, which participate in fundamental
biochemicalpathways,includingthefattyacidpathwayandtricarboxylicacidcycle
(Nelson&Cox,2000).
a,b-Unsaturated aldehydes are formed endogenously by lipid peroxidation
ofpolyu0nsaturatedfattyacids(Frankeletal.,1987),ortheycanbeingestedas
naturallyoccurringconstituentsoffood(Stofberg&Grundschober,1987;Nijssen
etal.,2003)and,toaminorextent,asaddedfavouringagents.Underconditions
of glutathione depletion and oxidative stress, high intracellular concentrations
of a,b-unsaturated aldehydes have been shown to form adducts with proteins
(Ichihashietal.,2001)andDNA(Frankeletal.,1987;Ederetal.,1993;Eisenbrand
etal.,1995;Golzeretal.,1996;Cadetetal.,1999;NationalToxicologyProgram,
2001a), resulting in cellular toxicity and DNA fragmentation during apoptosis.
At low intakes, a,b-unsaturated aldehydes undergo metabolic detoxication by
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 327
L1
T
a
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2
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c
c
u
r
r
i
n
g

p
e
r

d
a
y
)
c

p
e
r

d
a
y
)
d

f
o
o
d
s

(
k
g
)
e
B
u
t
y
l

2
-
d
e
c
e
n
o
a
t
e

(
1
3
4
8
)

E
u
r
o
p
e

0
.
1

0
.
0
1

0
.
0
0
0
2

0
.
0
0
0
0
7

U
S
A

0
.
3

0
.
0
0
4

0
.
0
0
1

N
A
2
-
D
e
c
e
n
a
l

(
1
3
4
9
)

E
u
r
o
p
e

1
9

0
.
0
4
5

U
S
A

4
5

0
.
0
9
9

3
4

1
1
1

7
5
8
2
-
D
o
d
e
c
e
n
a
l

(
1
3
5
0
)

E
u
r
o
p
e

1
0
9

1
6

0
.
2
5
9

U
S
A

1
3

0
.
0
2
9

6
4

5
E
t
h
y
l

a
c
r
y
l
a
t
e

(
1
3
5
1
)

E
u
r
o
p
e

1
1

0
.
0
2
6

0
.
0
1

U
S
A

0
.
7

0
.
0
1
1

0
.
0
0
5

3
8
6

7
7
E
t
h
y
l

2
-
n
o
n
y
n
o
a
t
e

(
1
3
5
2
)

E
u
r
o
p
e

N
D

N
D

N
D

U
S
A

0
.
9

0
.
0
1
5

0
.
0
0
5

N
A
2
-
H
e
x
e
n
a
l

(
1
3
5
3
)

E
u
r
o
p
e

5
4
2

7
9
1

1
3

U
S
A

1
0
3

4
0
9

1
5
5

3
3
5

5
0
2
-
H
e
x
e
n
-
1
-
o
l

(
1
3
5
4
)

E
u
r
o
p
e

7
6
5

3
9
5

U
S
A

2
0
9

2
9
1

9
8
9

4
(
E
)
-
2
-
H
e
x
e
n
-
y
l

a
c
e
t
a
t
e

(
1
3
5
5
)

E
u
r
o
p
e

3
9
7

1
9
9

U
S
A

4
2
6

5
6

0
.
6

4
2
4

1
328 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
T
a
b
l
e

2
.

(
c
o
n
t
d
)
F
l
a
v
o
u
r
i
n
g

a
g
e
n
t

(
N
o
.
)

M
o
s
t

r
e
c
e
n
t

I
n
t
a
k
e
b

I
n
t
a
k
e

o
f

I
n
t
a
k
e

o
f

A
n
n
u
a
l

C
o
n
s
u
m
p
t
i
o
n

a
n
n
u
a
l

m
g
/
d
a
y

m
g
/
k
g

b
w

a
l
c
o
h
o
l

a
l
d
e
h
y
d
e

v
o
l
u
m
e

i
n

r
a
t
i
o
f

v
o
l
u
m
e

(
k
g
)
a

p
e
r

d
a
y

e
q
u
i
v
a
l
e
n
t
s

e
q
u
i
v
a
l
e
n
t
s

n
a
t
u
r
a
l
l
y

(
m
g
/
k
g

b
w

(
m
g
/
k
g

b
w

o
c
c
u
r
r
i
n
g

p
e
r

d
a
y
)
c

p
e
r

d
a
y
)
d

f
o
o
d
s

(
k
g
)
e
M
e
t
h
y
l

2
-
n
o
n
y
n
o
a
t
e

(
1
3
5
6
)

E
u
r
o
p
e

1
6

0
.
0
3
8

0
.
0
0
8

U
S
A

1
5
9

2
1

0
.
3
4
9

0
.
0
6

N
A
M
e
t
h
y
l

2
-
o
c
t
y
n
o
a
t
e

(
1
3
5
7
)

E
u
r
o
p
e

1
4
9

2
1

0
.
3
5
4

0
.
0
8

U
S
A

2
8
6

3
8

0
.
6
2
8

0
.
1

N
A
M
e
t
h
y
l

2
-
u
n
d
e
c
y
n
o
a
t
e

(
1
3
5
8
)

E
u
r
o
p
e

N
D

N
D

N
D

U
S
A
g

0
.
2

0
.
0
4

0
.
0
0
1

0
.
0
0
0
1

N
A
2
-
T
r
i
d
e
c
e
n
a
l

(
1
3
5
9
)

E
u
r
o
p
e

0
.
6

0
.
0
1
0

U
S
A

0
.
7

0
.
0
1
1

N
A
t
r
a
n
s
-
2
-
H
e
p
t
e
n
a
l

(
1
3
6
0
)

E
u
r
o
p
e

4
4

0
.
1
0
5

U
S
A

2
3
1

3
0

0
.
5
0
7

6
1
4

3
3
t
r
a
n
s
-
2
-
H
e
x
e
n
o
i
c

a
c
i
d

(
1
3
6
1
)

E
u
r
o
p
e

1
2
8

1
8

0
.
3
0
4

U
S
A

2
7
7

3
6

0
.
6
0
8

N
A
2
-
N
o
n
e
n
a
l

(
1
3
6
2
)

E
u
r
o
p
e

1
2

0
.
0
2
9

U
S
A

0
.
4

0
.
0
0
7

4
1
3

1
8
0
4
2
-
O
c
t
e
n
a
l

(
1
3
6
3
)

E
u
r
o
p
e

2
7

0
.
0
6
4

U
S
A

0
.
9

0
.
0
1
5

0
4
6

2
9
2
2
-
P
e
n
t
e
n
a
l

(
1
3
6
4
)

E
u
r
o
p
e

0
.
9

0
.
0
1
4

U
S
A

0
.
1

0
.
0
0
2

6
0

6
0
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 329
L1 t
r
a
n
s
-
2
-
N
o
n
e
n
-
1
-
o
l

(
1
3
6
5
)

E
u
r
o
p
e

0
.
1

0
.
0
0
2

U
S
A

0
.
2

0
.
0
3

0
.
0
0
0
4

1
.
4

7
2
-
U
n
d
e
c
e
n
a
l

(
1
3
6
6
)

E
u
r
o
p
e

0
.
4

0
.
0
0
7

U
S
A

0
.
4

0
.
0
0
7

1
4

1
6
8

4
7
2
3
t
r
a
n
s
-
2
-
O
c
t
e
n
-
1
-
y
l

a
c
e
t
a
t
e

(
1
3
6
7
)

E
u
r
o
p
e

0
.
3

0
.
0
0
5

0
.
0
0
4

U
S
A
h

0
.
7

0
.
0
1
2

0
.
0
0
8

N
A
t
r
a
n
s
-
2
-
O
c
t
e
n
-
1
-
y
l

b
u
t
a
n
o
a
t
e

(
1
3
6
8
)

E
u
r
o
p
e

0
.
3

0
.
0
0
5

0
.
0
0
3

U
S
A
h

0
.
7

0
.
0
1
2

0
.
0
0
6

N
A
c
i
s
-
2
-
N
o
n
e
n
-
1
-
o
l

(
1
3
6
9
)

E
u
r
o
p
e

0
.
5

0
.
0
7

0
.
0
0
1

U
S
A
h

1
1

0
.
0
3
2

N
A
(
E
)
-
2
-
O
c
t
e
n
-
1
-
o
l

(
1
3
7
0
)

E
u
r
o
p
e

N
D

N
D

N
D

U
S
A
h

0
.
2

0
.
0
0
3

N
A
(
E
)
-
2
-
B
u
t
e
n
o
i
c

a
c
i
d

(
1
3
7
1
)

E
u
r
o
p
e

N
D

N
D

N
D

U
S
A
h

4
0

0
.
1
1
7

N
A
(
E
)
-
2
-
D
e
c
e
n
o
i
c

a
c
i
d

(
1
3
7
2
)

E
u
r
o
p
e

N
D

N
D

N
D

U
S
A
h

2
5

0
.
0
7
3

N
A
(
E
)
-
2
-
H
e
p
t
e
n
o
i
c

a
c
i
d

(
1
3
7
3
)

E
u
r
o
p
e

N
D

N
D

N
D

U
S
A
h

2
0

0
.
0
5
9

N
A
(
Z
)
-
2
-
H
e
x
e
n
-
1
-
o
l

(
1
3
7
4
)

E
u
r
o
p
e

N
D

N
D

N
D

U
S
A
h

5
5

1
0

0
.
1
6
1

N
A
t
r
a
n
s
-
2
-
H
e
x
e
n
y
l

b
u
t
y
r
a
t
e

(
1
3
7
5
)

E
u
r
o
p
e

N
D

N
D

N
D

U
S
A
h

2
1

0
.
0
6
1

0
.
0
4

N
A
330 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
T
a
b
l
e

2
.

(
c
o
n
t
d
)
F
l
a
v
o
u
r
i
n
g

a
g
e
n
t

(
N
o
.
)

M
o
s
t

r
e
c
e
n
t

I
n
t
a
k
e
b

I
n
t
a
k
e

o
f

I
n
t
a
k
e

o
f

A
n
n
u
a
l

C
o
n
s
u
m
p
t
i
o
n

a
n
n
u
a
l

m
g
/
d
a
y

m
g
/
k
g

b
w

a
l
c
o
h
o
l

a
l
d
e
h
y
d
e

v
o
l
u
m
e

i
n

r
a
t
i
o
f

v
o
l
u
m
e

(
k
g
)
a

p
e
r

d
a
y

e
q
u
i
v
a
l
e
n
t
s

e
q
u
i
v
a
l
e
n
t
s

n
a
t
u
r
a
l
l
y

(
m
g
/
k
g

b
w

(
m
g
/
k
g

b
w

o
c
c
u
r
r
i
n
g

p
e
r

d
a
y
)
c

p
e
r

d
a
y
)
d

f
o
o
d
s

(
k
g
)
e
(
E
)
-
2
-
H
e
x
e
n
y
l

f
o
r
m
a
t
e

(
1
3
7
6
)

E
u
r
o
p
e

N
D

N
D

N
D

U
S
A
h

4
0

0
.
1
1
7

0
.
0
8

N
A
t
r
a
n
s
-
2
-
H
e
x
e
n
y
l

i
s
o
v
a
l
e
r
a
t
e

(
1
3
7
7
)

E
u
r
o
p
e

N
D

N
D

N
D

U
S
A
h

2
0

0
.
0
5
9

0
.
0
3

N
A
t
r
a
n
s
-
2
-
H
e
x
e
n
y
l

p
r
o
p
i
o
n
a
t
e

(
1
3
7
8
)

E
u
r
o
p
e

N
D

N
D

N
D

U
S
A
h

2
2

0
.
0
7
7

0
.
0
4

N
A
t
r
a
n
s
-
2
-
H
e
x
e
n
y
l

p
e
n
t
a
n
o
a
t
e

(
1
3
7
9
)

E
u
r
o
p
e

N
D

N
D

N
D

U
S
A
h

2
5

0
.
0
7
0

0
.
0
4

N
A
(
E
)
-
2
-
N
o
n
e
n
o
i
c

a
c
i
d

(
1
3
8
0
)

E
u
r
o
p
e

N
D

N
D

N
D

U
S
A
h

1
5

0
.
0
4
4

N
A
(
E
)
-
2
-
H
e
x
e
n
y
l

h
e
x
a
n
o
a
t
e

(
1
3
8
1
)

E
u
r
o
p
e

N
D

N
D

N
D

U
S
A
h

0
.
5

0
.
0
9

0
.
0
0
1

0
.
0
0
0
5

N
A
(
Z
)
-
3

&

(
E
)
-
2
-
H
e
x
e
n
y
l

p
r
o
p
i
o
n
a
t
e

(
1
3
8
2
)

E
u
r
o
p
e

N
D

N
D

N
D

U
S
A

0
.
7

0
.
0
1
1

0
.
0
0
6

+
i

N
A
(
E
)
-
2
-
H
e
x
e
n
a
l

d
i
e
t
h
y
l

a
c
e
t
a
l

(
1
3
8
3
)

E
u
r
o
p
e

0
.
3

0
.
0
0
5

0
.
0
0
3

0
.
0
0
3

U
S
A
h

0
.
5

0
.
0
9

0
.
0
0
1

0
.
0
0
0
5

0
.
0
0
0
5

N
A
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 331
L1 2
-
U
n
d
e
c
e
n
-
1
-
o
l

(
1
3
8
4
)

E
u
r
o
p
e

N
D

N
D

N
D

U
S
A
g

0
.
5

0
.
0
0
8

N
A
T
o
t
a
l

E
u
r
o
p
e

1
0

2
4
0

U
S
A

0
9
4
N
A
,

n
o
t

a
v
a
i
l
a
b
l
e
;

N
D
,

n
o

i
n
t
a
k
e

d
a
t
a

r
e
p
o
r
t
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d
;

+
,

r
e
p
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r
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d

t
o

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c
c
u
r

n
a
t
u
r
a
l
l
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i
n

f
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d
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(
N
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j
s
s
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n

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t

a
l
.
,

2
0
0
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)
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b
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t

n
o

q
u
a
n
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a
t
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v
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d
a
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;

-
,

n
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p
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d

t
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c
c
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n
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332 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
enzymesofthehigh-capacityb-oxidationpathwayor,toalesserextent,bygluta-
thioneconjugation.
Itisanticipatedthathumanswillbiotransformsmallquantitiesof2-alkenolsand
2-alkenalsbyoxidationtothecorrespondingacids,whichmayundergob-oxidative
cleavage and complete metabolism via the tricarboxylic acid cycle.An alternate
minorpathwaymayinvolveconjugationoftheunsaturatedaldehydewithglutathi-
one,followedbyexcretionasthemercapturicacidderivative.
1.4 Application of the Procedure for the Safety Evaluation of
Flavouring Agents
Step 1. InapplyingtheProcedure,theCommitteeassignedall37ofthefavour-
ingagentsinthisgrouptostructuralclassI(Crameretal.,1978).
Step 2. All37favouringagents(Nos13481384)inthisgroupareexpectedto
bemetabolizedtoinnocuousproducts.Theevaluationofthesefavour-
ingagentsthereforeproceededviatheA-sideofthedecision-tree.
Step A3. Theestimateddailyintakesofall37favouringagentsinthisgroupin
Europe and the USA are below the threshold for concern for class I
(i.e. 1800mg/person).According to the Procedure, the safety of these
37 favouring agents raises no concern when they are used at their
estimatedcurrentintakes.
The intake considerations and other information used to evaluate the 37
aliphatic, linear, a,b-unsaturated aldehydes, acids and related alcohols, acetals
andestersinthisgroupaccordingtotheProcedurearesummarizedinTable1.
1.5 Consideration of secondary components
Asmanyofthefavouringagentsinthisgrouparesubjecttoconjugationwith
reducedglutathione,simultaneousconsumptionofthea,b-unsaturatedaldehydes,
atsuffcientlyhighconcentrations,couldtheoreticallydepleteglutathione,resulting
inlipidperoxidation.However,undernormalconditionsandattheestimatedcur-
rent intakes resulting from use as favouring agents, replenishable intracellular
concentrations of glutathione (approximately 110mmol/l) would be suffcient to
detoxifytheagentsinthisgroup.Additionally,sincethea,b-unsaturatedaldehydes
provide similar favouring characteristics, it is unlikely that all foods containing
these favouring agents will be consumed concurrently on a daily basis. On the
basisofestimatedcurrentintakesofa,b-unsaturatedaldehydesusedasfavouring
agents,andtheconstantreplenishmentofglutathionebybiosynthesis,theCom-
mittee therefore concluded that the combined intake of these favouring agents
wouldnotpresentasafetyconcern.
1.6 Consideration of combined intakes from use as favouring agents
Asmanyofthefavouringagentsinthisgrouparesubjecttoconjugationwith
reducedglutathione,simultaneousconsumptionofthea,b-unsaturatedaldehydes,
atsuffcientlyhighconcentrations,couldtheoreticallydepleteglutathione,resulting
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 333
L1
in lipid peroxidation. However, under normal conditions and at the estimated
currentintakesresultingfromuseasfavouringagents,replenishableintracellular
concentrations of glutathione (approximately 110mmol/l) would be suffcient to
detoxifytheagentsinthisgroup(Armstrong,1987,1991).Additionally,sincethe
a,b-unsaturatedaldehydesprovidesimilarfavouringcharacteristics,itisunlikely
thatallfoodscontainingthesefavouringagentswillbeconsumedconcurrentlyon
a daily basis. On the basis of estimated current intakes of a,b-unsaturated alde-
hydes used as favouring agents, and the constant replenishment of glutathione
by biosynthesis, the Committee therefore concluded that the combined intake of
thesefavouringagentswouldnotpresentasafetyconcern.
1.7 Conclusions
The Committee concluded that none of the favouring agents in this group
ofaliphatic,linear,a,b-unsaturatedaldehydes,acidsandrelatedalcohols,acetals
andesterswouldpresentsafetyconcernsatestimatedcurrentintakes.TheCom-
mittee noted that the available data on the toxicity and metabolism of these ali-
phatic,linear,a,b-unsaturatedaldehydes,acidsandrelatedalcohols,acetalsand
esterswereconsistentwiththeresultsofthesafetyevaluationconductedaccord-
ingtotheProcedure.
2. RELEVANT BACKGROUND INFORMATION
2.1 Additional considerations on intake
Quantitative data on natural occurrence and consumption ratios have been
reported for 2-decenal (No. 1349), 2-dodecenal (No. 1350), ethyl acrylate (No.
1351),2-hexenal(No.1353),2-hexen-1-ol(No.1354),(E)-2-hexen-ylacetate(No.
1355), trans-2-heptenal (No. 1360), 2-nonenal (No. 1362), 2-octenal (No. 1363),
2-pentenal(No.1364),trans-2-nonen-1-ol(No.1365),and2-undecenal(No.1366)
and demonstrate that consumption occurs predominantly from traditional foods
(i.e.consumptionratioof>1).2-Hexenal,thesubstancewiththehighestreported
annual volume of production in Europe and the USA, is a common component
of many foods. Intake of 2-hexenal from consumption of traditional foods ex-
ceeds intake as an added favouring agent by a factor of >100000 (Stofberg &
Grundschober, 1987) (see Table 2). The highest dietary exposure to 2-hexenal
occurs from fruits and vegetables, with an estimated daily intake of between
31and165mg/kgbw(Ederetal.,1999).
2.2 Biological data
2.2.1 Biochemical data
(a) Hydrolysis
(i) Acetals
Ingeneral,aliphaticacetalsundergohydrolysistotheircomponentaldehydes
andalcohols(Knoefel,1934;Morgareidge,1962).Studiesinvitrohaveshownthat
334 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
1,1-dimethoxyethane
1
, acetal
2
, and related acetals are hydrolysed within 15h
in simulated gastric fuid, and to a lesser extent in simulated intestinal fuid
(Morgareidge,1962).Indirectevidencereportedinastudyinwhichrabbitswere
given1,1-dimethoxyethane,acetal,andotheraliphaticacetalsinaqueoussuspen-
sion by stomach tube indicate that rapid hydrolysis occurs in the stomach (see
Figure 1) (Knoefel, 1934). A correlation was reported between narcotic effects,
which are observed at high doses of acetals, and resistance to acid hydrolysis
(Knoefel,1934).Itisanticipatedthataliphaticacetalswouldundergosimilarhydro-
lysisinhumans.
Aspartofastudytoinvestigatethefeasibilityofusingacetalsasprodrugs,2-
propylpentanalacetalsweresynthesizedandtheirmetabolicconversiontovalproic
acid (2-propylpentanoic acid), an anticonvulsant, was investigated.The acid and
alcohol of 2-propylpentanal were identifed in the supernatant and microsomal
fractions of rat liver incubated with the dimethyl, diethyl, and di-isopropyl ace-
tals of 2-propylpentanal. These fndings indicate that dimethoxy-, diethoxy-, and
diisopropyl-2-propylpentaneacetalshydrolysetoyieldthecorrespondingalcohols
andparentaldehyde2-propylpentanal(Vicchio&Callery,1989).
On the basis of this information, it can be concluded that acetals are readily
hydrolysedintheacidicenvironmentofthestomach,intestinalfuid,orintheliver
to yield the component alcohol and aldehyde.Therefore, trans-2-hexenal diethyl
acetal(No.1383)isexpectedtohydrolyseto2-hexenal(No.1353)andethanol.
Certainaspectsoftheabsorption,distribution,andexcretionoftheseacetalmetab-
oliteshavebeenstudiedinrodentsandhumans,respectively(Hald&Jacobsen,
1948;Wallgren&Barry,1970;Halstedetal.,1973;Lame&Segall,1986;Mitchell
&Petersen,1987).
Figure 1. Hydrolysis of acetal
O
CH
2
CH
3
CH
2
CH
3
C H
O
+ H
2
O
+HCl
C
H
3
C H
O
+
CH
3
CH
2
OH
Acetal Water Acetaldehyde
Ethanol
H
3
C 2
1

O
O
1,1- di me th ox ye th an e
2

O O
acetal
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 335
L1
(ii) Esters
In general, aliphatic esters formed from 2-alkenols or 2-alkenoic acids (Nos
1348, 1351, 1352, 13551358, 1367, 1368, 13751379, 1381, 1382) are rapidly
hydrolysedtotheircomponentalcoholsandcarboxylicacidsbycarboxylesterases
(see Figure 2) (Heymann, 1980; Graffner-Nordberg et al., 1998;Anders, 1989).
The substrate specifcity of B-carboxylesterase isoenzymes has been correlated
withthestructureofthealcoholandthecarboxylicacidcomponents(e.g.Rand
R , see Figure 1). Esters formed from 2-alkenols or 2-alkenoic acids are more
rapidlyhydrolysedthantheirsaturatedanalogues(Heymann,1980).
2-Propenyl esters are rapidly hydrolysed in vivo to yield 2-propenol (allyl
alcohol)andthecorrespondingcarboxylicacid(Silver&Murphy,1978).Hydrolysis
oftheseestersinvitrohasbeendemonstratedrepeatedly(Butterworthetal.,1975;
Grundschober, 1977; Longland et al., 1977; Silver & Murphy, 1978). 2-Propenyl
hexanoate
3
wasreadilyhydrolysedinartifcialpancreaticjuice(t
1/2
=1.98min),rat
liver(t
1/2
=3.96s),andratsmallintestinalmucosa(t
1/2
=0.096s),butmoreslowly
in artifcial gastric juice (t
1/2
= 1120min) (Longland et al., 1977).The tiglate ester,
ataconcentrationof400ml/l,wascompletelyhydrolysedinpigjejunumin<2hat
pH7.5(Leegwater&vanStraten,1974;Grundschober,1977).Dataontheacetate,
propionate, hexanoate, isobutyrate, isovalerate, and 2-ethyl hexanoate esters of
2-propenol indicate that the rate of hydrolysis of straight-chain 2-propenyl esters
isapproximately100timesgreaterthanthatofbranched-chain2-propenylesters
(Butterworthetal.,1975).Thealmostcompletesuppressionofthehydrolysisofa
seriesof2-propenylestersinratliverhomogenatebycarboxylesteraseinhibitors
(triorthotolyl phosphate and S,S,S-tributylphosphotrithioate) provides evidence
that hepatic carboxylesterases catalyse hydrolysis of 2-propenyl esters (Silver &
Murphy,1978).
The hydrolysis of esters formed from aliphatic alcohols and a,b-unsaturated
carboxylic acids is similar to that of the esters discussed above. In a study of
hydrolysisinvitro,aseriesofacrylate(2-propenoate)esters(1mmol/lperml)were
incubatedwithratliver,kidneyandlunghomogenates(seeTable3)(Milleretal.,
1981).Theratesofhydrolysisformethylacrylate
4
,ethylacrylate(No.1351),and
butylacrylate
5
intissuehomogenatesweresimilar,althoughtherateofhydrolysis
intheliverhomogenatewasapproximately20timesfasterthaninthekidneyand
lunghomogenates(Milleretal.,1981).Thethreeestersalsodisappearedrapidly
when added to rat blood in vitro; the half-life for the alpha and beta phases for
3

O
O 2-Propenyl hexanoate
4

O
O
methyl acrylate
5

O
O
bu ty l ac ry la te
336 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
ethylacrylatewere2.7and20.9min,respectively(Milleretal.,1981).Theshort-
chainacrylateesters(methylacrylate,V
max
=0.241mmol/lpermin;ethylacrylate,
V
max
= 0.568mmol/l per min) are hydrolysed more rapidly than butyl acrylate
(V
max
= 0.141mol/l per min) at enzyme-saturating levels of rat nasal mucosal
carboxylesteraseinvitro(Stott&McKenna,1985).Thespecifcactivityofratnasal
mucosal carboxylesterase is reported to be equivalent to that of rat liver and
greaterthanthatofratkidney,lungorblood(Stott&McKenna,1985).
Hydrolysis of 2- and 3-hexenyl esters has also been reported. Studies con-
ducted in vitro in simulated pancreatic fuid (100ml) incubated at 37C indicate
100%hydrolysisof0.015mmol/ltrans-2-hexenylpropionate(No.1378)tothecor-
respondingalcohol,2-hexen-1-ol(No.1354),andpropionicacidwithin2h(Bennett,
1998).Similarly,varyingconcentrations(0.015,0.020,or0.025mmol/l)ofarelated
unsaturatedester,cis-3-hexenylpropionate
6
,wereshowntobehydrolysedcom-
pletelytocis-3-hexenolwithin2hunderthesameexperimentalconditions(Bennett,
1998).
Once hydrolysed, the resulting alcohols, aldehydes, and carboxylic acids are
readilymetabolizedinwell-recognizedbiochemicalpathways.
(b) Absorption, distribution, and excretion
Dataona,b-unsaturatedaldehydes,carboxylicacidsandtheircorresponding
alcohols demonstrate that they are rapidly absorbed, distributed, metabolized
andexcretedintheurine,andtoalesserextentinthefaeces.Groupsof10male
Table 3. Hydrolysis of acrylate esters
Methylacrylate Ethylacrylate Butylacrylate
(nmol/min) (nmol/min) (nmol/min)
Liverhomogenate 17.2 26.8 23.6
Kidneyhomogenate 0.6 0.9 Negative
Lunghomogenate 1.2 1.3 Negative
Wholeblood 11.0 12.0 9.4
FromMilleretal.(1981)
6

O
O
ci s -3-hex en yl pr op ionate
R O
O
+
+
H
2
O
R, R'=alkyl
R'
carboxylesterase
hydrolysis
R OH
O
HO
Figure 2. Hydrolysis of esters
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 337
L1
Wistaralbinoratsweregiventhestructurallyrelatedsubstancestrans-2-nonenal
or trans-2-pentenal (in olive oil) as single oral dose at 100mg/kgbw.The control
groupreceivedthevehicleonly.Analysesbyprotonnuclearmagneticresonance
spectroscopy(
1
H-NMR)conductedonsamplesofurinecollectedbeforeandafter
administration of the test materials confrmed that trans-2-nonenal is absorbed
from the gastrointestinal tract into the systemic circulation and excreted in the
urinemainlyasC3mercapturateconjugateswithin24h.Traceamountsoftrans-
2-nonenalweredetectedinthefaeces.Similarresultswereobtainedfromtherats
giventrans-2-pentenal(Grootveld etal.,1998).
Inrodents,thea,b-unsaturatedaldehyde3,7-dimethyl-2,6-octadienalisrapidly
absorbed from the gastrointestinal tract and distributed throughout the body
(Phillips et al., 1976; Diliberto et al., 1988). In Fisher F344 rats given [
14
C
1
and
14
C
2
]3,7-dimethyl-2,6-octadienalatadoseof5,50,or500mg/kgbworallyor5mg/
kgbwintravenously,mostoftheradiolabelwasexcretedintheurine,faeces,and
expiredairasradiolabelledcarbondioxide(
14
CO
2
)or[
14
C]3,7-dimethyl-2,6-octadi-
enalwithin24h.At5mg/kgbwadministeredorally,>45%and>6%oftheadmin-
istereddosewasexcretedintheurineandfaeces,respectively,andapproximately
16% and 1% was excreted as expired
14
CO
2
and
14
C-labelled 3,7-dimethyl-2,6-
octadienal, respectively, within 24h. Production of
14
CO
2
essentially ceased 12h
afterdosing.Theexcretionproflesdidnotchangeattheotherdoses,indicating
thatdistributionisindependentofdose(Dilibertoetal.,1988).At5mg/kgbw,>75%
ofthedoseadministeredbyintravenousinjectionwasremovedfromthebloodin
the frst 2min. Elimination was essentially complete within 24h (Diliberto et al.,
1988).Inastudyontheeffectsofinductiononmetabolism,disposition,andexcre-
tion, rats were orally pre-treated with 3,7-dimethyl-2,6-octadienal at a dose of
5mg/kgbwperdayfor10days.Theywerethengivensingledosesofradiolabelled
3,7-dimethyl-2,6-octadienalatadoseof5mg/kgbworallyforthedispositionstudy
or5mg/kgbwintravenouslyforthebiliaryexcretionstudy.Afterintravenousdosing,
>20%oftotalradiolabelwasexcretedinthebilewithinthefrsthour.Fiveminutes
afteradministration,unmetabolized3,7-dimethyl-2,6-octadienalwasnotdetected
inthebile.Biliaryexcretionincreased34%inpre-treatedanimalscomparedwith
those receiving no pre-treatment, possibly indicating that some metabolism had
been induced; however, pre-treatment had no effect on the disposition of 3,7-
dimethyl-2,6-octadienal in rats (Diliberto et al., 1988). The authors concluded
that3,7-dimethyl-2,6-octadienalisrapidlyabsorbed,metabolizedandexcretedin
the urine, faeces, and expired air. Tissue distribution is widespread, but there is
no evidence to suggest that there is signifcant bioaccumulation (Diliberto et al.,
1988).
Theethylesterof2-propenoicacidisalsoreadilyabsorbed,metabolizedand
excreted(NationalToxicologyProgram,1986;DeBethizyetal.,1987;Ghanayem
etal.,1987;Fredericketal.,1992).Ethylacrylate(No.1351)givenorallytorats
is rapidly absorbed and distributed to all major tissues (Ghanayem et al., 1987).
Ratsgiven[2,3-
14
C]ethylacrylateatdosesof100to400mg/kgbwbyintragastric
instillationabsorbed>90%oftheradiolabelwithin4hofadministration.Thetissues
withthehighestconcentrationofradiolabelat4hafteradministrationwerethefore-
stomach,glandularstomach,intestine,liverandkidney.Generally,theconcentra-
tionofradiolabelinthebloodandtissueswasproportionaltothedose,exceptin
338 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
theforestomachwhereratsgiven[2,3-
14
C]ethylacrylateatadoseof400mg/kgbw
hadlowerconcentrationsofradiolabelat4hthanratsgivenadoseof200mg/kgbw
at the same time interval (Ghanayem et al., 1987).The major route of excretion
oflabelledethylacrylateisbyexhalationofCO
2
.
In a metabolic study, groups of three male Sprague-Dawley rats were given
[2,3-
14
C]ethyl acrylate as a single oral dose at 2, 20, or 200mg/kg (DeBethizy
etal.,1987).Totalradiolabelrecoveredattheendofthestudyat72hrangedfrom
73 to 108% (seeTable 4).Approximately 1015% of the residual radiolabel was
foundinfourmajortissuesliver,stomach,gastrocnemiusmuscle,andepididy-
mal fat. The dose did not affect the rate of expiration of
14
CO
2
, which was the
primary mode of elimination and accounted for 5261% of the original dose
excreted.Approximately4560%ofthetotal
14
CO
2
recoveredwasexcretedwithin
thefrst10hafterdoseadministration.Adose-dependentdecreaseinurinaryand
faecalexcretionofradiolabelwasreported(DeBethizyetal.,1987).
No ethyl acrylate was detected (limit of detection was 1mg/ml) in peripheral
bloodatupto60mininmaleandfemaleF344ratsgivenethylacrylateasasingle
doseat200mg/kgbygavage,indicatingeffcientabsorptionandrapidmetabolic
clearance (>95%) of ethyl acrylate after oral administration (National Toxicology
Program, 1986; Frederick et al., 1992). Plasma half-lives reported in male and
femaleF344ratsgivenethylacrylatewere14and11mininblood,74and94min
in forestomach tissue, 64 and 62min in glandular stomach tissue, and 49 and
68mininstomachcontents,respectively(NationalToxicologyProgram,1986).
Thesedatasupporttheconclusionthat2-alkenals,2-alkenoicacidsandrelated
2-alken-1-olsandestersarerapidlyabsorbed,metabolizedandexcreted.
(c) Metabolism
TheCommitteehaspreviouslyevaluatedagroupof26favouringagentsthat
included aliphatic, alicyclic, linear, a,b-unsaturated, di- and trienals and related
alcohols, acids and esters (Annex 1, reference 166) with similar metabolic
profles.
Table 4. Distribution of radiolabel (%) in male rats at 72 h after a single oral
dose of [2,3-
14
C]ethyl acrylate
Dose Radiolabel(%)
(mg/kgbw)
ExpiredCO
2
Urine Major Faeces Total
tissues recovered
2 61.1 28.4 13.0 5.9 108.4
20 56.8 13.5 14.9 3.7 88.8
200 52.3 8.4 10.4 1.8 72.8
FromDeBethizyetal.(1987).
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 339
L1
(i) Enzymatic conversion of aliphatic 2-alkenols and
a,b-unsaturated aldehydes to carboxylic acids
IsoenzymemixturesofNAD
+
/NADH-dependentalcoholdehydrogenase(ADH)
obtainedfromhumanlivermicrosomescatalysetheoxidationofaliphaticunsatu-
ratedalcohols(Pietruszkoet al.,1973).Inacomparisonofsaturatedandunsatu-
rated alcohols as substrates for human or horse ADH, the 2-alkenols exhibited
increasedbinding(lowerK
m
)thantheircorrespondingsaturatedanalogues.There
is also a correlation between increasing chain length (C1 to C6) of the alcohol
substrate and increasing binding affnity (lower K
m
) ofADH. However, maximum
rates of reaction (higher V
max
) for oxidation were essentially constant regardless
of the alcohol structure (Klesov et al., 1977), indicating that the binding or
release of the alcohol substrate is not the rate-limiting step for the reaction.The
metabolismof2-hexen-1-ol(No.1354)andthecorrespondingaldehydehasbeen
studied in mammals. Compared with six homologous saturated linear aliphatic
alcohols,2-hexen-1-olexhibitedthelowestK
m
(i.e.greaterenzyme-bindingaffnity)
andhighestV
m
(maximumreactionrate)duringoxidationcatalysedbyisoenzyme
mixtures of NAD
+
/NADH-dependentADH obtained from human liver (Pietruszko
etal.,1973).
Similarly, aldehyde dehydrogenase (ALDH) present predominantly in hepatic
cytosol (Lame & Segall, 1986) exhibits broad specifcity for the oxidation of ali-
phaticandaromaticaldehydestoyieldthecorrespondingcarboxylicacids(Feldman
& Weiner, 1972). trans-2-Hexenal (No. 1353) is readily oxidized to trans-2-
hexenoicacid(No.1361)inmousehepaticcytosolfractions(Lame&Segall,1986)
andinisoenzymesofratALDHpresentinmitochondrial,cytosolic,andmicrosomal
fractions(Mitchell&Petersen,1987).ALDHdemonstrateshighercatalyticactivity
invitroforhigherrelativemolecularmass,andmorelipophilicaldehydes(Nakayasu
et al., 1978). The molybdenum-containing enzymes xanthine oxidase and alde-
hydeoxidasealsocatalysetheoxidationofawiderangeofaldehydes(Beedham,
1988). Examination of the stomach contents of rats 16h after receiving trans-2-
nonenal at a dose of 100mg/kg showed that approximately 15% of the adminis-
tered dose had been oxidized to trans-2-nonenoic acid (Grootveld et al., 1998).
Thisassortmentofoxidativeenzymesprovidethenumerousmetabolicoptionsfor
therapidconversionof2-alkenolstoa,b-unsaturatedaldehydesandthentotheir
correspondinga,b-unsaturatedcarboxylicacids.
(ii) Metabolism of aliphatic linear a,b-unsaturated
carboxylic acids
Theresultinglineara,b-unsaturatedacids,suchastrans-2-hexenoicacid(No.
1361), participate directly in fatty acid metabolism. In the fatty acid pathway, the
a,b-unsaturatedcarboxylicacidisfrstcondensedwithcoenzymeA(CoA)(Nelson
&Cox,2000).Theresultingtrans-2,3-unsaturatedCoAester(trans-D
2
-enoylCoA)
isconvertedtothe3-ketothioester,whichundergoesb-cleavagetoyieldanacetyl
CoAfragmentandanewthioesterreducedbytwocarbons.
Cleavage of acetyl CoA units will continue along the carbon chain until the
positionofunsaturationisreached.Iftheunsaturationbeginsataneven-numbered
carbon as in an a,b-unsaturated acid, acetyl CoA fragmentation will eventually
340 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
yieldaD
2
-enoylCoAthatisasubstrateforfurtherfattyacidoxidation.Iftheregio-
chemistryofthedoublebondiscis,itisisomerizedtothetransdoublebondby
theactionof3-hydroxyacylCoAepimerasepriortoenteringthefattyacidoxidation
pathway. Even-numbered carbon acids continue to be cleaved to acetyl CoA.
AcetylCoAiscompletelymetabolizedinthecitricacidcycletoyieldCO
2
andwater
(Nelson&Cox,2000).
Studies with radiolabelled a,b-unsaturated acids support the conclusion that
a,b-unsaturatedacidsarecompletelymetabolizedtoCO
2
andwater.Between77
and85%ofasingleoraldoseof[1-
14
C](E,E)-2,4-hexadienoicacidofeither40or
8000mg/kgbw given to female mice is excreted as expired
14
CO
2
within 4 days.
Approximately 88% of the
14
CO
2
was recovered within the frst 24h. Between 4
and5%oftheoriginaldosewasexcretedintheurineas(E,E)-muconicacid
7
(i.e.
(E,E)-2,4-hexadienedioicacid)andunchanged(E,E)-2,4-hexadienoicacid,respec-
tively,accountingfor0.4and0.7%ofthetotalradiolabelpresentintheurinecol-
lectedoverthefrst24h.Onlyabout1%ofthe40mg/kgbwdosewasrecovered
in the faeces (Westoo, 1964). Regardless of dose, rats given [1-
14
C](E,E)-2,4-
hexadienoic acid at doses between 61 and 1213mg/kgbw excreted >85% as
exhaled
14
CO
2
within 10h. In the same period of time, approximately 2% of the
radiolabelwasdetectedintheurine.(E,E)-Muconicacidandunchanged(E,E)-2,4-
hexadienoicacidwerenotdetected(Fingerhut etal.,1962).
Similarresultsareavailableforestersformedfroma,b-unsaturatedcarboxylic
acids.Ratsgiven[2,3-
14
C]ethylacrylate(ethyl2-propenoate)atadoseof200mg/
kgbwexcretedapproximately27and70%asexhaled
14
CO
2
in4and24h,respec-
tively. A small amount of unchanged ethyl acrylate (1%) was also eliminated in
the expired air within 24h.Alternately, approximately 9 and 4% of the dose was
excretedintheurineandfaeceswithin24h,respectively(Ghanayemetal.,1987).
Approximately4%oftheoriginaldose(200mg/kgbw)wasexcretedinthebilewithin
6hofadministrationbygavage(Ghanayemetal.,1987).Inthedose-dependent
studydiscussedabove,mostofasingleoraldoseof[2,3-
14
C]ethylacrylateat2,
20,or200mg/kggiventoratswasexhaledas
14
CO
2
withapproximatelyhalf(45
60%)ofthetotal
14
CO
2
beingrecoveredwithinthefrst10h(DeBethizyetal.,1987).
Onthebasisofthesedata,itcanbeconcludedthatthepredominantpathwayfor
metabolism of 2,3-alkenols and a,b-unsaturated aldehydes involves oxidation to
yield the corresponding a,b-unsaturated carboxylic acid followed by complete
metabolisminthefattyacidpathwayandtricarboxylicacidcycle.
(iii) Glutathione conjugation of a,b-unsaturated aldehydes
a,b-Unsaturated aldehydes conjugate with glutathione (GSH) directly or
undergo allylic hydroxylation via lipid peroxidase to yield 4-hydroxyalkenals
(Esterbauer et al., 1982) that also conjugate with GSH (Esterbauer et al., 1975;
Winter et al., 1987).The GSH redox cycle maintains adequate levels of GSH in
7

H
O
H
O ( E,E) - muco ni c ac id
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 341
L1
animalcells(Nelson&Cox,2000;Schulzetal.,2000)andisamajorintracellular
mechanisminvolvedinthedetoxicationofa,b-unsaturatedaldehydes(Reedetal.,
1986).The addition of GSH across the electrophilic carboncarbon double bond
is catalysed by the enzyme glutathione S-transferase (GST), but can also occur
at a lower rate in a non-enzymatic reaction (Eisenbrand et al., 1995; Grootveld
etal.,1998).
Culturedprimaryrathepatocytes,whicharerichinGSHandGST,havebeen
shown to metabolize greater amounts of 2-alkenals than human lymphoblastoid
cells(Namalvacells)(Eisenbrandetal.,1995).ThelowlevelsofGSH,GST,and
deactivatingenzymesmakethehumanlymphoblastoidcellsmoresusceptibleto
thecytotoxiceffectsof2-alkenalsliketrans-2-hexenal.Inbothcelltypes,thecon-
sumption of 2-alkenals was directly related to the depletion of intracellular GSH
(Eisenbrandetal.,1995).A75%decreaseinthelevelsofliverGSHoccurredwhen
maleratsweregiventhestructurallyrelatedtrans,trans-muconaldehyde
8
atadose
of 36mmol/kgbw by intraperitoneal injection (Witz, 1989).Additionally, the report
that the presence of GSH reduces the cytotoxicity of a,b-unsaturated aldehydes
inS. typimuriumTA104invitroprovidesadditionalevidencethatGSHconjugation
playsanimportantroleinthedetoxicationprocess(Marnettetal.,1985).
Thehighlyreactivea,b-unsaturatedaldehydeacrolein(2-propenal)ismetabo-
lized via conjugation with GSH (Penttila et al., 1987) or other free thiol functions
(Ohno et al., 1985). Conjugation of acrolein with GSH occurs with or without
enzymecatalysis.TheGSHadductissubsequentlyreducedtothecorresponding
3-hydroxypropyl GSH conjugate, which is then excreted principally as the mer-
capturic acid or cysteine derivative. Metabolic precursors of 2-propenal produce
the same urinary metabolites as 2-propenal. 3-Hydroxypropylmercapturic acid
(611%) was isolated from the urine of male albino CFE strain of rats given
allyl alcohol (613mg), acrolein (606mg), allyl formate (758mg), allyl propionate
(1500mg), or allyl benzoate (dose not stated) by subcutaneous injection (Kaye,
1973). The mercapturic acid derivative was also the primary urinary metabolite
whenallylpropionatewasadministeredbyintraperitonealinjectionorbygavage.
Similar mercapturic acid conjugates have been reported for 2-butenol and 2-
butenal(Gray&Barnsley,1971)andthehigherhomologuesdiscussedbelow.
The major urinary metabolite isolated from the urine of male Wistar albino
rats given a 100mg/kgbw dose of trans-2-pentenal or trans-2-nonenal was
the mercapturic acid conjugate of the corresponding alcohol 3-S-(N-
acetylcysteinyl)pentan-1-olor3-S-(N-acetylcysteinyl)nonan-1-ol,respectively(see
Figure 3).Analysis of the faeces of animals dosed with 2-nonenal showed slight
amounts of the unchanged aldehyde, while analysis of the stomach contents
obtained 16h after dosing showed that approximately 15% of the administered
dosewaspresentastrans-2-nonenoicacid.Lowconcentrationsofglucuronicacid
conjugates were also detected in the urine. The authors suggested that these
8

O
O
H
H
tr an s , tr an s -muc on al de hy de
342 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
Figure 3. Glutathione conjugation of a,b-unsaturated aldehydes
O R
H
R = C
2
H
5
o r C
6
H
1 1
H S
N H C O C H
2
C H N H
2
C O
2
H
C O N H C H
2
C O
2
-
G l u t a t h i o n e ( G S H )
+
O
H
S
N H C O C H
2
C H N H
2
C O
2
H
C O N H C H
2
C O
2
-
R
G l u t a t h i o n e c o n j u g a t e
O
H
S
N H C O C H
3
C O
2
-
R
1 ) A c e t y l C o A , 2 H
2
O
2 ) A l c o h o l dehydrogenase + NADH
3 -S-( N- a c e t y l c y s t e i n y l ) p e n t a n - 1 - o l o r
3 - S- ( N- a c e t y l c y s t e i n y l ) n o n a n - 1 - o l
conjugatesarosefromasequentialpathwayinvolvingthiolconjugation,oxidation
orreductionofthealdehydefunctionalgroup,followedbyglucuronicacidconjuga-
tion of the resulting carboxylic acid or alcohol, respectively (Grootveld et al.,
1998).
IncreasedconjugationwithGSHisobservedina,b-unsaturatedaldehydesfor
which b-oxidation is inhibited. The mercapturic acid conjugate was the major
urinary excretion product isolated from rats given (E)-2-propyl 2,4-pentadienoic
acid
9
asasingleintraperitonealdoseat100mg/kgbw(Kassahunetal.,1991).
Alternatively, under conditions of oxidative stress (see section below), a,b-
unsaturatedaldehydesundergolipidperoxidationbeforereactionwithGSH.a,b-
Unsaturated aldehydes have been reported to undergo C4 allylic hydroxylation
catalysedbylipidperoxidase(Esterbaueretal.,1982)followedbyconjugationwith
GSH(Esterbaueretal.,1975).Within24hofreceiving5-(H
3
)-4-hydroxy-2-hexenal
at a dose of 15mg/kgbw by injection into the hepatic vein, Sprague-Dawley rats
eliminated most (79.35%) of the radiolabel in the urine as a mercapturic acid
metabolite(seeFigure4).ThemajorexcretionproductresultedfromMichaeladdi-
tionofGSHtotheb-positionof4-hydroxy-2-hexenalfollowedbyhemiacetalforma-
tion(Winteretal.,1987).
9

H O
O (E) -2-propyl 2,4-pentadienoic acid
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 343
L1
O
H
O H
H S
N H C O C H
2
C H N H
2
C O
2
H
C O N H C H
2
C O
2
-
g l u t a t h i o n e ( G S H )
O
H
O H
S
N H C O C H
2
C H N H
2
C O
2
H
C O N H C H
2
C O
2
-
G l u t a t h i o n e c o n j u g a t e
S
N H C O C H
3
C O
2
-
1 ) A c e t y l C o A , 2 H
2
O
2 ) A l c o h o l dehydrogenase + NADH
M e r c a p t u r i c a c i d o f t r a n s-4-hydroxy-2-hexenal
h e m i a c e t a l
O
H
l i p i d
p e r o x i d a s e
t r a n s - 2 - h e x e n a l
t r a n s - 4 - h y d r o x y - 2 - h e x e n a l
H
O
H O
Figure 4. Glutathione conjugation of a,b-unsaturated aldehydes under conditions
of oxidative stress
(iv) Endogenous formation of a,b-unsaturated aldehydes
Glutathione conjugation, oxidative stress, lipid peroxidation,
and apoptosis
a,b-Unsaturated aldehydes are formed endogenously by lipid peroxidation of
polyunsaturated fatty acids (Frankel et al., 1987). The levels of a,b-unsaturated
aldehydes obtained exogenously as naturally occurring constituents of food or
as added favouring agents are expected not to have a signifcant effect on the
endogenouslevelsofa,b-unsaturatedaldehydes.Itmaybeexpectedthat,under
normal conditions, intracellular concentrations of GSH, which range from 1 to
10mmol/l in mammalian cells (Armstrong, 1987, 1991), are suffcient to detoxify
bothendogenousandexogenousa,b-unsaturatedaldehydes.
It should be noted that, at suffciently high concentrations of a,b-unsaturated
aldehydes,levelsofcellularGSHmaybedepleted,resultinginastateofoxidative
stress,whichischaracterizedbytheformationofreactiveoxygenspeciesorfree
radicals that react with various cellular components, particularly polyunsaturated
fattyacids,leadingtotheformationofmorealdehydes.Inthealdehydefragmenta-
344 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
tionpathway,abstractionofadiallylichydrogenatomfromapolyunsaturatedfatty
acid (e.g. the C11 hydrogen of 9,12-octadecadienoic acid, linoleic acid) and
subsequent rearrangement yields a hydroperoxide intermediate. The unstable
hydroperoxidereadilydegradestoanalkoxyradicalthatispronetoundergoeither
b-scissionorhydrogenabstraction.b-Scissionyieldsshortened,conjugated,a,b-
unsaturatedaldehydessuchas2-butenal,trans-2-hexenal,4-hydroxy-2-nonenal,
and2,4-decadienal.Availabledatasuggestthattheformationofa,b-unsaturated
aldehydes during lipid peroxidation may be involved in the pathophysiological
effects associated with oxidative stress (Ichihashi et al., 2001). In addition to
forming reactive unsaturated aldehydes, lipid peroxidation disturbs the structural
integrity of the lipid bilayer, compromising cell permeability. This leads to mem-
braneleakage,Na
+
infux,K
+
effux,andinfuxofwaterleadingtocytotoxicoedema.
Additionally, aldehyde fragmentation products induce apoptotic cell death during
oxidativestress(Esterbaueretal.,1991;Eckletal.,1993;Dianzani,1998).
TheeffectivenessoftheGSHdetoxicationpathwayfora,b-unsaturatedalde-
hydes hinges on the ability of the cell to maintain the equilibrium between its
pro-oxidant and antioxidant systems (Nelson & Cox, 2000). A decrease in the
concentrationsofantioxidantsoranincreaseintheproductionofreactiveoxygen
species(e.g.oxygen,O
2
;hydrogenperoxide,H
2
O
2
;hydroxylradical,OH)canlead
to oxidative stress, a condition in which the cell is unable to maintain the level
of reactive oxygen species below a toxic threshold (Schulz et al., 2000). During
periods of oxidative stress, the ratio of GSH to glutathione disulfde decreases
owingtolossofGSHandaccumulationofglutathionedisulfde.Thelowlevelsof
cellular GSH render the detoxication pathway ineffcient and allow for increased
interaction between the a,b-unsaturated aldehydes and cellular components
(proteinsandDNA),eventuallyleadingtocytotoxicityandapoptosis(Ederetal.,
1993;Ichihashietal.,2001).
(v) Potential for macromolecular reactivity
Inaseriesofexperiments,theformationofproteinadductswithendogenous
andexogenoussourcesofa,b-unsaturatedaldehydeswasinvestigated(Ichihashi
etal.,2001).Whenbovineserumalbumin(1mg/ml)wasincubatedwith2-butenal
(crotonaldehyde)atalowconcentration(2.5mmol/l)for24h,thesumofthehisti-
dine and lysine residues lost on bovine serum albumin roughly corresponded to
the protein carbonyls formed. These data suggest that at low concentrations, 2-
butenal forms carbonyl adducts (Schiff bases) with histidine and lysine residues
on bovine serum albumin. At higher concentrations, non-carbonyl and carbonyl
adductsweredetected.Inadductsinwhichfreecarbonylswerepresent,theimid-
azolenitrogenonhistidineandfreeaminenitrogenonlysineresiduespresentin
bovine serum albumin formed a covalent bond in a Michael-type addition to the
betapositionof2-butenal.
Administrationof[2,3-
14
C]ethylacrylateatadoseof100,200,or400mg/kgbw
byintragastricinstillationresultedinahighlevelofproteinbindingintheforesto-
machat4h(Ghanayemetal.,1987).Bindingwasalsofoundintheliver.
In an effort to study the endogenous formation of a,b-unsaturated aldehydes
andsubsequentreactionwithprotein,amonoclonalantibodydirectedtoprotein-
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 345
L1
bound 2-butenal was developed (Ichihashi et al., 2001). The antibody showed
immunoreactivityforthe2-butenal-lysinemodifedproteinadductaswellasthose
formedfrom2-pentenaland2-hexenal.Subsequently,theproductionofimmuno-
reactive material was assessed in a model of renal carcinogenicity in which rats
weregivenFe
3+
-nitrilotriacetatemixturebyintraperitonealinjectiontoinduceacute
oxidativetissuedamageintherenalproximaltubules.Animalsweresacrifcedat
0,4,8,24,48,and72handtheirkidneyswereexcisedandpreparedforimmu-
nohistochemical study. Although incubation of stained kidney sections with the
antibodyshowedlittleimmunoreactivityattimesuptoandincluding24h,intense
immunoreactivitieswereobservedat48hinthecytoplasmandnuclei.Theauthors
notedthatthepatternofdistributionanddelayedonsetofadductformationinthe
ratkidneyisconsistentwiththeformationofmembranelipidperoxidationproducts
(aldehydes)withcytosolicproteins(Ichihashietal.,2001).
Intwoexperimentsconductedtolinktheformationof2-alkenal-proteinadducts
with lipid peroxidation, low-density lipoprotein (LDL) was incubated with 5mmol/l
Cu
2+
,andpolyunsaturatedfattyacidswereincubatedwithaniron-acsorbatefree
radical generating system to induce the formation of lipid peroxidation products.
Incubation of the antibody for the 2-butenal-lysine modifed protein adduct with
eithertheCu-oxidizedLDLproductortheiron/ascorbate(oxidant),linolenicacid,
bovineserumalbuminmixtureindicatedthepresenceof2-alkenal-lysinemodifed
protein. These data strongly suggest that 2-alkenals formed endogenously by
lipid peroxidation react with proteins in Schiff base and Michael addition-type
reactions.
Studies in vitro indicate that 2-alkenal-DNA adducts form under conditions of
oxidative stress. In cultured rat hepatocytes and human lymphoblastoid cells
(Namalva cells) treated with various 2-alkenals, DNA single-strand breaks were
detectableafterintracellularconcentrationsofGSHwerereducedtoapproximately
20% of those before treatment. Before incubation of Namalva cells and rat
hepatocytes with trans-2-hexenal, concentrations of GSH were measured to be
approximately1.6and80nmol/210
6
cellsineachrespectivecellline.Afterthe
1hincubation,concentrationsofGSHwerereducedtoapproximately10%ofthe
controlvalues.2-HexenalproducedDNAdamageintheNamalvacells,whichare
poorinGSH,atlowerconcentrationsthaninhepatocytes,whicharerichinGSH.
TheauthorsconcludedthatmetabolicallyprofcientcellscontainingGSHandGST
effcientlyprotectagainsttheeffectsof2-hexenal(Eisenbrandetal.,1995).
Studies using fuorescence spectroscopy revealed thata,b-unsaturated alde-
hydes bind DNA to form adducts in vitro and in vivo (Frankel et al., 1987; Eder
et al., 1993; Cadet et al., 1999; National Toxicology Program, 2001a). Trans-2-
Hexenal,aproductoflipidperoxidation,hasbeenshowntoreactwithdeoxyguano-
sinetoproducelowlevelsofexocyclic1,N
2
-propanoadductsincalfthymusDNA,
human lymphoblastoid Namalva cells, and in primary rat colon mucosa cells at
concentrationsof0.2and0.4mmol/l,respectively(Golzeretal.,1996).
InastudytoevaluatetheeffectofGSHdepletionontheoxidativeDNAbreak-
age, different alkenals (2-hexenal, 100mmol/l; cinnamaldehyde, 300mmol/l; 2,4-
hexadienal,300mmol/l,and2-cyclohexenone,300mmol/l)wereincubatedwithV79
cells for 1h. Under conditions in which levels of GSH were depleted to <20% of
thoseofcontrols,DNAdamagewasreportedforallfouralkenals.However,at3h
346 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
after treatment, DNA damage decreased and concentrations of GST were
increased. Using treatment with formamidopyrimidine DNA glycosylase (FPG) to
monitor oxidative DNA damage, FPG-sensitive sites were detected with hexenal
andcinnamaldehydebutnotwith2,4-hexadienalor2-cyclohexenone.Theauthors
concludedthatdose-dependentalkenaloxidativestressrelatedtoGSHdepletion
contributestocytotoxicandgenotoxiccelldamage(Janowskietal.,2003).
The formation in vivo of trans- and cis-isomers of 1-N
2
-propanodeoxyguano-
sineadductsof2-hexenalwasevaluatedinvariousorgansofgroupsoffourmale
Fischer344ratssacrifcedatdifferentintervals(8,24,48,and96h)afteradmin-
istrationof2-hexenalasasingleoraldoseat50,200,or500mg/kgbwbygavage
(Schuler&Eder,1999).Usinga
32
P-post-labellingtechniquewithalimitofquanti-
fcation of 0.03 adducts/10
6
nucleotides, no adducts were detected either in the
organsofuntreatedratsorintheorgansoftreatedratssacrifced8haftertreat-
ment. The highest levels of DNA adducts were detected 48h after treatment
andoccurredinorgansthatcameintodirectcontactwiththetestsubstance(fore-
stomach, oesophagus) or had high exposure (liver). In the groups receiving the
twohigherdoses,levelsofadductsdetermined2daysaftertreatmentat500mg/
kgbw (forestomach, 3.1 adducts/10
6
; liver, 1.7 adducts/10
6
; oesophagus, 1.1
adducts/10
6
) were disproportionately greater than those at 200mg/kgbw (fore-
stomach,0.42adducts/10
6
;liver,0.15adducts/10
6
;oesophagus,0.1adducts/10
6
).
Theauthorsanticipatedthathigherdosesof2-hexenaldepleteGSHandtherefore,
ahigherfractionofthedoseisavailableforDNAbinding.Theauthorsnotedthat
adduct levels in animals sacrifced at 4 days were signifcantly lower than those
sacrifcedat2days,suggestingthatsomeDNArepairwasoccurring.Exceptfor
the oesophagus (0.08 adducts/10
6
), no quantifable levels of DNA adducts were
detectedat50mg/kgbw,andonlytraceamountsofadducts(0.010.02adducts/10
6
)
couldbedetectedinotherorgans.Thisexperimentdidnotaccountfortheforma-
tion and reaction of 2-hexenal formed endogenously under conditions of GSH
depletionandoxidativestress.
In a similar experiment, groups of male Fischer rats were given 2-butenal as
single oral doses at 0, 200, or 300mg/kgbw by gavage and DNA adducts were
analysed using the same
32
P-post-labelling technique (Schuler & Eder, 1999).
Delayed(20h)formationofDNAadducts(2.9adducts/10
8
nucleotidesat200mg/
kgbwand3.4adducts/10
8
nucleotidesat300mg/kgbw)weredetectedintheliver.
Signifcantly lower levels were observed at 12h after the 200mg/kg dose. DNA
adducts(6.2adducts/10
8
nucleotidesor2.0adducts/10
8
nucleotides,respectively)
werealsodetectedinratsgiven2-butenalatadoseof1or10mg/kgbwbygavage
fvetimesperweekfor6weeks.Oneweekaftermaleratsweregiven2-butenal
fve times per week for 4 weeks, levels of DNA adducts were 69% of the peak
level measured 24h after the last dose. Two weeks after the last dose, adduct
levels were reduced to 18% of the peak level, suggesting that continued repair
wasoccurring.
Inastudytoassesstherelationshipbetweencytotoxicity,DNAdamage,and
GSH depletion, a series of a,b-unsaturated aldehydes (2-hexenal to 2-nonenal)
wereincubatedwithChinesehamsterfbroblastV79cellsorCaco-2humancolon
adenocarcinomacellsfor1h.Inbothcelllines,similarlevelsofcytotoxicitywere
reported,with2-nonenalbeingthemostcytotoxic.ThedegreeofDNAdamagein
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 347
L1
V79 and Caco-2 cell lines overlapped to a high extent with cytotoxic concentra-
tions,suggestingthatcytotoxicityisdirectlycorrelatedwithinteractionwithDNA.
After 1h, concentrations of GSH were approximately 20% of those of controls.
GSHdepletionoccurredatconcentrationsatleasttenfoldlowerthanthoserequired
forDNAdamage,alsosuggestingthatdepletionofcellularGSHisaprerequisite
forDNAdamage(Glaabetal.,2001).
Studiesusingthe
32
P-post-labellingprocedurehaveshownthattrans-2-hexenal
formscyclic1,N
2
-propanoadductswithdeoxyguanosineinprimarycolonmucosa
cells from rats and humans at concentrations as low as 0.4mol/l. Hexenal has
beendetectedinfavouredfoodsatconcentrationsofupto14mg/kg(0.14mmol/l),
which is comparable to its natural occurrence in some fruits and vegetables (up
to30mg/kg).Thisclearlyindicatesthathexenalinducesgenotoxiceffectsincells
from the rat and human gastrointestinal tracts at concentrations similar to those
foundinfood(Golzeretal.,1996).
InadditiontodirectreactionwithDNA,a,b-unsaturatedaldehydesacttoinduce
DNAfragmentationthroughapoptosis.Recentexperiments(Jietal.,2001)indicate
that depletion of GSH by a,b-unsaturated aldehydes (4-hydroxy-2-nonenal),
progressing in a dose- and time-dependent manner, induces poly-ADP-ribose
polymerase(PARP)cleavageandDNAfragmentation.Inoneofthepathwaysof
apoptosis,depletionofGSHresultsinthereleaseofmitochondrialcytochromec
tothecytosolwhereacascadeofcytosoliccysteineproteases(i.e.caspases)is
activated.Activationofcaspase-3causescleavageofcellularproteinsandPARP,
leadingtoDNAfragmentationandsubsequentcelldeath(Liuetal.,1996;Lietal.,
1997;Zouetal.,1997;Green&Reed,1998;Cainetal.,1999).
Insummary,itisconcludedthathighcellularconcentrationsof2-alkenalsmay
deplete GSH, leading to oxidative stress and the formation of protein and DNA
adducts.Undertheseconditions,alkenalsmayalsoformendogenouslyfromthe
increased lipid peroxidation of membrane polyunsaturated fatty acids. To some
extent, repair is observed after cessation of exposure. At the concentrations of
alkenalsthatarepresentinthediet,thereisnosignifcantpotentialforoxidative
stressortheformationofDNAadducts.
2.2.2 Toxicological studies
Typically,toxicologicalstudieswouldbeorganizedinthismonographaccording
to duration (i.e. short-term, long-term, and carcinogenicity), favouring agent and
then species. However, in the interest of preserving the continuity of the studies
performed by the NationalToxicology Program, the short-term studies of toxicity
andcarcinogenicitywillbediscussedinthesectiononlong-termstudies(see2.2.2
(c))inthesequenceinwhichtheywereconducted.
(a) Acute toxicity
Oral median lethal dose (LD
50
) values have been reported for 19 of the 37
substancesinthisgroup.Seventeenofthea,b-unsaturatedaldehydesandrelated
alcohols, acids, and esters used as favouring agents (Nos 13491353, 1356,
348 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
1357, 1359, 1360, 1362, 1369, 1371, 1375, 1377, 1378, 1381, and 1383) have
ratoralLD
50
sintherangeof767to>5000mg/kgbw(Pozzanietal.,1949;Br&
Griepentrog,1967;Smythetal.,1970;Gauntetal.,1971;Moreno,1972,1973a,
1973b,1973c,1976,1977a,1977b,1977c,1978a,1978b,1978c,1979;Freeman,
1980;Moreno,1980a,1980c;Mondino,1981;Moreno,1982).
Inmice,oralLD
50
values(Nos1353,1359,1367,and1368)areintherange
of1550to>8000mg/kgbw(Gauntetal.,1971;Pellmont,1974a,1974b;Moreno,
1980b), demonstrating that the oral acute toxicity of a,b-unsaturated aldehydes
andrelatedalcohols,acidsandestersislow(seeTable5).
(b) Short-term studies of toxicity
The results of short-term studies of toxicity on ethyl acrylate (No. 1351) and
2-hexenal(No.1353)aredescribedbelowandsummarizedinTable6.
(i) Ethyl acrylate (No. 1351)
Rats
Ina2-weekstudy,groupsof10maleF344/Nratsweregivenethylacrylateat
adoseof2,10,20,50,100or200mg/kgbwperdaybyintragastricinstillationin
cornoilfor5daysperweek,orweregivendrinking-watercontainingethylacrylate
at a concentration corresponding to a dose of 0, 23, 99, 197 or 369mg/kgbw
perdayfor7daysperweek.Concurrentcontrolgroupsweremaintained.Atstudy
termination, primary compound-related histopathological changes (i.e. hyperpla-
sia,hyperkeratosis,infammation,oedema,andulcers/erosions)wereobservedin
theforestomachoftheratstreatedbygavage.Theincidenceandseverityofthe
epithelialhyperplasiaincreasedinadose-relatedmanneratdosesof20mg/kgbw
per day administered by gavage. An increase in forestomach weight reaching
281% of the values for controls was reported in the group receiving a dose of
200mg/kgbw per day by gavage. No compound-related effects were observed
in the group receiving a dose of 10mg/kgbw per day by gavage. Rats given
drinking-water containing ethyl acrylate exhibited a lower incidence and severity
offorestomachirritationthandidratsdosedviagavage.Dose-dependentdiffuse
epithelialforestomachhyperplasiawasreportedatdosesof99mg/kgbwperday,
whileoedema,erosionsandulcerswerenotobservedinanyoftheanimalsgiven
drinking-water containing ethyl acrylate.A slight increase in forestomach weight
was reported only at the highest dose (369mg/kgbw per day). No compound-
relatedeffectswereobservedinthegroupsofratsgivendrinking-watercontaining
ethylacrylateatadoseof23mg/kgbwperday.Nolesionswereobservedinthe
glandular stomach of any treated animals. In comparison with controls, animals
treatedbygavageexhibitedminimaldepletionofnon-proteinsulfhydryl(NPSH)at
dosesof2050mg/kgbwperday,whileseveredepletionofNPSH(25%ofbase-
lineconcentration)wasobservedatdosesof100mg/kgbwperday.Incompari-
son,nosignifcantdepletionofNPSHwasnotedinanimalsreceivingdrinking-water
containing ethyl acrylate. On the basis of the results of this study, the authors
concludedthatcontinuedexposuretoethylacrylateatloworaldosesisnotassoci-
atedwithseveretissuetoxicityorcarcinogenicity(Fredericketal.,1990).
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 349
L1
Table 5. Studies of the acute toxicity of aliphatic, linear a,b-unsaturated
aldehydes, acids and related alcohols, acetals and esters administered orally
No. Flavouringagent Species Sex LD
50
Reference
(mg/kgbw)
1349 2-Decenal Rat NR 5000 Moreno(1977a)
1350 2-Dodecenal Rat M >5000 Moreno(1980a)
1351 Ethylacrylate Rat F 0.83ml/kg
a
Smythetal.(1970)
(767mg/kgbw)
b
1351 Ethylacrylate Rat M 1020 Pozzanietal.
(1949)
1352 Ethyl2-nonynoate Rat NR 2850 Moreno(1973a)
1353 2-Hexenal Rat NR 850 Moreno(1973b)
1353 2-Hexenal Rat M,F 780(M) Gauntetal.(1971)
1130(F)
1353 2-Hexenal Mouse M,F 1750(M) Gauntetal.(1971)
1550(F)
1356 Methyl2-nonynoate Rat M,F 1180(M) Freeman(1980)
870(F)
1356 Methyl2-nonynoate Rat NR 2220 Moreno(1973c)
1357 Methyl2-octynoate Rat M 2500 Moreno(1972)
1357 Methyl2-octynoate Rat NR 1530 Br&Griepentrog
(1967)
1359 2-Tridecenal Mouse NR >5000 Moreno(1980b)
1359 2-Tridecenal Rat NR >5000 Moreno(1979)
1360 trans-2-Heptenal Rat NR 1300 Moreno(1980c)
1360 trans-2-Heptenal Rat NR 1300 Moreno(1982)
1362 2-Nonenal Rat NR 5000 Moreno(1977b)
1367 trans-2-Octen-1-yl Mouse NR >8000 Pellmont(1974a)
acetate
1368 trans-2-Octen-1-yl Mouse NR >8000 Pellmont(1974b)
butanoate
1369 cis-2-Nonen-1-ol Rat M,F >5000 Mondino(1981)
1371 (E)-2-Butenoicacid Rat NR 1000 Br&Griepentrog
(1967)
1375 trans-2-Hexenyl Rat NR >5000 Moreno(1978a)
butyrate
1377 trans-2-Hexenyl Rat NR >5000 Moreno(1978b)
isovalerate
1378 trans-2-Hexenyl Rat NR >5000 Moreno(1976)
propionate
1381 (E)-2-hexenyl Rat NR >5000 Moreno(1978c)
hexanoate
1383 (E)-2-Hexenaldiethyl Rat NR 860 Moreno(1977c)
acetal
F,female;M,male;NR,notreported.
a
Ethylacrylatewasprovidedasamixture,togetherwithethylacetateorformalin.
b
Calculatedusingdensityofethylacrylate=0.924g/ml(availableatwww.sigmaaldrich.
com).
350 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
T
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ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 351
L1
Groupsof40maleand20femaleF344/Nratsweregivendrinking-watercon-
tainingethylacrylateataconcentrationof0,200,1000,2000or4000mg/kgfor7
days per week for 13 weeks.These concentrations correspond to average daily
dosesof0,20,100,200and400mg/kgbw,respectively(FoodandDrugAdminis-
tration,1993).Clinicalsignsoftoxicityweremonitoreddaily,whilefoodandwater
consumptionandbodyweightweremonitoredweekly.Atweeks1,2,4and13,a
selectnumberofratsfromeachgroupwasnecropsiedandsubjectedtoacomplete
histopathological examination. All treated animals survived until the end of the
study period. No clinical signs of systemic toxicity were observed at any of the
doses examined. Decreased consumption of water was observed in all treated
animals.Althoughdecreasedfoodconsumptionandbodyweightwerereportedin
alltreatedmalesanddecreasedfoodconsumptionwasreportedinfemalesat100,
200and400mg/kgbwperday,theauthorsdidnotconsiderthistobeanindication
ofsystemictoxicity,butratherasecondaryresponsetothedecreasedconsump-
tion of water. Similarly, although variations in weights of the kidney and liver
were noted at weeks 4 and 13, these were deemed to be secondary to body-
weightchangesandtoxicologicallyinsignifcantonthebasisofthelackofadose-
dependentresponseandthemagnitudeofthechanges.Increasedweightsofthe
stomach were reported in males and females at 100, 200 and 400mg/kgbw per
day throughout the study. This increase was accompanied by histopathological
changes,includingminimal(100mg/kgbwperday)tomoderate(400mg/kgbwper
day) hyperplasia and hyperkeratosis (200 and 400mg/kgbw per day) of the
forestomach.Acuteirritationwaspresentintheforestomachofmalesandfemales
at 200 and 400mg/kgbw per day during the frst 2 weeks, but not after week 4.
No histopathological changes were reported in the glandular stomach of males
andfemalesatanydose.Nocompound-relatedchangeswerereportedat20mg/
kgbwperdaycomparedwiththecontrols(Bernackietal.,1987a).
Groupsof20maleF344/Nratsweregivenethylacrylate(dissolvedincornoil)
atadoseof0,20,100or200mg/kgbwperdaybyintragastricinstillation,5days
perweek,for13weeks.Anadditionalrecoverygroupof10ratswasgivenethyl
acrylateatadoseof200mg/kgbwperdayforthefrst4weeksofthestudyonly
(administration was subsequently discontinued for the remainder of the study
period, i.e. 9 weeks). Animals were monitored daily for clinical signs of toxicity.
Foodconsumptionandbodyweightsweremonitoredweekly.Atweeks4and13
(only at week 13 for the recovery group), 10 animals from each treatment group
were necropsied and subjected to a complete histopathological examination. No
clinical signs of toxicity and no early deaths were reported throughout the study.
A slight decrease in body weight was reported at 200mg/kgbw per day at week
13. Only incidental, non-dose-dependent liver and kidney weight changes were
identifed.Signifcantlyincreased(p<0.05),generallyinadose-dependentmanner,
absoluteandrelativeweightsofthestomachwerereportedinthegroupsreceiv-
ingadoseof100and200mg/kgbwperdayafter4weeksofexposureandinall
treatedgroupsafter13weeksofexposure.Theincreasedstomachweightswere
accompanied at every dose by histopathological changes in the forestomach,
includingdiffusehyperplasiaandhyperkeratosisofthesquamousepithelium.Evi-
denceofsustainedirritationoftheforestomachwaspresentat100(minimalonly)
and 200mg/kgbw per day at week 4, and at 200mg/kgbw per day at week 13.
352 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
Focal papillomatous hyperplasia, overlaying sites of focal infammation, was
reportedatweeks4and13,butonlyatthehighestdose(200mg/kgbwperday),
aswereincidencesoffocaleschar,epithelialhaemorrhageandulcersofthelimit-
ingridgeatweek4.Nohistopathologicalchangeswerereportedintheglandular
stomachofmalesandfemalesatanydose.Increasedstomachweightsandthe
accompanying histopathological changes were not reported in males or females
fedethylacrylateatadoseof200mg/kgbwperdayfor4weeksandallowedto
recover for 9 weeks. On the basis of these results, the authors concluded that
earlyhistopathologicalchangesintheforestomachofratsresultingfromtreatment
withethylacrylatearereversible(Bernackietal.,1987b).
Inastop-exposurestudy,groupsofmaleF344/Nratsweregivenethylacrylate
(incornoil)atadoseof100or200mg/kgbwperdaybyintragastricinstillation5
daysperweek,for13weeks.Aconcurrentcontrolgroupwasmaintainedoncorn
oil. At study termination, necropsies were performed on 1011 rats from each
groupat24hafterthelastdosewasadministered,andcompletegrossandhisto-
pathologicalevaluationsoftheforestomach,glandularstomachandtheliverwere
alsocarriedout.Additionalgroupsof10and2635remainingratsweresimilarly
examined at necropsy following 8-week and 19-month recovery periods, respec-
tively,whichfollowedimmediatelyafterthelastdoseofethylacrylatewasadmin-
istered.Resultsshowedthatcompound-relatedtoxicityoccurredasaresultofthe
interactionofethylacrylatewiththetissuesdirectlyexposedtothetestmaterial.
At the end of the 13 weeks of treatment, moderate (at 100mg/kgbw per day) to
severe (at 200mg/kgbw per day) forestomach hyperplasia was reported in all
treated rats, presented as a slight thickening accompanied by occasional foci at
thelowestdose(100mg/kgbwperday),andasfocalandmultifocalraisednodules
at the highest dose (200mg/kgbw per day). No lesions were observed in the
glandular stomach or liver. Following the 8- and 19-month recovery periods, a
signifcant decline in the incidence and severity of forestomach hyperplasia was
reported.Theauthorsnotedthatmostofthetreatedratshadhistologicallynormal
forestomachs. These results indicate that sustained forestomach hyperplasia in
ratsresultingfromprolongedexposuretohighdosesofethylacrylateadministered
bygavagecanregressaftercessationofdosing(Ghanayemetal.,1991a).
Groups of male F344 rats were given ethyl acrylate (in corn oil) at a dose of
100 or 200mg.kgbw per day by intragastric instillation 5 days per week for 13
weeks.A concurrent control group was maintained on corn oil only.At week 13,
1011ratsfromeachgroupwerenecropsied24hafterthelastdosewasadmin-
istered and their stomachs were subjected to histopathological evaluation.Addi-
tionally,5ratsand1215ratsfromeachgroupweresimilarlyexaminedfollowing
8-weekand19-monthrecoveryperiods,respectively.Attheendofthe13weeks
of treatment, a slight thickening of the forestomach mucosa accompanied by
occasional foci was observed at the lowest dose and focal and multifocal raised
nodularproliferationsoftheforestomachwerefoundatthehighestdose.Afterthe
8-weekand19-monthrecoveryperiods,theincidencesofforestomachhyperplasis
werereportedtobe20%and0%,respectively,atthelowestdoseand100%and
27%,respectively,atthehighestdose.Therefore,regressionoftheforestomach
hyperplasiainducedbyethylacrylateoccurreduponcessationofdosing(Ghanayem
etal.,1991b).
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 353
L1
(ii) 2-Hexenal (No. 1353)
Rats
Groupsof15maleand15femaleCFWratsweremaintainedondietscontain-
ing2-hexenalataconcentrationof0,260,640,1600or4000mg/kgfor13weeks
(Gauntetal.,1971).Theseconcentrationscorrespondtoaveragedailydosesof
0,13,32,80and200mg/kgbw,respectively(FoodandDrugAdministration,1993).
Bodyweightsandfoodintakeweremeasuredweekly.Samplesofbloodandurine
werecollectedatweeks6and7,respectively,andagainattheendofthestudy.
Attermination,grossexaminationswerecarriedoutonalltreatedratsandarange
of organs (brain, pituitary, thyroid, heart, liver, spleen, adrenal glands, kidneys,
andgonads)wereweighed.Tissuesectionstakenfromweighedorgansandother
tissues(lymphnodes,thymus,urinarybladder,stomach,duodenum,ileum,colon,
caecum,rectum,pancreas,uterus,andskeletalmuscle)ofthecontrolratsandthe
ratsat200mg/kgbwperdaywerestainedformicroscopicexamination.Nodiffer-
enceingeneralhealthandbehaviourwasreportedinanyofthetreatedanimals.
Theslight,notstatisticallysignifcant,decreaseingrowthrateat200mg/kgbwper
daywasassociatedwitha10%reductioninfoodintakeattributabletodecreased
palatability of the diet. In males, statistically signifcant but not dose-dependent
decreaseswerereportedinconcentrationsofhaemoglobinat80mg/kgbwperday
and in erythrocytes at 32 and 80mg/kgbw per day; these, however, were not
associatedwithincreasedkidneyweightsoranychangesinhistology.Nochanges
inhaematologywerereportedinanytreatedfemales.Theresultsofurineanalysis
were unremarkable, with the exception of a signifcant reduction in the specifc
gravityoftheurineobservedinmaleatthehighestdose(200mg/kgbwperday).
Comparedwithcontrols,a2030%,non-dosedependentincreaseinrelativeand
absoluteweightsoftheovarywasreportedinfemalesateverydose,withoutany
accompanying changes in ovarian histology. Subsequent to these results, addi-
tionalgroupsoffemalerats(10pergroup)werefeddietscontaining2-hexenalat
aconcentrationof0or4000mg/kg(approximately200mg/kgbwperday)fromthe
sampleusedinthemainstudyorfromadifferentsamplefor13weeks.Theeffects
observed in the frst study were not observed in the second study regardless of
the source sample of 2-hexenal. Furthermore, histopathological examination of
theovaries,uterus,pituitary,andadrenalglandsrevealednoadverseeffects.The
authors concluded that the increased ovary weight seen in the frst study was
accidentalandthattheno-observed-effect-level(NOEL)for2-hexenalinratswas
approximately80mg/kgbwperday(Gauntetal.,1971).
Rabbits
In a subsequent study at the same laboratory, groups of 10 female New
Zealandwhiterabbitsweregiventrans-2-hexenalatadoseof0or200mg/kgbw
perdaybygavageincornoil(2ml/kg)for13weeks.Asingleearlydeath,attributed
togavageerror,wasreported.Weeklymeasurementofbodyweightsshowedthat
treatedanimalsgainedlessweightcomparedwithcontrolsearlyinthestudy,but
thedifferencewasnotstatisticallysignifcant.Atnecropsy,bloodsamplesforhae-
matologicalevaluationwereobtainedatthesameintervalsasinthestudyinrats,
354 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
and organ weight measurements and histopathological examinations were per-
formedonthesameorgansandtissuesasinthestudyinrats.Weightsofstom-
achs of treated rabbits were signifcantly increased (p < 0.001) compared with
thoseofcontrols,buttherewerenodifferencesintheweightsofanyotherorgans,
includingtheovaries,betweentestandcontrolanimals.Microscopicexamination
revealedhaemorrhageandsmallacuteulcersinthreeofthetreatedanimals.The
authors concluded that the gastric damage in rabbits treated by gavage was
due to the route of administration, which produced high stomach concentrations
(600mg/100ml) of an irritating aldehyde, providing conditions for ulceration of
thegastricmucosa.Thelackofulcerativeeffectsinthestomachsofratsfeddiets
containing2-hexenalatadoseof200mg/kgbwperdaysupportedthisconclusion.
Nootherhistopathologicalchangeswerereported.Mildanaemiawasreportedin
treated rabbits, as evidenced by a statistically signifcant decrease in concentra-
tionsofhaemoglobin,whichtheauthorsassociatedwiththepresenceofstomach
ulcers(Gauntetal.,1971).
(c) Long-term studies of toxicity and carcinogenicity
Theresultsoflong-termstudiesoftoxicityandcarcinogenicitywithethylacry-
late(No.1351)aredescribedbelowandsummarizedinTable6.
(i) Ethyl acrylate (No. 1351)
Several studies were conducted in mice and rats to determine a mode of
administration that would deliver suffcient amounts of ethyl acrylate to produce
systemic toxicity without producing severe irritation at the site of administration
(National Toxicology Program, 1986). Histopathological changes and lesions in
the forestomach and glandular stomach have been reported in mice and rats
givenethylacrylateatdosesof>100mg/kgbwperdaybygavagefor214days
(Ghanayem et al., 1986; National Toxicology Program, 1986; Ghanayem et al.,
1991b).
Mice
Nocompound-relatedeffectswereseeninmaleandfemaleB6C3F
1
micegiven
ethylacrylate(incornoil)atadoseof0,1.5,3,6,12or25mg/kgbwperdayby
intragastricinstillation,5daysperweekfor13weeks(NationalToxicologyProgram,
1986).Consequently,asecondstudywasundertakeninwhichgroupsof10male
and10femaleB6C3F
1
miceweregivenethylacrylate(incornoil)atadoseof0,
12, 25, 50 or 100mg/kgbw per day by gavage, 5 days per week for 13 weeks
(NationalToxicologyProgram,1986).Aconcurrentcontrolgroupwasmaintained.
The animals were observed twice daily for clinical signs of toxicity and body
weights were recorded weekly.At termination, necropsies were performed on all
treated animals and complete histopathology was performed on animals in the
groupreceivingthehighestdose(100mg/kgbwperday)andthecontrolgroups.
Nocompound-relatedeffectswerereportedinsurvivalorbodyweightineithersex
atanydose.Nocompound-relatedgrossormicroscopicchangeswereobserved
inanyofthetreatedgroupswhencomparedwiththecontrols(NationalToxicology
Program,1986).
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 355
L1
Groupsof50maleand50femaleB6C3F
1
miceweregivenethylacrylate(in
corn oil) at doses of 0, 100 or 200mg/kgbw per day by intragastric instillation,
5 days per week for 103 weeks. A concurrent control group was maintained.
Theanimalswereobservedtwicedailyformortalityandsignsofmorbidity.Body
weights were recorded weekly for the frst 12 weeks and monthly thereafter.At
termination,necropsieswereperformedonalltheanimalsthatsurvivedtotheend
ofthestudyandonallanimalsfounddeadduringthestudy.Nosignifcantdiffer-
encesinsurvivalwereobservedbetweenanytreatedgroupsofthesamesex.The
meanbodyweightsofalltreatedanimalsweresimilartothecontrolsthroughout
thestudy,exceptforanunexplaineddecreaseinthefemalesatthelowestdose
(100mg/kgbw per day). No compound-related clinical fndings were observed in
anytreatedanimals.Adose-relatedincreaseintheincidencesofhyperkeratosis,
hyperplasia,andinfammationoftheforestomachwerereportedinbothsexesof
treatedmice(seeTable7).Statisticallysignifcantpositivetrendsintheincidence
of squamous cell neoplasms were reported in the forestomachs of treated male
andfemalemice(NationalToxicologyProgram,1986).
On the basis of these fndings, the National Toxicology Program concluded
that:
...undertheconditionsofthesestudies,ethylacrylatewascarcinogenicfortheforestomach
ofB6C3F
1
micecausingsquamouscellcarcinomasinmalemice,squamouscellpapillomas
inmalemice,andsquamouscellpapillomasorcarcinomas(combined)inmaleandfemale
mice.Evidenceforcarcinogenicitywasgreaterinmalesthaninfemales.Ethylacrylatealso
causedirritationoftheforestomachmucosainmaleandfemalemice.
Rats
Groups of 10 male and 10 female F344/N rats were given ethyl acrylate (in
cornoil)atadoseof0,7,14,28,55or110mg/kgbwperdaybygavage,5days
perweekfor13weeks.Aconcurrentcontrolgroupwasmaintained.Theanimals
were observed twice daily for clinical signs of toxicity and body weights were
recordedweekly.Attermination,necropsieswereperformedonalltreatedanimals
and complete histopathology was performed on the animals at the highest dose
(110mg/kgbw per day) and in the control groups. No compound-related effects
werereportedinsurvivalorbodyweightineithersexatanydose.Reddeningin
theduodenumandprominentbloodvesselsinthecardiacregionofthestomach
were seen in three males fed with ethyl acrylate at a dose of 110mg/kgbw per
day.Nocompound-relatedclinicalsignsoftoxicityandnohistopathologicaleffects
were observed in any of the treated groups when compared with the controls
(NationalToxicologyProgram,1986).
Groups of 50 male and 50 female F344/N rats were given ethyl acrylate (in
cornoil)atadoseof0,100or200mg/kgbwperdaybygavage,5daysperweek
for 103 weeks. A concurrent control group was maintained. The animals were
observed twice daily for mortality and signs of morbidity. Body weights were
recorded weekly for the frst 12 weeks and monthly thereafter. At termination,
necropsies were performed on all animals that survived to the end of the study
andonanyanimalsfounddeadduringthestudy.Nosignifcantdifferencesinsur-
vivalwereobservedbetweengroupsofthesamesex.Themeanbodyweightsof
356 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
the treated groups were comparable to those of the controls and no compound-
related clinical signs of toxicity were observed in the treated animals throughout
thestudy.Dose-relatedincreasesintheincidenceofnon-neoplasticlesionswere
observedintheforestomachofmalesandfemales(seeTable8).Statisticallysig-
nifcantpositivetrendsintheincidenceofsquamouscellpapillomaswerereported
in males and females. Statistically signifcant positive trends in the incidence of
squamouscellcarcinomaswerereportedinmales(NationalToxicologyProgram,
1986).
On the basis of these fndings, the National Toxicology Program concluded
that:
under the conditions of these studies, ethyl acrylate was carcinogenic for the forestomach
ofF344/Nrats,causingsquamouscellcarcinomasinmalerats,squamouscellpapillomas
inmaleandfemalerats,andsquamouscellpapillomasorcarcinomas(combined)inmale
and female rats. Evidence for carcinogenicity was greater in males than in females. Ethyl
acrylatealsocausedirritationoftheforestomachmucosainmaleandfemalerats.
Table 7. Incidences of neoplasms and non-neoplastic lesions of the
forestomach in B6C3F
1
mice given ethyl acrylate by gavage
Neoplasmor Dose(mg/kgperday)
non-neoplasticlesion
0(vehiclecontrol) 100 200
Males
Hyperkeratosis,incidence/ 0/48(0%) 19/47(40%) 28/50(56%)
No.ofanimalsnecropsied(%)
Epithelialhyperplasia,incidence/ 0/48(0%) 17/47(36%) 26/50(52%)
No.ofanimalsnecropsied(%)
Squamouscellpapilloma/ 0/48(0%) 4/47(9%) 9/50(18%)
No.ofanimalsnecropsied(%)
Squamouscellcarcinoma/ 0/48(0%) 2/47(4%) 5/50(10%)
No.ofanimalsnecropsied(%)
Squamouscellpapillomaor 0/48(0%) 5/47(11%) 12/50(24%)
carcinoma/No.ofanimals
necropsied(%)
Females
Hyperkeratosis,incidence/ 2/50(4%) 14/49(29%) 32/48(67%)
No.ofanimalsnecropsied(%)
Epithelialhyperplasia, 3/50(6%) 12/49(24%) 30/48(63%)
incidence/No.ofanimals
necropsied(%)
Squamouscellpapilloma/ 1/50(2%) 4/49(8%) 5/48(10%)
No.ofanimalsnecropsied(%)
Squamouscellcarcinoma/ 0/50(0%) 1/49(2%) 2/48(4%)
No.ofanimalsnecropsied(%)
Squamouscellpapillomaor 1/50(2%) 5/49(10%) 7/48(15%)
carcinoma/No.ofanimals
necropsied(%)
FromNationalToxicologyProgram(1986).
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 357
L1
Studies have indicated that concentrations of GSH are depleted in the rat
forestomach after dosing with ethyl acrylate by gavage (DeBethizy et al., 1987;
Frederick et al., 1990). Saturation of the GSH pathway is suggested from the
observationthat,asdoseofethylacrylateincrease,excretionofmercapturicacid
conjugatesdecreases(DeBethizyetal.,1987;Fredericketal.,1990).
Tissuetoxicityasaresultoftheaccumulationofethylacrylateisafunctionof
therateofethylacrylatedeliverytothetissueandtheeffciencyofthemetabolic
detoxication process in the tissue. Studies have shown that the rapid delivery of
largequantitiesofethylacrylatetoatissuedepletesGSHfasterthanitcanbere-
synthesized.Thismethodofdosingalsodepletestheavailablenon-lethalprotein-
binding sites (Frederick et al., 1992). The depletion of GSH observed in the rat
forestomachisassociatedwiththetoxicityobservedinratsgivenethylacrylatein
largedosesbygavage(Fredericketal.,1990).Previousstudieshavealsoshown
Table 8. Incidences of neoplasms and non-neoplastic lesions of
the forestomach in F344/N rats given ethyl acrylate by gavage
Neoplasmor Dose(mg/kgperday)
non-neoplasticlesion
0(vehiclecontrol) 100 200
Males
Hyperkeratosis,incidence/ 0/50(0%) 37/50(74%) 46/50(92%)
No.ofanimalsnecropsied(%)
Epithelialhyperplasia,incidence/ 1/50(2%) 41/50(82%) 46/50(92%)
No.ofanimalsnecropsied(%)
Squamouscellpapilloma/ 1/50(2%) 15/50(30%) 29/50(58%)
No.ofanimalsnecropsied(%)
Squamouscellcarcinoma/ 0/50(0%) 5/50(10%) 12/50(24%)
No.ofanimalsnecropsied(%)
Squamouscellpapillomaor 1/50(2%) 18/50(36%) 36/50(72%)
carcinoma/No.of
animalsnecropsied(%)
Females
Hyperkeratosis,incidence/ 0/50(0%) 24/50(48%) 46/50(92%)
No.ofanimalsnecropsied(%)
Epithelialhyperplasia,incidence/ 0/50(0%) 34/50(68%) 49/50(98%)
numberanimals
necropsied(%)
SquamousCellPapilloma/ 1/50(2%) 6/50(12%) 9/50(18%)
Numberanimals
necropsied(%)
Squamouscellcarcinoma/ 0/50(0%) 0/50(0%) 2/50(4%)
numberanimals
necropsied(%)
Squamouscellpapillomaor 1/50(2%) 6/50(12%) 11/50(22%)
carcinoma/numberanimals
necropsied(%)
FromNationalToxicologyProgram(1986).
358 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
no changes in the histopathology of the forestomach at doses of 10mg/kg per
day,mildhyperplasiaatdosesofbetween20and50mg/kgbwperdayandsevere
hyperplasia, infammation and ulceration at doses of 100mg/kg. These toxic
responses were attributed to ineffcient metabolic detoxifcation at higher doses
(Fredericketal.,1990).
No carcinogenic effects on the skin were reported when 100% ethyl acrylate
as25mldoseswasappliedtotheskinofagroupof40maleC3H/HeJmicethree
timesperweekinalifetimestudy(408days)(DePassetal.,1984)orwhenmice
orratswereexposedtovapoursofethylacrylateatconcentrationsof75mg/lfor
6hperday,5daysperweekfor27monthsand225mg/lfor6hperday,5days
perweekfor6months(Milleretal.,1985).Theseresultssupporttheconclusion
that the carcinogenic potential of ethyl acrylate is associated with the route of
administration (i.e. in corn oil, by gavage) when doses administered to the test
animalsexceedthemaximumtolerateddose.
Groupsof25femaleand25maleyoungalbinoWistarratsweregivendrinking-
water containing ethyl acrylate at a concentration of at 0, 6, 60 or 2000mg/l for
2 years (Borzelleca et al., 1964). The National Toxicology Program determined
that the concentration of 2000mg/l in drinking-water corresponded to a dose of
approximately170or120mg/kgbwperdayformaleandfemalerats,respectively
(National Toxicology Program, 1986). Decreased body weights were reported in
maleandfemaleratsreceivingethylacrylateat2000mg/l.Haematologicalevalu-
ations (i.e. erythrocyte volume fraction, haemoglobin, total white and differential
whitecellcounts)andurineanalysis(i.e.protein,reducingsubstances)conducted
at 3-month intervals showed normal ranges for the parameters studied in all
treated animals throughout the study. At termination, histopathology revealed
no compound-related neoplastic or non-neoplastic lesions in treated animals of
eithersex.
Dogs
Groups of two male and two female pure-bred beagle dogs were given corn
oilgelatincapsulescontainingethylacrylateatadietaryequivalentconcentration
of0,10,100or1000mg/kgfor2years.Thiscorrespondstoaveragedailyintakes
of0.75,7.5and75mg/kgbw,respectively(FoodandDrugAdministration,1993).
Reducedbody-weightgainsreportedindogsreceivingethylacrylateatadoseof
75mg/kgbw per day were correlated with decreased food consumption. Haema-
tologicalevaluations(i.e.erythrocytevolumefraction,haemoglobin,totalanddif-
ferential leukocyte counts) and urine analysis (i.e. protein, reducing substances)
conductedat3-monthintervalsshowednormalrangesfortheparametersstudied
inalltreatedanimalsthroughoutthestudy.Attermination,histopathologyrevealed
nocompound-relatedneoplasticornon-neoplasticlesionsineithersexoftreated
animals(Borzellecaetal.,1964).
(ii) Related substance, trans,trans-2,4-hexadienal (No. 1175)
Trans,trans-2,4-Hexadienal(No.1175),whichwaspreviouslyevaluatedbythe
Committeeatitssixty-frstmeeting(Annex1,reference166),isstructurallysimilar
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 359
L1
totrans-2-hexenal(No.1353).Thetwosubstancesdifferonlyinthepresenceof
anadditionaldoublebondatthe3,4positionofhexadienal.Beinga,b-unsaturated
aldehydesofthesamecarbonchainlength,bothsubstancesarerapidlyabsorbed
andmetabolizedviathesamepathways.Athighconcentrations,bothareirritating
aldehydesthatexhibittoxiceffectsuponrepeateddirectcontactwithtissuessuch
asthegastricmucosaoftheforestomach.
Thestructurallyrelateda,b-unsaturatedaldehyde,trans,trans-2,4-hexadienal,
hasbeenthesubjectofarecentNationalToxicologyProgrambioassay(National
Toxicology Program, 2001a) in mice and rats. The results of these studies were
presented in a previous review (Annex 1, reference 166). Therefore, a concise
summary of the results of these studies and the evaluation of the effects in the
contextof2-hexenal(No.1353)willbepresented.
Groups of 50 male and 50 female B6C3F
1
mice and 50 male and 50 female
F344/Nratsweregiven2,4-hexadienal(incornoil)atadoseof0,30,60or120
and0,22.5,45or90mg/kgbwperday,respectively,bygavage5daysperweek
for104weeks(NationalToxicologyProgram,2001a).Onthebasisofthesefnd-
ings,theNationalToxicologyProgram concludedthat:
...there was clear evidence of carcinogenic activity of 2,4-hexadienal in male and female
B6C3F
1
miceandinmaleandfemaleF344/Nratsbasedonincreasedincidencesofsqua-
mouscellneoplasmsoftheforestomach.
Relevance of forestomach tumours
Therelevanceoftheappearanceofforestomachtumoursinrodentstopoten-
tial carcinogenic targets in humans has been the subject of much investigation
(Grice,1988;Wester&Kroes,1988;Claysonetal.,1990).AnInternationalAgency
forResearchonCancerworkinggroup(IARC,2003)concludedthatinevaluating
therelevanceoftheinductionofforestomachtumoursinrodentsforhumancancer,
theexposureconditionsintheexperimentshavetobeconsidered.Theexposure
conditionsduringoraladministrationareunusual(particularlyifdosingbygavage
isemployed)inthatphysicaleffectsmayresultinhighlocalconcentrationsoftest
substances in the forestomach and prolonged exposure of the epithelial tissue.
Agents that only produce tumours in the forestomach in rodents after prolonged
treatment through non-DNA reactive mechanisms may be of less relevance to
humans, since human exposure to such agents would need to surpass time-
integrateddosethresholdsinordertoelicitthecarcinogenicresponse.
Therefore, the appearance of forestomach lesions in the 2-year bioassays in
rodentsinwhichethylacrylate(No.1351)ortrans,trans-2,4-hexadienal(No.1175)
wereadministeredathighconcentrationsbygavagehasnorelevancetohumans,
giventhattheresultsareattributabletotheirritatingeffectofhighbolusdosesof
ethylacrylateortrans,trans-2,4-hexadienaldeliveredtothecontactsite(forestom-
ach)bygavage,andnottheeffectsofsystemicconcentrationsinthewholeanimal.
On biochemical grounds and in analogy to trans,trans-2,4-hexadienal, trans-2-
hexenal (No. 1353), therefore, would also be expected not to have any carcino-
genicpotentialinhumans.
360 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
(d) Genotoxicity
(i) In vitro
Testing in vitro for genotoxicity has been performed with representative
members of the group of aliphatic, linear, a,b-unsaturated alcohols, aldehydes,
carboxylicacidsandrelatedestersusedasfavouringagents(seeTable9).Testing
formutagenicityinbacteriawitha,b-unsaturatedaldehydesisproblematicowing
totheirhighbacterialtoxicity.Thecytotoxicityofthesesubstancesisbelievedto
arise from their interactions with protein sulfhydryl and amino groups (Marnett
et al., 1985; Eder et al., 1992). Owing to the nature of the GSH conjugation
pathway, studies of genotoxicity in which a,b-unsaturated aldehydes are applied
athighconcentrationsarelikelytopromoteoxidativestress.Itisanticipatedthat
cells exposed to a,b-unsaturated aldehydes at high concentrations will rapidly
deplete GSH, eventually leading to cellular damage and decreased cell viability,
andsubsequentreactionwithcellularproteinsandDNA.
Since the majority of assays were performed with ethyl acrylate, a series of
homologous a,b-unsaturated aldehydes, or unsaturated acids and esters, the
discussion of the results of these tests are organized according to substance
(e.g. ethyl acrylate) or group of substances (2-pentenal, 2-hexenal, 2-heptenal,
2-octenal).
Ethyl acrylate (No. 1351)
NegativeresultswerereportedinAmesassayswhenSalmonella typhimurium
strains (TA97, TA98, TA100, TA1535, TA1537 and TA1538) were incubated with
ethyl acrylate (No. 1351) at a concentration of up to 10000mg/plate (Ishidate
etal.,1981;Waegemaekers&Bensink,1984;Tennantetal.,1987;Zeigeretal.,
1992).
Noevidenceofmitoticchromosomallosswasobtainedwhenethylacrylateat
concentrationsofupto1095mg/mlwereincubatedwithSaccharomyces cerevisiae
strainD61.M.Underacoldshockregimen,evidenceofmitoticrecombinationwas
reported(Zimmermann&Mohr,1992).
In the standard assay for forward mutation in mouse lymphoma cells, ethyl
acrylategaveuniformlypositiveresultsintheabsenceofmetabolicactivationwhen
incubated with mouse lymphoma L5178Y Tk
+
cells at cytotoxic concentrations
(37.550mg/ml)(Tennantetal.,1987;McGregoretal.,1988;Mooreetal.,1988;
Ciaccioetal.,1998).Inthemostrecentstudy(Ciaccioetal.,1998),therelation-
shipbetweencytotoxicityandmutationfrequencyintheassayforforwardmutation
in mouse lymphoma cells, the ethyl acrylate-induced mutagenic response was
found to be directly related to the time- and concentration-dependent decrease
in non-protein sulfhydryl levels and subsequent mitochondrial membrane
impairment.
Inassaysforclastogenicity,ethylacrylateelicitedincreasesinsisterchromatid
exchanges (SCE) and chromosomal aberrations (CA) in Chinese hamster ovary
cells with metabolic activation, but showed no evidence of clastogenicity in the
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 361
L1
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362 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
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ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 369
L1
absenceofmetabolicactivation(Lovedayetal.,1990).Theclastogenicpotential
was unaffected by changes in harvest time (Loveday et al., 1990). In a dose-
dependent manner, beginning at 20mg/ml, ethyl acrylate induced an increase
inSCEandCAinmouselymphomacells(Mooreetal.,1988)intheabsenceof
metabolicactivation.AnincreaseinSCEandCAinChinesehamsterovarycells
wasreportedwithethylacrylateatconcentrationsof150and299mg/ml,respec-
tively, with metabolic activation (Tennant et al., 1987). There was no evidence
of clastogenicity in the absence of metabolic activation (Tennant et al., 1987).
IncreasesinCAwerereportedat9.8mg/mlinChinesehamstercellswithorwithout
metabolicactivation(Ishidateetal.,1981).
Linear a,b-unsaturated aldehydes
Inastudyusingtesterstrains(TA104)ofS. typhimurium thataremoresensi-
tive than other strains in identifying a,b-unsaturated aldehydes as mutagens, a
seriesofa,b-unsaturatedaldehydeswereincubatedwithTA104.Inthismodifed
Ames assay using liquid pre-incubation protocols (i.e. addition of a GSH chase
at the end of an incubation of 20 min inTA104), signifcant increases in reverse
mutationsintheabsenceofmetabolicactivationwerereportedwhenS. typhimurium
strainTA104wasincubatedwith2-hexenal(No.1353)atconcentrationsof>196mg/
plate(Marnettetal.,1985).a,b-Unsaturatedaldehydesofhigherrelativemolecular
mass were too toxic to test.At the concentrations tested, 2-heptenal (No. 1360)
(upto101mg/plate),2-octenal(No.1363)(upto101mg/plate),and2-nonenal(No.
1362) (up to 1mg/plate) gave no evidence of mutagenicity when incubated with
TA104withoutmetabolicactivation.S. typhimuriumstrainTA104containsanon-
sense mutation (TAA) at the site of reversion and is much more sensitive to
carbonylmutagenesisthanstandardSalmonellastrains.IncreasedTA104sensitiv-
ityisrelatedtothedeletionoftheuvrBgene,whichencodesforanerror-freeDNA
excision repair and incorporation of the pKM101 plasmid, which encodes for an
error-prone DNA polymerase involved in bypass replication of lesions (Marnett
et al., 1985). TA104 is also sensitive to cytotoxicity. To reduce cytotoxicity, GSH
wasincorporatedintotheAmesassay.Themaximumnon-toxicdoseof2-hexenal
tested increased from 196 to >491mg/plate after the addition of reduced GSH at
aconcentrationof10mmol/lattheendofthepre-incubationperiod;however,its
mutagenic potential remained unaltered. The authors proposed that the addition
ofGSHreducedtoxicitybypreventingexcessaldehyde,presentafterincubation,
from reacting with protein sulfhydryl groups. No mutagenicity was reported for
2-heptenal(No.1360)(upto494mg/plate)or2-octenal(No.1363)(upto505mg/
plate)whenGSHat10mmol/lwasadded.Also,noevidenceofmutagenicitywas
reported when the six 2-alkenals were incubated with TA102, which contains
theuvrBgenethatencodesforanerror-freeDNAexcisionrepair(Marnett etal.,
1985).
In other modifedAmes assays, changes in methodology have been used to
evaluate mutagenic potential in the presence of signifcant cytotoxicity. InAmes
pre-incubation assays, using strain TA100, a,b-unsaturated aldehydes were
incubated with the standard bacterial cell density or three times the standard
bacterialcelldensity(Eder etal.,1992,1993).Underusualconditionsinvolvinga
370 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
pre-incubation period of 30min, and a standard cell density, the high cytotoxicity
demonstrated by simple linear aldehydes may limit the detection of mutagenic
responses(e.g. butenal,pentenal,hexenal,heptenal);however,atthreetimesthe
standard cell density and an increased pre-incubation time of 90min, butenal,
pentenal,hexenal,orhexadienalincubation,withorwithoutmetabolicactivation,
producedaspontaneousreversionfrequencyofatleasttwicethatobservedunder
standard conditions. Under the specifed conditions, results obtained using S.
typhimuriumstrainTA100werefoundtobeconsistentwithreportsofmutagenicity
caused by a,b-unsaturated aldehydes as demonstrated in tester strainTA104 in
the presence of GSH (Marnett et al., 1985).Among the aldehydes investigated,
increasedcytotoxicityandmutagenicitycorrelatedwithincreasedlipophilicity.The
effectofdetoxicationuponadditionofmetabolicactivationwasindicatedbyashift
tohighernon-cytotoxicdosesandhigherpeakrevertantfrequencies.
2-Hexenal (No. 1353) was tested for mutagenicity in the Ames assay using
different strains of S. typhimurium (e.g. TA98, TA100, TA1535, TA1537) in the
presenceorabsenceofmetabolicactivation.Noevidencewasfoundformutagen-
icityatconcentrationsupto3mmol/plate(294mg/plate)(Florinetal.,1980).
Negative results were reported with E. coli strains PQ37 and PQ243 (SOS
chromotest) incubated in the presence of 2-pentenal (No. 1364), 2-hexenal
(No.1353),or2-heptenal(No.1360)atconcentrationsupto37,43or30mg/plate,
respectively (Eder et al., 1992). The authors noted that high bacterial toxicity
interferedwiththeperformanceofthetest.
Theabilityofa,b-unsaturatedaldehydestoinduceSCE,numericalandstruc-
tural CA, and formation of micronuclei was investigated in cell lines that are low
in GSH and detoxication enzymes (i.e. human blood lymphocytes and Namalva
celllines)(Dittberneretal.,1995).trans-2-Butenalat5250mmol/l,2-hexenal(No.
1353)at5250mmol/l,ortrans-2-cis-6-nonadienalat550mmol/lwereseparately
incubated with human lymphocyte and Namalva cells. The number of SCE
increasedsignifcantly(p<0.05)atconcentrationsof10mmol/l(0.7mg/ml),40mmol/
l (3.9mg/ml) and 20mmol/l (2.8mg/ml) for 2-butenal, 2-hexenal, and trans-2-cis-6-
nonadienal, respectively, in lymphocytes, and 20mmol/l for 2-butenal (1.4mg/ml)
and2-hexenal(2.0mg/ml),and10mmol/lfortrans-2-cis-6-nonadienal(1.4mg/ml)in
Namalvacells.IntheCAexperimentusingthesamerangesofconcentrations,the
number of structural chromosomal aberrations in human blood lymphocytes sig-
nifcantlyincreasedonlyfor2-butenalatconcentrationsof10mmol/l.InNamalva
cells, which contain lower concentrations of GSH and detoxication enzymes, in-
creasesinchromosomalaberrationswerereportedatconcentrationsof100mmol/l
(7.0mg/ml) for 2-butenal, 100mmol/l (9.8mg/ml) for 2-hexenal (No. 1353), and
5mmol/l(0.7mg/ml)fortrans-2-cis-6-nonadienal.Theincidenceofmicronucleiwas
signifcantly increased at minimum concentrations of 50mmol/l for 2-butenal and
2-hexenal (No. 1353) in lymphocytes, and 40mmol/l for 2-butenal and 150mmol/l
for2-hexenalinNamalvacells.Theincidenceofformationofmicronucleiinblood
lymphocytesandNamalvacellswassignifcantlyincreasedatminimumconcentra-
tionsoftrans-2-cis-6-nonadienalof20mmol/l(2.8mg/ml)and40mmol/l(5.5mg/ml),
respectively. trans-2-cis-6-Nonadienal exhibited severe toxicity at concentrations
of>50mmol/l.Theauthorsconcludedthatundertheconditionsoftheexperiment,
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 371
L1
2-butenal is clastogenic. On the basis of the observations that chromosome
breaks were not signifcantly increased and that micronuclei were positive for a
centromere-specifcDNA,2-hexenal(No.1353)andtrans-2-cis-6-nonadienalwere
classifedasaneugens,notclastogens.Intheabovestudy,noattemptsweremade
toassessatwhatconcentrationslysosomalbreakdownoccurredintheassaysfor
SCE and CA. It has been previously established that increases in the incidence
of SCE and CA near or at observable levels of cytotoxicity may be refective of
secondary effects resulting from lysosome breakdown and release of DNAase
(Zajac-Kaye&Tso,1984;Bradley etal.,1987).
Thepotentialmutagenicityof2-pentenal(No.1364),2-hexenal(No.1353),2-
heptenal(No.1360),2-octenal(No.1363),and2-nonenal(No.1362)wastested
in Chinese hamster V79 cells at concentrations ranging from 0.003mmol/l to
0.3mmol/l (Canonero et al., 1990). All fve alkenals induced a dose-dependent
increaseinthefrequencyof6-thioguanine-resistantmutantsandtheirmutagenic
potency was found to increase with the length of the carbon chain. 2-Heptenal
producedanincreaseinthenumberofmutationstoouabainresistance,butthese
increasesweresignifcantlydifferentfromcontrolsonlyatthehighestdosetested
(0.10mmol/l)(Canoneroetal.,1990).
Cultured rat hepatocytes were incubated with 0.1, 1.0, 10 or 100mmol/l of
trans-2-nonenal(No.1362)for3h(Esterbaueretal.,1990).Signifcantincreases
in the incidence of micronuclei formation were reported at 10 and 100mmol/l,
but not at 0.1 or 1.0mmol/l. There was no statistically signifcant increase in the
incidence of chromosomal aberrations at any concentration tested. In a similar
studyconductedbyEckletal.(1993),signifcantincreasesinSCEswerereported
with trans-2-nonenal at concentrations of 0.1, 10, and 100mmol/l; however, no
signifcant induction of chromosomal aberrations or micronuclei formation was
demonstrated.
In an assay for unscheduled DNA synthesis, cultured rat hepatocytes (60 to
600nmol/10
6
cells) were incubated with trans-2-hexenal (No. 1353) or trans-2-
nonenal (No. 1362) for 20h (Griffn & Segall, 1986). Cytotoxicity was evaluated
by measurement of lactate dehydrogenase release. Increases in unscheduled
DNAsynthesisactivity,asmeasuredbyanincreaseinnetgraincounts(nuclear-
cytoplasmic grain counts), increased in a dose-dependent manner beginning at
120nmol/10
6
cellsfor2-hexenaland60nmol/10
6
cellsfor2-nonenal.Theincreases
correlatedcloselywithincreasedreleaseofLDH.
High concentrations of a series of a,b-unsaturated aldehydes induced single
strandbreaksasdeterminedbythealkalineelutionassayusingmouseleukaemia
L1210 cells (Eder et al., 1993). In almost all cases, strand breaks occurred at
or near cytotoxic concentrations: 600800mmol for 2-pentenal (No. 1364), 250
500mmolfor2-hexenal(No.1353),400500mmolfor2-heptenal(No.1360),and
350mmolfor2-octenal(No.1363).Withtheexceptionof2-pentenalat600mmol,
2-hexenalat250mmol,and2-heptenalat400500mmol,cytotoxicitywasobserved
at all concentrations inducing strand breaks. Additionally, trans-2-pentenal and
trans-2-hexenalreactedwithnucleosidesandnucleotides.WhentheDNAadducts
were investigated, both aldehydes produced 1,2-cyclic deoxyguanosine, but no
7,8-cyclicguanosineadductsorevidenceofcross-linkedadductswereobserved.
372 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
Theauthorsconcludedthata,b-unsaturatedaldehydes mayinducestrandbreaks
eitherbydirectDNAinteraction,orbyprogrammedcelldeath,whichinvolvesthe
releaseofendonucleolyticenzymes(Eder etal.,1993).
Subsequent studies investigated the infuence of GSH and detoxication
enzymeson2-alkenal-inducedDNAdamageinprimaryrathepatocytesandhuman
Namalvacells,thelatterhavinglowerGSHcontentandGSTactivity(Eisenbrand
et al., 1995). DNA single strand breaks were induced at lower concentrations in
Namalvacellsthaninhepatocytes.
Methyl 2-nonynoate (No. 1356) and methyl 2-octynoate
(No. 1357)
In standard assay for mutation in S. typhimurium, methyl 2-nonynoate (No.
1356) and methyl 2-octynoate (No. 1357) were not mutagenic in S. typhimurium
strainsTA98,TA100,TA1535,TA1537,andTA1538whentestedatconcentrations
ofupto3600mg/plate,withandwithoutmetabolicactivation(Wildetal.,1983).
(E)-2-Butenoic acid (No. 1371)
(E)-2-Butenoicacid(No.1371)wastestedformutagenicityintheAmesassay
using S. typhimurium strainsTA98,TA100,TA1535,TA1537, andTA1538 in the
presenceorabsenceofmetabolicactivation.Therewasnoevidenceofmutagenic-
ityatconcentrationsofupto1000mg/plate(Lijinsky&Andrews,1980).However,
using the liquid pre-incubation method, positive results were obtained for (E)-2-
butenoicacidinS. typhimuriumstrainTA100withorwithoutmetabolicactivation
(Lijinsky&Andrews,1980).Intheabsenceofmetabolicactivation,positiveresults
were frst observed with (E)-2-butenoic acid at concentrations as low as 10mg/
plate,whileinthepresenceofmetabolicactivation,signifcantmutagenicactivity
was frst observed with (E)-2-butenoic acid at a concentration of 250mg/plate.
According to the authors, the addition of the metabolic activation system (S9)
partiallydetoxifesthecompound,producingamutagenthatisdifferentfromthat
detectedwithouttheaddedactivation.Inasimilarassay,therewasnoevidence
for mutagenicity at concentrations ranging from 0.1mg/plate to 1000mg/plate in
strainTA100(Rapsonetal.,1980).
A slight dose-dependent increase in SCEs was observed in vitro for (E)-2-
butenoic acid in human lymphocytes, at concentrations ranging from 2.5 to
10.0mmol/l(215to861mg/ml)(Sipietal.,1992).However,asignifcantincrease
inSCEsrelativetocontrolswasnotedonlyatthehighestdosetested(10mmol/l);
atthisdose,adecreaseinthepHofthemedium(by0.40.68pHunits)compared
withthatofcontrolswasalsoobserved.
(ii) In vivo
Ethyl acrylate (No. 1351)
Singleoraldosesofethylacrylateatconcentrationsofupto4%wereadmin-
istered to male F344 rats (Morimoto et al., 1990). The forestomachs exhibited
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 373
L1
oedema and infammation, but no DNA damage was detected by alkaline
elution.
In an in vivo-in vitro assay for clastogenicity, C57BL/6 male mice were given
ethyl acrylate at a dose of 0, 125, 250, 500 or 1000mg/kgbw by intraperitoneal
injection (Kligerman et al., 1991). Twenty-fourh later, animals were sacrifced,
splenocyteswereisolated,andconcanavalinAwasaddedtostimulatecelldivision.
Analysis for chromosomal aberrations in frst division cells and SCE in second
divisioncellsrevealednoevidenceofclastogenicity.Atthehighestdose(1000mg/
kgbw),ethylacrylatedidinduceasmallincreaseinbinucleatedcellmicronuclei;
however,thisdosewasfvefoldhigherthanthehighestdoseusedintheNational
ToxicologyProgramstudy(seebelow)(Kligermanetal.,1991).
InanassayformutagenicityinvivoinDrosophila melanogaster,therewasno
evidenceofanincreaseinsex-linkedrecessivelethalsinthreesuccessivebroods
obtained from Basc virgin females mated with male Canton-S wild-type males
either injected with ethyl acrylate at a concentration of 20000mg/kg or fed a
solution containing ethyl acrylate at a concentration of 40000mg/kg for 3 days
(Valencia et al., 1985). In a second experiment, there was no evidence of muta-
genicitywhenD. melanogasterwerefedasolutioncontainingethylacrylateata
concentrationof18000or20000mg/kg(Valenciaetal.,1985).
Although an early report (Przybojewska et al., 1984) indicated that ethyl
acrylate was genotoxic in a standard assay for micronucleus formation in mice,
subsequentstudies(Ashbyetal.,1989;Kligermanetal.,1991;Haraetal.,1994;
Moritaetal.,1997)confrmedthatethylacrylateexhibitsnogenotoxicpotentialin
thisassay.Anincreaseintheincidenceofmicronucleiinbone-marrowpolychro-
maticerythrocyteswasreportedwhenBALB/Cmalemiceweregivenethylacrylate
atdosesof2251800mg/kgbwbyintraperitonealinjectionintwoseparatedoses
(Przybojewska et al., 1984). However, there was no evidence of an increase in
micronuclei collected 24h after groups of six BDF
1
male mice were given ethyl
acrylate as a single dose at 0, 188, 375 or 750mg/kgbw by oral gavage. There
werealsonoclastogeniceffectsobservedwhenethylacrylateatadoseof0,188,
375 or 750mg/kgbw was administered by double oral gavage, or when ethyl
acrylate as a single dose at 0, 375, 500 or 750mg/kgbw was administered by
intraperitonealinjection(Haraetal.,1994).Inanotherassayformicronucleusfor-
mation,groupsoffvemaleandfvefemaleC57BL/6miceweregivenethylacrylate
asasingleintraperitonealdoseat461or738mg/kgbwandsampleswerecollected
at 24, 48 (738mg/kgbw dose only), and 72h (738mg/kgbw dose only) (Ashby
etal.,1989).Insubsequentexperimentsduplicatingconditionsusedinanearlier
study(Przybojewskaetal.,1984),groupsof510maleC57BL/6andBALB/cmice
weregivenethylacrylateatadoseof738or812mg/kgbwintwodosesadminis-
tered by intraperitoneal injection within 24h, and erythrocytes were sampled
at 30h. In none of these experiments was there any evidence of an increase in
theformationofmicronucleiinbonemarrowofmice(Ashbyetal.,1989).Negative
results were obtained for micronucleus induction when groups of six male BDF
1

mice were given ethyl acrylate either as a single oral dose (188, 375 or 750mg/
kgbw)orasingleintraperitonealdose(188or375mg/kgbw)andsamplesofbone
marrowwerecollectedafter24h(Moritaetal.,1997).
374 ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES
L1
2-Hexenal (No. 1353)
Human volunteers rinsed their mouths with 100ml of an aqueous solution of
trans-2-hexenal(No.1353)ataconcentrationof10mg/kgfourtimesperdayfor
3 consecutive days and exfoliated buccal mucosa cells were then evaluated for
induction of micronuclei (Dittberner et al., 1997).An increase of about threefold
inthefrequencyofformationofmicronucleiwasobservedondays6and7after
administration.Noincreaseswereobservedonprecedingdays.Slowlyeatingfve
tosixcompletelyyellowbananas,whichcontainhexenal,producedasimilar,but
weakereffect.
Methyl 2-nonynoate (No. 1356) and methyl 2-octynoate
(No. 1357)
The potential of methyl 2-nonynoate and methyl 2-octynoate to induce sex-
linkedrecessivelethalmutationsinadultD. melanogasterwerestudiedintheBasc
testforgenotoxicity.Mutationfrequencywasunaffectedwhensolutionsofmethyl
2-nonynoate and methyl 2-octynoate, at 2.5 and 1.0mmol/l (421 and 154mg/ml,
respectively)respectively,werefedtothefiesfor3days(Wildet al.,1983).
In a test for micronucleus formation, groups of four male and female NMRI
miceweregivenmethyl2-nonynoateasasingleintraperitonealdoseat168,336
or505mg/kgbw,ormethyl2-octynoateat154,231or308mg/kgbw.Noincrease
inmicronucleatederythrocytesinbonemarrowsamplesobtained30hafteradmin-
istrationwasobservedforeithersubstance(Wildetal.,1983).
(iii) Conclusion
Testing of a,b-unsaturated aldehydes in standardized Ames assays using
a variety of strains (TA97, TA98, TA100, TA102, TA104, TA1535, TA1537 and
TA1538)hasshownnoevidenceformutagenicityinbacteria(Florin etal.,1980;
National Toxicology Program, 2001a). However, alternative protocols have been
developedtoavoidcompetingcytotoxicityofa,b-unsaturatedaldehydes.Inthese
studies,positiveresultswerereportedinmodifedAmesassayswithpre-incubation
conditionsconducivetodepletionofmetabolicdetoxicationpathways(Eder etal.,
1992,1993).Positiveevidenceofgenotoxicityalsowasreportedinotherassays
(SCE, CA, micronucleus formation) performed in cell lines low in detoxication
capacity (Namalva cells and human lymphocytes) (Dittberner et al., 1995). The
high concentrations of a,b-unsaturated aldehydes (2040mmol/l) used in these
studiesresultedinsingleDNAstrandbreaksbutnocross-linking.Theconditions
oftheexperiments(highconcentrationsofaldehydeincelllinespoorindetoxica-
tioncapacity)providedopportunityforeitherdirectinteractionofa,b-unsaturated
aldehydes with DNA or indirect formation of DNA adducts because of oxidative
stress.Itisnowwellrecognizedthathighconcentrationsofa,b-unsaturatedalde-
hydesdepleteGSH,leadingtoreleaseofnucleocytolyticenzymesthatinduceDNA
fragmentation,cellulardamageandapoptosis(seediscussioninsection2.2.1(c)).
Nonetheless, evidence also has indicated that at low concentrations, such as
those resulting from intake of favouring substances, a,b-unsaturated aldehydes
arerapidlymetabolizedinthehigh-capacityb-oxidationpathway.Inaddition,there
ALIPHATIC, LINEAR a,b-UNSATURATED ALDEHYDES 375
L1
isnoconvincingevidencethata,b-unsaturatedaldehydesexhibitsignifcantgeno-
toxicpotentialinvivo.
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L1
385
MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS, KETONES AND
RELATED ESTERS
First draft prepared by
Professor I.G. Sipes
1
and Dr A.G.A.C. Knaap
2
1
Department of Pharmacology, College of Medicine, University of Arizona,
Tucson, AZ, USA; and
2
Center for Substances and Risk Assessment, National Institute of Public
Health and the Environment, Bilthoven, Netherlands
Evaluation ............................................................................... 385
Introduction......................................................................... 385
Estimateddailyintake........................................................ 394
Absorption,distribution,metabolismandelimination........ 394
ApplicationoftheProcedurefortheSafetyEvaluation
ofFlavouringAgents.................................................... 398
Considerationofsecondarycomponents.......................... 398
Considerationofcombinedintakesfromuseas
favouringagents......................................................... 398
Conclusions........................................................................ 399
Relevantbackgroundinformation............................................. 399
Additionalconsiderationsonintake................................... 399
Biologicaldata.................................................................... 399
Biochemicaldata......................................................... 399
Hydrolysis.............................................................. 399
Absorption,distributionandexcretion................... 400
Metabolism............................................................ 401
Toxicologicalstudies.................................................... 407
Acutetoxicity......................................................... 407
Short-termstudiesoftoxicity................................. 408
Long-termstudiesoftoxicityandcarcinogenicity. 418
Genotoxicity........................................................... 418
Reproductivetoxicity............................................. 423
References............................................................................... 425
1. EVALUATION
1.1 Introduction
The Committee evaluated a group of 32 monocyclic and bicyclic secondary
alcohols,ketonesandrelatedesters(seeTable1)bytheProcedurefortheSafety
Evaluation of Flavouring Agents (see Figure 1, p 192). The Committee has not
previouslyevaluatedanyofthemembersofthisgroup.
Nineteenofthe32favouringagents(Nos13851389,1391,13941400,1403,
1404,1407,1412,1414and1416)havebeenreportedtooccurnaturallyinfoods.
Theyhavebeendetectedinbutter,beef,beer,parmesanandothercheeses,wine,
fruit,herbs,spices,mints,andcocoa(Nijssenetal.,2003).
386 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
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e

n
o
t
e
s

N
o

s
a
f
e
t
y

(
+
)
-
B
o
r
n
y
l
i
s
o
v
a
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a
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e
O
O
H
o
B
-
)
-
(
r
n
y
l
a
v
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s
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l
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r
a
t
e
O H
O

E
u
r
o
p
e
:

0
.
1

a
n
d

c
o
n
c
e
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n

U
S
A
:

0
.
5
I
s
o
b
o
r
n
y
l

i
s
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v
a
l
e
r
a
t
e

1
3
9
4

7
7
7
9
-
7
3
-
9

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

(
+
)
-
I
s
o
b
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n
y
l
i
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v
a
l
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a
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e
H
O
O
o
s
I
-
)
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(
b
o
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y
l
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O

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p
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d

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S
A
:

0
.
0
8

F
e
n
c
h
y
l

a
l
c
o
h
o
l

1
3
9
7

1
6
3
2
-
7
3
-
1

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

p
l
a
h
a
-
F
e
n
c
h
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l
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H
b
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t
a
-
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n
c
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H

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u
r
o
p
e
:

6
4

c
o
n
c
e
r
n

U
S
A
:

1
7
MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS 389
L1
1
,
3
,
3
-
T
r
i
m
e
t
h
y
l
-
2
-

1
3
9
9

1
3
8
5
1
-
1
1
-
1

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y
n
o
r
b
o
r
n
a
n
y
l

a
c
e
t
a
t
e

p
l
a
h
a
-
f
o
r
m
O
A
c
H
b
e
t
a
-
f
o
r
m
H
O
A
c

E
u
r
o
p
e
:

a
n
d

c
o
n
c
e
r
n

U
S
A
:

0
.
0
7
2
(
1
0
)
-
P
i
n
e
n
-
3
-
o
l

1
4
0
3

5
9
4
7
-
3
6
-
4

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

O
H
H
s
i
c
-
f
o
r
m
O
H
H
s
n
a
r
t
-
f
o
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m

E
u
r
o
p
e
:

0
.
0
1

a
n
d

c
o
n
c
e
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n

U
S
A
:

0
.
0
1
V
e
r
b
e
n
o
l

1
4
0
4

4
7
3
-
6
7
-
6

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

O
H
H
s
i
c
o
f
-
r
m
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H
H
s
n
a
r
t
-
f
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m

E
u
r
o
p
e
:

0
.
3

a
n
d

c
o
n
c
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n

U
S
A
:

0
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2
3
-
l
-
M
e
n
t
h
o
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p
r
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n
e
-
1
,
2
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1
4
0
8

8
7
0
6
1
-
0
4
-
9

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

d
i
o
l

O
O
H
O
H

E
u
r
o
p
e
:

N
D

a
n
d

c
o
n
c
e
r
n

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S
A
:

7
8
9
b
-
I
o
n
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l

a
c
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t
a
t
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1
4
0
9

2
2
0
3
0
-
1
9
-
9

N
o

S
e
e

n
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t
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s

N
o

s
a
f
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t
y

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A
c

E
u
r
o
p
e
:

N
D

a
n
d

c
o
n
c
e
r
n

U
S
A
:

9
390 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
L1
T
a
b
l
e

1
.

(
C
o
n
t
d
)
F
l
a
v
o
u
r
i
n
g

a
g
e
n
t

N
o
.

C
A
S

N
o
.

a
n
d

S
t
e
p

A
3
a
,
b

D
o
e
s

C
o
m
m
e
n
t
s

C
o
n
c
l
u
s
i
o
n

s
t
r
u
c
t
u
r
e

i
n
t
a
k
e

e
x
c
e
e
d

t
h
e

b
a
s
e
d

o
n

c
u
r
r
e
n
t

t
h
r
e
s
h
o
l
d

f
o
r

i
n
t
a
k
e

h
u
m
a
n

i
n
t
a
k
e
?
a
-
I
s
o
m
e
t
h
y
l
i
o
n
y
l

a
c
e
t
a
t
e

1
4
1
0

6
8
5
5
5
-
6
1
-
3

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

O
A
c

E
u
r
o
p
e
:

N
D

a
n
d

c
o
n
c
e
r
n

U
S
A
:

9
3
-
(
l
-
M
e
n
t
h
o
x
y
)
-
2
-
m
e
t
h
y
l

1
4
1
1

1
9
5
8
6
3
-
8
4
-
4

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

p
r
o
p
a
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e
-
1
,
2
-
d
i
o
l

O
O
H
O
H

E
u
r
o
p
e
:

N
D

a
n
d

c
o
n
c
e
r
n

U
S
A
:

8
8
B
o
r
n
y
l

b
u
t
y
r
a
t
e

1
4
1
2

1
3
1
0
9
-
7
0
-
1

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

(
+
)
-
B
o
r
n
y
l
b
u
t
y
r
a
t
e
O
O
H
o
B
-
)
-
(
r
n
y
l
a
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y
t
u
b
t
e
O H
O

E
u
r
o
p
e
:

N
D

a
n
d

c
o
n
c
e
r
n

U
S
A
:

9
d
,
l
-
M
e
n
t
h
o
l
(

)
-
p
r
o
p
y
l
e
n
e

1
4
1
3

1
5
6
3
2
4
-
8
2
-
2

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y
g
l
y
c
o
l

c
a
r
b
o
n
a
t
e

O

O

O

O
H

+

E
u
r
o
p
e
:

N
D

a
n
d

c
o
n
c
e
r
n

U
S
A
:

1
4
0

O

O

O

O
H

MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS 391
L1
l
-
M
o
n
o
m
e
n
t
h
y
l

g
l
u
t
a
r
a
t
e

1
4
1
4

2
2
0
6
2
1
-
2
2
-
7

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

O
O
H
O
O

E
u
r
o
p
e
:

N
D

a
n
d

c
o
n
c
e
r
n

U
S
A
:

1
3
2
p
-
M
e
n
t
h
a
n
e
-
3
,
8
-
d
i
o
l

1
4
1
6

4
2
8
2
2
-
8
6
-
6

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

O
H
H
O

E
u
r
o
p
e
:

N
D

c
o
n
c
e
r
n

U
S
A
:

1
8
S
t
r
u
c
t
u
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a
l

c
l
a
s
s

I
I
d
-
C
a
m
p
h
o
r

1
3
9
5

4
6
4
-
4
9
-
3

N
o

S
e
e

n
o
t
e
s

1
,

N
o

s
a
f
e
t
y

(
d
+
(
-
)
)
-
C
a
m
p
h
o
r
O

E
u
r
o
p
e
:

5
8

5
,

a
n
d

c
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n
c
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n

U
S
A
:

3
9
6
d
-
F
e
n
c
h
o
n
e

1
3
9
6

4
6
9
5
-
6
2
-
9

N
o

S
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e

n
o
t
e
s

1
,

N
o

s
a
f
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t
y

(
d
+
(
-
)
)
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n
c
h
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e
O

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r
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p
e
:

5
,

a
n
d

c
o
n
c
e
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n

U
S
A
:

5
N
o
o
t
k
a
t
o
n
e

1
3
9
8

4
6
7
4
-
5
0
-
4

N
o

S
e
e

n
o
t
e
s

1
,

N
o

s
a
f
e
t
y

E
u
r
o
p
e
:

1
5
2

5
,

6
,

a
n
d

c
o
n
c
e
r
n

U
S
A
:

2
0
392 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
L1
T
a
b
l
e

1
.

(
C
o
n
t
d
)
F
l
a
v
o
u
r
i
n
g

a
g
e
n
t

N
o
.

C
A
S

N
o
.

a
n
d

S
t
e
p

A
3
a
,
b

D
o
e
s

C
o
m
m
e
n
t
s

C
o
n
c
l
u
s
i
o
n

s
t
r
u
c
t
u
r
e

i
n
t
a
k
e

e
x
c
e
e
d

t
h
e

b
a
s
e
d

o
n

c
u
r
r
e
n
t

t
h
r
e
s
h
o
l
d

f
o
r

i
n
t
a
k
e

h
u
m
a
n

i
n
t
a
k
e
?

M
e
t
h
y
l

j
a
s
m
o
n
a
t
e

1
4
0
0

1
2
1
1
-
2
9
-
6

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

O
O
O

E
u
r
o
p
e
:

3
1

a
n
d

c
o
n
c
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n

U
S
A
:

0
.
4
C
y
c
l
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p
t
a
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c
a
-
9
-
e
n
-
1
-

1
4
0
1

5
4
2
-
4
6
-
1

N
o

S
e
e

n
o
t
e
s

1
,

N
o

s
a
f
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t
y
o
n
e

C
(
H
2
)
7
C
(
H
2
)
7
O
s
n
a
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t
-
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s
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m
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r

E
u
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p
e
:

0
.
3

5
,

a
n
d

c
o
n
c
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n

U
S
A
:

0
.
0
5
3
-
M
e
t
h
y
l
-
1
-

1
4
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2

5
4
1
-
9
1
-
3

N
o

S
e
e

n
o
t
e
s

1
,

N
o

s
a
f
e
t
y
c
y
c
l
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p
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t
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d
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c
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e

C
(
H
2
)
1
2
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p
e
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0
.
4

5
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n
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c
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c
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S
A
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0
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0
0
9
7
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l
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4
,
4
a
,
5
,
6
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1
4
0
5

3
4
5
4
5
-
8
8
-
5

N
o

S
e
e

n
o
t
e
s

1
,

N
o

s
a
f
e
t
y
t
e
t
r
a
h
y
d
r
o
-
2
(
3
H
)
-

E
u
r
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p
e
:

N
D

5
,

a
n
d

c
o
n
c
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r
n
n
a
p
h
t
h
a
l
e
n
o
n
e

U
S
A
:

0
.
0
4
3
-
M
e
t
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y
l
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2
-
(
n
-
p
e
n
t
a
n
y
l
)
-

1
4
0
6

1
1
2
8
-
0
8
-
1

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y
MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS 393
L1
2
-

c
y
c
l
o
p
e
n
t
e
n
-
1
-
o
n
e

E
u
r
o
p
e
:

0
.
4

a
n
d

c
o
n
c
e
r
n

U
S
A
:

0
.
2
D
i
h
y
d
r
o
n
o
o
t
k
a
t
o
n
e

1
4
0
7

2
0
4
8
9
-
5
3
-
6

N
o

S
e
e

n
o
t
e
s

1
,

N
o

s
a
f
e
t
y

E
u
r
o
p
e
:

0
.
7

5
,

6
,

a
n
d

c
o
n
c
e
r
n

U
S
A
:

0
.
9

S
t
r
u
c
t
u
r
a
l

c
l
a
s
s

I
I
I
l
-
M
e
n
t
h
y
l

m
e
t
h
y
l

e
t
h
e
r

1
4
1
5

1
5
6
5
-
7
6
-
0

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

E
u
r
o
p
e
:

N
D

a
n
d

c
o
n
c
e
r
n

U
S
A
:

5
3
C
A
S
:

C
h
e
m
i
c
a
l

A
b
s
t
r
a
c
t
s

S
e
r
v
i
c
e
;

N
D
:

N
o

i
n
t
a
k
e

d
a
t
a

r
e
p
o
r
t
e
d
.
a

S
t
e
p

2
:

A
l
l

t
h
e

a
g
e
n
t
s

i
n

t
h
i
s

g
r
o
u
p

c
a
n

b
e

p
r
e
d
i
c
t
e
d

t
o

b
e

m
e
t
a
b
o
l
i
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e
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394 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
L1
1.2 Estimated daily intake
Thetotalannualvolumeofproductionofthe32monocyclicandbicyclicsec-
ondaryalcohols,ketonesandrelatedestersinthisgroupisapproximately11000kg
in Europe (International Organization of the Flavor Industry, 1995) and 14000kg
intheUSA(NationalAcademyofSciences,1970,1982,1987;Lucasetal.,1999)
(Table 2). Approximately two-thirds of the total annual volume of production in
Europeisaccountedforbyoneagentinthegroup,isobornylacetate(No.1388),
whileborneol(No.1385)andnootkatone(No.1398)accountforanadditional20%
of the total volume.Approximately 80% of the total annual volume of production
in the USA is accounted for by three agents, isobornyl acetate (No. 1388), d-
camphor (No. 1395) and 3-l-menthoxypropane-1,2-diol (No. 1408). Daily intakes
inEuropeandtheUSAwerecalculatedtobe1039and236mg/personforisobornyl
acetate(No.1388),155and23mg/personforborneol(No.1385),152and20mg
for nootkatone (No. 1398), and 58 and 396mg/person for d-camphor (No. 1395),
respectively. For 3-l-menthoxypropane-1,2-diol (No. 1408) and d,l-menthol-
propyleneglycolcarbonate(No.1413),thedailyintakesintheUSAarecalculated
tobe789and140mg/person,respectively.Thedailyintakesoftheotherfavouring
agentsinthegroupwereintherangeof0to132mg/person,withmostofthevalues
beingatthelowerendofthisrange.Theestimateddailypercapitaintakeofeach
agentinEuropeandintheUSAisreportedinTable1.
1.3 Absorption, distribution, metabolism and elimination
Studiesinhumans,dogs,andrabbitshaveshownthatthemono-andbicyclic
secondary alcohols and ketones in this group are rapidly absorbed, distributed,
metabolizedandexcretedmainlyintheurine.Smallamountsmaybeeliminated
inexhaledair.Inhumans,theesterswithinthisgroupareexpectedtobehydro-
lysedtotheircomponentsecondaryalcoholandcarboxylicacid.
Themajormetabolicpathwayfortheketonesinvolvesreductiontothecorre-
sponding secondary alcohols, which are subsequently excreted, primarily as the
glucuronicacidconjugates(Williams,1959;Lington&Bevan,1994;Toppingetal.,
1994).Metabolitescontainingadoublebondthatareexcretedintothebilemaybe
reducedtothecorrespondingdihydroderivativesbythegutmicrofora(Krasavage
etal.,1982).Inadditiontoreductivepathways,alicyclicketonesand,toalesser
extent,secondaryalcoholscontaininganalkylside-chain,undergooxidationofthe
side-chaintoformpolarpoly-oxygenatedmetabolitesthatareexcretedmainlyin
theurine,eitherunchangedorastheglucuronideorsulfateconjugates.
Formorelipophilicketones(e.g.nootkatone,No.1398)orthosewithsterically
hinderedfunctionalgroups(e.g.d-camphor,No.1395),oxidationofaringposition
by cytochrome P450 (CYP) may compete with reduction of the ketone group or
oxidation of the alcohol group (Asakawa et al., 1986; Nelson et al., 1992). For
example, bicyclic ketones tend to show greater lipophilicity and steric hindrance
of the carbonyl function than do short-chain aliphatic or monocyclic ketones.As
such, bicyclic ketones are expected to be poor substrates for cytosolic reducing
enzymes.Consequently,thepredominantdetoxicationrouteisCYP-mediatedring
hydroxylationtoyieldpolar,excretablepoly-oxygenatedmetabolites.
MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS 395
L1
Table 2. Annual volumes of production of monocyclic and bicyclic secondary
alcohols, ketones and related esters used as favouring agents in Europe and
the USA
Flavouringagent(No.) Mostrecent Intake
b
Annual Consumption

annual
mg/day mg/kgbw
volumein ratio
d

volume(kg)
a
perday
naturally

occurring
foods(kg)
c
Borneol(1385)
Europe 1084 155 3
USA 172 23 0.4 863 5
Isoborneol(1386)
Europe 170 24 0.4
USA 0.5 0.07 0.001 + NA
Bornylacetate(1387)
Europe 146 21 0.3
USA 23 3 0.05 424 18
Isobornylacetate(1388)
Europe 7278 1039 17
USA 1792 236 4 + NA
Bornylformate(1389)
Europe 10 1 0.02
USA
f
0.5 0.09 0.001 + NA
Isobornylformate(1390)
Europe 5 0.7 0.01
USA
f
2 0.4 0.006 - NA
Isobornylpropionate(1391)
Europe 21 3 0.05
USA 0.05 0.007 0.0001 + NA
Bornylvalerate(1392)
Europe ND ND ND
USA
f
30 5 0.09 - NA
Bornylisovalerate,endo-(1393)
Europe 1 0.1 0.002
USA
f
3 0.5 0.009 - NA
Isobornylisovalerate(1394)
Europe 0.1 0.01 0.0002
USA
f
0.5 0.08 0.001 + NA
d-Camphor(1395)
Europe 408 58 1
USA 3007 396 7 + NA
d-Fenchone(1396)
Europe 52 7 0.1
USA 40 5 0.09 + NA
Fenchylalcohol(1397)
Europe 451 64 1
USA 132 17 0.3 873 7
Nootkatone(1398)
Europe 1067 152 3
USA 154 20 0.3 1051 7
1,3,3-Trimethyl-2-norbornanylacetate(1399)
Europe 24 3 0.06
USA 0.5 0.07 0.001 + NA
396 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
L1
Table 2. (Contd)
Flavouringagent(No.) Mostrecent Intake
b
Annual Consumption

annual
mg/day mg/kgbw
volumein ratio
d

volume(kg)
a
perday
naturally

occurring
foods(kg)
c
Methyljasmonate(1400)
Europe 217 31 0.5
USA 3 0.4 0.007 37 12
Cycloheptadeca-9-en-1-one(1401)
Europe 2 0.3 0.005
USA 0.4 0.05 0.0009 - NA
3-Methyl-1-cyclopentadecanone(1402)
Europe 3 0.4 0.01
USA
f
0.05 0.009 0.0001 - NA
2(10)-Pinen-3-ol(1403)
Europe 0.1 0.01 0.0002
USA 0.1 0.01 0.0002 + NA
Verbenol(1404)
Europe 2 0.3 0.005
USA
f
1 0.2 0.003 + NA
7-Methyl-4,4a,5,6-tetrahydro-2(3H)-naphthalenone(1405)
Europe ND ND ND
USA 0.3 0.04 0.0007 - NA
3-Methyl-2-(n-pentanyl)-2-cyclopenten-1-one(1406)
Europe 3 0.4 0.007
USA 1.4 0.2 0.003 - NA
Dihydronootkatone(1407)
Europe 5 0.7 0.01
USA
e
5 0.9 0.01 + NA
3-l-Menthoxypropane-1,2-diol(1408)
Europe ND ND ND
USA 5987 789 13 - NA
b-Ionylacetate(1409)
Europe ND ND ND
USA
e
50 9 0.1 - NA
a-Isomethyllionylacetate(1410)
Europe ND ND ND
USA
e
50 9 0.1 - NA
3-(l-Menthoxy)-2-methylpropane-1,2-diol(1411)
Europe ND ND ND
USA
e
500 88 1 - NA
Bornylbutyrate(1412)
Europe ND ND ND
USA
e
50 9 0.1 +
g
NA
d,l-Menthol-()-propyleneglycolcarbonate(1413)
Europe ND ND ND
USA
e
800 140 2 - NA
l-Monomenthylglutarate(1414)
Europe ND ND ND
USA
e
750 132 2 +
h
NA
l-Menthylmethylether(1415)
Europe ND ND ND
USA
e
300 53 0.9 - NA
MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS 397
L1
Table 2. (Contd)
Flavouringagent(No.) Mostrecent Intake
b
Annual Consumption

annual
mg/day mg/kgbw
volumein ratio
d

volume(kg)
a
perday
naturally

occurring
foods(kg)
c
p-Menthane-3,8-diol(1416)
Europe ND ND ND
USA
e
100 18 0.3 +
i
NA
Total
Europe 10949
USA 13955
NA,notavailable;ND,nointakedatareported;+,reportedtooccurnaturallyinfoods
(Nijssenetal.,2003),butnoquantitativedata;-,notreportedtooccurnaturallyinfoods
a
FromInternationalOrganizationoftheFlavourIndustry(1995)andLucasetal.(1999)or
NationalAcademyofSciences(1970,1982,1987).
b
Intakeexpressedasmg/personperdaywascalculatedasfollows:
[(annualvolume,kg)(110
9
mg/kg)/(populationsurveycorrectionfactor365
days)],wherepopulation
(10%,eatersonly)=3210
6
forEuropeand2610
6
fortheUSA.Thecorrection
factor=0.6forEuropeand0.8fortheUSArepresentingtheassumptionthatonly60%
and80%oftheannualproductionvolumeofthefavour,respectively,wasreportedin
thepoundagesurveys(InternationalOrganizationoftheFlavourIndustry,1995;Lucaset
al.,1999;NationalAcademyofSciences,1970,1982,1987)orintheanticipatedannual
volumeofproduction.
Intakeexpressedasmg/kgbwperdaywascalculatedasfollows:
[(mg/personperday)/bodyweight],wherebodyweight=60kg.Slightvariationsmay
occurfromrounding.
c
QuantitativedatafortheUSAreportedbyStofberg&Grundschober(1987).
d
Theconsumptionratioiscalculatedasfollows:
(annualconsumptionviafood,kg)/(mostrecentlyreportedvolumeasafavouringagent,
kg)
e
Thevolumecitedistheanticipatedannualvolume,whichwasthemaximumamountof
favouringagentestimatedtobeusedannuallybythemanufactureratthetimethe
materialwasproposedforfavouruse.
f
AnnualvolumereportedinpreviousUSAsurveys(NationalAcademyofSciences,1970;
1982;1987).
g
Frattinietal.(1981)
h
NaturaloccurrencedatareportedinaprivatecommunicationtoFlavorandExtract
ManufacturersAssociation(2003)
i
Nishimuraetal.(1984)
Thepathwaysbywhichfusedringandmacrocyclicketonesaredetoxifedare
similartothoseforthebridgedbicyclicsubstances.Activatedringpositions(e.g.
tertiaryandallylicpositions)andringsubstituentsareoxidizedprimarilybyCYP,
introducing additional polar groups into the molecule. The resulting metabolites
arethenexcreted,mainlyintheurine.
398 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
L1
1.4 Application of the procedure for the safety evaluation of
favouring agents
Step1. InapplyingtheProcedure,theCommitteeassigned22(Nos13851394,
1397,1399,1403,1404,14081414,1416)ofthe32agentstostructural
classI.Nineoftheseagents(Nos1395,1396,1398,14001402,1405
1407)wereassignedtostructuralclassII,andtheremainingagent(No.
1415)wasassignedtostructuralclassIII(Crameretal.,1978).
Step2. Allthefavouringagentsinthisgroupareexpectedtobemetabolizedto
innocuousproducts.TheirevaluationthereforeproceededviatheA-side
ofthedecision-tree.
Step3. Theestimateddailyintakesofall22ofthefavouringagentsinstructural
classI,allnineoftheagentsinstructuralclassIIandtheagentinstruc-
tural class III are below the thresholds of concern (i.e. 1800mg/person
for class I, 540mg/person for class II, and 90mg/person for class III).
According to the Procedure, the safety of these 32 favouring agents
raisesnoconcernwhentheyareusedatestimatedcurrentintakes.
Theintakeconsiderationsandotherinformationusedtoevaluatethe32mono-
cyclic and bicyclic secondary alcohols, ketones and related esters in this group
accordingtotheProcedurearesummarizedinTable1.
1.5 Consideration of secondary components
Six members (Nos 1386, 1398, 1407, 1409, 1413 and 1414) of this group of
favouringagentshaveminimumassayvaluesof<95%.Informationonthesafety
ofthesecondarycomponentsofthesesixcompoundsissummarizedinAnnex5
(Summaryofthesafetyevaluationofsecondarycomponentsforfavouringagents
withminimumassayvaluesoflessthan95%).ThesecondarycomponentsofNo.
1407(aceticacidandb-ionol)wereevaluatedbytheCommitteeatitsforty-ninth
meetingandffty-frstmeetings(Annex1,references131and 137),respectively.
ThesecondarycomponentsofNo.1413,d,l-menthol2-propyleneglycolcarbonate,
andofNo.1414,dimenthylglutarateandglutaricacid,havenotbeenpreviously
evaluated.However,d,l-menthol2-propyleneglycolcarbonateanddimenthylglu-
tarate are structurally related to the primary favouring agents in this group and
areexpectedtosharethesamemetabolicfate.Glutaricacidisstructurallyrelated
to valeric acid, which was evaluated by the Committee at its forty-ninth meeting
(Annex 1, reference 131). The secondary components of Nos 1386, 1398 and
1407 (borneol, dihydronootkatone and nootkatone) were evaluated as favouring
agentsbytheCommitteeatitscurrentmeeting.Onthebasisoftheseevaluations,
thesecondarycomponentsforthesesixfavouringagentswereconsiderednotto
presentasafetyconcernatcurrentestimatedintakes.
1.6 Consideration of combined intakes from use as favouring agents
In the unlikely event that all 22 agents in structural class I were consumed
concurrently on a daily basis, the estimated combined intake would not exceed
MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS 399
L1
the human intake threshold for class I (1800mg/person per day). In the unlikely
eventthatallnineagentsinstructuralclassIIwereconsumedconcurrentlyona
daily basis, the estimated combined intake would not exceed the human intake
thresholdforclassII(540mg/personperday).Overallevaluationofthedataindi-
catedthatcombinedintakeoftheagentsinthisgroupwouldnotpresentasafety
concern.
1.7 Conclusions
The Committee concluded that none of the favouring agents in this group of
monocyclic and bicyclic secondary alcohols, ketones and related esters would
raiseasafetyconcernatcurrentestimatedintakes.Availabledataonthetoxicity
andmetabolismofthesesubstanceswereconsistentwiththeresultsofthesafety
evaluation.
2. RELEVANT BACKGROUND INFORMATION
2.1 Additional considerations on intake
The majority of members in this group of monocyclic and bicyclic secondary
alcoholsandketonesareterpenemonocyclicandbicyclicketones(e.g.camphor;
No. 1395), secondary alcohols (e.g. borneol; No. 1385), and related esters (e.g.
isobornyl acetate; No. 1388). Therefore, it is expected that many are common
constituentsofplants.Insomecases,differentstereoisomersofasubstancemay
be major constituents of the volatile portion of different plants. For instance, (+)-
borneol(No.1385)isfoundasaconstituentofthymeoilandrosemaryoilatcon-
centrationsofupto40%,while(-)-borneol(No.1385)anditsacetateester(No.
1387)arepresentinfrandpineoils(Abies,Pinus,andPiceaspecies)atconcen-
trationsofupto45%(Nijssenetal.,2003).
2.2 Biological data
2.2.1 Biochemical data
(a) Hydrolysis
Theesterswithinthisgroupareexpectedtobehydrolysedinhumanstotheir
component alcohols and aliphatic carboxylic acids. Subsequently, the carboxylic
acids are completely metabolized through recognized biochemical pathways
(Nelson & Cox, 2000). Ester hydrolysis is catalysed by classes of enzymes rec-
ognized as carboxylesterases (Heymann, 1980; White et al., 1990), the most
importantofwhicharetheB-esterases.Inmammals,theseenzymesoccurinmost
tissues(Heymann,1980;Anders,1989),butpredominateinhepatocytes(Heymann,
1980).
Inrabbits,>90%ofanoraldoseofd-,l-,ordl-bornylacetate(No.1387)was
excreted in the urine as the glucuronic acid conjugate of hydrolysed borneol
(Williams, 1959). In two separate in vitro hydrolysis studies l-menthol ethylene
400 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
L1
glycol carbonate (No. 443) and l-menthol propylene glycol carbonate (No. 1413)
werehydrolysedfollowingincubationwithratliverhomogenate(Emberger,1998).
Incubationofb-ionylacetate(No.1409)inthepresenceofsimulatedgastricjuice
orintestinalfuidresultedin43%and>60%hydrolysistob-ionolwithin4h,respec-
tively(Bennett,1998).Approximately75%ofd,l-mentholpropyleneglycolcarbon-
ate (No. 1413) was hydrolysed to menthol when incubated for 4h with liver
homogenate (Emberger, 1998). It is anticipated that d,l-mentholethylene glycol
carbonate would be hydrolysed in a similar manner. Incubation of the mandelic
acidesterof3,3,5,5-tetramethylcyclohexanol,astructurallyrelatedester,withrat
liver microsomes resulted in >80% hydrolysis within 2min (White et al., 1990).
cis-andtrans-p-1(7),8-Menthadien-2-ylacetate(No.1098)wasalsorapidlyhydro-
lysed in vitro in the presence of rat liver homogenate. Incubation of the ester
resultedin92%hydrolysisafter15minand100%after60min(Salzer,1998).On
thebasisofthesedata,itisanticipatedthattheestersofthisgroupwillberapidly
hydrolysedinthedigestivetractorintheliver.
(b) Absorption,distributionandexcretion
(i) Bicyclicderivatives
Studiesinhumans,dogsandrabbits,haveshownthatthesecondaryalcohols
and ketones of this group are rapidly absorbed, distributed, metabolized, and
excreted mainly in the urine as glucuronide conjugates. Small amounts may be
expiredinexhaledair.Previouslyreviewed(Annex1,reference138)dataforother
cyclicterpenesecondaryalcoholsandketonesincludingmenthol(No.427),men-
thone(No.429),andcarvone(Nos380a,380b)supportthisconclusion.
Casereports,inwhichingestionofcamphor(No.1395)resultedintoxicityin
bothadultsandchildrenwithinminutesofexposure(Jacobziner&Raybin,1962;
Phelan, 1976; Kopelman et al., 1979; Gibson et al., 1989), demonstrate rapid
absorption of this substance. Rabbits given d-camphor (No. 1395) at a dose of
1.93.5mmol/kgbw(289533mg/kgbw)bygavageexcreted59.1%oftheadmin-
istereddoseconjugatedwithglucuronicacidintheurinewithin24h(Robertson&
Hussain, 1969).A group of 50 Sprague-Dawley rats was given 40% camphor in
cottonseed oil as a single dose at 1000mg/kgbw (approximately 400mg of
camphor) by gavage, and killed at 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0, or
10.0haftertreatment.Bloodsamplesweretakenbeforedeath.Peakbloodcon-
centrationofcamphoroccurredat96min,withanabsorptionhalf-lifeof38minand
aplasmaeliminationhalf-lifeof142min.Theauthorsconsideredthatthesedata
comparedfavourablywiththoseinhumans(Deanetal.,1992).
The toxicokinetics of d,l-camphor (No. 1395 for the d-form) were studied in
B6C3F
1
mice and F344 rats. In mice, camphor was rapidly eliminated from the
plasmaafterasingleintravenousinjectionat50mg/kgbwwithaneliminationrate
constant of 0.0337 and 0.0335min
-1
for males and females, respectively, and a
half-life of 21min. In rats, camphor underwent biphasic elimination from plasma
afterasingleintravenousinjectionat6mg/kgbwwithaneliminationrateconstant
of 0.0038 and 0.0059min
-1
for males and females, respectively, and half-lives of
185and118minformalesandfemales,respectively(Grizzleetal.,1996).
MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS 401
L1
Inacasereport,apregnantwoman(week40ofgestation)accidentallyingested
12g of camphorated oil (% camphor not specifed) and 36h later gave birth to a
cyanoticbabyexhibitingnorespiration.Thebabydiedwithin30min.Thepresence
of camphor was noted at 15min in maternal circulation, at 20h in amniotic fuid,
andat36hincordblood,infantbrain,liverandkidneys(Riggsetal.,1965).
Approximately80%ofanorallyadministereddoseof2000mgofd-borneol(No.
1385)giventohumans(sexandnumbernotspecifed)wasexcretedwithin10h
(Williams,1959).
(ii) Monocyclicderivatives
Monocyclic ketones and alcohols in this group follow a fate similar to that of
bicyclic derivatives. Five female and fve male Sprague-Dawley rats pre-treated
with3-l-menthoxypropane-1,2-diol(No.1408)atadailyoraldoseof29.4mg/kgbw
for 7 days were given [3-
14
C]3-l-menthoxypropane-1,2-diol as a single oral dose
at 30mg/kgbw on day 8, and urine, faeces, expired air, and cage washes were
collected over the next 120h. Total recovery of the radiolabelled substance was
95.2%formalesand94.4%forfemaleswithmostofthedose(72.0%formales
and 64.6% for females) being recovered within the frst 24h.The primary routes
of excretion were the urine (56.4% for males and 61.7% for females) and the
faeces(34.5%formalesand26.5%forfemales).Lessthan3%wasrecoveredas
radiolabelledcarbondioxideforbothsexes(Ferdinandi,1993a).
Four male beagle dogs pre-treated with 3-l-menthoxypropane-1,2-diol (No.
1408) at daily oral doses of 49.9mg/kgbw for 7 days, were given [3-
14
C]3-l-
menthoxypropane-1,2-diol as a single oral dose of 49.6mg/kgbw on day 8, and
theurine,faeces,expiredair,andcagewasheswerecollectedoverthenext120h.
Total recovery of the radiolabelled substance was 91.9%, with most of the dose
(63.7%) being recovered within the frst 24h. As in rats, the primary routes of
excretionweretheurine(58.2%)andthefaeces(28.1%)(Ferdinandi,1993b).
In summary, the esters of monocyclic and bicyclic secondary alcohols are
readily hydrolysed. The resulting secondary alcohols and the corresponding
ketones are then rapidly absorbed, metabolized, and excreted primarily as gluc-
uronicacidconjugatesintheurine.
(c) Metabolism
Themajormetabolicpathwayfortheketonesinvolvesreductiontothecorre-
sponding secondary alcohols, which are subsequently excreted primarily as the
glucuronicacidconjugates(Williams,1959;Lington&Bevan,1994;Toppingetal.,
1994).Metabolitesthatareexcretedintothebileandthatcontainadoublebond
may be reduced to the corresponding dihydro derivatives by the gut microfora
(Krasavageetal.,1982).Inadditiontoreductivepathways,alicyclicketonesand,
to a lesser extent, secondary alcohols containing an alkyl side-chain undergo
oxidation of the side-chain to form polar poly-oxygenated metabolites that are
excreted either unchanged or as the glucuronide, or sulfate conjugates mainly
intheurine.
402 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
L1
Formorelipophilicketones(e.g.nootkatone,No.1398)orthosewithsterically
hinderedfunctionalgroups(e.g.d-camphor,No.1395)oxidationofaringposition
by nonspecifc CYP mixed function oxidases may compete with reduction of the
ketone functional group or oxidation of the alcohol functional group (Asakawa
et al., 1986; Nelson et al., 1992). For example, bicyclic ketones tend to show
greater lipophilicity and steric hindrance of the carbonyl function than do short-
chain aliphatic or monocyclic ketones, which are primarily reduced to the corre-
sponding secondary alcohol.As such, bicyclic ketones are expected to be poor
substratesforcytosolicreducingenzymes.Consequently,thepredominantdetoxi-
cation route is CYP-mediated ring hydroxylation to yield polar, excretable poly-
oxygenatedmetabolites.
As shown in Figure 1, in humans ingestion of 600010000mg of camphor
(No. 1395) resulted in urinary excretion of 3-, 5-, 8-, and 9-hydroxycamphor, 5-
ketocamphor and the carboxylic acid of either 8- or 9-hydroxycamphor, unconju-
gated or conjugated with glucuronic acid (Kppel et al., 1982).A minor amount
wasexhaledinexpiredair.Hydroxylationproducts,predominantly5-endo-and5-
exo-hydroxycamphorandacompoundresembling3-endo-hydroxycamphor,have
alsobeenreportedwhencamphorwasadministeredorallytodogs(1000mgper
animal,ingelatincapsules,fourtimesperdayfor7days)orrabbits(300mgper
animal,singledoseadministeredbygavage)(Leibman&Ortiz,1973).Thesame
camphorhydroxylationproducts,withasmallamountof2,5-bornanedione,were
similarlyidentifedinvitroafterincubationwithratandrabbitliverfractions(Leibman
10
9
8 7
6
5
4
3
2
1
O
O
O
OH
H
O
O
O
O
HO
OH
O
+
Borneol
?
8- (or 9-) Hydroxycamphor
carboxylic acid
COOH
HOH
2
C
CH
2
OH
5-Ketocamphor
9-Hydroxycamphor 8-Hydroxycamphor
+
+
5-Hydroxycamphor
3-Hydroxycamphor
Camphor
Figure 1. Metabolism of camphor in humans
MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS 403
L1
&Ortiz,1973).Similarhydroxylationproducts(4-and5-hydroxyfenchoneandp-
apofenchone-3-carboxylicacid)weredetectedintheurineofdogsfedd-fenchone
(No.1396)(Reinartz&Zanke,1936).Themetabolismofd-fenchonealsodemon-
strates that hydroxylation of ring methyl substituents leads to the corresponding
carboxylicacidderivatives.
In rabbit liver cytosol, d-camphor (No. 1395) was reduced via an NADPH-
dependent pathway to borneol and a small amount of isoborneol (Robertson &
Hussain,1969;Leibman&Ortiz,1973).Inratliver,camphorinducedmembersof
the CYPIIB subfamily, which were most likely to be CYPb and/or CYPe (Austin
et al., 1988). Female Swiss albino mice given camphor at a dose of 50, 150, or
300mg/kgbwperdayinoliveoilbygavagefor20daysshowedastatisticallysig-
nifcantincreaseinCYPandcytochromeb
5
,arylhydrocarbonhydrolase,andglu-
tathioneS-transferaseactivitiesonlyatthehighestdose(Banerjeeetal.,1995).
Data for bicyclic ketones structurally related to d-camphor indicate that ring
hydroxylationisamajorpathwayofmetabolismforsuchcompounds.Forexample,
at 18h after oral administration of cis-3-pinanone (100mg/kgbw) to male albino
Swiss-Webstermice,themajormetabolitesexcretedintheurinewereconjugated
(glucuronide or sulfate) 2-hydroxy-cis-3-pinanone, two other hydroxylated cis-3-
pinanones,andunconjugated2(8)-dehydro-cis-3-pinanone.Mouseorhumanliver
microsomes or human CYP3A4 containing NADPH were incubated with either
cis-3-pinanoneortrans-3-pinanone.Forcis-3-pinanone,2-hydroxy-cis-3-pinanone,
themajormetabolite,andtwootherminorhydroxylatedcis-3-pinanonemetabolites
wereidentifedinincubationswithmicrosomalfractionsandwithCYP3A4.Mouse
livermicrosomesproducedmore2-hydroxy-cis-3-pinanonethandidhumanmicro-
somes or CYP3A4. For trans-3-pinanone, two hydroxy-trans-3-pinanones were
identifed.Thecis-3-pinanonemetaboliteswereidenticaltothoseobtainedinvivo.
Mice were given a lethal dose of cis-3-pinanone or trans-3-pinanone at 250mg/
kgbwbyintraperitonealinjectionandsacrifcedatdifferenttimesupto80min.The
braintissuecontained2-hydroxy-cis-3-pinanoneasthemajormetaboliteofcis-3-
pinanone.The maximum amount of metabolite was reached within 1020min of
dosing.Metabolitesidentifedfortrans-3-pinanonesinvivowerethesameasthose
identifedinthestudyinlivermicrosomesinvitro(Hldetal.,2002).
Fused ring and macrocyclic ketones are detoxicated by pathways similar to
thoseforthebridgedbicyclicsubstances.Activatedringpositions(e.g.tertiaryand
allylicpositions)andringsubstituentsareoxidizedprimarilybyCYPtointroduce
additionalpolarfunctionalitiesintothemolecule.Theresultingmetabolitesarethen
excretedmainlyintheurine.
Gasliquidchromatography(GLC)analysisof3-dayurinesamplestakenfrom
rabbitsgivennootkatone(No.1380)inlargedoses(6000mg)byoraladministra-
tion, identifed nootkatone-13,14-diol and nootkatone-13,14-diol monoacetate as
metabolites(Asakawaetal.,1986).Nootkatone-13,14-diolisaneutralmetabolite,
which is most likely to be the result of epoxidation of the side-chain isopropenyl
group followed by hydration, as shown in Figure 2, while nootkatone-13,14-diol
monoacetate probably results from the subsequent acetylation of nootkatone-
13,14-diolduringisolationofthediolmetabolite(Asakawaetal.,1986).
404 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
L1
Althoughnootkatone(No.1380)containsa,b-unsaturation,noglutathionecon-
jugation in a Michael-type addition has been observed for this fused ring ketone
orothermoncyclica,b-unsaturatedketones.Thepresenceofringcarbonsoralkyl
substituents at the b-position inhibits glutathione conjugation (Portoghese et al.,
1989). Structurally related a,b-unsaturated monocyclic ketones isophorone (No.
1112) and carvone (No. 380) have been evaluated previously by the Committee
(Annex1,references138and161,respectively)andmetabolismstudiesindicate
little evidence of extensive glutathione conjugation. Rather, side-chain oxidation
and ketone reduction are the reported metabolic pathways leading to poly-
oxygenatedmetabolitessimilartothatreportedfornootkatone.
Samplesofurinecollectedover4daysfromrabbitsgivenisophorone(astruc-
turallyrelatedsubstance;No.1112,3,5,5-trimethyl-2-cyclohexen-1-one)atadose
of1000mg/kgbwbygavagecontainedseveralmetabolites:themajormetabolite,
5,5-dimethyl-1-cyclohexene-3-one-1-carboxylic acid formed by oxidation of the
methyl group at an exocyclic allylic position; 3,5,5-trimethyl-2-cyclohexen-1-ol
(isophorol) formed by reduction of the ketone group and then conjugation with
glucuronicacid;3,5,5-trimethylcyclohexanone(dihydroisophorone)formedbyhydro-
genationoftheendocyclicdoublebond;cis-andtrans-3,5,5-trimethylcyclohexanol
formed by hydrogenation of the endocyclic double bond and reduction of the
ketonegroup(seeFigure3)(Truhautetal.,1970;Dutertre-Catella,1978).
Carvone(No.380,2-methyl-5-(1-methylethenyl)-2-cyclohexen-1-one),astruc-
turallyrelateda,b-unsaturatedketone,waspartiallyexcretedastheparentcom-
pound in both humans and rats (Tamura et al., 1962; Zlatkis et al., 1973).Allylic
oxidation products, namely 9-hydroxycarvone, have also been detected in rats,
(Williams,1959;Ishidaetal.,1989).Inmice,carvoneinducescytosolicglutathione
transferase activity in mice (Zheng et al., 1992) suggesting that carvone may
undergo some detoxication via glutathione conjugation at the b-position
(Portogheseetal.,1989).Inrabbits,carvonewasmainlyreducedtoyieldcarveol,
which was then converted to the glucuronic acid conjugate and excreted in the
urine (Fisher & Bielig, 1940). Unchanged dihydrocarveol (Fisher & Bielig, 1940)
and the glucuronic acid conjugate of dihydrocarveol (Hmlinen, 1912) were
additional metabolites of carvone detected in the urine of rabbits treated with
carvone.
Structurallyrelatedfusedringketonesalsoundergooxidationofringpositions
that are remote from the ketone function (Asakawa et al., 1986).An example of
this is cyclocolorenone, a tricyclic, a,b-unsaturated ketone, which is metabolized
toyieldtwohydroxyketonemetabolites(Asakawaetal.,1986).TheC9(methylene)
andC10(methane)ringpositionsarehydroxylated,asshowninFigure4.Similar
Figure 2. Metabolism of nootkatone in rabbits
O
Nootkatone
O
OH
OH
Nootkatone-13,14-diol
1. Epoxidation
2. Hydration
MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS 405
L1
pathwaysofoxidationofthedouble-bondringpositionsandringalkylsubstituents
areexpectedinhumans.
Thebicyclicsecondaryalcoholsarerapidlyconjugatedwithglucuronicacidin
humans,dogs,andrabbitsandexcretedviatheurine.Inhumans(Figure5),81%
and 94% of the orally administered dose of borneol (No. 1385) at 1000 and
2000mg,respectively,wereexcretedastheglucuronicacidconjugatewithin24h
(Wagreichetal.,1941).At10hafteringestionof2000mgofborneol,81%ofthe
administereddosewasdetectedastheglucuronicacidconjugateinhumanurine
(Quick,1928).Atahigherdose(i.e.3500mgofborneol),69%oftheadministered
dosewasdetectedinhumanurineafter6h(Quick,1928).Similarconjugationhas
beenreportedindogs(Quick,1927;Pryde&Williams,1934).Anincreasedlevel
of b-glucuronidase activity has been reported in several tissues of dogs given
borneol by oral administration (Fishman, 1940).At oral doses of 100mg/kg per
day,ratsfedborneolover10daysshowedanincreaseintheurinaryconcentra-
tions of total glucuronic acid, o-glucuronide, and ascorbic acid (Tamura et al.,
O
O OH
CO
2
H
O
dihydroisophorone
isophorone
isophorol
5,5-dimethyl-2-cyclohexen-1-one-3-carboxylic acid
Figure 3. Metabolic fate of isophorone in rabbits
O
O
OH
OH
O
cyclocolorenone
10-hydroxy
cyclocolorenone
9-hydroxy
cyclocolorenone
Figure 4. Metabolism of cyclolorenone in rabbits
406 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
L1
1962). Fenchyl alcohol (No. 1397) administered by gavage to rabbits was also
excreted via urine as a glucuronide conjugate (Hmlinen, 1912). Glucuronic
conjugatesofverbenol(No.1404)and2(10)-pinen-3-ol(No.1403)wereidentifed
intheurineofrabbitsgiven a-pinenebyoraladministration(Ishidaetal.,1981).
Cis-andtrans-verbenol(No.1404)havealsobeendetectedinhumanurineafter
occupationalinhalationexposuretoa-andb-pineneandd-3-carene(Eriksson&
Levin,1990).
Inrats,treatmentwithborneol(No.1385)for3days(intraperitonealordietary
exposure), causes increases (of approximately 25%) in the activities of biphenyl
4-hydroxylase, glucuronyl transferase, 4-nitrobenzoate reductase, and in hepatic
CYP(Parke&Rahman,1969).Inanotherstudy,groupsoffourratsgivenl-borneol
atadoseof250mg/kgbwperdaybyintraperitonealinjectionfor3daysshowed
no signifcant increase in liver UDP-glucuronosyltransferase (UDPGT) activity.
After daily treatment for up to 4 weeks, slight increases in this activity were
observed.Theauthorsconcludedthatovershortperiodsofexposure,detoxication
of borneol does not require the induction of UDPGT; however, longer exposure
periods, at high doses, necessitate induction of UDPGT (Boutin et al., 1983).
Conversely,inratsintubatedwithborneolatadoseof3mmol/kgbw(463mg/kgbw,
inoliveoil),theactivityofhepaticS-3-hydroxy-3-methylglutarylcoenzymeAreduc-
tase was decreased by approximately 50% at 17h after dosing (Clegg et al.,
1980).
CYP2B1wasinducedinlivermicrosomesisolatedfromratsinjectedintraperi-
toneallywithborneolatadoseof300mg/kgbw(Hiroietal.,1995),indicatingthat
oxidationmayoccurtoalimitedextent.Ratsinjectedintraperitoneallywithisobor-
nylacetate(No.1388)atadoseof1000mg/kgbwfor3daysshowedaminimum
increase of twofold in the activities of N-demethylase and NADPH cytochrome c
reductase,andinCYPcontent,indicatingthatisobornylacetateinducesthemicro-
somalmixed-functionoxidasesystem(Cintietal.,1976),whichalsosuggeststhat
oxidationofringpositionsandringsubstituentsmayoccur.
Other minor routes of metabolism of the bicyclic secondary alcohols include
hydroxylation of an allylic position and oxidative cleavage of the strained ring in
thebicyclicsubstance.Verbenol(No.1404)containsbothanallylicmethylgroup
andastrained(cyclobutane)ringsystem.Therefore,verbenolmayundergooxida-
tion of the allylic methyl group in a manner similar to that reported for trans-
sobrerol,astructurallyrelatedterpenoid(Venturaetal.,1985),aswellascleavage
Urine
H
glu-o
Borneol
HO
H
Glu = Glucuronic acid
Figure 5. Metabolism of borneol in humans
MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS 407
L1
ofthecyclobutaneringtoyieldmonocyclicpolarmetabolites,ashasbeenreported
forstructurallyrelatedring-strainedbicyclicaldehydes(Ishidaetal.,1989).
As with bicyclic alcohols, the metabolism of the menthol derivatives included
inthegroupdemonstratesthatconjugationwithglucuronicacidisamajorpathway
ofexcretion.FivefemaleandfvemaleSprague-Dawleyrats,pre-treatedwith3-l-
menthoxypropane-1,2-diolatanoraldoseof29.4mg/kgperdayfor7days,were
given [3-
14
C]3-l-menthoxypropane-1,2-diol (No. 1408) as a single oral dose at
30mg/kgbwonday8.Themajorurinarymetaboliteinbothsexes(56.5%inmales
and 73.3% in females) was the glucuronic acid conjugate of the parent diol
(Ferdinandi, 1993a). Four male beagle dogs, pre-treated with 3-l-menthoxypro-
pane-1,2-diol(No.1408)atanoraldoseof49.9mg/kgbwperdayfor7days,were
given[3-
14
C]3-l-menthoxypropane-1,2-diolasasingleoraldoseat49.6mg/kgbw
onday8.Theparentdiolanditsglucuronicacidconjugateaccountedfor78.9%
of the radiolabelled substance recovered within the frst 48h (Ferdinandi,
1993b).
Theabovedatademonstratethattheestersinthisgrouparereadilyhydrolysed
tothecorrespondingmono-orbicyclicsecondaryalcoholsandaresubsequently
conjugatedwithglucuronicacidandexcretedintheurine.Otherminormetabolic
routesincludeoxidationofringpositionsandsubstituentstoyieldpoly-oxygenated
metabolitesthatarealsoreadilyexcreted.Themono-andbicyclicketonesinthe
group undergo reduction of the corresponding secondary alcohol followed by
conjugationwithglucuronicacidandexcretionintheurine.However,ifthebicylic
ketone is sterically hindered and exhibits increased lipophilicity, then oxidation
of the ring positions and substituents competes favourably with the reduction of
theketonefunctionalgroup.Inthecaseoffusedring(nootkatone;No.1398)and
macrocyclicketones,oxidationofside-chainalkylgroupsubstituentsandreduction
oftheketonefunctionyieldpolarexcretablemetabolites.Thesepathwaysarealso
operativefora,b-unsaturatedketones.
2.2.2 Toxicological studies
(a) Acutetoxicity
Oralmedianlethaldoses(LD
50
)havebeenreportedfor18ofthe32substances
in this group and are summarized in Table 3. In rats, LD
50
values ranged from
1220mg/kgbw for 7-methyl-4,4a,5,6-tetrahydro-2(3H)-naphthalenone (No. 1405)
to>10000mg/kgbwforisobornylacetate(No.1388),demonstratingthattheacute
toxicity of these monocyclic and bicyclic secondary alcohols and ketones when
administered orally is low (Fogleman & Margolin, 1970; Keating, 1972; Denine,
1973; Moreno, 1973, 1974; Levenstein, 1975; Moreno, 1975, 1976a, 1976b,
1977a, 1977b, 1977c; Gabriel, 1980; Mallory et al., 1982; Sedlacek, 1985;
Watanabe & Kinosaki, 1989; Driscoll, 1993; Kajiura & Kinosaki, 1995; Oh et al.,
1997;Gilman,1998;Yajima&Tanaka,2001).
For 3-methyl-2-(pentanyl)-2-cyclopenten-1-one (No. 1406), an oral LD
50
of
between4000and8000mg/kgbwwasreportedinmice(Engler&Bahler,1983),
and >2000mg/kgbw in dogs (You et al., 1997), confrming the low acute toxicity
ofthesubstancesinthisgroupwhenadministeredorally.
408 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
L1
Groups of eight rabbits were given a single dose of camphor (No. 1395) at
1000,1300,1400,1600,1800,2000,3000,or4000mg/kgbwincottonseedoilby
gavage.Additionally,groupsoftworabbitsweregivencamphoratadoseof1600
or1800mg/kgbwinalcoholbygavage.Alltreatedrabbitsexhibitedtonic(rigidity
andhyperextensionoftheforelegs)andclonic(violentshakingmotionsofentire
body)convulsionswithin540minoftreatment.Timetoconvulsionwasrelatedto
dose. Surviving rabbits were killed and examined microscopically, revealing no
signifcant lesions of the kidneys, lungs, heart, liver, pancreas, spleen, brain, or
spinal cord. Congestion and small focal haemorrhages were reported in the
oesophagealandgastricmucosaofseveralrabbits(Smith&Margolis,1954).
In an assay for competitive binding in vitro, which was developed to predict
male rat-specifc a
2m
globulin nephropathy, four male and four female rats were
intubateddailywithborneol(No.1385)atadoseof1mmol/kgbw(154mg)for3
days, after which the rats were killed, and their kidneys removed, weighed and
examinedhistologicallyforhyalinedroplets(Lehman-McKeeman&Caudill,1999).
Treatmentwithborneolwasreportedtoincreasehyalinedropletformationsignif-
cantlyatanincidencethatwasapproximatelyhalfofthatreportedford-limonene
(evaluatedbytheCommitteein1993;Annex1,reference107),whichwasadmin-
isteredunderthesameconditions.
(b) Short-termstudiesoftoxicity
Short-termstudiesoftoxicityconductedtoexaminethepotentialtoxicityofthe
monocyclicandbicyclicsecondaryalcohols,ketonesandrelatedesterswereavail-
ableforsevenrepresentativemembersofthisgroup(Nos1385,1388,1395,1396,
1402, 1408, and 1411). The results of these studies are summarized in Table 4
anddescribedbelow.
(i) Borneol(No.1385)
Dogs
Inastudyonthemetabolismofglucuronicacid,threedogsweregivenborneol
at a dose of approximately 526mg/kgbw per day in 1% agar by gavage for 31
days.Noadverseeffectswerereported(Milleretal.,1933).
Inanotherstudy,agroupofthreedogswasfedborneolatadoseofapproxi-
mately312mg/kgbwperday,whichwasgraduallyincreasedto1300mg/kgbwper
day within 2 months. Mucin (approximately 625mg/kgbw per day) was added to
the diet in order to offset any toxic effects of administration of borneol at high
doses. During the third month, the dogs were fed borneol at a dose of 1300mg/
kgbwperdayfor24days,butmucinwasnotaddedtothediet.Onedogdeveloped
distemperandwaskilled.After17days,aseconddogdiedafteradropinthelevel
ofglucuronicacidexcretedanditsdeathwasconsideredbytheauthorstobedue
to toxicity caused by borneol.The third dog was fasted for 7 days after 21 days
oftreatmentwithborneolatahighdoseanddiedwithin3daysafterfasting.The
dog showed signs of toxicity, including a drop in the level of glucuronic acid
excreted.Theauthorsconsideredthattheresultsindicatedthatmucin,asasource
ofglucuronicacid,didprovideprotectivepropertiesagainsttoxicityattributableto
MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS 409
L1
Table 3. Studies of the acute toxicity of monocyclic and bicyclic secondary
alcohols, ketones and related esters administered orally
No. Flavouringagent(No.) Species Sex LD
50
(mg/kgbw) Reference
1386 Isoborneol Rat NR 5200 Moreno(1977a)
1388 Isobornylacetate Rat NR >10000 Fogleman&Margolin
(1970)
1390 Isobornylformate Rat NR >5000 Levenstein(1975)
1391 Isobornylpropionate Rat NR >5000 Moreno(1973)
1393 Bornylisovalerate Rat NR >5000 Denine(1973)
(endo-)
1395 d-Camphor Rat NR >5000 Moreno(1976a)
1397 Fenchylalcohol Rat NR ND Moreno(1976b)
1398 Nootkatone Rat NR >5000 Moreno(1977b)
1398 Nootkatone Rat M,F >2000 Gilman(1998)
1399 1,3,3-Trimethyl-2- Rat NR >5000 Moreno(1975)
norbornanylacetate
1400 Methyljasmonate Rat M,F >5000 Gabriel(1980)
1401 Cycloheptadeca-9- Rat NR >5000 Moreno(1974)
en-1-one
1402 3-Methyl-1- Rat NR >5000 Moreno(1977c)
cyclopentadecanone
1402 3-Methyl-1- Dog M,F >2000 Youetal.(1997)
cyclopentadecanone
1402 3-Methyl-1- Rat M,F >5000 Ohetal.(1997)
cyclopentadecanone
1405 7-Methyl-4,4a,5,6- Rat M,F 1220 Malloryetal.(1982)
tetrahydro-2(3H)-
naphthalenone
1406 3-Methyl-2-(n-pentanyl)- Rat NR 2500 Keating(1972)
2-cyclopenten-1-one
1406 3-Methyl-2-(n-pentanyl)- Mouse M >4000,but Engler&Bahler
2-cyclopenten-1-one <8000 (1983)
1407 Dihydronootkatone Rat NR >5ml/kg Sedlacek(1985)
1408 3-l-Menthoxypropane- Rat M,F 5800(M); Watanabe&Kinosaki
1,2-diol 5600(F) (1989)
1408 3-l-Menthoxypropane- Rat M,F >2000 Yajima&Tanaka
1,2-diol (2001)
1413 d,l-Menthol-()- Rat M,F >2000 Driscoll(1993)
propyleneglycol
carbonate
1416 p-Menthane-3,8-diol Rat M,F >2000 Kajiura&Kinosaki
(1995)
F,female;M,male;NR,notreported
high doses of borneol and even with the withdrawal of mucin from the diet, the
body was capable of storing large quantities of glucuronic acid, which provided
someextendedabilitytodetoxifyborneol(Milleretal.,1933).
Finally, a third group of fve dogs was fasted and fed 5g of borneol daily
(approximately500mg/kgbwperday)for37days.Onepregnantdogdiedafter2
410 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
L1
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.
MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS 411
L1
weeks of fasting.A second dog showed signs of toxicity after 2 weeks and was
then given a diet supplemented with mucin. The third dog survived the 37-day
treatment period without supplementation with mucin. The two other dogs were
fasted, but also fed 10g of mucin daily.At necropsy, the dogs showed signs of
gastritis (more marked in the pyloric area) and marked duodenitis (Miller et al.,
1933).
(ii) Isobornylacetate(No.1388)
Rats
Groupsof15maleand15femaleCFEratswereorallyintubatedwithisobornyl
acetateatadoseof0(control),15,90or270mg/kgbwperdayincornoilfor13
weeks.Additional groups of fve male and fve female rats were orally intubated
withisobornylacetateatadoseof0,90or270mg/kgbwperdayfor2or6weeks.
Animalswereobservedthroughoutthestudyforsignsoftoxicityandoverallcondi-
tion. Weekly measurements of body weight, and food and water intake were
obtained.Haematologicalevaluationwasperformedattheendofthestudyperiod,
whilecompleteurineanalysiswasconductedatweek6andatstudytermination.
Atweek2,onlyurinaryvolumeandspecifcgravityweredetermined.Atstudyter-
mination,bloodsamplesweretakenforclinicalchemistryevaluationandanimals
werekilledandnecropsied.Animalswereexaminedmacroscopically,andselected
organs were weighed and tissues preserved for histopathological evaluation. No
deaths or abnormal appearance or behaviour were reported. Body weight mea-
surements revealed no differences in body-weight gain between controls and
treatedrats,butaslightdecreaseintheweightofmalesatthehighestdosewas
signifcantafterthe24-hfastbeforedeathatboth6and13weeks.Comparedwith
controls,waterconsumptioninmalesatthehighestdosewasincreasedthrough-
outthestudy(p<0.01).Nodifferencesinfoodintakewerereported.Haematologi-
calexaminationatweek2showedstatisticallysignifcantincreasesinhaemoglobin
concentration in females at the highest dose (p < 0.01) and in total leukocyte
countsinmalesatthehighestdose(p<0.05).Additionally,signifcantlyelevated
erythrocyte counts were observed in and males at the intermediate and highest
doses. These changes were not reported at 6 or 13 weeks. Reticulocyte counts
inall(controlandtreated)youngerratswerehigherthanintheiroldercounterparts
anderythrocytesshowedamarkedpolychromasia.Serumchemistryresultswere
similar in treated and control animals, showing no signifcant doseresponse
effects. Urine analysis results showed that urine was of normal colour and was
freefromglucose,blood,bile,andketones.Intermsofconcentrationandresults
oftestsforurinarycellexcretion,treatedfemalesatweeks2,6,and13andtreated
malesatweek2werenotdifferentfromcontrolsthroughoutthestudy;however,
males treated for 6 or 13 weeks had signifcantly increased cell excretion at the
highest dose and males treated for 13 weeks also showed this effect at 90mg/
kgbwperday.Malesatthehighestdosealsoexhibitedimpairmentofurinecon-
centrating ability; seen at week 6 only after 1620h of water deprivation and at
week13afterdehydrationfor6h.Absoluteweightsoftheliver(p<0.001),kidney
(p<0.05)andcaecum(p<0.001)weresignifcantlyincreasedinfemalesatthe
highestdoseafter13weeks.Signifcantlyincreasedabsoluteweightofthecaecum
(p < 0.05) also was reported in groups of males treated at the highest dose.At
412 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
L1
week2,relativebrainweightwassignifcantlyincreasedinfemalesatthehighest
dose(p<0.01).Atweek6,relativeweightofthekidney(p<0.01)wassignifcantly
increasedinmalesatthehighestdoseandrelativeweightofthegonads(p<0.05)
was signifcantly increased in females at the highest dose.At week 13, relative
weightsoftheliver(p<0.05),kidney(p<0.01)andcaecum(p<0.001)inratsat
thehighestdose(bothsexes)weresignifcantlyincreased.Histologicalexamina-
tionrevealedamildpulmonaryinfectioninallrats(controlandtreated).Theonly
histologicalfndingsattributedbytheauthorstoexposuretoisobornylacetatewere
reportedinthekidneyofanimalsatthehighestdoseanimalsandconsistedofan
increasedincidenceoffocaltubulardegenerationandatrophy(bothsexes)anda
vacuolation of the tubular epithelium (males only). In addition, vacuolation of the
epithelial cells of the intrahepatic bile ducts of males at the highest dose was
reported.Onthebasisofrenalandhepaticeffectsobservedinmalesattheinter-
mediateandhighestdosesandtheabsenceofanyeffectinfemalesatthelowest
andintermediatedoses,theno-observed-effectlevel(NOEL)was15and90mg/
kgbwperdayformalesandfemales,respectively(Gauntetal.,1971).
(iii) d-Camphor(No.1395)
Rats
Sage oil, which is estimated to contain approximately 30% camphor (Millet
et al., 1981), was orally administered to groups of fve rats (strain and sex not
reported)atadoseof0,250,500,1000or1200mg/kgbwperday(approximately
equivalent to camphor at a dose of 0, 75, 150, 300 and 360mg/kgbw per day,
respectively)for8weeks.Administrationof250mg/kgbwperdayofsageoilwas
reportedtobewelltoleratedbytherats,astheyshowednoclinicalsignsoftoxicity
andtheirbodyweightswerecomparabletothoseofthecontrols.Ratsinthegroup
receiving a dose of 500mg/kgbw per day exhibited occasional clonic seizures
immediately after dosing, as well as slightly lower body-weight gains compared
withthoseofcontrols.Oneratgivenadoseof500mg/kgbwperdaydiedonday
22oftreatmentduringoneofthesecramp-likeattacks.Deathswerealsoreported
inthemajorityoftheanimalsat1000mg/kgbwperday,andinalltheratsatthe
highestdose(1200mg/kgbwperday)(Skramlik,1959).
(iv) Nootkatone(No.1398)
Rats
In a 28-day study designed to investigate the systemic toxicity of two a,b-
unsaturated ketones, nootkatone and verbenone
1
, groups of 10 male and 10
femaleSprague-DawleyCrl:CD(SD)IGSBRratsweregivennootkatoneorver-
1

O
verbenone
MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS 413
L1
benone(inArachisoilBP)atadoseof10mg/kgbwperdaybygavagefor28days.
Tenmaleand10femaleratsservedasvehiclecontrols.Allanimalswereexamined
forclinicalsignsoftoxicityimmediatelybeforedosingand15hafterdosingduring
theworkingweekand1hafterdosingonweekends.Bodyweightswererecorded
onday0andweeklythereafter.Foodconsumptionwasmeasuredatweeklyinter-
vals and water intake was measured daily. Haematology and blood chemical
investigationswereperformedatday28forallanimals.Atterminationallanimals
were subjected to gross examination. Organ weights were obtained and histo-
pathological examination was performed for all major tissue types and on any
lesions. No signs of clinical toxicity were observed throughout the study. Water
consumption, food consumption, food effciency and weight gain were similar in
testandcontrolgroups.Bloodbiochemistryrevealednodifferencesbetweentest
groupsandcontrols.Femalesinbothstudygroupsshowedincreasedconcentra-
tionsoftotalprotein.Theauthorsviewedthisintergroupdifference,whichdidnot
occurinmales,tobeofnotoxicologicalimportance.Nogrossabnormalitieswere
attributedtotheadministrationofnootkatoneorverbenone.Therewerenotreat-
ment-relatedchangestoorganweights.
Histopathologyfndingsrevealedglomularaccumulationsofeosinophillicmate-
rialinthekidneytubularepitheliumofmaleratstreatedwithnootkatoneandver-
benone.Theauthorsnotedthattheobservationswereconsistentwiththepresence
ofa
2m
globulinnephropathy.
Given that a
2m
globulin is a well characterized phenomenon that is specifc to
male rats (Capen et al., 1999), the authors concluded that no adverse effects
occurredatadoseof10mg/kgbwperdayfornootkatoneorverbenonewhenorally
administereddailyfor28days(Jonesetal.,2004).
(v) 3-Methyl-1-cyclopentadecanone(No.1402)
Rats
Groupsof10maleand10femaleSprague-Dawleyratsweregiven3-methyl-
1-cyclopentadecanoneatadoseof0(vehiclecontrol),10,100or1000mg/kgbw
perdayinTween80bygavagefor30days.Ratswereobserveddailyforclinical
signsoftoxicityandwereweighedtwiceperweek.Intakesoffeedandwaterwere
measuredtwotothreetimesperweek.Atstudytermination,samplesofbloodand
urineweretakenforserumchemistry,haematology,andurineanalysisdetermina-
tions.Subsequently,alltheratswerekilled,necropsied,andexaminedhistopatho-
logically. No signifcant differences from controls were reported in any of the
parametersexamined.Relativetocontrols,theonlystatisticallysignifcantfnding
was an increase in the absolute and relative weight of the liver in males and
females at the highest dose.According to the authors, this effect may be due to
enzyme induction, since hepatic CYP and associated activities were elevated
in rats at the highest dose. However, owing to the absence of histopathological
or haematological fndings to support compound-related toxicity, these effects
were not considered to be toxicologically signifcant. The NOEL for 3-methyl-1-
cyclopentadecanone assigned by the authors was therefore >1000mg/kgbw per
day(Ohetal.,1997).
414 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
L1
Dogs
Nine male and nine female beagle dogs were given 3-methyl-1-
cyclopentadecanoneatadoseof0,0.2,2.0or20mg/kgbwperdayinTween80
byoraladministration(specifcroutenotstated)for4weeks.Dogswereobserved
dailyforclinicalsignsoftoxicity,andbodyweightswererecordedtwiceperweek.
Intakes of feed and water were measured daily. Ophthalmoscopic examinations
wereconductedatthestartofthestudyandthenweeklythereafter.Bloodsamples
weretakenbeforethestartofthestudyandatstudyterminationfordetermination
ofhaematologicalparametersandserumchemistry.Urinesampleswerecollected
on the frst and last days of treatment. At study termination, dogs were killed,
necropsied, and examined histopathologically. Some temporary and sporadic
decreases in feed intake and an increased incidence of diarrhoea were noted in
treated rats; however, no compound-related effects were reported in any of the
parameterstested,includingorganweightchanges(Youetal.,1997).
(vi) 3-l-Menthoxypropane-1,2-diol(No.1408)
Rats
Ina14-dayscreenfortoxicity,groupsoffvemaleandfvefemaleF344rats
were fed diets containing 3-l-menthoxypropane-1,2-diol, to provide an intended
doseof1000mg/kgbwperday.Onthebasisoffeedconsumption,themeandose
of 3-l-menthoxypropane-1,2-diol was calculated to be approximately 738.1 and
809.2mg/kgbwperdayformalesandfemales,respectively.Ratswereobserved
daily for clinical signs of toxicity, and body weights were recorded 1 day before
studyinitiation,aswellasondays6and14ofthestudy.Feedintakewasdeter-
mined on days 7 and 14.At study termination, rats were killed, necropsied and
examined for gross pathology and renal and hepatic histopathology. With the
exceptionofastatisticallysignifcantincreaseinabsoluteandrelativeweightsof
the liver in rats treated with 3-l-menthoxypropane-1,2-diol relative to controls, no
compound-related effects were reported in any of the parameters examined. In
addition,nocompound-relatedmacroscopicormicroscopiclesionswerenotedin
theliveroranyotherorgansoftreatedrats(Weaver&VanMiller,1989).
Groups of fve male and fve female Sprague-Dawley rats were given diets
containing3-l-menthoxypropane-1,2-diolatadoseof0,250,500,1000,2000or
5000mg/kgbw per day for 28 days. Parameters evaluated included daily clinical
observation, weekly measurement of body weights and food consumption, and
ophthalmoscopic, electrocardiographic and clinical pathology (i.e. haematology,
serum chemistry, and urine analysis) examinations at week 4.All animals were
sacrifcedattheendofthestudy,andsubjectedtomacroscopicandmicroscopic
histopathological examination, and absolute and relative organ weight measure-
ments.Adose-relateddecreaseinmeanbodyweightswasnotedintreatedrats;
however,relativetocontrols,thisdifferencereachedstatisticalsignifcanceonlyat
thehighestdose(5000mg/kgbwperday).Comparedwithcontrols,meanweekly
and total food consumption was signifcantly decreased in males at the highest
dose (5000mg/kgbw per day). No compound-related changes were reported in
the clinical, haematological, and ophthalmological examinations of the animals.
MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS 415
L1
Whilenocompound-relatedalterationsinserumchemistryandurineanalysiswere
reportedinfemalerats,maleratsexhibitedsignifcantdose-relateddecreasesin
serum glucose and blood urea nitrogen concentrations compared with those of
controls.Atthehighestdose,ratsexhibitedsignifcantlydecreasedmeanabsolute
weights of the heart compared with controls. In addition, signifcant dose-related
increases in absolute and relative weights of the liver were reported in animals
of both sexes. It was suggested that the reductions in serum concentrations of
glucoseobservedinmaleratswereassociatedwiththedose-relatedincreasesin
theincidenceandseverityofdiffusehepatocellularenlargementandeosinophilic
inclusionsreportedatalldosesexcept250mg/kgbwperday.Diffusehepatocellu-
larenlargementwasalsoreportedinfemalesat2000and5000mg/kgbwperday.
Nootherhistopathologicaleffectswerenotedthatcouldbeattributedtothecom-
poundadministered(Wolfe,1992a).Theliversofmalesandfemalesatthehighest
dose were later examined by electron microscopy (Cockrell, 1992a). Myelinoid
bodies, and moderate proliferation of the smooth endoplasmic reticulum were
observed in the liver of males at the highest dose, while moderate proliferation
andminorwhorlingofthesmoothendoplasmicreticulumwasnotedintheliverof
femalesatthehighestdose.Formationofmyelinoidbodieswaspresumedtobe
theresultofmetabolicadaptationinwhichcontinuousenzymeinductionstimulated
proliferationofthesmoothendoplasmicreticulum,similarobservationshavebeen
reported in the liver of dogs treated with butylated hydroxyanisole (Ikeda et al.,
1986).Thelaboratoryreportedthattheseeffectsappearedtobereversibleupon
cessation of treatment (Ghadially, 1982), and should not be considered to be a
degenerativechangeinthehepatocyte(Cockrell,1992a).
Inasubsequent91-daystudy,groupsof20maleand20femaleCDratswere
maintained on diets containing 3-l-menthoxypropane-1,2-diol at a dose of of 0
(control), 30, 200 or 1000mg/kgbw. Daily clinical observation, weekly measure-
mentofbodyweightsandfoodconsumption,measurementofwaterconsumption
every 2 weeks, and ophthalmoscopic and electrocardiographic examinations at
week12wereperformed.Allanimalswerereportedtosurvivetheentireduration
of the study. Male and female rats showed hunched posture, and females at
1000mg/kgbw per day showed signifcant decreases in body weight and body-
weightgain.Inthefemalesatthehighestdose,thebody-weightvariationswere
accompaniedbyslightdecreasesinfoodconsumption.Sporadic,statisticallysig-
nifcant differences in food consumption were observed at various time-points in
mostothergroups.Haematologicalexaminations,bloodchemicaldeterminations,
andurineanalyseswereperformedatweeks4and13,andwiththeexceptionof
a signifcant increase in g-glutamyl transferase activity reported in males at the
highest intake, no other variations were observed. In males and females at the
intermediate and highest doses, signifcant increases were reported in absolute
weightsoftheliver.Relativeweightsoftheliverandratiosoflivertobrainweight
wereincreasedatallthreedosesinfemales,andat200and1000mg/kgbwper
dayinmales.Theincreasesobservedinweightoftheliverweredose-dependent
inbothsexes.Slighttoseverediffusehepatocellularenlargementwasreportedin
malesat1000mg/kgbwperdayandmidzonalandcentrilobularenlargementwas
reportedinfemalesat1000mg/kgbwperday.Eosinophiliccytoplasmicinclusions
werenotedintheliversoftwoandtenmalesattheintermediateandhighestdoses,
416 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
L1
respectively.Absoluteandrelativeweightsofthekidneyweresignifcantlyincreased
inmalesat1000mg/kgbwperdayandinfemalesat200mg/kgbwperday.Rela-
tiveweightsofthekidneywerealsosignifcantlyelevatedinfemalesatthehighest
dose. Increased ratios of kidney to brain weight were signifcant in males at the
highestdoseandinfemalesat30and200mg/kgbwperday.Inmales,theincrease
in absolute weights of the kidney was noted to be dose-related, as was the
increase in relative weights of the kidney in females. Granular casts were noted
inthekidneysoftwomalesatthehighestdose;however,thiswasnotassociated
with an increased presence of hyaline droplets in stained tissues. Overall, histo-
pathologydidnotrevealanycompound-relatedvariationsinanimalsofeithersex
atthelowestdoseorinfemalesattheintermediatedose.Onthebasisofhisto-
pathological examination, the NOEL was 30mg/kgbw per day in males and
200mg/kgbwperdayinfemales(Wolfe,1992b).
Dogs
Groupsoftwomaleandtwofemalebeagledogsweregivengelatincapsules
containing3-l-menthoxypropane-1,2-diolatadoseof0,250,500,1000,2000or
5000mg/kgbwperdaybyoraladministrationfor28days.Parametersevaluated
includeddailyclinicalobservation,weeklymeasurementofbodyweightsandfood
consumption, and electrocardiographic examinations at weeks 2 and 4. Clinical
pathology evaluations (haematology, serum chemistry, and urine analysis) were
performedonallanimalsbeforeinitiationofthestudy,onalldogasatthehighest
dose(5000mg/kgbwperday)duringweek2,ononefemaledogat2000mg/kgbw
perdayduringweek3,andonallsurvivingdogsattheendofthestudy.Statistical
evaluation of the clinical pathology data was not performed owing to the limited
sample size. All surviving animals were sacrifced at the end of the study, and
subjected to macroscopic and microscopic histopathological examination, and
measurement of absolute and relative organ weights. Clinical signs of toxicity,
including soft or mucoid faeces, emesis, and salivation, were noted primarily in
dogs given 3-l-menthoxypropane-1,2-diol at a dose of 1000, 2000 and 5000mg/
kgbwperday.Treateddogsalsoexhibitedlacrimation,tremors,nasaldischarge,
ataxia, and decreased activity. Food consumption and mean body weights of all
survivingtreateddogswerereportedtobesimilartothoseofcontrolsthroughout
thestudy,withtheexceptionofonefemaleat500mg/kgbwperdaythatexhibited
progressivebody-weightreductionwithaccompanyingdecreaseinfoodconsump-
tion.Nocompound-relatedeffectsonelectrocardiographictracings,haematology,
and urine analysis were observed. Dogs at 200, 500, 1000, 2000 and 5000mg/
kgbwperdayexhibitedincreasedmeanconcentrationsofcholesterol,andslight,
dose-related reductions in serum concentrations of glucose. Increased absolute
andrelativeweightsoftheliverwerereportedinbothsexesat250,500,1000and
2000mg/kgbw per day. Male dogs at 1000 and 2000mg/kgbw per day showed
decreased absolute and relative weights of the thymus. Relative to controls,
decreasedabsoluteandrelativeweightsoftheprostateandtestisofmalesat250,
500,1000and2000mg/kgbwperday,andoftheuterusinfemalesat1000and
2000mg/kgbwperdaywereobserved.Alldogsatthehighestdose,twodogsat
2000mg/kgbwperday,andonefemaleat1000mg/kgbwperdayweresacrifced
inextremisbeforetheendofthestudy.Thesedogswerereportedtoshowclinical
MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS 417
L1
signsoflethargy,depressionsinfoodconsumptionwithprogressivebody-weight
losses,elevatedconcentrationsoftotalcholesterolandtriglycerideconsistentwith
theemesisnotedclinically,andincreasedalkalinephosphataseactivityandcon-
centrations of total protein, albumin, sodium, potassium, and chloride, indicative
ofdehydration.Thecauseofthemoribundconditionofthethreefemales(at1000,
2000 and 5000mg/kgbw per day) sacrifced in extremis was not determined;
however, the death of three males (at 2000 and 5000mg/kgbw per day) was
attributed to pulmonary infammation caused by aspiration of vomit. While the
death of one female at the highest dose was attributed to severe liver necrosis,
nosuchliveralterationswereobservedinanyoftheothertreatedanimals.Clinical
pathological and histopathological examination of all of the treated animals was
reportedtorevealnoorganeffectsormorphologicaltissuealterationsthatcould
be related to the administration of 3-l-menthoxypropane-1,2-diol. The NOEL for
3-l-menthoxypropane-1,2-diol administered orally was 1000mg/kgbw per day in
malesand500mg/kgbwperdayinfemales(Dalgard,1993).
Groupsoffourmaleandfourfemalebeagledogsweregivencapsulescontain-
ing3-l-menthoxypropane-1,2-diolatadoseof0,10,50or250mg/kgbwperday
orallyfor91days.Dailyclinicalobservation,weeklymeasurementofbodyweights
and food consumption, measurement of water consumption every 2 weeks, and
ophthalmoscopic and electrocardiographic examinations at week 12 were per-
formed and revealed no signifcant difference between test and control animals.
Haematologicalexaminations,bloodchemicaldeterminationsandurineanalyses
wereperformedatweeks4and13,andshowedaslight(lessthantwofoldgreater
than that of controls) but signifcant (p 0.05) increase in alkaline phosphatase
activity for the group receiving the highest dose (250mg/kgbw per day).A non-
specifcincreaseinabsoluteandrelativeweightsoftheliverwasreportedforthe
groupatthehighestdose,accompaniedbyadiffusehepatocellularenlargement
in fve out of eight dogs in this group.A dose-related increase in weights of the
thyroid and/or parathyroid and decrease in weights of the prostate were noted;
however, these observations did not reach statistical signifcance, and were not
accompanied by histopathological evidence. Therefore, these alterations were
notconsideredtobebiologicallysignifcant.TheNOELwas50mg/kgbwperday
(Dalgard,1994).Electronmicroscopyperformedonliversofmalesandfemalesa
the highest dose revealed proliferation of smooth endoplasmic reticulum associ-
atedwiththepresenceofmyelinoidbodies(i.e.lipidcytosomes)(Cockrell,1992b).
Thesamelaboratoryobservedmyelinoidbodiesandproliferationofsmoothendo-
plasmicreticulumintheliverofdogstreatedwithbutylatedhydroxyanisole(Ikeda
etal.,1986).Thelaboratoryreportedthatthesechangesappeartobereversible
(Ghadially, 1982), and should not be considered a degenerative change in the
hepatocyte(Cockrell,1992b).
(vii) 3-l-Menthoxy-2-methylpropan-1,2-diol(No.1411)
Rats
In a 28-day single dose screening study, groups of fve male and fve
female Sprague-Dawley CD rats were given diets containing 3-l-menthoxy-2-
methylpropan-1,2-diolatadoseof1000mg/kgbwperday.Ratswereindividually
418 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
L1
housedandgivenaccesstofoodandwateradlibitumRatswereobservedtwice
dailyforclinicalsignsoftoxicity,bodyweightsandfoodconsumptionwererecorded
weekly,andfeedeffciencywascalculated.Necropsywasperformedonday29,
and brain, kidney and liver were weighed.Tissues from these organs as well as
thoseexhibitinganymacroscopicabnormalitieswerepreservedforhistopathologi-
calexamination.Allanimalswerereportedtosurviveuntiltheendofthestudyand
physical examination did not reveal any compound-related adverse effects.
Althoughmeanbodyweightswerenotedtobegenerallylowerintreatedanimals
than in controls (i.e. at study termination, mean body weights of treated females
were8%lowerthanthoseofcontrols),nostatisticallysignifcantdifferenceswere
observed. However, at weeks 3 and 4, females exhibited a signifcantly reduced
body-weightgaincomparedwiththatofcontrolanimals.Decreasedfoodconsump-
tionwasreportedintreatedmalesandfemalesduringthefrstweekofthestudy
andwaspartiallyattributedtotheinitialpoorpalatabilityofthediet.Meanvalues
forfoodconsumptionwerecomparabletothoseforthecontrolsduringtheremain-
der of the study. Similarly, feed utilization in treated animals was reduced during
the frst week, but was comparable to that of controls thereafter. The authors
reportedastatisticallysignifcantincreaseinthemeanweightoftheliver(p<0.01)
oftreatedmales,andinratiosoflivertoterminalbodyweightandbrainweight(p
<0.01)comparedwiththoseforthecontrols.Thestatisticallysignifcantdecrease
inmeanweightofthebrain,aswellastheincreasesinratiosofkidneytoterminal
body weight and brain weight were reported to be refective of normal variability
andwere,therefore,consideredtobeunrelatedtocompoundadministration.His-
topathologyrevealedhepatocellularhypertrophyinbothsexes.Infemales,itwas
central lobular and minimal to slight in severity, while in males it was diffuse
(pan-lobular)andmoderatetomoderatelysevere.Hepatocellularhypertrophyisa
common response associated with the increased metabolic requirements
(Crampton et al., 1977; ONeill et al., 2003). On the basis of microscopic varia-
tionsobservedinbothsexes,theNOELwas<1000mg/kgbwperday(Madarasz
&Bolte,1997).
(c) Long-termstudiesoftoxicityandcarcinogenicity
No information on long-term studies of toxicity and carcinogenicity was
available.
(d) Genotoxicity
Tests for genotoxicity in vitro and in vivo using standardized protocols have
beenusedtostudysevenrepresentativemembers(Nos1385,1391,1395,1408,
1411,1413and1416)ofthemonocyclicandbicyclicsecondaryalcohols,ketones
andrelatedestersgroupusedasfavouringagents(seeTable5).
(i) Invitro
Seven members of this group (borneol, No. 1385; isobornyl propionate,
No. 1391; d-camphor, No. 1395; 3-l-menthoxypropane-1,2-diol, No. 1408; 3-l-
menthoxy-2-methylpropan-1,2-diol, No. 1411; d,l-menthol 1- and 2-propylene
MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS 419
L1
glycol carbonate; No. 1413 and p-menthan-3,8-diol; No. 1416) consistently gave
negative results in theAmes assay when incubated at a concentration of up to
5000mg/plate with a variety of Salmonella typhimurium strains including TA97,
TA98, TA100, TA102, TA1535, TA1537 and TA1538 with or without metabolic
activation (Simmon et al., 1977; Anderson & Styles, 1978; Wild et al., 1983;
Watanabe & Morimoto, 1989; National Toxicology Program, 1992a; King, 1993;
Azizan & Blevins, 1995; Kajiura, 1995, 1996; Marzin, 1998; Shirai & Sasaki,
2000).
Borneol(No.1385),3-l-menthoxypropane-1,2-diol(No.1408),3-l-menthoxy-2-
methylpropan-1,2-diol (No. 1411) and p-menthan-3,8-diol (No. 1416) showed no
mutagenic activity when tested in Escherichia coli WP2 uvrA at concentrations
of up to 5000mg/plate (Yoo, 1986; Watanabe & Morimoto, 1989; Kajiura, 1995;
Kajiura, 1996; Shirai & Sasaki, 2000), although cytotoxicity was reported at
concentrationsexceeding1250mg/plate(Shirai&Sasaki,2000).
IntheRec
-
assay,borneol(No.1385)wasreportedtoinducegrowthinhibition
inBacillussubtilisstrainM45
-
whentestedatconcentrationsofupto10mg/disc
(Yoo,1986).
When tested by the National Toxicology Program, d-camphor (No. 1395) at
concentrationsreaching1500mg/mldidnotinducesisterchromatidexchangesin
Chinese hamster ovary cells in the presence or absence of metabolic activation
derived from the livers of rats induced with Aroclor 1254 (National Toxicology
Program,1992a).Cytotoxicitywasreportedatconcentrationsexceeding750mg/ml
without metabolic activation and at concentrations exceeding 550mg/ml with
metabolicactivation.
Structurally related a,b-unsaturated alicyclic ketones isophorone (No. 1112)
andcarvone(No.380)showednomutagenicpotentialinthestandardAmesassay
when incubated at concentrations of 3.3333mg/plate with various strains of S.
typhimurium(TA98,TA100,TA1535,TA1537)withorwithoutmetabolicactivation
fromS9(Mortelmansetal.,1986).WhenincubatedwithBacillussubtilisH17(rec
+
)
or M45 (rec
-
) in the rec
-
assay at a dose of 0.6 ml/disc, carvone (unspecifed
stereochemistry) was negative both in the presence and absence of metabolic
activationfromS9(Matsuietal.,1989).
Theresultsoftheassayforforwardmutationinmouselymphomacellsinvitro
werenegativewithisophoroneatconcentrationsof1301300mg/mlwithoutmeta-
bolic activation from S9 and with isophorone at concentrations of 67890mg/ml
with metabolic activation (McKee et al., 1987; ODonoghue et al., 1988). An
increaseinthefrequencyofmutationwasreportedwithisophoroneatconcentra-
tionsof400and800mg/mlinmouselymphomaL5178YTk
+/-
cellswithoutmeta-
bolicactivation(McGregoretal.,1988).
Isophorone,testedinChinesehamsterovarycells,producedequivocalresults.
In one study, no chromosomal aberrations were induced at concentrations up to
1600mg/ml with or without metabolic activation (Gulati et al., 1989). In another
study,isophorone,ataconcentrationof1250mg/mlwithoutmetabolicactivationor
ataconcentrationof1500mg/mlwithmetabolicactivation,inducedchromosomal
aberrations(Matsuokaetal.,1996);however,lowerconcentrationsof2501000mg/
420 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
L1
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422 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
L1
mlwithoutmetabolicactivationdidnotinducechromosomalaberrations.Isopho-
rone only induced sister chromatid exchanges when tested at concentrations of
5001000mg/mlinChinesehamsterovarycellswithoutmetabolicactivation,and
thenonlyafteradelayedharvest,duetothecytostaticeffectofisophorone(Gulati
etal.,1989).Atlowerconcentrationswithoutmetabolicactivation,oratconcentra-
tionsofupto1600mg/mlwithmetabolicactivation,isophoronedidnotinducesister
chromatidexchanges(Gulatietal.,1989).InanassayforunscheduledDNAsyn-
thesisinrathepatocytes,therewasnoevidenceforgenotoxicitywithisophorone
at concentrations of up to 0.2 ml/ml (McKee et al., 1987; ODonoghue et al.,
1988).
d-Carvone at concentrations of 0.167502mg/ml induced slight increases in
sisterchromatidexchangeandchromosomalaberrationinChinesehamsterovary
cellseitherwithorwithoutmetabolicactivationfromS9(liverfrommaleSprague-
DawleyratsinducedwithAroclor1254).Theresultsofthisassaywerestatistically
positive in two out of three assays for sister chromatid exchanges, but there
was no correlation of dose with response.Also, the number of sister chromatid
exchanges per cell was less than 1.5-times that of solvent controls. A dose
responserelationshipwasnotconfrmedinasecondtrialoftheassayforchromo-
some aberration, conducted without metabolic activation; chemical-induced cell
cycledelaywasreportedinthistrial(NationalToxicologyProgram,1990).
(ii) Invivo
Thepotentialofisobornylpropionate(No.1391)toinducesex-linkedrecessive
lethalmutationsinadultDrosophilamelanogasterwasstudiedinaBasctest.No
increasedfrequencyofmutationwasobservedinfiesfedwithisobornylpropionate
(No.1391)ina10mmol/lsolutionfor3days(Wildetal.,1983).
In the test for micronucleus formation, groups of NMRI mice given isobornyl
propionate(No.1391)atadoseof841,1893or2944mg/kgbwbyintraperitoneal
administration showed no increase in micronucleated erythrocytes in samples of
bonemarrow,30hafteradministration(Wildetal.,1983).
Topical application of d,l-camphor at a dose of up to 1000mg/kgbw over 90
days(atotalof65treatments)didnotinducemicronucleusformationintheperiph-
eralblooderythrocytesofB6C3F
1
mice(NationalToxicologyProgram,1999).
Whenthestructurallyrelatedmonocyclica,b-unsaturatedketone,isophorone
(No. 1112) was fed to adult D. melanogaster for 3 days, no mutations were
observed(Fouremanetal.,1994).Inaddition,negativeresultswereobtainedwhen
D. melanogaster were injected with a single dose of 12500mg of isophorone
(Fouremanetal.,1994).
Therewasnoincreaseinthefrequencyofmicronucleatedpolychromaticeryth-
rocytes in the bone marrow of male or female CD-1 mice given isophorone at a
doseof540mg/kgbwbyintraperitonealinjection(McKeeetal.,1987;ODonoghue
etal.,1988).
MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS 423
L1
(iii) Conclusion
The testing of these representative monocyclic and bicyclic secondary alco-
hols,ketonesandrelatedestersinbacterial(Amesassay)andmammalian(micro-
nucleus formation) in-vivo systems showed no evidence of genotoxic potential,
andtheseresultsarefurthersupportedbythelackofpositivefndingsintheDro-
sophila Basc test. These data are fortifed by the lack of genotoxic potential of
relateda,b-unsaturatedmonocyclicketones,isophoroneandcarvone.
(e) Reproductivetoxicity
(i) d-Camphor(No.1395)
Rats
Groupsof20pregnantSprague-Dawleyratsweregivend-camphor
2
atadose
of 0 (vehicle control), 216, 464 or 1000mg/kgbw per day in propylene glycol by
gavageduringdays617ofgestation.Damswereobservedforanysignsoftoxic-
ity,andbodyweightandfoodintakewererecordeddaily.Onday20ofgestation,
dams were killed and examined macroscopically. Fetuses were examined for
external, skeletal and visceral anomalies. No adverse effects were reported in
damsgivend-camphoratadoseof216mg/kgbwperday.Atthetwohigherdoses,
salivation and reduced feed intake were reported, with clonic convulsion, pilo-
erection,reducedmotility,andreducedbody-weightgainreportedindamsatthe
highest dose. Necropsy revealed ulcers in the cardiac region of the stomach of
twodamsattheintermediatedoseandfvedamsatthehighestdose.Inaddition,
one dam at the highest dose was reported to have a thickened rough cardiac
epithelium.Inthefetuses,noeffectonprenataldevelopmentwasreportedandno
variations,retardations,ormalformationswerereportedatanydose,eventhose
causingmaternaltoxicity(Leuschner,1997).
Groups of 2629 pregnant Sprague-Dawley rats were given d-camphor at a
doseof0(vehiclecontrol),100,400or800mg/kgbwperdayincornoilbygavage
duringdays615ofgestation.Thedamswereobservedforclinicalsignsoftoxicity,
and body weights, feed and water consumption were recorded. On day 20 of
gestation,damswerekilledandbody,liverandintactuterusweightswererecorded,
andcorporaluteawerecounted.Numbersofimplantationsites,resorptions,dead
fetuses,andlivefetusesweredetermined.Livefetuseswereweighed,andexam-
ined for external, visceral and skeletal abnormalities. No maternal deaths were
reported.Initially,foodconsumptionwassignifcantlydecreasedatthetwohigher
doses, but was reported to recover by the end of the study. Water intake was
increasedinalltreatedgroups,reachingstatisticalsignifcanceatthetwohigher
doses at various time-points during the study. In the group receiving the lowest
dose, water intake was signifcantly increased only on days 69 of gestation.
During the treatment period, a dose-dependent reduction in weight gain was
2
TheauthorsdescribedthetestmaterialasD-camphor,CASNo.76-22-2.TheCASNo.
ford-camphor[(+)-camphor]is464-49-3;whiletheCASNo.for()-camphoris76-22-2.
424 MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS
L1
reported, which reached levels of statistical signifcance in dams at the highest
dose.Additionally,slight(10%)butsignifcantanddose-dependentincreasesin
absolute and relative weights of the liver were reported at the intermediate and
highestdoses.Exposuretod-camphorproducednoeffectonfetalgrowth,viability,
ormorphologicaldevelopment,evenatdosescausingmaternaltoxicity(National
ToxicologyProgram,1992b).
Rabbits
Groupsof12pregnantHimalayanrabbitsweregivend-camphoratadoseof
0(vehiclecontrol),147,316or681mg/kgbwperdayinpropyleneglycolbygavage
duringdays618ofgestation.Doeswereobservedfortoxicity,andbodyweight
andfeedintakewererecordeddaily.Onday29ofgestation,doeswerekilledand
examined macroscopically. Fetuses were examined for external and skeletal
anomalies.Noeffectswerereportedindoesatthelowest(147mg/kgbwperday)
and intermediate (316mg/kgbw per day) doses; while reduced body-weight gain
andfoodintakewerereportedindoesatthehighestdose(681mg/kgbwperday).
At necropsy, no pathological fndings were reported at any dose. In the fetuses,
no effect on prenatal development was reported and no variations, retardations,
ormalformationswerereportedatanydose,eventhosecausingmaternaltoxicity
(Leuschner,1997).
Groupsof26pregnantNewZealandwhiterabbitsweregivend-camphorata
doseof0(vehiclecontrol),50,200or400mg/kgbwperdayincornoilbygavage
duringdays619ofgestation.Twodoeswereremovedfromthegroupreceiving
thehighestdoseowingtogavageerror.Doeswereobservedforclinicalsignsof
toxicity and fetuses were examined for possible effects on growth, viability, and
morphologicaldevelopment.Nocompound-relatedmaternaldeathswerereported.
Maternal body weights and feed intake were comparable to those of controls;
however,maternalbody-weightgaintendedtodecrease(signifcanttrendtest)in
adose-relatedmanner:13,5and59%reductioninthe50,200and400mg/kgbw
per day groups, respectively, compared with controls. Gravid uterine and liver
weights (absolute and relative) for does treated with d-camphor were not signif-
cantlydifferentfromthoseofcontrols.Noeffectsonresorptionsperlitter(%),late
fetal deaths per litter (%), non-live implants per litter (%), adversely affected
implants per litter (%), the proportion of litters with one or more resorptions, late
fetal deaths, non-live implants or adversely affected implants, live litter size,
average fetal body weight, the overall incidence of malformations, the incidence
ofexternal,skeletalandvisceralmalformations,andtheincidenceofanatomical
variations or defects were reported.The NOEL ford-camphor for developmental
andmaternaltoxicitywas400mg/kgbwperday,thehighestdosetested.
(ii) 3-l-Menthoxypropane-1,2-diol(No.1408)
Rats
Groupsof30pregnantCharlesRiverCDratsweregiven3-l-menthoxypropane-
1,2-diol(No.1408)asasingledailyoraldosesat0,100,500or1500mg/kgbwin
MONOCYCLIC AND BICYCLIC SECONDARY ALCOHOLS 425
L1
cornoilbygavageondays615ofgestation.Caesareansectionswereperformed
on all females surviving to day 20 of gestation.Two mortalities were reported in
the group receiving the highest dose (1500mg/kgbw per day); one of these rats
wassacrifcedinextremisonday6ofgestation.Nogrosslesionswereobserved
upon necropsy examination of this rat; however, discolouration of the lung was
noted upon necropsy of a second dam at the highest dose that died on day 13
ofgestation.Clinicalobservationsnotedinthesetwoanimalsincludeprostration,
lossofrightingrefex,gasping,increasedsalivation,convulsions,andmoribundity.
Absoluteandrelativeweightsoftheliverweresignifcantlyhigherindamsatthe
highestdosethaninthecontrols;however,nocompound-relatedliverlesionswere
noted at necropsy. Body-weight gain of dams at the highest dose, which was
slightlydecreasedondays69ofgestation,wassignifcantlyhigherondays912
of gestation than in control animals. Relative to controls, food consumption was
signifcantlydecreasedatthehighestfromdays612ofgestation.Notreatment-
related differences in maternal or fetal parameters were observed. No fetal mal-
formations or developmental variations were reported. The NOEL for maternal
toxicity was 500mg/kgbw per day and the NOEL for developmental toxicity was
>1500mg/kgbwperday(York,1993).
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435
AMINO ACIDS AND RELATED SUBSTANCES
First draft prepared by
Dr P.J. Abbott
1
and Mrs E. Vavasour
2
1
Food Standards Australia New Zealand, Canberra, Australian Capital
Territory, Australia; and
2
Food Directorate, Health Canada, Ottawa, Ontario, Canada
Evaluation ............................................................................... 435
Introduction......................................................................... 435
Estimateddailyintake........................................................ 436
Absorption,distribution,metabolismandelimination........ 437
ApplicationoftheProcedurefortheSafety
EvaluationofFlavouringAgents.................................. 437
Considerationofsecondarycomponents.......................... 437
Considerationofcombinedintakesfromuse
asfavouringagents.................................................... 437
Conclusion.......................................................................... 444
Relevantbackgroundinformation............................................. 444
Explanation......................................................................... 444
Additionalconsiderationsonintake................................... 444
Biologicaldata.................................................................... 445
Biochemicaldata......................................................... 445
Absorption,distribution,andexcretion.................. 445
Metabolism............................................................ 452
Toxicologicalstudies.......................................................... 458
Acutetoxicity................................................................ 458
Short-termstudiesoftoxicity................................. 460
Long-termstudiesoftoxicityand
carcinogenicity................................................ 468
Genotoxicity........................................................... 477
Reproductivetoxicity............................................. 480
References............................................................................... 480
1. EVALUATION
1.1 Introduction
The Committee evaluated a group of 20 favouring agents comprising amino
acidsandrelatedsubstances.Thegroupincluded16a-aminoacids(someL-form
and some D,L-form) (Nos 14191424, 1426, 14281432, 1434, 14371439) and
one a-imino acid (No. 1425, L-proline), which are normally found in protein, and
twob-aminoacids(b-alanine,No.1418,andtaurine,No.1435)andtheS-methyl
sulfonium salt of methionine (D,L-(3-amino-3-carboxypropyl)dimethylsulfonium
chloride, No. 1427), which are not normally found in protein (see Table 1). L-
Glutamicacid(No.1420)wasevaluatedbytheCommitteeatitsthirty-frstmeeting
(Annex1,reference77)andanADInotspecifedwasestablishedforL-glutamic
436 AMINO ACIDS AND RELATED SUBSTANCES
acid and its ammonium, calcium, magnesium, monosodium, and potassium
salts.
TheCommitteewasoftheopinionthattheuseoftheProcedurefortheSafety
Evaluation of FlavouringAgents (Annex 1, reference 131) was inappropriate for
12 members of this group, namely, the 11 L-form a-amino acids (L-cysteine,
No. 1419; L-glutamic acid, No. 1420; glycine, No. 1421; L-leucine, No. 1423; L-
phenylalanine, No. 1428; L-aspartic acid, No. 1429; L-glutamine No. 1430; L-
histamine,No.1431;L-tyrosine,No.1434;L-arginine,No.1438;L-lysine,No.1439)
andtheonea-iminoacid(L-proline,No.1425).Thesesubstancesaremacronutri-
ents and normal components of protein and, as such, human exposure through
foodisordersofmagnitudehigherthantheanticipatedlevelofexposurefromuse
asfavouringagents.
TheCommitteealsonotedthataminoacidsmayreactwithotherfoodconstitu-
entsuponheating.Themixturesthusformedarecommonlyreferredtoasprocess
favours.Thesafetyofprocessfavourshasnotbeenreviewedduringthisevalu-
ationandmaybeconsideredatafuturemeeting.Thepresentevaluationisthere-
foreonthebasisthatthesefavouringagentsarepresentinanunchangedform
atthepointofconsumption.
For the remaining eight members of the group, namely, the D,L-amino acids
(D,L-isoleucine, No. 1422; D,L-methionine, No. 1424; D,L-valine, No. 1426; D,L-
phenylalanine,No.1432;D,L-alanine,No.1437),thetwob-aminoacids(b-alanine,
No. 1418, and taurine, No. 1435) and the S-methyl sulfonium salt of methionine
(D,L-(3-amino-3-carboxypropyl)dimethylsulfonium chloride, No. 1427) (see Table
1), the evaluations were conducted according to the Procedure for the Safety
EvaluationofFlavouringAgents(seeFigure1,p192).AlthoughtheD-formofthe
a-aminoacidsandtheotherthreecompoundsarenotfoundinprotein,theyare
naturalcomponentsoffood.Fortheseeightmembersofthegroup,theevaluation
has been conducted only in relation to their use as favouring agents leading to
thecurrentestimatedintakes.
1.2 Estimated daily intake
Thetotalannualvolumeofproductionforuseasfavouringagentsonlyofthe
20 substances in this group is approximately 11200kg in Europe (International
Organization of the Flavor Industry, 1995) and 21100kg in the USA (National
AcademyofSciences,1987;Lucasetal.,1999).Theannualvolumesofproduction
are equivalent to a total daily intake of 1600mg/person in Europe and 2800mg/
personintheUSA.
Approximately 74% of the total annual volume of production in Europe is
accounted for by four favouring agents: L-cysteine (No. 1419), 40%; L-glutamic
acid(No.1420),20%;glycine(No.1421),10%;and D,L-alanine(No.1437),8%.
Approximately83%ofthetotalannualvolumeofproductionintheUSAisaccounted
for by fve favouring agents in the group: L-cysteine (No. 1419) 11%; L-glutamic
acid(No.1420),10%;L-asparticacid(No.1429),45%;L-histidine(No.1431),9%);
andtaurine(No.1435),8%.Theestimateddailypercapitaintakeofeachfavour-
ingagentisreportedinTable1.
AMINO ACIDS AND RELATED SUBSTANCES 437
1.3 Absorption, distribution, metabolism and elimination
Amino acids are absorbed readily through the intestinal mucosa, distributed
throughthebloodstreamandtransportedintocellsbyavarietyofcarriersystems.
TheD-isomersandthoseL-aminoacidsthatarenotneededfornewproteinsyn-
thesisundergocatabolism,primarilyintheliver.Thereisnomechanismforstorage
ofaminoacidsinhumans.Aminoacidsundergooxidativedeamination,inwhich
amino acids are deaminated to yield a-ketoacids that are either completely oxi-
dizedtocarbondioxide(CO
2
)andwater,orprovidethreeorfourcarbonunitsthat
are converted via gluconeogenesis to yield glucose, or undergo ketogenesis to
yieldketonebodies.
The S-methyl sulfonium salt of methionine (No. 1427) is demethylated to
methionineorconvertedtohomoserinebythelossofdimethylsulfde.
1.4 Application of the procedure for the safety evaluation of
favouring agents
Step 1. In applying the Procedure, the Committee assigned seven of the eight
favouring agents (D,L-isoleucine, No. 1422; D,L-methionine, No. 1424;
D,L-valine,No.1426;D,L-phenylalanine,No.1432;D,L-alanine,No.1437)
andthetwob-aminoacids(b-alanine,No.1418,andtaurine,No.1435)
tostructuralclassI.Theremainingfavouringagent(D,L-(3-amino-3-car
boxypropyl)dimethylsulfoniumchloride,No.1427)wasassignedtostruc-
turalclassIII(Crameretal.,1978).
Step 2. TheeightfavouringagentsevaluatedusingtheProcedurewereallpre-
dictedtobemetabolizedtoinnocuousproducts.Theirevaluationthere-
foreproceededviatheA-sideofthedecision-tree.
Step A3. Theestimateddailyintakesofallthefavouringagentsinstructuralclass
IandthatoftheonefavouringagentinstructuralclassIIIarebelowthe
thresholdsfordailyhumanintakefortheirrespectiveclasses(1800mg/
personperdayforclassI,and90mg/personperdayforclassIII).Accord-
ingtotheProcedure,theuseoftheseeightfavouringagentsraisesno
safetyconcernsatestimatedcurrentintakes.
Theintakeconsiderationsandotherinformationusedtoevaluatethe20amino
acidsandrelatedsubstancesaresummarizedinTable1.
1.5 Consideration of secondary components
Nofavouringagentsinthisgrouphaveminimumassayvaluesof<95%.
1.6 Consideration of combined intakes from use as favouring agents
The eight favouring agents evaluated using the Procedure are effciently
metabolized and eliminated, and the overall evaluation of the data indicates that
combined intake would not raise any safety concerns at estimated current
intakes.
438 AMINO ACIDS AND RELATED SUBSTANCES
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s
e
d

s
t
r
u
c
t
u
r
e

D
o
e
s

i
n
t
a
k
e

o
n

c
u
r
r
e
n
t

i
n
t
a
k
e

e
x
c
e
e
d

t
h
e

t
h
r
e
s
h
o
l
d

f
o
r

h
u
m
a
n

i
n
t
a
k
e
?
a
,
b
S
t
r
u
c
t
u
r
a
l

c
l
a
s
s

I
b
-
A
l
a
n
i
n
e

1
4
1
8

1
0
7
-
9
5
-
9

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

H
2
N
O
H
O

E
u
r
o
p
e
:

N
D

U
S
A
:

1
3
d
,
l
-
I
s
o
l
e
u
c
i
n
e

1
4
2
2

4
4
3
-
7
9
-
8

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

O
H
O
N
H
2

E
u
r
o
p
e
:

U
S
A
:

2
2
d
,
l
-
M
e
t
h
i
o
n
i
n
e

1
4
2
4

5
9
-
5
1
-
8

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

S
O
H
N
H
2
O

E
u
r
o
p
e
:

9
7

U
S
A
:

3
5
d
,
l
-
V
a
l
i
n
e

1
4
2
6

5
1
6
-
0
6
-
3

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

H
2
N
O
O
H

E
u
r
o
p
e
:

4
1

U
S
A
:

4
8
AMINO ACIDS AND RELATED SUBSTANCES 439
d
,
l
-
P
h
e
n
y
l
a
l
a
n
i
n
e

1
4
3
2

1
5
0
-
3
0
-
1

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

O O
H
N
H
2

E
u
r
o
p
e
:

U
S
A
:

0
.
7
T
a
u
r
i
n
e

1
4
3
5

1
0
7
-
3
5
-
7

N
o

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

O
H
S
O
O
H
2
N

E
u
r
o
p
e
:

N
D

U
S
A
:

2
1
7
d
,
l
-
A
l
a
n
i
n
e

1
4
3
7

3
0
2
-
7
2
-
7

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

O
O
H
N
H
2

E
u
r
o
p
e
:

1
3
4

U
S
A
:

1
S
t
r
u
c
t
u
r
a
l

c
l
a
s
s

I
I
I
d
,
l
-
(
3
-
A
m
i
n
o
-
3
-

1
4
2
7

1
1
1
5
-
8
4
-
0

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n
c
a
r
b
o
x
y
p
r
o
p
y
l
)
d
i
m
e
t
h
y
l
s
u
l

S
+

O

O
H

N
H
2

C
l

E
u
r
o
p
e
:

N
D
f
o
n
i
u
m

c
h
l
o
r
i
d
e

U
S
A
:

7
5
l
-
C
y
s
t
e
i
n
e

1
4
1
9

5
2
-
9
0
-
4

E
u
r
o
p
e
:

6
4
2

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

H
2
N
S
H
O
O
H

U
S
A
:

2
9
3
440 AMINO ACIDS AND RELATED SUBSTANCES
A
m
i
n
o

a
c
i
d
s

n
o
t

e
v
a
l
u
a
t
e
d

b
y

t
h
e

P
r
o
c
e
d
u
r
e
F
l
a
v
o
u
r
i
n
g

a
g
e
n
t

N
o
.

C
A
S

N
o
.

a
n
d

D
a
i
l
y

p
e
r

c
a
p
i
t
a

C
o
n
c
l
u
s
i
o
n

b
a
s
e
d

s
t
r
u
c
t
u
r
e

i
n
t
a
k
e
b

o
n

c
u
r
r
e
n
t

i
n
t
a
k
e
l
-
G
l
u
t
a
m
i
c

a
c
i
d

1
4
2
0

5
6
-
8
6
-
0

E
u
r
o
p
e
:

3
1
3

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

N
H
2

(
S
)

O

O

H

O

O

U
S
A
:

2
7
3
G
l
y
c
i
n
e

1
4
2
1

5
6
-
4
0
-
6

E
u
r
o
p
e
:

1
5
8

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

H
2
N
O
H
O

U
S
A
:

5
l
-
L
e
u
c
i
n
e

1
4
2
3

6
1
-
9
0
-
5

E
u
r
o
p
e
:

1
4

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

N
H
2
(
S
)
O
O
H

U
S
A
:

2
4
l
-
P
r
o
l
i
n
e

1
4
2
5

1
4
7
-
8
5
-
3

E
u
r
o
p
e
:

4
9

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

H N
(
S
)
O O
H

U
S
A
:

1
1
5
AMINO ACIDS AND RELATED SUBSTANCES 441
l
-
P
h
e
n
y
l
a
l
a
n
i
n
e

1
4
2
8

6
3
-
9
1
-
2

E
u
r
o
p
e
:

2
0

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

H
2
N
(
S
)
O O
H

U
S
A
:

2
8
l
-
A
s
p
a
r
t
i
c

a
c
i
d

1
4
2
9

5
6
-
8
4
-
8

E
u
r
o
p
e
:

7
9

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

N
H
2
O
O
H
O
O
H

U
S
A
:

2
4
0
l
-
G
l
u
t
a
m
i
n
e

1
4
3
0

5
6
-
8
5
-
9

E
u
r
o
p
e
:

1
6

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

N
H
2
O
H
2
N
O
O
H

U
S
A
:

1
0
l
-
H
i
s
t
i
d
i
n
e

1
4
3
1

7
1
-
0
0
-
1

E
u
r
o
p
e
:

1
1

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

N
H
2
(
S
)
N
H
N
O
O
H

U
S
A
:

2
5
9
l
-
T
y
r
o
s
i
n
e

1
4
3
4

6
0
-
1
8
-
4

E
u
r
o
p
e
:

1
2

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

N
H
2
O
H
O O
H

U
S
A
:

4
442 AMINO ACIDS AND RELATED SUBSTANCES
A
m
i
n
o

a
c
i
d
s

n
o
t

e
v
a
l
u
a
t
e
d

b
y

t
h
e

P
r
o
c
e
d
u
r
e

(
C
o
n
t
d
)
F
l
a
v
o
u
r
i
n
g

a
g
e
n
t

N
o
.

C
A
S

N
o
.

a
n
d

D
a
i
l
y

p
e
r

c
a
p
i
t
a

C
o
n
c
l
u
s
i
o
n

b
a
s
e
d

s
t
r
u
c
t
u
r
e

i
n
t
a
k
e
b

o
n

c
u
r
r
e
n
t

i
n
t
a
k
e
l
-
A
r
g
i
n
i
n
e

1
4
3
8

7
4
-
7
9
-
3

E
u
r
o
p
e
:

N
D

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

N
H
2
H N
N
H
H
2
N
O
O
H

U
S
A
:

5
7
l
-
L
y
s
i
n
e

1
4
3
9

5
6
-
8
7
-
1

E
u
r
o
p
e
:

N
D

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

H
2
N
(
S
)
H
2
N
O O
H

U
S
A
:

5
7
C
A
S
:

C
h
e
m
i
c
a
l

A
b
s
t
r
a
c
t
s

S
e
r
v
i
c
e
;

N
D
:

N
o

i
n
t
a
k
e

d
a
t
a

r
e
p
o
r
t
e
d
.
a

S
t
e
p

2
:

A
l
l

e
i
g
h
t

f
a
v
o
u
r
i
n
g

a
g
e
n
t
s

i
n

t
h
i
s

g
r
o
u
p

e
v
a
l
u
a
t
e
d

u
s
i
n
g

t
h
e

P
r
o
c
e
d
u
r
e

a
r
e

e
x
p
e
c
t
e
d

t
o

b
e

m
e
t
a
b
o
l
i
z
e
d

t
o

i
n
n
o
c
u
o
u
s

p
r
o
d
u
c
t
s
.

T
h
e

e
v
a
l
u
a
t
i
o
n

o
f

t
h
e
s
e

f
a
v
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u
r
i
n
g

a
g
e
n
t
s

t
h
e
r
e
f
o
r
e

p
r
o
c
e
e
d
e
d

v
i
a

t
h
e

A
-
s
i
d
e

o
f

t
h
e

d
e
c
i
s
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o
n
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r
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e
.
b

T
h
e

t
h
r
e
s
h
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l
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f
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r

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u
m
a
n

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n
t
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k
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s

1
8
0
0

m
g

p
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r

d
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y

f
o
r

s
t
r
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c
t
u
r
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l

c
l
a
s
s

a
n
d

9
0

m
g
/
p
e
s
o
n

p
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r

d
a
y

f
o
r

c
l
a
s
s

I
I
I
.

A
l
l

i
n
t
a
k
e

v
a
l
u
e
s

a
r
e

e
x
p
r
e
s
s
e
d

i
n

m
g
/
p
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r
s
o
n

p
e
r

d
a
y
.

T
h
e

c
o
m
b
i
n
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d

i
n
t
a
k
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o
f

t
h
e

f
a
v
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r
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g

a
g
e
n
t
s

i
n

s
t
r
u
c
t
u
r
a
l

c
l
a
s
s

i
s

1
5
9
4

m
g
/
p
e
r
s
o
n

p
e
r

d
a
y

i
n

E
u
r
o
p
e

a
n
d

2
7
0
1

m
g
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p
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r
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n

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d
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y

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n

t
h
e

U
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A
.

T
h
e

i
n
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f

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e

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c
l
a
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s

7
5

m
g
/
p
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r
s
o
n

p
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r

d
a
y

i
n

t
h
e

U
S
A
.
N
o
t
e
s
:
1

D
e
a
m
i
n
a
t
e
d

t
o

f
o
r
m
y
l
a
c
e
t
i
c

a
c
i
d

a
n
d

m
e
t
a
b
o
l
i
z
e
d

i
n

t
h
e

c
i
t
r
i
c

a
c
i
d

c
y
c
l
e
.
2

D
e
a
m
i
n
a
t
e
d

t
o

f
o
r
m

a
c
e
t
y
l

c
o
e
n
z
y
m
e

(
C
o
A
)
,

p
r
o
p
i
o
n
y
l

C
o
A
,

s
u
c
c
i
n
y
l

C
o
A
,

a
n
d

m
e
t
a
b
o
l
i
z
e
d

i
n

c
i
t
r
i
c

a
c
i
d

c
y
c
l
e
.
3

D
e
a
m
i
n
a
t
e
d

t
o

f
o
r
m

h
o
m
o
c
y
s
t
e
i
n
e
,

p
r
o
p
i
o
n
y
l

C
o
A
,

s
u
c
c
i
n
y
l

C
o
A
,

a
n
d

m
e
t
a
b
o
l
i
z
e
d

i
n

c
i
t
r
i
c

a
c
i
d

c
y
c
l
e
.
4

D
e
a
m
i
n
a
t
e
d

t
o

f
o
r
m

p
r
o
p
i
o
n
y
l

C
o
A
,

s
u
c
c
i
n
y
l

C
o
A
,

a
n
d

m
e
t
a
b
o
l
i
z
e
d

i
n

c
i
t
r
i
c

a
c
i
d

c
y
c
l
e
.
5

C
o
n
v
e
r
t
e
d

t
o

t
y
r
o
s
i
n
e
,

t
h
e
n

d
e
a
m
i
n
a
t
e
d

t
o

f
o
r
m

a
c
e
t
o
a
c
e
t
y
l

C
o
A

a
n
d

m
e
t
a
b
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l
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z
e
d

i
n

t
h
e

c
i
t
r
i
c

a
c
i
d

c
y
c
l
e
.
6

D
e
a
m
i
n
a
t
e
d

t
o

p
y
r
u
v
a
t
e

a
n
d

a
c
e
t
y
l

C
o
A

a
n
d

m
e
t
a
b
o
l
i
z
e
d

i
n

t
h
e

c
i
t
r
i
c

a
c
i
d

c
y
c
l
e
.
7

D
e
m
e
t
h
y
l
a
t
e
d

t
o

m
e
t
h
i
o
n
i
n
e

o
r

d
e
a
m
i
n
a
t
e
d

t
o

h
o
m
o
s
e
r
i
n
e

b
y

l
o
s
s

o
f

d
i
e
m
e
t
h
y
l
s
u
l
f
d
e
.
AMINO ACIDS AND RELATED SUBSTANCES 443
1.7 Conclusion
InviewofthefactthattheL-formofthe11a-aminoacidsandtheonea-imino
acidinthisgrouparemacronutrientsandnormalcomponentsofprotein,theuse
ofthesesubstancesasfavouringagentswouldnotraiseanysafetyconcernsat
estimatedcurrentintakes.TheCommitteealsoconcludedthattheuseoftheother
eightsubstancesinthegroupleadingtotheestimatedcurrentintakeswouldnot
raiseanysafetyconcerns.
TheADInotspecifedforL-glutamicacidanditsammonium,calcium,magne-
sium,monosodiumandpotassiumsaltswasmaintained.
2. RELEVANT BACKGROUND INFORMATION
2.1 Explanation
Thebackgroundinformationsummarizeskeydatarelevanttothesafetyevalu-
ationofthe20aminoacidsandrelatedsubstancesusedasfavouringagents.
2.2 Additional considerations on intake
Thetotalannualvolumeofproductionofthe19aminoacids(Nos14181426,
14281432, 1434, 1435, 14371439) and S-methyl sulfonium salt of methionine
(No. 1427) obtained from industry-wide surveys is approximately 180600kg in
Europe(InternationalOrganizationoftheFlavorIndustry,1995),and316100kgin
theUSA(NationalAcademyofSciences,1989;Lucasetal.,1999)(seeTable2).
However,mostofthetotalannualvolumeofproductionofaminoacidsisusedas
starting material in the production of process favourings. Process favours are
polyheteroaromatic substances produced by the heated reaction of amino acids
and simple sugars and these favours do not contain signifcant amounts of free
amino acids. The percentage of use of an individual amino acid as favouring
agentswillvary,butoverallitisestimatedthat<10%ofthetotalreportedannual
volumeofproductionofaminoacidsisintendedforuseasfavouringagentsper
se.Basedonestimatesoftherelativeamountofeachaminoacidusedexclusively
asafavouringagent,thetotalannualvolumeofproductionofaminoacidsforuse
as favouring agents is 11200kg in Europe and 21000kg in the USA (seeTable
2). The estimated total daily intake of amino acids added to food as favouring
agents is approximately 1.5 and 3mg/person per day in Europe and the USA,
respectively.
a-L-Aminoacidsarenormalconstituentsofproteinandthereforedietaryintake
canbeestimatedfromanalysisoftheproteincontentofthenormaldiet.Dietary
referenceintakesforaminoacidshavebeendeterminedbytheInstituteofMedi-
cine(2002).Thedietaryintakefromfoodvariedfrom1010mg/dayforcysteineto
15270mg/day for glutamic acid. Dietary intakes for the other amino acids were
between2000and6000mg/day.Dietaryexposuretothe15aminoacidsanalysed
wassignifcantlygreater(bythreetofourordersofmagnitude)inthenormaldiet
thanfromtheiruseasfavouringagents.
444 AMINO ACIDS AND RELATED SUBSTANCES
Eightaminoacidscannotbesynthesizedbytheadultbodyandareconsidered
to be essential in the human diet those used as favouring agents are: L-
phenylalanine, L-methionine, L-valine, L-leucine, L-isoleucine, L-histidine and L-
lysine. The recommended daily requirements for these amino acids are in the
rangeof1330to2940mg/day(InstituteofMedicine,2002).Theselevelsofintake
aresignifcantlygreaterthanthedailypercapitaintakefromtheiruseasfavouring
agents.
Aminoacidsareusedasfavouringagentsinavarietyoffoodcategories(Hall
& Oser, 1965; Oser & Hall, 1972; Oser & Ford, 1975; 1978; Oser et al., 1984;
Smithetal.,1996;Newberneetal.,1998).Typicaluselevelsrangefrom5mg/kg
forL-arginineinprocessedvegetablesto4000mg/kgforL-alanineinseasonings
andfavours(Newberneetal.,1998).Itisclaimedthataminoacidssuchasglycine,
alanine, valine, leucine, isoleucine, and lysine reduce the unpalatable astringent
favourofzincandaluminiumsaltsinfood(Godfrey,1987,1993).
Amino acids are also used as dietary supplements. In the USA, amino acids
canbeusedtoamaximumpercentageofthedietthisrangesfrom2.3%forL-
cysteine/L-cystineto8.8%forL-leucine,asspecifedintheCodeofFederalRegu-
lationsoftheUSA(Title21,21CFR172.320,2003).Theselevelsareconsiderably
higherthanthecomparablerecommendedlevelsofuseofthesameaminoacids
as favouring agents, namely, 0.01% for L-cysteine/L-cystine and 0.005% for L-
leucine(Oser&Hall,1972).
2.3 Biological data
2.3.1 Biochemical data
a-Amino acids are present in animal- and plant-based foods and are normal
constituentsofahealthydiet.a-Aminoacidsarereferredtoaseitheressentialor
non-essential. Animals do not synthesize the essential amino acids and must
ingestthemaspartofanormaldiet.Essentialaminoacidsaremetabolicprecur-
sorstothesynthesisofotheraminoacids.Theessentialaminoacidsareisoleucine
(No.1422),leucine(No.1423),methionine(No.1424),valine(No.1426),phenyl-
alanine (Nos 1428 and 1432), histidine (No. 1431), arginine (No. 1438)
1
, lysine
(No.1439),threonine,andtryptophan.
Excess dietary amino acids are neither stored nor excreted; rather, they
undergoconversiontocommonmetabolicintermediates(e.g.pyruvate,oxaloac-
etate,a-ketoglutarate).Therearenumerousstudiesontheabsorption,distribution,
metabolism,andexcretionofaminoacidsintheliterature,onlysomeofwhichare
summarizedbelow.
(a) Absorption, distribution, and excretion
Freea-aminoacids,whetheringestedassuchintheformoffavouringagents
or released after the digestion of proteins by proteolytic enzymes, are absorbed
1
Arginineisonlyanessentialaminoacidforyoung,growinganimals,notforadults.
AMINO ACIDS AND RELATED SUBSTANCES 445
T
a
b
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e

2
.

A
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(
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8
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A

1
0
0

1
0
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1
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1
3

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A
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(
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446 AMINO ACIDS AND RELATED SUBSTANCES
T
a
b
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e

2
.

(
C
o
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d
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8
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(
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(
1
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AMINO ACIDS AND RELATED SUBSTANCES 447
T
a
u
r
i
n
e

(
1
4
3
5
)

E
u
r
o
p
e

N
/
D

N
/
D

N
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D

N
/
D

N
/
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A
h

2
5
0

2
0

6
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2
1
7

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A

N
A
d
,
l
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A
l
a
n
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e

(
1
4
3
7
)

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p
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1
8

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4
5

9
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3
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2
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2

6
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(
1
4
3
8
)

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N
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N
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A
h

6
0
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4
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5
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9

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(
1
4
3
9
)

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h

6
0
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T
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3
1
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7
1

7
7
5
N
A
,

n
o
t

a
v
a
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l
a
b
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;

N
/
D
,

n
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O
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F
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(
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9
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a
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.

(
1
9
9
9
)

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S
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n
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s

(
1
9
7
0
,

1
9
8
2
,

1
9
8
7
)
.

T
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e

t
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a
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a
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v
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448 AMINO ACIDS AND RELATED SUBSTANCES
primarilythroughtheintestinalmucosaandentertheportalblood.Onceabsorbed,
a variety of carrier systems transport a-amino acids into cells (Kilberg, 1982).
These amino-acid carriers are mostly sodium ion-dependent systems that are
specifctoaparticularclassofa-aminoacids(e.g.neutralaminoacidswithshort
side-chains, neutral amino acids with branched or aromatic side-chains, basic
aminoacids,anddicarboxylicaminoacids).Thecarriersystemsareadaptiveand
under hormonal regulatory control.Although small amounts of protein and poly-
peptides may be absorbed by a transport system involving membrane-bound g-
glutamyltransferase,mostaminoacidsenterthecellsunchanged(Nelson&Cox,
2000).
Afterabsorption,a-aminoacidsareusedinproteinsynthesisorrapidlymetabo-
lizedtointermediatesinthecitricacidcycle,asevidencedbythepresenceofonly
traceamountsofa-aminoacidsintheplasma.Theexcretionofa-aminoacidsis
regulated by renal tubular reabsorption, in which the proximal tubules conserve
a-aminoacids.Thedailyexcretionofa-aminoacidsintheurineamountstoonly
20150mg/dayinhumans
2
(Tietz,1986).Minimallossofa-aminoacidsoccursin
theurineandfaeces.
b-Amino acids are also rapidly absorbed but they are not incorporated into
proteinsrathertheyaremetabolisedviaoxidativedeaminationtoyieldshorter
chain acids that are either completely oxidized in the fatty acid pathway and
tricarboxylicacidcycle,orexcretedprimarilyintheurine.
(i) a-Amino acids (Nos 14191426, 14281432, 1434,
14371439)
Thesimplesta-aminoacid,glycine,israpidlyabsorbedanddistributedtocells
whereitiseithermetabolizedorenterstheaminoacidpool.Inametabolicstudy,
20healthyvolunteersconsumedafruitbeveragecontainingglycineatadoseof
0.14mg/kgbw.A maximum plasma concentration of glycine of 4.18mg/100ml of
plasma was attained in 45min. Subsequently, glycine was rapidly removed from
theplasmaandat180minwasdetectedatconcentrationsofonly0.06mg/100ml
of plasma (Craft et al., 1968). In another study with glycine, patients were given
8mg(100mC
i
)of[
14
C-carboxy]glycineviaintravenousinjection;after3h,anaverage
of 25% (1336%) of the radiolabel was eliminated in expired CO
2
. Over the fol-
lowing55days,8392%oftheremainingradiolabelledglycinewasremoved.Only
5%wasexcretedintheurineduringthefrst14daysofthestudy,while2%was
retainedbyerythrocytesforthedurationoftheirlifespan(approximately120days)
(Berlinetal.,1951).
In a study comparing liver function in healthy volunteers and in patients with
hepaticcirrhosis,controlvolunteersandcirrhoticpatientsreceivedL-alanine(No.
1437) intravenously at an infusion rate of 0.007mg/kgbw per min for 2h after a
primingdoseof0.623mg/kgbwof[
15
N]L-alanine.Tenminafterthe2-hperfusion,
2
Duringpregnancyinhumans,excretionofproteinmayincreaseharmlesslyto200300mg/
day(Tietz,1986).
AMINO ACIDS AND RELATED SUBSTANCES 449
the rate at which radiolabelled alanine appeared in the control volunteers was
slightly greater than 50% of the rate of infusion (3.8 0.2mmol/kg per min or
0.004mg/kg per min) and the rate of clearance was approximately four times
(16 1.0mmol/kg per min) the rate of appearance, indicating rapid clearance of
L-alaninefromtheblood(Schrickeretal.,1995).
Ratesofintestinalabsorptionofleucine(No.1423)andvaline(No.1426)were
measuredin11normalhumanvolunteers(Adibi,1969).Theintestinalabsorption
of leucine (10100mmol/l) and valine (5100mmol/l) was reported to be directly
proportionaltotheconcentrationofaminoacid,withsaturationofintestinalabsorp-
tionoccurringwithleucineat>50mmol/l(Adibi,1969).
Inanothermetabolicstudy,miceweregiven2.0mgof
14
C-labelledleucine(No.
1423)viaintravenousinjection.Almostnoleucinecouldbedetectedintheblood
10min after injection, while 47% was accounted for in the viscera after 30min,
100%wasdistributedthroughoutthecarcasswithin4h,and57%wasexhaledas
14
CO
2
withinthesameperiodof4h(Borsooketal.,1950).
Inastudyin12healthy,fastedvolunteers,peakserumconcentrationsofglu-
tamatewerereported3045minaftertheyweregivenabouillondrinkcontaining
glutamicacidsodiumsaltatadoseof43mg/kgbw.Whenthedosewasincreased
to64mg/kgbw,thepeakserumconcentrationofglutamicacidalsoincreasedand
theareaunderthecurve(AUC)morethandoubled,suggestingnearsaturationof
distributionandmetabolicprocesses(Ghezzietal.,1985).
Inastudyofthreecystinuriaphenotypesdifferentiatedbyurinaryexcretionof
aminoacids,threehumanvolunteersweregivenlysine(No.1439)orarginine(No.
1438) as a single oral dose at 0.5mmol/kgbw. Plasma concentrations of lysine,
arginine and ornithine, an arginine metabolite, increased by 110, 75, and 100%,
respectively,overbaselinelevels.Concentrationsoflysineandarginineintheurine
remainedconstantoverthe6hafteradministration.However,urinaryexcretionof
ornithine, an amino acid intermediate in urea synthesis, increased by 175% (de
Sanctus,2001).
Inastudyinmice,[
14
C]lysine(No.1439)or[
14
C]histidine(No.1431)atadose
of1.46mgor1.47mg,respectively,weregiventomiceviaintravenousinjection.
Mostoftheradioactivitywasremovedfromthebloodwithinthefrst10min.Histi-
dine and lysine were detected at 30 and 45%, respectively, in the non-protein
fractionofthevisceraafter10min,while58and92%weredistributedthroughout
thecarcasswithin4h,respectively.Inthe4hafteradministration,28%ofhistidine
and43%oflysinewereexhaledas
14
CO
2
(Borsooketal.,1950).
In a multi-species study, a single dose of [2-
14
C]L-histidine was administered
intravenouslytoMacaquemonkeys(2.5mg),orallyto23normalandschizophrenic
human volunteers (14mg), or intraperitoneally to Sprague-Dawley rats (7.4mg).
Lessthan10%oftheradiolabelwasexcretedintheurinewithin5hinbothmonkey
and humans, and within 24h in rats. Measurement of tissue radioactivity in the
monkey indicated that L-histidine concentrated in the liver after 2h, the liver and
muscle after 7 days, and was then widely distributed to all tissues after 11 days
(Brownetal.,1960).
450 AMINO ACIDS AND RELATED SUBSTANCES
Twohealthyvolunteers,onemaleandonefemale,weregivenL-[3,3-
2
H
2
,1,3-
15
N
2
]histidineasasingleoraldoseat100mg(approximately1.5mg/kgbw).Radio-
abelled L-histidine was rapidly absorbed and maximum plasma concentrations
(C
max
)(1.06and1.64mg/ml)werereachedat30and60mininthemaleandfemale,
respectively.Theplasmahalf-life(t
1/2
)forlabelledL-histidinewas1.0and1.9hfor
themaleandfemale,respectivelyandtheAUCwas2.4timesgreaterinthefemale
than in the male. The total amount of radiolabelled histidine recovered from the
24-hurinesamplewasminor(Furutaetal.,1996).
GroupsoffvemaleSprague-DawleyorHolzman-Rolfsmeyeralbinoratsgiven
dietscontainingD,L-methionine(No.1424)at0,0.6,or2.5%for4weeksexcreted
onlyminoramountsintheirdailyurine(64,667,or1833mgofD,L-methionineper
day,respectively)(DeBeyetal.,1952).
In another metabolic study, Wistar rats (two of each sex) were given a diet
supplementedwith4.7%L-methionineandeither6%or12%caseinfor18days.
Urineanalysisshowedthatonly2.1and3.4%ofthecalculatedintakeofmethio-
nine remained unchanged for males and females, respectively. Both groups
excreted minor amounts of L-methionine in the faeces (Hoshino & Miyazaki,
1964).
(ii) D,L-(3-amino-3-carboxypropyl)dimethylsulfonium
chloride (No. 1427)
Inametabolicstudyinhumans,fourmalevolunteersweregivenD,L-(3-amino-
3-carboxypropyl)dimethylsulfoniumchloride(MMSC),theS-methylsulfoniumsalt
ofmethionine(No.1427),asasingleoraldoseat750mg/kgbw.Plasmaconcen-
trations of radiolabelled MMSC were 7.1mg/ml at 30min, 9.2mg/ml after 2h, and
then decreased by more than 50% to 3.93mg/ml over the next 4h. At 2h, the
maximum concentration of labelled MMSC in the blood was reached and repre-
sentedapproximately12%oftheadministereddose(Suzueetal.,1975).
(iii) b-Amino acids (Nos 1418 and 1435)
Inastudytoexaminetheabsorptionandexcretionofb-alanine,anunspecifed
numberofalbinoratsweregivenb-alaninelabelledwith
14
Catposition1,2,or3
atadoseof6.257.78mg/kgbwbyintraperitonealinjection.Exhaledairrevealed
that the carboxyl carbon atom (C
1
) was excreted rapidly, with maximal rate at
30minafterinjection.Within2h,89%ofthe1-
14
CwaseliminatedasexhaledCO
2
.
This is consistent with metabolic oxidative decarboxylation of b-alanine. The a-
carbon (C2), had the slowest rate of excretion, with maximum rate reported at
60min after injection. The b-carbon (C3), was reported to display intermediate
kineticsofelimination.Thecumulativeexcretionof
14
Cat5hwas93,60,and77%
of the total dose of [1-, 2-, or 3-
14
C]b-alanine, respectively (Pihl & Fritzson,
1955).
As part of a study of the metabolism of malondialdehyde, an intermediary
metaboliteofb-alanine,anunspecifednumberofmaleandfemaleSwissalbino
micewereinjectedwith[1-
14
C]-or[3-
14
C]-labelledb-alanineat200or600mmol.It
AMINO ACIDS AND RELATED SUBSTANCES 451
was reported that male and female mice expired the carboxy-labelled carbon as
CO
2
within the frst 1.5h.After 4h, male and female mice injected with [1-
14
C]b-
alanineat200mmoleliminated89and90%,respectively,ofthetotalradiolabelas
expiredCO
2
.Inaddition,maleandfemalemiceeliminated66and74%ofthe[3-
14
C]b-alanine, respectively, as expired CO
2
within a period of 3h after injection.
The rapid oxidation of b-alanine was accompanied by limited excretion of
unchangedb-alanineintheurine(Marnettetal.,1985).Onthebasisofthesedata,
it was concluded that b-alanine is rapidly absorbed and completely oxidized to
CO
2,
water,andammonia.
(b) Metabolism
Undernormalconditions,a-aminoacidsthatarenotrequiredfornewprotein
synthesis undergo catabolism primarily in the liver, with the exception of the
branchedaminoacids,whichundergodegradationinmuscle,adipose,kidneyand
brain tissues. There is no storage of amino acids in humans. The amino acids
undergo a process called oxidative deamination in which most amino acids are
transformed to a-ketoacids that are completely oxidized to CO
2
and water or
providethreeorfourcarbonunitsthatareconvertedviagluconeogenisistoyield
glucose,orviaketogenesistoyieldketonebodies(Nelson&Cox,2000).
The amino groups resulting from transamination of most of the amino acids
are transferred by transaminases to a-ketoglutarate to form L-glutamate in the
cytosolofhepatocytes.L-Glutamatethenundergoesdeaminationinthemitochon-
driayieldingNH
4
+
viaL-glutamatedehydrogenase(seeFigure1).Theammonium
ion is either used in other metabolic pathways or converted to urea for
excretion.
The carbon unit skeletons of the amino acids are all broken down to one of
fve products, each of which enters the citric acid cycle.These fve products are
acetyl-coenzyme A (acetyl-CoA), a-ketoglutarate, succinyl-CoA, fumarate, and
oxaloacetate. These pathways are illustrated in Figures 25 and discussed
below.
(i) D,L-Alanine (No. 1437), L-cysteine (No. 1419) and
glycine (No. 1421)
Theseaminoacidsaredegradedtoacetyl-CoAviapyruvate(seeFigure2).D-
Alanine is converted to pyruvate upon transamination. Cysteine is converted to
pyruvateintwosteps,thefrstbeingremovalofsulfurandthesecondtransamina-
tiontoremovetheaminogroup.Glycineisconvertedfrsttotheaminoacidserine
via serine hydroxymethyltransferase, or undergoes oxidative cleavage yielding
CO
2
,NH
4
+
,andamethylenegroup(Nelson&Cox,2000).
452 AMINO ACIDS AND RELATED SUBSTANCES
(ii) D,L-Isoleucine (No. 1422), L-leucine (No. 1423),
L-phenylalanine (No. 1428), L-tyrosine (No. 1434),
L-lysine (No. 1739)
These fve amino acids enter the citric acid cycle via acetyl-CoA (see Figure
3) following deamination. Unlike phenylalanine, tyrosine and lysine, leucine and
isoleucinearedegradedinextrahepatictissuesthatcontainaspecifcaminotrans-
ferase that removes the amino group of branched chain amino acids to produce
the corresponding a-ketoacid. In addition to contributing carbon atoms to acetyl-
CoA,phenylalanine,tyrosine,andisoleucinealsodonatecarbonatomstopyruvate
or to other intermediates in the citric acid cycle. For example, phenylalanine is
hydroxylated to form tyrosine that is subsequently broken down into two carbon
units,thefour-carbonunityieldingfreeacetoacetateandanotherfour-carbonunit
yielding fumarate, with one carbon being converted to CO
2
(Nelson & Cox,
2000).
O

O
O

O
O
a -Ket og lu ta ra te
H
3
N
O
HO
O

O
Gl ut am at e
NH
3
O
O
Alani ne
O
O
O

Py ru vate
H
3
N
O
H
2
N
O
O
Gl ut am in e
NH
4
O
O
R
H
H
3
N
O
O
R
O
Am ino ac id s
a -K et o acid s
NH 4
+
, ur ea, or ur ic ac id
Cellula r pr ot ei n
Li ver
Am ino acid s
fr om i ngest ed
prot ei n
Al an in e
fr om
mu scl e
Glut am in e
fr om muscl e a nd
ot he r tissu e
Tr an sa mi na tion
De am in at io n
Deam in atio n
De am in at io n
Figure 1. Catabolism of amino groups
FromNelson&Cox(2000)
AMINO ACIDS AND RELATED SUBSTANCES 453
Figure 2. Catabolic pathways for alanine, cysteine and glycine
H
3
N
O
O
Glycine
H
3
N
OH
O
O
Serine
H
3
N
SH
O
O
Cysteine
NH
3
O
O
Alanine
O
O
O
Pyruvate
S-CoA
O
N
5
,N
10
-MethyleneH
4
folate
H
4
folate
serine
hydroxymethyl-
transferase
Pyridoxal phosphate (PLP)
H
2
O
serine
dehydratase
H
2
O
CO
2
CoA-SH
pyruvate
dehydrogenase
Acetyl-CoA
-Ketogluta a rate
Glutamate
a
la
n
in
e a
m
in
o
tra
n
s
fe
r
a
s
e
2
s
te
p
s
NH
4
PLP
FromNelson&Cox(2000)
CoA,coenzymeA;PLP,pyridoxalphosphate
454 AMINO ACIDS AND RELATED SUBSTANCES
Figure 3. Catabolic pathways for lysine, phenylalanine, tyrosine, leucine and
isoleucine
a
C O
2
C o A - S H
O

O
O
O

O
H
3
N H
3
N O
O
N H
3
O
O
O O
S - C o A H O
N A D
N A D H
C O
2
S - C o A
O
S - C o A
O
S - C o A
O
S - C o A
O O
C O
2
N H
3
O
O
O O
O
C O
2
C o A - S H
H O
O
S - C o A
O
O
O
O
O
N H
3
O H O
O
H
3
N
O
O
L y s i n e
- K e t o a d i p a t e
L e u c i n e
P h e n y l a l a n i n e
T y r o s i n e
F u m a r a t e
A c e t o a c e t a t e
G l u t a r y l - C o A
A c e t o a c e t y l - C o A
A c e t y l - C o A
I s o l e u c i n e
P r o p i o n y l - C o A
S u c c i n y l - C o A
4 s t e p s
4 s t e p s
6 s t e p s
A c e t y l - C o A
5 s t e p s
6 s t e p s
3 s t e p s
FromNelson&Cox(2000)
AMINO ACIDS AND RELATED SUBSTANCES 455
(iii) L-Glutamic acid (No. 1420), L-proline (No. 1425), L-
glutamine (1430), L-histidine (No. 1431), and L-arginine
(No. 1438)
Thesefveaminoacidsenterthecitricacidcycleasa-ketoglutarate(seeFigure
4).Proline,whichhasafve-memberedring,isoxidizedtotheSchiffbase,which
is then hydrolysed to yield glutamate g-semialdehyde. This aldehyde undergoes
further oxidation to glutamate, which is subsequently deaminated to yield a-
ketoglutarate.Glutamineisconvertedtoglutamateviathelossofitsamidenitro-
gen(Nelson&Cox,2000).
Although arginine and histidine are C6 amino acids, they are also converted
to C5 a-ketoglutarate via the transamination of glutamate, but the removal of
theadditionalcarbonattachedrequiresadditionalsteps.Arginineisconvertedto
ornithine in the urea cycle, which is subsequently deaminated to glutamate g-
semialdehyde. Histidine is degraded by non-oxidative deamination to urocanate
followed by hydration of both double bonds and hydrolytic ring opening resulting
informiminoglutamate.Theformiminomoietyistransferredtotetrahydrofolateand
theglutamateisdeaminatedtoa-ketoglutarate(Nelson&Cox,2000).
Arecentstudyillustratesthepathwayforcatabolismofhistidine.Twohealthy
volunteers, one male and one female received L-[3,3-
2
H
2
,1,3-
15
N
2
]histidine as a
single oral dose at 1.5mg/kgbw after a 12-h overnight fast. Urocanic acid was
detected in the plasma within 5min for the male volunteer and within 15min for
thefemalevolunteer.Traceamountsofurocanicacidwereexcretedintheurine
(Furutaetal.,1996).
(iv) D,L-Isoleucine (No. 1422), D,L-methionine (No. 1424),
and D,L-valine (No. 1426)
The degradation products of these amino acids yields succinyl-CoA, which
is an intermediate in the citric acid cycle (see Figure 5). After deamination, the
branchedaminoacidisoleucineundergoesoxidativedecarboxylationyieldingCO
2
,
acetyl-CoAandpropionyl-CoA,whichisaprecursorofsuccinyl-CoA.Methionine
donates a methyl group through S-adenosylmethionine, while the remaining
carbonsareconvertedtoa-ketobutyratewhichissubsequentlydecarboxylatedto
formpropionyl-CoA.Valine,anotherbranchedchainaminoacid,undergoesdeami-
nationinextrahepatictissuesfollowedbydecarboxylationandoxidationeventually
yieldingpropionyl-CoA.
(v) D,L-(3-amino-3-carboxypropyl)dimethylsulfonium chloride
(No. 1427)
InastudyofthemetabolismoftheS-methylsulfoniumsaltofmethionine,male
volunteersweregiventheracemicformof(3-amino-3-carboxypropyl)dimethylsulf
oniumchloride(No.1427)atadoseof750mg/kgbworallyandsamplesofblood
weretakenat0.5,1,2,3,and6h.Onlysmallamounts(0.52%)ofthedemethyla-
tion metabolite, methionine, and the metabolite, homoserine, formed by loss
of dimethylsulfde were measured in the blood at 2h, this was expected since
456 AMINO ACIDS AND RELATED SUBSTANCES
Figure 4. Catabolic pathways for proline, glutamic acid, glutamine, arginine
and histidine
a
g
d
a
H
3
N
H N
N H
H
3
N
O
O
A r g i n i n e
H
3
N
H
3
N
O
O
O r n i t h i n e
N H
3
H N
N
O
O
H i s t i d i n e
H
3
N
O
O
O
O
G l u t a m i c a c i d
H
3
N
O
H
2
N
O
O
G l u t a m i n e
O
O
O
O
O
- K e t o g l u t a r a t e
H
2
N O
O
P r o l i n e
H
3
N
O
H
O

O
G l u t a m a t e
- s e m i a l d e h y d e
H
N O
O
D
1
- P y r r o l i n e -
5 - c a r b o x y l a t e
H
2
O U r e a
- K e t o -
g l u t a r a t e G l u t a m a t e
a r g i n a s e
o r n i t h i n e
- a m i n o t r a n s f e r a s e
H
2
O
H
2
O
1 / 2 O
2
H
2
O
p r o l i n e
o x i d a s e
N A D ( P ) H + H
g l u t a m a t e
s e m i a l d e h y d e
d e h y d r o g e n a s e
H
2
O
H
4
f o l a t e
N
5
- f o r m i m i n o -
H
4
f o l a t e
H
2
O H
2
O N H
4
h i s t i d i n e
a m m o n i a
l y a s e
u r o c a n a t e
h y d r a t a s e
i m i d a z o l o n e -
p r o p i o n a s e
g l u t a m a t e
f o r m i m i n o
t r a n s f e r a s e
N A D ( P ) H + H
N H
4
g l u t a m a t e
d e h y d r o g e n a s e
N H
4
N A D ( P )
N A D ( P )
FromNelson&Cox(2000)
AMINO ACIDS AND RELATED SUBSTANCES 457
methionine formed in the liver would be quickly incorporated into proteins, and
homoserinefurthermetabolized(Suzueetal.,1975).
(vi) Aspartic acid (No. 1429)
Aspartic acid undergoes transamination with a-ketoglutarate to oxaloacetate,
whichisanintermediateinthecitricacidcycle.
(vii) b-Alanine (No. 1418)
b-Alanine has been reported to be deaminated to formylacetic acid and then
metabolized rapidly and extensively to CO
2
and water (Pihl & Fritzson, 1955;
Marnett et al., 1985). It is also incorporated into two dipeptides, carnosine
3
(b-
alanyl-L-histidine) and anserine
4
(b-alanyl-1-methyl-L-histidine), in vertebrate
muscletissueatreportedconcentrationsof0.50and1.90mg/g,respectively.When
fvepartiallyhepatectomizedratsweregivenb-alanineatadoseof22mmol/lper
100g, 22h after surgery, synthesis of carnosine and anserine increased (Harms
&Winnick,1954).
2.4 Toxicological studies
Numerous studies of toxicity with amino acids were available, the majority
being of short duration, employing limited protocols and conducted in the 1950s
and1960s.Thesestudiesweredesignedtoevaluatenutritionalfactorsrelatedto
intake of amino acids rather than potential toxicity. This monograph focuses on
studies of longer duration and those that provide more detailed descriptions of
protocol.
2.4.1 Acute toxicity
Oralmedianlethaldose(LD
50
)valueshavebeenreportedforsevenofthe20
favouringagentsinthisgroup(Table3).Inrats,LD
50
valueswereintherangeof
1890to12400mg/kgbw(Bregliaetal.,1973;Takasakietal.,1973;Sprinceetal.,
1974).Inmice,oralLD
50
valueshavebeenreportedforfourfavouringagentsof
the20inthisgroup.Thevaluesrangedfromgreaterthan1000to8380mg/kgbw
(Bersin et al.,1956; Doull et al., 1962;Takasaki et al., 1973; Llobet et al., 1988).
TheseLD
50
valuesindicatethattheoralacutetoxicityofthesefavouringagents
islow.
3

N
H
N
C O
2
H
H N
N H
2
O
carnosine ( -alanyl-L-histidine)
4

anserine (b
N
N
C O
2
H
H N
N H
2
O
-alanyl-1-methyl-L-histidine)
458 AMINO ACIDS AND RELATED SUBSTANCES
Figure 5. Catabolic pathways for isoleucine, methionine and valine
a
a
NH
3
O
O
CO
2
S-CoA
O
O
O
O
NH
3
HS
O
O
S-CoA
O
NH
3
S
O
O
O
O
S-CoA
O
O S-CoA
O O
CO
2
NAD
CoA-SH
HCO
3
CO
2
NH
3
O
O
Methionine
Homocysteine
-Ketobutyrate
Isoleucine
Valine
Acetyl-CoA
Propionyl-CoA
Succinyl-CoA
Methylmalonyl-CoA
3 steps
PLP
Serine
cystathionine
g-lyase
Cysteine
PLP
cystethionine
-synthase
NADH + H
-keto acid
dehydrogenase
2 steps
methylmalonyl-
CoA mutase
coenzyme B
12
7 steps
6 steps
FromNelson&Cox(2000)
CoA,coenzymzeA;PLP,pyridoxalphosphate
AMINO ACIDS AND RELATED SUBSTANCES 459
(a) Short-term studies of toxicity
Short-termstudiesoftoxicityhavebeenundertakenforanumberoftheamino
acidsandrelatedsubstancesinthisgroup.Inthosecaseswhereano-observed-
effectlevel(NOEL)canbederived,theresultsareprovidedinTable4.
(i) b-Alanine (No. 1418) and L-glutamic acid (No. 1420)
Rats
In a study designed to examined the effect of b-alanine on the natural levels
of taurine in heart and retinal tissues, groups of 15 or 18male Sprague-Dawley
rats were given drinking-water containing b-alanine at 0 or 3% (approximately
3000mg/kgbwperday)forupto6weeks.Fourtreatedandthreecontrolratswere
killedattheendofweeks1,2and3,andtheremainingsixtreatedandsixcontrol
ratswerekilledattheendofweek6.Treatedratsexhibitedatransient,butsignif-
cantdecreaseinlevelsoftaurineintheheartatweeks1,2,and3.Nodifference
betweenthetestandcontrolgroupwasnotedatweek6.Retinalcontentoftaurine
was unaffected by treatment with b-alanine. Compared with the age-matched
controls, body-weight gain was reduced among the treated animals at all time-
points;however,thedecreasewasnotstatisticallysignifcant.Orallyadministered
b-alaninewasnotconsideredtohaveatoxicologicallysignifcanteffectontaurine
levels(Lake&DeMarte,1988).
Two weanling male albino rats were given a diet containing a mixture of L-
glutamic acid (No. 1420) at 0.7% (700mg/kgbw per day) and b-alanine at 0.4%
Table 3. Studies of acute toxicity with amino acids and related substances
administered orally
No. Flavouringagent Species Sex LD
50
Reference
(mg/kgbw)
1419 l-Cysteine Mouse M,F 3550(M) Takasakietal.(1973)
4200(F)
1419 l-Cysteine Rat M,F 6350(M) Takasakietal.(1973)
5580(F)
1419 l-Cysteine Rat NR 1890 Sprinceetal.(1974)
1419 l-Cysteine Mouse M 1502 Llobetetal.(1988)
1421 Glycine Rat M,F 3340 Bregliaetal.(1973)
1424 d,l-Methionine Mouse M >2000 Doulletal.(1962)
1427 dl-(3-Amino-3- Mouse NR 8380 Bersinetal.(1956)
carboxypropyl)
dimethylsulfoinium
chloride
1435 Taurine Mouse M >1000 Doulletal.(1962)
1438 l-Arginine Rat M,F 12400 Bregliaetal.(1973)
1439 l-Lysine Rat M,F 10130 Bregliaetal.(1973)
F,female;M,male;NR,notreported
460 AMINO ACIDS AND RELATED SUBSTANCES
T
a
b
l
e

4
.

R
e
s
u
l
t
s

o
f

s
h
o
r
t
-
t
e
r
m

s
t
u
d
i
e
s

o
f

t
o
x
i
c
i
t
y

a
n
d

l
o
n
g
-
t
e
r
m

s
t
u
d
i
e
s

o
f

t
o
x
i
c
i
t
y

a
n
d

c
a
r
c
i
n
o
g
e
n
i
c
i
t
y

w
i
t
h

a
m
i
n
o

a
c
i
d
s

a
n
d

r
e
l
a
t
e
d

s
u
b
s
t
a
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c
e
s

u
s
e
d

a
s

f
a
v
o
u
r
i
n
g

a
g
e
n
t
s
N
o
.

F
l
a
v
o
u
r
i
n
g

a
g
e
n
t

S
p
e
c
i
e
s
;

s
e
x

N
o
.

o
f

t
e
s
t

R
o
u
t
e

D
u
r
a
t
i
o
n

(
d
a
y
s
)

N
O
E
L

(
m
g
/
k
g

b
w

R
e
f
e
r
e
n
c
e

g
r
o
u
p
s
a
/
n
o
.

p
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r

d
a
y
)

p
e
r

g
r
o
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p
b
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h
o
r
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r
m

s
t
u
d
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e
s

o
f

t
o
x
i
c
i
t
y
1
4
1
9

l
-
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y
s
t
e
i
n
e

M
o
u
s
e
;

M
,

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/
1
6

2
4

G
a
v
a
g
e

3
0

<
2
0
0

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a
k
a
s
a
k
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t

a
l
.

(
1
9
7
3
)
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1
9

l
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t
;

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2
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a
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1
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(
1
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AMINO ACIDS AND RELATED SUBSTANCES 461
(400mg/kgbw per day) for 56 days. Compared with a control group of four rats,
maintainedforonly45days,treatedanimalsshowedareductioninweightgain.
Noskeletaloraorticlesionswereobservedasaresultoftheadditionof0.7%L-
glutamicacidand0.4%b-alaninetothediet(Wawzoneketal.,1955).
(ii) L-Cysteine (No. 1419)
Mice
Ina30-daystudy,groupsofmaleandfemaleICR-JCLmice(812ofeachsex
pergroup)weregivensuspensionsofL-cysteinein1%CMCsolution atadoseof
0,200,1000or3000mg/kgbwperdayorallyviagavage.Throughoutthecourse
oftheexperiment,generalcondition,body-weightgain,andfoodandwatercon-
sumptionweremonitored.After30days,allsurvivingmiceweresubjectedtoclini-
calchemistrytestsandthennecropsied.Allanimalswereexaminedmacroscopically,
andselectedorganswereweighedandtissuespreservedforhistologicalevalua-
tion. No clinical symptoms of toxicity and no early deaths were reported at the
lowest dose (200mg/kgbw per day). Two mice (one of each sex) in the group
receiving the intermediate dose (1000mg/kgbw per day) died before the end of
thestudy.Animalsinthegroupreceivingthehighestdose(3000mg/kgbwperday)
exhibitedlethargyandonlythreemice(twofemaleandonemale)survivedtothe
completionofthestudy.Thesesurvivingmicewerereportedtobetwo-thirdsthe
weightofthecontrolmice,asaresultofaninitialweightloss.Atthelowestdose
(200mg/kgbw per day), animals were reported to gain slightly less weight than
controlswhileweightgainofanimalsattheintermediatedose(1000mg/kgbwper
day)wascomparabletothatofcontrols.Waterandfoodconsumptionoftreated
animals was generally less than that of the controls. Clinical chemistry indicated
a signifcant decrease in concentrations of blood urea nitrogen and glucose,
transaminaseactivityinmalesattheintermediatedose(1000mg/kgbwperday),
andanincreaseintotalproteinconcentrationsinfemalesattheintermediatedose.
Haematology analysis indicated no differences between the control and test
animals.Analysisoforganweightsindicatedadecreaseinabsoluteweightsofthe
adrenalandanincreaseinabsoluteweightsofthethymusinfemalesattheinter-
mediateandlowestdoses,respectively,andadecreaseinabsoluteweightofthe
seminalvesiclesinmalesatthelowestandintermediatedoses.Necropsyofthe
surviving animals revealed no signifcant changes; however, haemorrhage and
blood congestion in the internal organs and in the brain were reported in the
animalsthatdied.Histopathologyindicatedalltreatedmicehadbloodcongestion
and haemorrhage in the lungs. Mice receiving cysteine at a dose of 1000 or
3000mg/kgbwperdayshowedcongestionofthetissuessurroundingthefollicles
inthespleen,accompaniedbyatrophyofthefolliclesatthehighestdose.Approxi-
mately2530%ofmiceatthelowestdoseand<25%ofthemiceat3000mg/kgbw
perdayexhibiteddisappearanceofhepaticcellnuclei.Congestionoftheliverwas
alsoreportedinthecontrolgroup(Takasakietal.,1973).
462 AMINO ACIDS AND RELATED SUBSTANCES
Rats
Ina30-daystudy,groupsofmaleandfemaleSD-JCLrats(912ofeachsex
pergroup)weregivenL-cysteineatadoseof0,200,1000or5000mg/kgbwper
day orally via gavage. The protocol was the same as for the study in mice,
describedabove.Allmalesandfemalesatthehighestdosediedbeforetheend
of the study. Food consumption for animals at the highest dose was slightly
decreased.Waterconsumptionofbothsexeswasdecreasedattheintermediate
dose.Withtheexceptionofanincreaseinbloodureanitrogenandglucosecon-
centrations,andadecreaseintotalproteinconcentrationsinmalesatthelowest
andintermediatedoses,respectively,theresultsoftestsforhaematologyandliver
functionwereotherwiseunremarkable.Absoluteweightsoftheliverweresignif-
cantly elevated in females at the lowest and intermediate doses and in males at
intermediatedose.Inbothmalesandfemalesattheintermediatedose,asignif-
cantincreaseinrelativeweightsofthekidneywasobserved.Absoluteweightsof
thethymuswereincreasedinfemalesat5000mg/kgbwperday.Atnecropsy,no
obvious changes were observed in the rats that survived the experiment.Those
thatdiedprematurelyshowedbloodcongestionandhaemorrhageintheinternal
organs.Morespecifcally,histologicalevaluationrevealedbloodcongestioninthe
lungs,liver,kidneys,andcerebrumofratsat5000mg/kgbwperday.Attheinter-
mediatedose(1000mg/kgbwperday),congestionofthelungsandliveralsowere
reported.Animalsat200mg/kgbwperdaywerereportedtohavebloodcongestion
intheliver.Thestructureofthefascicularlayeroftheadrenalcortexwasreported
tobeindefniteinanimalsat200or1000mg/kgbwperday,andalsointhecontrol
group(Takasakietal.,1973).
Groupsof1012maleSD-JCLratsweregivenL-cysteineatadoseof0,100,
500or2000mg/kgbwperdayasa20%suspensionwith1%CMCviagavage,6
daysaweek,for6months.Allratssurvivedtotheendofthestudy,andnosignif-
cant differences in behaviour, body-weight gain or food and water consumption
wereobserved.Aslight,butsignifcantdecreaseinbloodureanitrogenandtotal
proteinconcentrationswerereportedinratsat500and2000mg/kgbwperday.A
reductionintotalproteinconcentrationswaslimitedtotheintermediatedosegroup.
Aslightenlargementoftheliverwasobservedinratsat100and500mg/kgbwper
day,whileanincreaseinabsoluteweightsofthekidneywasreportedattheinter-
mediate and highest doses (500 and 2000mg/kgbw per day, respectively). At
necropsy,histologicalexaminationoftreatedratsshowedbloodcongestioninthe
liver, lungs, spleen and kidneys, indefnite structure of the fascicular layer of the
adrenal glands, and haemosiderin pigment in the spleen. However, these same
changes, except for the congestion of the kidneys, also were observed in the
controlanimals.Indefnitestructureoflivercellsalsowasreportedinratsreceiving
L-cysteineatadoseof500or2000mg/kgbwperday.Noobviouschangeswere
notedinotherinternalorgansandtheauthorsfounditdiffculttoassignanysig-
nifcancetothefndingsdiscussedabove(Takasakietal.,1973).
AMINO ACIDS AND RELATED SUBSTANCES 463
(iii) D,L-Isoleucine (No. 1422)
Rats
Groupsof10maleand10femaleF344rats(aged4weeks)weregivendiets
containing0,1.25,2.5,5.0,or8.0%D,L-isoleucine(equivalenttoadoseofapproxi-
mately0,1250,2500,5000or8000mg/kgbwperday)for13weeks.Bodyweights
andfoodconsumptionweremeasuredweekly.Noclinicalsignsoftoxicityormor-
tality related to administration of isoleucine were observed throughout the test
period.Bodyweightsandfoodconsumptionwerecomparableintestandcontrol
animals.Urineanalysisatweek13revealedadose-relatedincreaseinurinarypH
inmalesreceivingdietscontaining2.5%D,L-leucine,andsignifcantlyincreased
urinevolumeinanimalsreceivingdietcontaining8%D,L-leucine.Haematological
examinationandbloodchemicaldeterminationsatweek13revealedadecrease
inglutamicpyruvictransaminaseactivityatdietaryconcentrationsof2.5,5.0and
8.0%inbothsexes,aslightincreaseinalkalinephosphataseactivityinmalesat
5 and 8%, a statistically signifcant decrease in total protein in females at 5 and
8% (p < 0.05), and several changes in serum electrolytes in both males and
femalesat2.5%andgreater.Signifcantincreasesinrelativekidneyweightswere
observedinbothmalesandfemalesatthe8%dietarylevel.Grosspathologywas
conductedatnecropsyandhistologicalexaminationperformedonallmajororgans
and all grossly visible lesions. Histological examination revealed no treatment-
related abnormalities. The NOEL was 1250mg/kgbw per day in rats (Kawabe
etal.,1996).
(iv) D,L-Methionine (No. 1424)
Rats
Twelve male Holtzman rats were given a diet providing 2% D,L-methionine
(equivalenttoapproximately1000mg/kgbwperday)forupto12weeks.Necrop-
sieswereperformedafter2,8and12weeksofexposure.Signifcantlydecreased
weight gain was observed at 2 weeks (p < 0.01), but was comparable to that in
controlanimalsby8and12weeks.Asignifcant,buttransientenlargementofthe
kidneys (p < 0.01) and of the adrenals (p < 0.05) was reported at 2 weeks. At
weeks8and12,however,organweightsdidnotdifferfromthoseofthecontrols
(Horger&Gerheim,1958).
MaleSprague-Dawleyratsweregivendietscontaining0or4%D,L-methionine
(approximately2000mg/kgbwperday)for10weeks.Thetreatedratswerecom-
paredwiththecontrolsatweek5andatstudytermination.Bodyweightsoftreated
animalswereslightlydecreasedatbothtime-points.After10weeks,theanimals
were sacrifced and the enzyme activities and fat content of the liver were ana-
lysed.Therewasamarkeddepressionofgrowthintreatedanimalscomparedwith
thecontrols.Thelevelsofneutralfatintheliversincreasedalongwiththeactivities
oftryptophanpyrrolase,arginase,glutamatepyruvatetransaminaseandglutamate
oxaloacetatetransaminase,howevertherewasamarkeddecreaseintheconcen-
tration of nicotinamide adenine dinucleotide (NAD
+
). The decrease in NAD
+
was
464 AMINO ACIDS AND RELATED SUBSTANCES
attributed to the accumulation of lipids, as NAD
+
is a necessary cofactor for the
oxidationoflipids(Klainetal.,1963).
(v) L-Proline (No. 1425)
Rats
In a 30-day study, a group of seven white female Sprague-Dawley rats were
givenL-prolineatadoseof50mg/kgbwperdayinwater.Agroupof10ratsserved
as the control group.After 30 days, all animals were weighed, necropsied, and
subjectedtoacompletegrossexamination.Histopathologyandmicroscopicexam-
inationsoftheliverandkidneyswereconducted.Samplesofserumwereobtained
for determination of enzyme activities, and concentrations of creatinine and total
protein.There were no treatment-related effects in rats given L-proline at 50mg/
kgbwperdaycomparedwiththecontrols(Kampeletal.,1989).
(vi) L-Phenylalanine (No. 1428)
Rats
Inalimitedstudyoftoxicityinrats,youngWistarratsweregivenwatercontain-
ing 7% L-phenylalanine by gavage from postnatal days 1 to 21.At day 21, they
wereplacedonasoliddietsupplementedwith7%L-phenylalaninefor7days.This
dietary level corresponds to an average daily intake of of L-phenylalanine of
approximately 7000mg/kgbw.There were no concurrent controls in this study.A
high rate of mortality was reported and the rats demonstrated signs of toxicity
whichincludedlesionsoftheeyes,swollentoesandsometoeatrophy,aswellas
diffcultyinurinating.Necropsyrevealedswellingofthebladderandobstructionof
theurethrainthemoreseverecases(numberunspecifed).Theurinecontained
small white crystals, which were identifed as primarily tyrosine, a metabolite of
phenylalanine(Dolan&Godin,1966).
(vii) Taurine (No. 1435)
Rats
In an 8-week feeding study, a group of six male weanling Wistar rats were
givenadietcontainingeither0or5%taurine(correspondingtoanaveragedaily
intake of 0 or 5000mg/kgbw). Food and water were available ad libitum. Body
weightsofthetaurine-treatedrats,recordedaftereachfeeding,werecomparable
tothoseofthecontrolsthroughouttheentirestudyperiod.Bloodsamplesdrawn
at2-weekintervalsforanalysisofaspartatetransaminase,alaninetransaminase
and alkaline phosphatase activities revealed no signifcant differences between
controlandtreatedanimals.Attermination,nosignifcantdifferencesintherelative
weightsofliverandkidneywerereportedbetweenthecontrolandtreatedanimals
(Hwangetal.,2000).
AMINO ACIDS AND RELATED SUBSTANCES 465
(viii) D,L-Alanine (No. 1437)
Rats
In a 26-week feeding study, groups of eight male and eight female weanling
Wistarratsweregivendietscontaining0,5,10or20%alanine(equivalentto0,
5000,10000or20000mg/kgbwperday).Foodandwaterconsumptionandbody
weightsrecordedeverytwoweeksrevealedadose-relatedincreaseinfoodcon-
sumption in males.All rats appeared to be healthy throughout the study period.
Ratsatthehighestdosegained2030%lessweightthanthoseatthelowerdoses.
Pooled samples of urine collected at weeks 1314 and at weeks 2526 showed
no signifcant differences between test and control groups; however, increased
intake of alanine was associated with a 1001000-fold increase in the urinary
excretionofthisaminoacid.Theauthorssuggestedthatgroupsgiventhehighest
dosemayhaveexperiencedhyperalaninaemia,possiblyleadingtoanincreasein
concentrationsofserumglucoseandinsulin.Attermination,bloodsampleswere
collected,agrosspathologicalexaminationwasperformedandtheliverandkidney
weightswererecorded.Serumconcentrationsofalanineincreasedonlyinmales
andfemalesfed20%D,L-alanine.Increasedconcentrationsofammoniainmales
and a decrease in concentrations of pyruvate in both sexes at the highest dose
were observed.At necropsy, gross examination did not reveal any abnormalities
orlesions.Althoughabsoluteweightsofthekidneyintreatedratswerecomparable
tothoseofcontrols,increasedrelativeweightsofthekidneywerereportedinmales
and females fed D,L-alanine at at dietary concentration of 20%. There were no
treatment-related effects in rats fed diets containing up to 10% D,L-alanine
(10000mg/kgbwperday)(Chowetal.,1976).
(ix) L-Lysine (No. 1439)
Rats
Ina30-daystudy,agroupofsixwhitefemaleSprague-Dawleyratsweregiven
L-lysineatadoseof50mg/kgbwperdayintapwater.Anadditionalgroupof10
untreated,healthyratsservedasthecontrolgroup.Allanimalswereprovidedwith
accesstofoodandtapwateradlibitum.After30days,allanimalswereweighed,
necropsied, and subjected to a complete gross examination. Histopathology and
microscopic examinations of the liver and kidneys were conducted. Samples of
serumwereobtainedfordeterminationofenzymeactivities,andconcentrationsof
creatinineandtotalprotein.Comparedwithcontrols,nochangeswereobserved
infnalbodyweight,andnogrossabnormalitieswereidentifed.Histopathological
examination of the liver was unremarkable; however, severe proximal tubular
dystrophy and necrosis, accompanied by glomerular mesangial crescents, were
reported in samples of kidney. Electron microscopic studies, however, indicated
the extracellular matrix of the kidney was without pathological fndings. Serum
analysis revealed a statistically signifcant increase in alkaline phosphatase and
in creatinine levels (p < 0.05) The style of reporting of this study makes a clear
interpretationoftheresultsdiffcult(Kampeletal.,1989).
466 AMINO ACIDS AND RELATED SUBSTANCES
(x) Monosodium glutamate
Short-term studies of toxicity with glutamic acid and its salt were previously
considered by the Committee at its thirty-frst meeting (Annex 1, reference 77).
Morerecentstudiesaresummarizedbelow.
Rats
Ina13-weekstudy,groupsof10weanlingSPF-bredmalealbinoWistarrats
weregiveneitheracereal-basedstockdietoranacidcasein-basedpurifeddiet
containing 6% monosodium glutamate (equivalent to approximately 6000mg/
kgbw).Toinvestigatethemechanismoftoxicityattributabletomonosodiumgluta-
mate, additional groups of rats were fed with diets containing 6% monosodium
glutamateplus1.6%NaHCO
3
or1.0%NH
4
Cl,or2.5%KHCO
3
alone.Animalswere
provided with access to food and water ad libitum for the duration of the experi-
ment. Food intake and body weights were monitored weekly, with water intake
measured during weeks 3, 6, 8, and 11. Urine was collected for analysis during
thefrst2hofthelight/darkcycleatseveraltime-points.Allanimalsweresacrifced
andexaminedforgrossabnormalitiesattheendofweek13.Theurinarytissues
and organs were preserved for histopathological analysis. Rats fed the purifed
diet containing 6% monosodium glutamate exhibited a decrease in fnal body
weights and in body weights evaluated on day 28, compared with animals not
receiving monosodium glutamate. No variation in body weight was observed
amonganyofthegroupsmaintainedonthestockdiet,norwereanydifferences
in food intake reported. Relative weights of the kidney were increased in groups
feedingondietssupplementedwithmonosodiumglutamateonly,reachinglevels
ofstatisticalsignifcanceinanimalsreceivingthepurifeddietcontainingmonoso-
dium glutamate. Feeding with diets that containined monosodium glutamate
causedtheurinetobecomemarkedlymorealkaline.However,ratsfedthepurifed
dietproducedurineofhigheraciditythantheratsfedthestockdiet,afndingthat
was attributed to the greater excess of base in the stock diet. Rats receiving
monosodiumglutamateinthestockdietshowedaclearlyincreasedincidenceand
degreeofhyperplasiaoftheurinarybladderepithelium.Suchhyperplasticchanges
also occurred in rats fed the purifed diet supplemented with monosodium gluta-
mate,butonlyinasingleanimalandonlytoaminimalorslightdegree.Hyperplasia
inratsfedthepurifeddietwasnotassociatedwithcystformation.Simultaneous
feedingwithNH
4
Clsaltreducedtheurinarybladderhyperplasiaobservedinrats
fedthestockdietcontainingmonosodiumglutamate.Hyperplasiaoftheepithelium
liningtherenalpelvisorrenalpapillawasobservedinseveralratsfedmonosodium
glutamateormonosodiumglutamateplusNaHCO
3
ineitherofthetwodiets.The
authorsconcludedthattheurinarybladderchangesinducedbymonosodiumglu-
tamateareattributabletoitsalkalizingpropertiesratherthantomonosodiumglu-
tamateperse(deGrootetal.,1988).
Groupsof10maleF344rats(aged4weeks)weregivenadietsupplemented
with 5.83% monosodium glutamate (equivalent to 5830mg/kgbw per day) for 10
weeks.Ratsweregivenaccesstofoodandwateradlibitumthroughoutthecourse
of the study. Water and food consumption, and body weight were monitored
throughoutthestudyperiod.Urinewascollecteddailyduringweeks4and10of
AMINO ACIDS AND RELATED SUBSTANCES 467
the study. After 10 weeks, animals were necropsied, and the urinary bladder,
kidneys and stomach subjected to microscopic examination. At week 8, body
weightsoftreatedratsweresignifcantlyreducedcomparedwiththoseofuntreated
controls.An amorphous precipitate containing calcium phosphate was observed
in the urine of some rats, with a larger amount being present at week 4 than at
week10.TheurinarypHoftreatedrats(pH8.0)washigherthanthatofthecontrols
(pH7.0).Asignifcant(p<0.05)decreaseinurinaryconcentrationsofcreatinine
was reported in the group fed monosodium glutamate (95 16mg/dl) compared
withthecontrols(257100mg/dl),butthiswasconcludedtobeadilutionaleffect
corresponding to an increase in urinary volume (increase not statistically signif-
cant).Oneof10ratsreceivingmonosodiumglutamateshowedaslighthyperplasia
ofthelimitingridgeoftheforestomach.Simpleurothelialhyperplasiaoftherenal
papillaatthefornixwasobservedinthreeratsgivenmonosodiumglutamateand
was associated with calcifcation (Cohen et al., 1995). However, subsequent
studies have demonstrated that the formation of urinary calculi in rats generally
occurs after administration of a variety of sodium salts at high doses (Capen
etal.,1999;Cohen,1999).
(b) Long-term studies of toxicity and carcinogenicity
(i) Glycine (No. 1421)
Rats
Groupsof50maleand50femaleF344DuCrjrats(aged6weeks)weregiven
drinking-watercontaining0,2.5or5%glycine(equivalenttoapproximately0,2500
or5000mg/kgbwperday)for108weeks.Theanimalswereweighedevery1or
2weeksanddietaryconsumptionwasdeterminedevery4weeks.Haematological
examination,bloodchemicaldeterminations,andurineanalysisperformedatstudy
termination revealed no signifcant differences between the treated and control
animals.At the highest dose tested, concentrations of haemoglobin were signif-
cantly increased in males and decreased in females. In females, a signifcant
decreaseinerythrocytevolumefractionwasalsonoted.Bloodchemistryrevealed
adecreaseinserumcreatinephosphokinaseactivityatbothdosesinmalesand
females,andanincreaseinbloodureanitrogenalsoinbothmalesandfemales,
butonlyatthehighestdose.Malesatbothdosesexhibitedareductioninconcen-
trations of creatinine. The fnal mean body weights of males and females given
drinking-watercontaining5%glycineweresignifcantlylower(p <0.05)thanthose
of the controls.Absolute weights of the liver were signifcantly lower in males at
2.5 and 5%. A complete histopathological evaluation performed at necropsy
revealed a high incidence of renal calcifcation in treated and control groups of
females(control,16outof41;2.5%,15outof46;5%,13outof31).Alowinci-
denceofkidneypapillomaswasreportedinfemalestreatedwithglycine(2.5%,4
outof46;5%,2outof31),butnotinmales.Arenalcellcarcinomawasreported
inonemale(1outofof40)treatedwith5%glycine.Furtherdetailedhistopathol-
ogyoftheurinarysystemrevealedhyperplasiaofthetransitionalepitheliumofthe
renalpelvisin2outof46ofthefemalesatthe2.5%levelonly.Anincreasedinci-
denceofnecrosisoftherenalpapillaewasreportedintreatedmales(2.5%,2out
468 AMINO ACIDS AND RELATED SUBSTANCES
of45;5%,3outof40)andfemales(2.5%,14outof46;5%,10outof31)com-
paredwiththecontrols(0outof40inmales;0outof41infemales).Atumourof
thetransitionalepitheliumoftherenalpelviswasseeninonemalecontrolrat(1
out of 40). Owing to the organ distribution and the histological characteristics of
theneoplasticlesionsobservedinthetreatedanimals,withtheexceptionofthose
in the renal pelvis, the authors concluded that they were spontaneous and not
related to administration of glycine. These spontaneous tumours are known to
occurinthisstrainofrats(Kitahorietal.,1994).Lesionsoftheurinarytractgener-
allyoccurasaresultoftheformationofurinarytractcalculiinducedbyexposure
at high doses, which causes subsequent chronic irritation and toxicity (Capen
etal.,1999;Cohen,1999).
(ii) Histidine (No. 1431)
Groups of 50 male and 50 female F344 rats were given diets containing L-
histidinemonohydrochlorideataconcentrationof0,1.25or2.5%for104weeks
(equivalentto0,0.47and0.96g/kgbwperdayinmales,and0,0.56and1.1g/kg
bwperdayinfemales).Inthegroupreceiving2.5%L-histidine,therewassignif-
cantdepressionofbody-weightgainduringweeks76105formalesandinweeks
84104 for females. There was a slight decrease in survival rate in both males
and females at 2.5% compared with controls. In females, relative weights of the
brain and adrenals were signifcantly higher at 2.5% than those of the controls,
whiletheabsoluteweightswerecomparableinallgroups.Thereweresignifcant
increasesinerythrocytecount,concentrationofhaemoglobin,erythrocytevolume
fractionandplateletcountinmalesat2.5%.Therewasanincreaseinincidence
of tumours in all groups, but there was no treatment-related increase in the inci-
denceofanytumour(Ikezakietal.1996).
(iii) Taurine (No. 1435)
GroupsofninemaleandsevenfemaleWistarrats(aged7week)weregiven
diets containing 0, 0.5 or 5.0% taurine daily (equivalent to approximately 0, 500
or 5000mg/kgbw per day) for 18 months. No differences in appearance and
behaviour were observed between treated and control animals throughout the
study.Aslight,statisticallyinsignifcantdepressionofgrowthwasobservedinthe
animalsgivendietcontaining5%taurinewhencomparedwiththecontrols,butno
differenceinaveragefoodconsumptionwasreportedbetweenthesetwogroups.
Atstudytermination,theanimalswerenecropsiedandperipheralbloodsamples
werecollectedforhaematologicalexamination.Nosignifcantdifferencesinorgan
weightsandhaematologicalvalues,orhistologicalchangeswereobservedbetween
the control and treated animals. The haematological values obtained for treated
animalswerecomparabletothoseforthecontrols.Malesandfemalesgivendiet
containing5%taurineexhibitedmoderateproliferationofthebileducts;however,
this was not accompanied by any other histological changes. The NOEL was
500mg/kgbwperday(Takahashietal.,1972).
AMINO ACIDS AND RELATED SUBSTANCES 469
(iv) Monosodium glutamate
Long-termstudiesoftoxicitywithglutamicacidanditssaltswereconsidered
previouslybytheCommitteeatitsthirty-frstmeeting(Annex1,reference77).More
recentstudiesaresummarizedbelow.
Rats
Groups of 50 male and 50 female Fisher 344 rats (aged 5 weeks) were fed
diets containing 0, 0.6, 1.25, 2.5 or 5.0% monosodium glutamate daily (equal to
0,231,481,975and1982mg/kgbwperdayinmales,and0,268,553,1121and
2311mg/kgbwperdayinfemales)for104weeks.Allanimalswereexaminedtwice
dailyforgeneralhealthandsignsoftoxicity.Bodyweightsweremeasuredweekly
forthefrst14weeksandevery2weeksthereafteruntilstudytermination.Food
consumption was measured over a 2-day period before each weighing.Ten rats
fromeachgroupwererandomlychosenforurineanalysisatweek1,andmonths
1, 3, 6, 12, 18 and 24. At study termination, haematological evaluations were
conductedonallsurvivinganimals.Atnecropsy,agrosspathologicalexamination
was performed and the organ weights of major organs (i.e. brain, heart, liver,
spleen, kidneys) were measured. Extensive microscopic examinations were per-
formedonallmajororgantissuesobtainedfromanimalsinthecontrolgroupand
atthehighestdose,aswellasallanimalsthatdiedorwerefoundtobeinamori-
bundconditionduringthestudyperiod.Microscopicevaluationofallotheranimals
was limited to the stomach, liver, kidneys, urinary bladder, and all gross lesions.
Therewerenodifferencesinphysicalappearance,behaviour,foodconsumption,
ormortalitybetweentreatedanimalsandcontrolsthroughoutthestudy.Nodiffer-
encesbetweentreatedandcontrolanimalswerereportedinanyhaematological
parameterevaluated.Adecreaseinfnalbodyweightwasreportedinmalesat5%
compared with concurrent controls. Urine analysis revealed increased pH and
concentrationsofNa
+
,anddecreasedconcentrationsofK
+
inmalesandfemales
at2.5and5%.Manyanimalmodelshavebeenusedtodemonstratethatlong-term
variationsinconcentrationsofNa
+
orK
+
andinpHarerelatedtocellproliferation
and the eventual development of tumours of the urinary bladder. However, the
changesinelectrolytesandpHinthisstudywerenotassociatedwithanyevidence
for the development of tumours of the urinary bladder. These urine conditions
promotetumourdevelopmentonlyunderspecifcconditions.Asignifcantincrease
(p<0.05)inrelativeweightoftheurinarybladderobservedinmalesat5%was
attributedtodistensionofthebladderasaresultofincreasedvolumeofurine.The
signifcantlyincreased(p <0.05)relativeweightsofthekidneyobservedinratsof
bothsexesfedwithdietscontaining5%monosodiumglutamatewereattributedto
theincreasedintakeofNa
+
.Transitionalcellhyperplasiaoftherenalpelvisassoci-
atedwithmoderatetoseverechronicnephropathywasincreasedinthemalesat
1.25 and 5%, but not at 2.5% monosodium glutamate. No difference in the inci-
denceoftumourswasreportedincontrolandtreatedanimalsofeithersexatany
dietarylevel(Shibataetal.,1995).
470 AMINO ACIDS AND RELATED SUBSTANCES
(c) Genotoxicity
Testingforgenotoxicityhasbeenperformedfor17representativeaminoacids
in this group. The results of these tests are summarized in Table 5 and are
describedbelow.
5
(i) In vitro
IntheassayforreversemutationinSalmonella typhimurium,negativeresults
werereportedforL-glutamicacid(No.1420),glycine(No.1421),methionine(No.
1424), and L-proline (No. 1425) at concentrations of up to 1000mg/ml, with and
without metabolic activation, in several strains of S. typhimurium (TA92, TA97,
TA98, TA100, TA102, TA1530, TA1531, TA1532, TA1535, TA1537, TA1538, and
TA1964) (Green & Savage, 1978; Baker & Bonin, 1981; Brooks & Dean, 1981;
Ichinotsubo et al., 1981a; MacDonald, 1981; Nagao & Takahashi, 1981; Richold
&Jones,1981;Rowland&Severn,1981;Trueman,1981;Vennitt&Crofton-Sleigh,
1981;Haworthetal.,1983;Fujitaetal.,1994).
When evaluated in various strains of Escherichia coli, methionine (No. 1424)
atconcentrationsofupto6000mg/plate(or1000000mg/ml),withorwithoutmeta-
bolicactivationdidnotshowanyevidenceofmutagenicactivity(Flucketal.,1976;
Ichinosubo et al., 1981b; Matsushima et al., 1981; Mohn et al., 1981; Venitt &
Crofton-Sleigh,1981).WhenE. coliWP2uvrAwasincubatedwithmethionineat
a concentration of 10, 100 or 1000mg/ml in a non-standard microtitre assay for
fuctuation,ahighernumberofrevertantswasobservedintheabsenceofmeta-
bolic activation at the highest dose tested, but not in the presence of metabolic
activation(Gatehouse,1981).Theauthorssuggestedthatexcessmethioninehas
an inhibitory effect on auxotrophs that require tryptophan, including this strain of
E. coli, allowing spontaneous revertants to dominate the culture (Gatehouse,
1981).When E. colistrainsWP2andWP67uvrApolA,andCM871uvrArecAlexA
were incubated with methionine at concentrations of up to 2500mg/ml, slight
increasesinthenumberofrevertantswerereportedinthepresenceofmetabolic
activation.The authors, however, were of the opinion that these results were an
artefactofenhancedbacterialgrowthcausedbythepresenceofahighconcentra-
tion of methionine and the metabolic activation system, S9, and, therefore, con-
cludedthattheresultsofthetestwerenegative(Green,1981).Inasimilarstudy,
usingthesamestrainsofE. coli(WP2,WP67uvrApolAandCM871uvrArecAlexA)
D,L-methioninegavenegativeresultswithandwithoutmetabolicactivationatcon-
centrationsofupto1000mg/ml(Tweats,1981).
NoevidenceofmutagenicitywasreportedwhenvariousstrainsofE. coliwere
incubated with valine (No. 1426), L-histidine (No. 1431), or L-tyrosine (No. 1434)
atconcentrationsofupto5000mg/platewithoutmetabolicactivation(Flucketal.,
1976;Martinezetal.,2000).
In a Rec assay using Bacillus subtilis H17 and M45, glycine (No. 1421),
D,L-isoleucine (No. 1422), D,L-methionine (No. 1424), D,L-valine (No. 1426), and
5
Forconversionsused,seeTable4.
AMINO ACIDS AND RELATED SUBSTANCES 471
D,L-alanine(No.1437)consistentlygavenegativeresultswhenincubatedatcon-
centrationsupto10000mg/mlwithandwithoutmetabolicactivation(Kada,1981;
Kurodaetal.1984).
In a test designed to investigate potential induction of aneuploidy in Saccha-
romyces cerevisiae strain D6, D,L-methionine (No. 1424) at a concentration of
50mg/ml was considered to produce a false positive response (Parry & Sharp,
1981).Theauthorsconcludedthattheeffectwascausedbyselectivestimulation
ofgrowthofmonosomiccellsinamethionine-richenvironment,attheexpenseof
theoriginaldiploidcells,owingtolossofthewild-typealleleoftheMet13genein
themonosomiccells.Althoughmethionine(dissolvedindimethylsulfoxide)atcon-
centrationsofuptoandincluding750mg/mlcausedadoublingofthenumberof
revertantscomparedwithcontrolvaluesinatestevaluatingmitoticgeneconver-
sion in S. cerevisiae strain JD1; the difference was not statistically signifcant
(Sharp&Parry,1981).Additionally,whendistilledwaterwasusedasthesolvent,
thenumbersofrevertantsobservedwithmethionineatconcentrationsofuptoand
including750mg/mlwerecomparabletothoseinthecontrols.Overall,theauthors
concludedthatmethioninedoesnotinducemitoticaneuploidyorgeneconversion
intheyeaststrainstested(Parry&Sharp,1981;Sharp&Parry,1981).InS. cere-
visiaestrainsD7andD4,D,L-methionineatconcentrationsupto1600mg/mland
333mg/plate, respectively, tested negative for mitotic gene conversion with and
without metabolic activation (Jagannath et al., 1981; Zimmermann & Scheel,
1981).
InanassayforforwardmutationinmouselymphomaL5178YTk
+/-
cells,D,L-
methionine(No.1424)andL-tyrosine(No.1434)didnotinducemutationsatcon-
centrationsupto3000mg/mland271.8mg/ml,respectively(Jotz&Mitchell,1981;
Garbergetal.,1988).
Sixteenaminoacids(L-cysteine,No.1419;L-glutamicacid,No.1420;glycine,
No. 1421; L-isoleucine, No. 1422; leucine, No. 1423; L-methionine, No. 1424; L-
proline,No.1425;L-valine,No.1426;L-phenylalanine,No.1428;L-asparticacid,
No. 1429; L-glutamine, No. 1430; L-histidine, No. 1431; L-tyrosine, No. 1434; L-
alanine, No. 1437; L-arginine, No. 1438; L-lysine, No. 1439) at concentrations of
10, 50 and 100mg/ml produced slight increases in sister chromatid exchanges
(SCEs)inhumanlymphocytes.TheelevatedfrequenciesofSCEweresimilarat
all three doses and were considered to be metabolic rather than genotoxic
responses(Xing&Na,1996).Furthermore,D,L-methionine(1005000mg/ml)and
taurine (No. 1435) (125mg/ml) showed no evidence of SCE in Chinese hamster
ovarycells,withorwithoutmetabolicactivation(Evans&Mitchell,1981;Natarajan
&vanKesteren-vanLeeuwen,1981;Perry&Thomson,1981;Cozzietal.,1995).
Also, no SCEs were observed when Chinese hamster V79 cells were incubated
withL-cysteine(No.1419)(12.1121mg/ml)(Speitetal.,1980).
L-Cysteine (No. 1419), D,L-methionine (No. 1424), L-glutamine (No. 1430), L-
tyrosine (No. 1434), and taurine (No. 1435) produced uniformly negative results
inassaysforchromosomalaberrationwhenincubatedatconcentrationsofupto
5000mg/mlinChinesehamsterovarycells(Natarajan&vanKesteren-vanLeeuwen,
1981; Stich et al., 1981; Cozzi et al., 1995; Tavares et al., 1998). Similarly, in
ChinesehamsterlungV79fbroblastsincubatedwithmethionineatconcentrations
472 AMINO ACIDS AND RELATED SUBSTANCES
T
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AMINO ACIDS AND RELATED SUBSTANCES 473
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t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
A
1
5
3
5
,

1
0

0
0
0

m
g
/
p
l
a
t
e

N
e
g
a
t
i
v
e
b

B
a
k
e
r

&

B
o
n
i
n

(
1
9
8
1
)

(
u
n
s
p
e
c
i
f
e
d
)

T
A
1
5
3
7
,
T
A
1
5
3
8
,

T
A
9
8

&

T
A
1
0
0
1
4
2
4

M
e
t
h
i
o
n
i
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
A
1
5
3
5
,

0
.
2
,

2
,

2
0
,

2
0
0

o
r

N
e
g
a
t
i
v
e
b
,
c

B
r
o
o
k
s

&

D
e
a
n

(
1
9
8
1
)

(
u
n
s
p
e
c
i
f
e
d
)

T
A
1
5
3
7
,

T
A
1
5
3
8
,

0
0
0

m
g
/
p
l
a
t
e

T
A
9
8
,

T
A
9
2

&

T
A
1
0
0
1
4
2
4

M
e
t
h
i
o
n
i
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
A
1
5
3
5
,

4
,

2
0
,

1
0
0
,

5
0
0

o
r

N
e
g
a
t
i
v
e
b

T
r
u
e
m
a
n

(
1
9
8
1
)

(
u
n
s
p
e
c
i
f
e
d
)

T
A
1
5
3
7
,

T
A
1
5
3
8
,

T
A
9
8

5
0
0

m
g
/
p
l
a
t
e

&

T
A
1
0
0
1
4
2
4

M
e
t
h
i
o
n
i
n
e

R
e
v
e
r
s
e

m
u
t
a
t
i
o
n

S
.

t
y
p
h
i
m
u
r
i
u
m

T
A
9
8

&

1
0
,

2
0
,

5
0
,

1
0
0
,

2
0
0

N
e
g
a
t
i
v
e
b

V
e
n
i
t
t

&

C
r
o
f
t
o
n
-
S
l
e
i
g
h

(
1
9
8
1
)

(
u
n
s
p
e
c
i
f
e
d
)

T
A
1
0
0

o
r

5
0
0

m
g
/
p
l
a
t
e
1
4
2
4

M
e
t
h
i
o
n
i
n
e

M
u
t
a
t
i
o
n

E
.

c
o
l
i

5
0
0

m
g
/
p
l
a
t
e

N
e
g
a
t
i
v
e
b

V
e
n
i
t
t

&

C
r
o
f
t
o
n
-
S
l
e
i
g
h

(
1
9
8
1
)

(
u
n
s
p
e
c
i
f
e
d
)

W
P
2

&

W
P
2

u
v
r
A
1
4
2
4

M
e
t
h
i
o
n
i
n
e

M
u
t
a
t
i
o
n

E
.

c
o
l
i

J
C
2
9
2
1
,

J
C
9
2
3
9
,

0
0
0

0
0
0

m
g
/
m
l

N
e
g
a
t
i
v
e
f

I
c
h
i
n
o
s
u
b
o

e
t

a
l
.

(
1
9
8
1
b
)

(
u
n
s
p
e
c
i
f
e
d
)

J
C
8
4
7
1
,

J
C
5
5
1
9
,

J
C
7
6
8
9
,

J
C
7
6
2
3
1
4
2
4

M
e
t
h
i
o
n
i
n
e

M
u
t
a
t
i
o
n

E
.

c
o
l
i

N
R

N
e
g
a
t
i
v
e
c
,
h

M
a
t
s
u
s
h
i
m
a

e
t

a
l
.

(
1
9
8
1
)

(
u
n
s
p
e
c
i
f
e
d
)

W
P
2

u
v
r
A

&

W
P
2

u
v
r
A
(
p
K
M
1
0
1
)
1
4
2
4

M
e
t
h
i
o
n
i
n
e

M
u
t
a
t
i
o
n

E
.

c
o
l
i

3
4
3
/
1
1
3
/
u
v
r
B

&

0
0
0

m
g
/
m
l

N
e
g
a
t
i
v
e
b

M
o
h
n

e
t

a
l
.

(
1
9
8
1
)

(
u
n
s
p
e
c
i
f
e
d
)

3
4
3
/
1
1
3
/
u
v
r
B
/
l
e
u
8
1
4
2
4

M
e
t
h
i
o
n
i
n
e

M
u
t
a
t
i
o
n

E
.

c
o
l
i

W
P
2
,

W
P
6
7

2
5
0
,

5
0
0

o
r

N
e
g
a
t
i
v
e
b

T
w
e
a
t
s

(
1
9
8
1
)

(
u
n
s
p
e
c
i
f
e
d
)

u
v
r
A
p
o
l
A

&

C
M
8
7
1

0
0
0

m
g
/
m
l

u
v
r
A
r
e
c
A
l
e
x
A
1
4
2
4

M
e
t
h
i
o
n
i
n
e

M
u
t
a
t
i
o
n

E
.

c
o
l
i

W
P
2
,

W
P
6
7

5
0
0

m
g
/
m
l

N
e
g
a
t
i
v
e
b

G
r
e
e
n

(
1
9
8
1
)

(
u
n
s
p
e
c
i
f
e
d
)

u
v
r
A
p
o
l
A

&

C
M
8
7
1

u
v
r
A
r
e
c
A
l
e
x
A
1
4
2
4

d
,
l
-
M
e
t
h
i
o
n
i
n
e

M
u
t
a
t
i
o
n

E
.

c
o
l
i

P
3
4
7
8

0
0
0

m
g
/
p
l
a
t
e

N
e
g
a
t
i
v
e
e

F
l
u
c
k

e
t

a
l
.

(
1
9
7
6
)
474 AMINO ACIDS AND RELATED SUBSTANCES
T
a
b
l
e

5
.

(
C
o
n
t
d
)
N
o
.

F
l
a
v
o
u
r
i
n
g

a
g
e
n
t

E
n
d
-
p
o
i
n
t

T
e
s
t

s
y
s
t
e
m

D
o
s
e

o
r

c
o
n
c
e
n
t
r
a
t
i
o
n

R
e
s
u
l
t

R
e
f
e
r
e
n
c
e
1
4
2
4

d
,
l
-
M
e
t
h
i
o
n
i
n
e

M
u
t
a
t
i
o
n

E
.

c
o
l
i

W
P
2

u
v
r
A

1
0

0
0
0

m
g
/
m
l

P
o
s
i
t
i
v
e
e
,
f
,
i

G
a
t
e
h
o
u
s
e

(
1
9
8
1
)
1
4
2
4

d
,
l
-
M
e
t
h
i
o
n
i
n
e

M
u
t
a
t
i
o
n

E
.

c
o
l
i

W
P
2

u
v
r
A

1
0

0
0
0

m
g
/
m
l

N
e
g
a
t
i
v
e
f
,
i

G
a
t
e
h
o
u
s
e

(
1
9
8
1
)
1
4
2
4

d
,
l
-
M
e
t
h
i
o
n
i
n
e

R
e
c
o
m
b
i
n
a
t
i
o
n

B
.

s
u
b
t
i
l
i
s

H
1
7
r
e
c
+

&

2
0

m
l
/
d
i
s
k

N
e
g
a
t
i
v
e
j

K
a
d
a

(
1
9
8
1
)

M
4
5
r
e
c
-
1
4
2
4

d
,
l
-
M
e
t
h
i
o
n
i
n
e

M
i
t
o
t
i
c

g
e
n
e

S
.

c
e
r
e
v
i
s
i
a
e

D
4

0
.
3

3
3
3

m
g
/
p
l
a
t
e

N
e
g
a
t
i
v
e
b

J
a
g
a
n
n
a
t
h

e
t

a
l
.

(
1
9
8
1
)

c
o
n
v
e
r
s
i
o
n
1
4
2
4

d
,
l
-
M
e
t
h
i
o
n
i
n
e

A
n
e
u
p
l
o
i
d
y

S
.

c
e
r
e
v
i
s
i
a
e

D
6

5
0

m
g
/
m
l

N
e
g
a
t
i
v
e
b
,
k

P
a
r
r
y

&

S
h
a
r
p

(
1
9
8
1
)
1
4
2
4

d
,
l
-
M
e
t
h
i
o
n
i
n
e

M
i
t
o
t
i
c

g
e
n
e

S
.

c
e
r
e
v
i
s
i
a
e

D
7

6
0
0

m
g
/
m
l

N
e
g
a
t
i
v
e
f

Z
i
m
m
e
r
m
a
n

&

S
c
h
e
e
l

(
1
9
8
1
)

c
o
n
v
e
r
s
i
o
n
1
4
2
4

d
,
l
-
M
e
t
h
i
o
n
i
n
e

M
i
t
o
t
i
c

g
e
n
e

S
.

c
e
r
e
v
i
s
i
a
e

J
D
1

7
5
0

m
g
/
m
l

N
e
g
a
t
i
v
e
b

S
h
a
r
p

&

P
a
r
r
y

(
1
9
8
1
)

c
o
n
v
e
r
s
i
o
n
1
4
2
4

d
,
l
-
M
e
t
h
i
o
n
i
n
e

F
o
r
w
a
r
d

m
u
t
a
t
i
o
n

M
o
u
s
e

l
y
m
p
h
o
m
a

4
6
.
9

0
0
0

m
g
/
m
l

N
e
g
a
t
i
v
e
b

J
o
t
z

&

M
i
t
c
h
e
l
l

(
1
9
8
1
)

L
5
1
7
8
Y

c
e
l
l
s
1
4
2
4

d
,
l
-
M
e
t
h
i
o
n
i
n
e

F
o
r
w
a
r
d

m
u
t
a
t
i
o
n

M
o
u
s
e

l
y
m
p
h
o
m
a

0
.
0
0
0
5

0
.
0
1
5

m
o
l
/
l

N
e
g
a
t
i
v
e

f
,
i

G
a
r
b
e
r
g

e
t

a
l
.

(
1
9
8
8
)

L
5
1
7
8
Y

T
k
+
/
-

(
7
4
.
6

2
3
8

m
g
/
m
l
)
m
1
4
2
4

l
-
M
e
t
h
i
o
n
i
n
e

S
i
s
t
e
r

c
h
r
o
m
a
t
i
d

H
u
m
a
n

l
y
m
p
h
o
c
y
t
e
s

2
1
,

6
1

o
r

1
1
1

m
g
/
m
l
c

N
e
g
a
t
i
v
e
d
,
e

X
i
n
g

&

N
a

(
1
9
9
6
)

e
x
c
h
a
n
g
e
1
4
2
4

d
,
l
-
M
e
t
h
i
o
n
i
n
e

S
i
s
t
e
r

c
h
r
o
m
a
t
i
d

C
h
i
n
e
s
e

h
a
m
s
t
e
r

o
v
a
r
y

0
.
1
,

1
,

1
0

o
r

1
0
0

m
g
/
m
l

N
e
g
a
t
i
v
e
b

P
e
r
r
y

&

T
h
o
m
s
o
n

(
1
9
8
1
)

e
x
c
h
a
n
g
e

c
e
l
l
s
1
4
2
4

d
,
l
-
M
e
t
h
i
o
n
i
n
e

S
i
s
t
e
r

c
h
r
o
m
a
t
i
d

C
h
i
n
e
s
e

h
a
m
s
t
e
r

o
v
a
r
y

6
7
0
,

3
0
0

o
r

N
e
g
a
t
i
v
e
g

N
a
t
a
r
a
j
a
n

&

v
a
n

e
x
c
h
a
n
g
e

c
e
l
l
s

0
0
0

m
g
/
m
l

K
e
s
t
e
r
e
n
-
v
a
n

L
e
e
u
w
e
n

(
1
9
8
1
)
1
4
2
4

d
,
l
-
M
e
t
h
i
o
n
i
n
e

S
i
s
t
e
r

c
h
r
o
m
a
t
i
d

C
h
i
n
e
s
e

h
a
m
s
t
e
r

o
v
a
r
y

2
1
.
8
8

3
5
0

m
g
/
m
l

N
e
g
a
t
i
v
e
b

E
v
a
n
s

&

M
i
t
c
h
e
l
l

(
1
9
8
1
)

e
x
c
h
a
n
g
e

c
e
l
l
s
1
4
2
4

d
,
l
-
M
e
t
h
i
o
n
i
n
e

C
h
r
o
m
o
s
o
m
a
l

C
h
i
n
e
s
e

h
a
m
s
t
e
r

o
v
a
r
y

6
7
0
,

3
0
0

o
r

N
e
g
a
t
i
v
e
f

N
a
t
a
r
a
j
i
n

&

v
a
n

K
e
s
t
e
r
e
n
-
v
a
n

a
b
e
r
r
a
t
i
o
n
s

c
e
l
l
s

0
0
0

m
g
/
m
l

L
e
e
u
w
e
n

(
1
9
8
1
)
1
4
2
4

d
,
l
-
M
e
t
h
i
o
n
i
n
e

C
h
r
o
m
o
s
o
m
a
l

R
a
t

h
e
p
a
t
o
c
y
t
e
s

5
0
,

1
0
0

o
r

2
0
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9
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1
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(
1
9
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1
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s
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s
AMINO ACIDS AND RELATED SUBSTANCES 475
1
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4

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(
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476 AMINO ACIDS AND RELATED SUBSTANCES
T
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5
.

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478 AMINO ACIDS AND RELATED SUBSTANCES
of45to1494mg/ml,noincreaseinthefrequencyofchromosomeaberrationswas
reported(Swenbergetal.,1976).Inastudyevaluatingthepotentialinductionof
chromosomal aberrations in rat hepatocytes treated with methionine at doses of
upto200mg/ml,resultswerereportedtobeinconclusive(Dean,1981).
No unscheduled DNA synthesis (UDS) was observed when human fbroblast
cells were incubated with D,L-methionine (No. 1424) at a concentration of up to
1000mg/mlwithmetabolicactivation(Agrelo&Amos,1981).HeLaS3cellsincu-
batedwithD,L-methionineataconcentrationsofupto100mg/mlalsoshowedno
evidenceofUDS(Martin&McDermid,1981).Furthermore,noUDSwasobserved
inWI-38humanfbroblastsincubatedwithD,L-methionineatconcentrationsof63
to1000mg/mlintheabsenceofmetabolicactivation.However,inthepresenceof
metabolicactivation,aweakUDSresponsewasobserved,whichappearedtobe
dose-relatedatconcentrationsof125to2000mg/ml(Robinson&Mitchell,1981).
(ii) In vivo
ThreemaleandthreefemaleWistarratsweregivenglutamine(No.1430)as
asingledoseat600mg/kgbwbygavage.Preparationsofmetaphasecellswere
obtainedfromsamplesofbonemarrow.Whencomparedwithuntreatedcontrols,
ratsreceivingglutaminedidnotexhibitanincreaseinthenumberofchromosomal
aberrations(Tavaresetal.,1998).
MaleCBA/Jmiceweregivenmethionine(No.1424)atadoseof0,1,10,100
or1000mg/kgbwbyintraperitonealinjection.Partialhepatectomywasperformed
onhalfoftheanimals,whichwerethengivenupto54htoallowforliverregenera-
tion.At termination, the mice were sacrifced and bone marrow and hepatocytes
wereharvestedforanalysis.Itwasreportedthat,overall,methioninedidnotinduce
SCE in the bone marrow of intact or partially hepatectomized mice (Paika et al.,
1981).
Inamodifedassayformicronucleusformationinbonemarrow,B6C3F
1
mice
wereinjectedintraperitoneallywithmethionineatadoseof35mg/kgbwat0and
24h.Samplesofbonemarrowwereharvestedat48,72and96h,andanalysed
forinductionofmicronuclei.Methioninewasreportedtocauseincreasedformation
of micronuclei in samples harvested at 72h only. However, in a subsequent test
conductedunderthesameconditions(samplesofbonemarrowobtainedonlyat
48 and 72h), methionine at a dose of 3.7, 17.5 or 35mg/kgbw did not increase
theformationofmicronuclei(Salamoneetal.,1981).Inanothertestforinduction
of micronuclei, CD-1 mice were given methionine at a dose of 0, 250, 500 or
1000mg/kgbw as two intraperitoneal injections separated by an interval of 24h.
The mice were sacrifced 6h after the last injection, and the bone marrow was
harvestedforanalysis.Therewasnoincreaseinthefrequencyofmicronucleated
polychromaticerythrocytes(Tsuchimoto&Matter,1981).
(iii) Conclusion
Theaminoacidstestedwerefoundtobenon-genotoxicinvitroandinvivoin
avarietyoftestsystems.
AMINO ACIDS AND RELATED SUBSTANCES 479
(d) Reproductive toxicity
AstudywasperformedtodeterminetheeffectofexcessL-cysteineonrepro-
ductioninrats.GroupsoffourpregnantCDNorwegianhoodedinbredMiddleAston
ratswerefeddietscontainingL-cysteineataconcentrationofeither0or3500mg/
kgofdietdaily(equivalenttoapproximately0and175mg/kgbwperday,respec-
tively)forthedurationofgestationandthroughoutweaningofthepups.Atweaning,
thedamsandallexcepttwomaleandthreefemaleoffspring(retainedforbreed-
ing)fromeachlitterweresacrifced.Thenumberofpupsborn,litterweightatbirth
andweaning,andnumbersatweaningwererecorded.Aftersacrifce,theweights
of the carcass, liver and kidney were recorded, and an examination for gross
lesionswasperformed.Thisprotocolwasfollowedforthesecondandthirdgenera-
tions.Atthefourthgeneration,12malesand24femaleswereretainedfromeach
dietandbred.Thesixthgenerationwasdividedintofourgroupsthatweregiven
L-cysteineataconcentrationof0,35,350or3500mg/kgofdietdaily(equivalent
toapproximately0,1.75,17.5and175mg/kgbwperday,respectively).Thestudy
wasterminatedattheseventhgeneration.Grossandmicroscopicexaminationof
animals from the ffth generation revealed no evidence of pathology. There was
an increase in absolute weights of the liver and kidney for animals in the group
receivingthehighestdose,butnosignifcantdifferencesinrelativeweightsofthe
liverandkidneywerereportedforthisgroup.Theauthorsconcludedthatnosig-
nifcantabnormalitiesrelatedtotheadministrationofcysteineoversixgenerations
wereobserved(Frapeetal,1971).
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K2
487
TETRAHYDROFURAN AND FURANONE DERIVATIVES
First draft prepared by
Professor G. Williams
1
and Dr D.G. Hattan
2
1
Environmental Pathology and Toxicology, New York Medical College,
Valhalla, NY, USA; and
2
Offce of Food Additives Safety, Center for Food Safety and Applied
Nutrition, Food and Drug Administration, College Park, MD, USA
Evaluation ............................................................................... 487
Introduction......................................................................... 487
Estimateddailyintake........................................................ 488
Absorption,distribution,metabolismandelimination........ 488
ApplicationoftheProcedurefortheSafety
EvaluationofFlavouringAgents................................. 497
Considerationofsecondarycomponents.......................... 497
Considerationofcombinedintakesfromuseas
favouringagents......................................................... 498
Conclusions........................................................................ 498
Relevantbackgroundinformation............................................. 498
Additionalconsiderationsonintake................................... 498
Biologicaldata.................................................................... 499
Biochemicaldata......................................................... 499
Hydrolysis.............................................................. 499
Absorption,distributionandexcretion................... 499
Metabolism............................................................ 499
Toxicologicalstudies.................................................... 501
Acutetoxicity......................................................... 501
Short-termstudiesoftoxicity................................. 502
Long-termstudiesoftoxicity
andcarcinogenicity......................................... 512
Genotoxicity........................................................... 513
Otherrelevantstudies:anti-tumourstudies.......... 519
References............................................................................... 519
1. EVALUATION
1.1 Introduction
TheCommitteeevaluatedagroupof18tetrahydrofuranandfuranonefavour-
ing agents (Table 1) by the Procedure for the Safety Evaluation of Flavouring
Agents (see Figure 1, p 192). The Committee has not previously evaluated any
memberofthegroup.
Twelveofthe18favouringagentsinthisgroup(Nos1443,1446,14481457)
havebeenreportedtooccurnaturallyinvariousfoods.Theyhavebeendetected
instrawberries,pineapple,mango,otherfruits,shoyu,cookedbeefandpork,fried
488 TETRAHYDROFURAN AND FURANONE DERIVATIVES
K2
chicken, roasted hazelnuts and peanuts, cocoa, mat, black and green teas,
smokedfsh,popcorn,andSwisscheese(Nijssenetal.,2003).
1.2 Estimated daily intake
Thetotalannualvolumeofproductionofthe18tetrahydrofuranandfuranone
derivatives is approximately 40000kg in both Europe (International Organization
oftheFlavorIndustry,1995)andintheUSA(NationalAcademyofSciences,1982,
1987; Lucas et al., 1999) (see Table 2). Approximately 92% of the total annual
volumeofproductioninEuropeandapproximately98%intheUSAisaccounted
for by 4-hydroxy-2,5-dimethyl-3(2H)-furanone (DMHF; No. 1446). The estimated
daily intake of DMHF was 5300mg/person in Europe and 5200mg/person in the
USA.Thedailyintakesofalltheotherfavouringagentsinthisgroupwereinthe
rangeof0.001to238mg/person,withmostvaluesbeingatthelowerendofthis
range (National Academy of Sciences, 1982, 1987; Lucas et al., 1999). The
estimateddailypercapitaintakeofeachagentinEuropeandtheUSAisreported
inTable1.
1.3 Absorption, distribution, metabolism and elimination
Thefourestersinthischemicalgroup(Nos1442,1444,1445and1447)are
expected to be hydrolysed to tetrahydrofurfuryl alcohol and the corresponding
carboxylicacids.Tetrahydrofuranderivativesarerapidlyabsorbedandeliminated
primarilyintheurineinlaboratoryanimals.
Hydroxyl-substitutedtetrahydrofuranandfuranonederivativesarepredictedto
form glucuronic acid conjugates, which are primarily excreted in the urine. In
humansfedwithfreshstrawberries,DMHF(No.1446)israpidlyabsorbed,conju-
gatedintheliverwithglucuronicacidandexcretedintheurine.Themetabolism
of the tetrahydrofurfuryl alcohol derivatives is anticipated to be similar to that of
the furfuryl alcohol derivatives. These compounds will not form epoxides. After
hydrolysisofthetetrahydrofurfurylesters,theresultingprimaryalcoholisoxidized
tothecorrespondingcarboxylicacid,conjugatedandexcretedintheurine(Nomeir
etal.,1992).Theremainingtetrahydrofurfurylalcohol,linalooloxide(No.1454)is
atertiaryalcoholthatisconjugatedwithglucuronicacidandexcretedintheurine
(Parkeetal.,1974).
Thealkyl-substitutedtetrahydrofuranderivativesaresubjectedtoringorside-
chainhydroxylationcatalysedbyhumancytochromeP450(CYP)toyieldringor
side-chain-substituted alcohols that may be conjugated with glucuronic acid and
excreted,orfurtheroxidized,conjugated,andexcretedintheurine(Whiteetal.,
1979; Guengerich et al., 1984; Kremers & Beaune, 1987; Ortiz de Montellano,
1995).
Genotoxicityobservedwithsomemembersofthegroup(Nos1446,1449and
1450)wasconsideredtobeaneffectcausedbyhighdoseandrelatedtoamecha-
nism involving reactive oxygen species, rather than the generation of a reactive
metabolite, such as an epoxide. DMHF (No 1446) showed no evidence of carci-
nogenicityina2-yearstudyinwhichratsweregivenadoseofupto400mg/kgbw
perday(Kelly&Bolte,2003).
TETRAHYDROFURAN AND FURANONE DERIVATIVES 489
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K2
494 TETRAHYDROFURAN AND FURANONE DERIVATIVES
K2
Table 2. Annual volumes of production of tetrahydrofuran and furanone
derivatives used as favouring agents in Europe and the USA
Flavouring Mostrecent Intake
b
Annualvolumein Consumption
agent(No.)

annual
mg/day mg/kgbw
naturallyoccurring ratio
d

volume(kg)
a
perday
foods(kg)
c
2-Hexyl-4-acetoxytetrahydrofuran(1440)
Europe ND ND ND
USA
e
4.00 0.7 0.01 - NA
2-(3-Phenylpropyl)tetrahydrofuran(1441)
Europe 0.01 0.001 0.00002
USA 5 0.7 0.01 - NA
Tetrahydrofurfurylacetate(1442)
Europe 5 0.7 0.01
USA 64 8 0.1 - NA
Tetrahydrofurfurylalcohol(1443)
Europe 270 39 0.6
USA 168 22 0.4 + NA
Tetrahydrofurfurylbutyrate(1444)
Europe 0.1 0.01 0.0002
USA
e
1 0.2 0.003 - NA
Tetrahydrofurfurylpropionate(1445)
Europe 0.4 0.06 0.001
USA 37 5 0.08 - NA
4-Hydroxy-2,5-dimethyl-3(2H)-furanone,DMHF(1446)
Europe 36818 5254 88
USA 39500 5203 87 45601 1
Tetrahydrofurfurylcinnamate(1447)
Europe ND ND ND
USA
e
0.05 0.01 0.0001 - NA
2-Methyltetrahydrofuran-3-one(1448)
Europe 171 24 0.4
USA 68 9 0.1 2409 35
2-Ethyl-4-hydroxy-5-methyl-3(2H)-furanone,HEMF(1449)
Europe 1666 238 4
USA 100 13 0.2 + NA
4-Hydroxy-5-methyl-3(2H)-furanone(1450)
Europe 391 56 0.9
USA 0.5 0.07 0.001 5 10
2,5-Dimethyl-4-methoxy-3(2H)-furanone(1451)
Europe 100 14 0.2
USA
f
5 0.9 0.01 699 140
2,2-Dimethyl-5-(1-methylpropen-1-yl)tetrahydrofuran(1452)
Europe 78 11 0.2
USA
e
0.2 0.04 0.0006 + NA
2,5-Diethyltetrahydrofuran(1453)
Europe 0.1 0.01 0.0002
USA
f
0.5 0.09 0.001 + NA
Linalooloxide(1454)
Europe 594 85 1
USA 109 14 0.2 6233 57
TETRAHYDROFURAN AND FURANONE DERIVATIVES 495
K2
Table 2. (Contd)
Flavouring Mostrecent Intake
b
Annualvolumein Consumption
agent(No.)

annual
mg/day mg/kgbw
naturallyoccurring ratio
d

volume(kg)
a
perday
foods(kg)
c
5-Isopropenyl-2-methyl-2-vinyltetrahydrofuran(1455)
Europe 9 1 0.02
USA
e
0.2 0.04 0.0006 + NA
4-Acetoxy-2,5-dimethyl-3(2H)furanone(1456)
Europe ND ND ND
USA
f
45 8 0.1 +
g
NA
()-2-(5-Methyl-5-vinyltetrahydrofuran-2-yl)propionaldehyde(1457)
Europe ND ND ND
USA
e
5 0.9 0.01 +
h
NA
Total
Europe 40103
USA 40112
NA,notavailable;ND,nointakedatareported;+,reportedtooccurnaturallyinfoods
(Nijssenetal.,2003),butnoquantitativedata;-,notreportedtooccurnaturallyinfoods.
a
FromInternationalOrganizationoftheFlavorIndustry(1995)andLucasetal.(1999)or
NationalAcademyofSciences(1982,1987).
b
Intakeexpressedasmg/personperdaywascalculatedasfollows:[(annualvolume,kg)
(110
9
mg/kg)]/[populationsurveycorrectionfactor365days],wherepopulation
(10%,eatersonly)=3210
6
forEuropeand2610
6
fortheUSA.Thecorrection
factor=0.6forEurope,and0.8fortheUSA,representingtheassumptionthatonly60
and80%oftheannualvolumeofthefavour,respectively,wasreportedinthepoundage
surveys(NationalAcademyofSciences,1982,1987;InternationalOrganizationofthe
FlavorIndustry,1995;Lucas etal.,1999).
Intakeexpressedasmg/kgbwperdaywascalculatedasfollows:[(mg/personperday)/
bodyweight],wherebodyweight=60kg.Slightvariationsmayoccurfromrounding.
c
QuantitativedatafortheUSAreportedbyStofberg&Grundschober(1987).
d
Theconsumptionratioiscalculatedasfollows:(annualconsumptionviafood,kg)/(most
recentlyreportedvolumeasafavouringagent,kg).
e
Thevolumecitedistheanticipatedannualvolume,whichwasthemaximumamountof
favourestimatedtobeusedannuallybythemanufactureratthetimethematerialwas
proposedforfavouruse.Subsequentnationalsurveys(NationalAcademyofSciences,
1982,1987;Lucasetal.,1999),ifapplicable,revealednoreporteduseofthesubstance
asafavouringredient.
f
AnnualvolumereportedinpreviousUSAsurveys(NationalAcademyofSciences,1982,
1987).
g
PersonalcommunicationfromFlavorandExtractManufacturersAssociationoftheUSA
(1994).
h
vanDortetal.(1993).
496 TETRAHYDROFURAN AND FURANONE DERIVATIVES
K2
1.4 Application of the procedure for the safety evaluation
of flavouring agents
Step 1. InapplyingtheProcedure,theCommitteeassigned11ofthe18agents
(Nos1446,14481457)tostructuralclassII.Sevenagents(Nos1440
1445 and 1447) were assigned to structural class III (Cramer et al.,
1978).
Step 2. All18tetrahydrofuranandfuranonederivatives(Nos14401457)inthis
groupareexpectedtobemetabolizedtoinnocuousproducts.Theevalu-
ation of these 18 favouring agents therefore proceeded via theA-side
ofthedecision-tree.
Step A3. TheestimateddailyintakesinEuropeandtheUSAof10ofthe11fa-
vouring agents in structural class II, and of all seven of the favouring
agents in structural class III are below the threshold of concern (i.e.
540mg/person for class II, and 90mg/person for class III).According to
theProcedure,thesafetyofthese17favouringagentsraisesnoconcern
whentheyareusedattheirestimatedcurrentintakes.Oneofthefavour-
ingagentsinstructuralclassII,DMHF(No.1446),exceedsthethreshold
ofconcernforthatclass.ThedailyintakeofDMHFwas5300mg/person
inEuropeand5200mg/personintheUSA.AccordingtotheProcedure,
theevaluationofthisfavouringagentproceededtostepA4.
Step A4. DMHF(No.1446)oritsmetabolitesarenotendogenous.Therefore,the
evaluationofthisfavouringagentproceededtostepA5.
Step A5. For DMHF (No. 1446), the no-observed-effect level (NOEL) of 200mg/
kgbw per day from a 2-year dietary study in rats (Kelly & Bolte, 2003)
is>2300timesgreaterthantheestimateddailypercapitaintakeofthis
agent from its use as a favouring agent in Europe or the USA. The
Committee therefore concluded that the safety of this agent would not
beaconcernattheestimatedcurrentintake.
Theintakeconsiderationsandotherinformationusedtoevaluatethe18tetra-
hydrofuranandfuranonederivativesinthisgroupaccordingtotheProcedureare
summarizedinTable1.
1.5 Consideration of secondary components
Two members of this group of favouring agents (Nos 1456 and 1457) have
minimumassayvaluesof<95%.Informationonthesafetyofthesecondarycom-
ponents of these two compounds is summarized in Annex 5 (Summary of the
safety evaluation of secondary components for favouring agents with minimum
assayvaluesoflessthan95%).ThesecondarycomponentofNo.1456(DMHF,
No.1446)wasevaluatedbytheCommitteeatitspresentmeetingandwascon-
siderednottobeaconcernatcurrentestimatedintakes.Thesecondarycompo-
nentofNo.1457(6-hydroxy-2,6-dimethyl-2,7-octadienal)hasnotbeenpreviously
evaluated by the Committee. The Committee did evaluate a structurally related
compound(hydroxycitronellal,No.611)atitsffty-thirdmeeting(Annex1,reference
143) and concluded that it did not present a safety concern at estimated current
TETRAHYDROFURAN AND FURANONE DERIVATIVES 497
K2
intakes.Onthisbasis,theCommitteeconsideredthat6-hydroxy-2,6-dimethyl-2,7-
octadienaldidnotposeasafetyconcernatcurrentestimatedintakes.
1.6 Consideration of combined intakes from use as favouring agents
IntheunlikelyeventthatallsevenagentsinstructuralclassIIIwereconsumed
concurrently on a daily basis, the estimated combined intake would not exceed
theintakethresholdforclassIII(90mg/personperday).Intheunlikelyeventthat
all 11 agents in structural class II were consumed concurrently on a daily basis,
theestimatedcombinedintakewouldexceedthehumanintakethresholdforclass
II(540mg/personperday).Nevertheless,allthesefavouringagentsareexpected
tobeeffcientlymetabolizedandwouldnotsaturatemetabolicpathways.Overall
evaluation of the data indicated that combined intake would not raise a safety
concern.
1.7 Conclusions
The Committee concluded that none of the favouring agents in this group of
tetrahydrofuran and furanone derivatives would present safety concerns at esti-
matedcurrentintakes.TheCommitteenotedthattheavailabledataonthetoxicity
andmetabolismofthesetetrahydrofuranandfuranonederivativeswereconsistent
withtheresultsofthesafetyevaluationusingtheProcedure.
2. RELEVANT BACKGROUND INFORMATION
2.1 Additional considerations on intake
Tetrahydrofuran and furanone derivatives have been detected in a variety of
foodsincludingstrawberries,fruit,shoyu,cookedbeef,friedchicken,cookedpork,
smoked fsh, coffee, green and black tea, cocoa, mat, roasted hazelnuts and
peanuts,popcorn,andSwisscheeses(Nijssenetal.,2003).AsshowninTable2,
12ofthefavouringagentsinthisgrouphavebeenreportedtooccurnaturallyin
foods(Nijssenetal.,2003).Quantitativedataonnaturaloccurrenceandconsump-
tionratioshavebeenreportedforfvesubstancesinthegroupanddemonstrate
thattheirconsumptionoccurspredominantlyfromtraditionalfoods(i.e.consump-
tionratio,>1)(Stofberg&Kirschman,1985;Stofberg&Grundschober,1987)(see
Table2).
The principal member of the group, 2,5-dimethyl-4-hydroxy-3(2H)-furanone
1

(DMHF, No. 1446), is consumed as a naturally occurring constituent of approxi-

mately 20 foods. Quantitative data on natural occurrence are available for its
presenceinpineapple,raspberry,strawberry,mango,coffee,teaandSwisscheese
(Nijssen et al., 2003). On the basis of its presence in pineapples, raspberries,
strawberries,andcoffee(Stofberg&Grundschober,1987),intakeofDMHFfrom
consumptionoffoodisslightlygreaterthanfromitsconsumptionasanaddedfa-
vouring agent. On the basis of more recent data on natural occurrence (Nijssen
etal.,2003),theintakefromconsumptionoffoodexceedsthatfromintakeasan
addedfavouringagentbyatleast50%.
498 TETRAHYDROFURAN AND FURANONE DERIVATIVES
K2
2.2 Biological data
2.2.1 Biochemical data
(a) Hydrolysis
Thefourestersinthischemicalgroup(Nos.1442,1444,1445and1447)are
expected to be hydrolysed to tetrahydrofurfuryl alcohol and the corresponding
carboxylic acids. In animals, the hydrolysis of esters is catalysed by classes of
enzymesknownascarboxylesterasesoresterases(Heymann,1980).
Thehalf-livesoffurfurylestersincubatedinartifcialpancreaticfuidwithpan-
creatinrangedfrom<0.01minforaliphaticlinearcomponentcarboxylicacids,such
asfurfurylacetate,to5.10.4minforaliphaticbranched-chaincarboxylicacids,
suchasfurfurylisopentanoate.Thehalf-lifeforhydrolysisoffurfurylpropionatein
ratliverhomogenatewasalsoextremelyrapid(t
1/2
<0.01min)(Buck&Renwick,
2000). By analogy, the tetrahydrofurfuryl esters in this group are anticipated to
undergorapidhydrolysisinvivo.
(b) Absorption, distribution and excretion
Thetetrahydrofuranandfuranonederivativeswouldbeexpectedtoberapidly
absorbed and eliminated primarily in the urine of animals. DMHF (No. 1446) in
aqueoussolutionwasadministeredintraperitoneallytofourorfveICRmiceasa
singledoseat500mg/kgbw,ororallyasasingledoseat1000mg/kgbw(Hiramoto
et al., 1998). The maximum plasma concentration of DMHF, 170270mg/ml, for
thegrouptreatedintraperitoneallywasattained15minafterdosing,andgradually
disappearedafter2h.MicegivenDMHForallyshowedamaximumplasmacon-
centrationof100150mg/mlafter1545min,followedbycompletedisappearance
after2h(Hiramotoetal.,1998).
In a similar experiment, 4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone
2
(HEMF,
No.1449)atadoseof500or1000mg/kgbwwasgivenintraperitoneallyororally
toagroupofnineorfveICRmice,respectively(Hiramotoetal.,1998).Plasma
concentrations reached a maximum 15min after intraperitoneal administration
(75330mg/ml) or 1545min after oral administration (50100mg/ml). Essentially
alloftheplasmaHEMFdisappearedwithin2haftereitherintraperitonealororal
administration(Hiramotoetal.,1998).Onthebasisofthesedata,itcanbecon-
cluded that the two furanone derivatives are rapidly cleared from the plasma of
ICRmice.
WistarratsweregivenDMHFatadoseof500mg/kgbwviaintragastrictube
(Lietal.,1990).TheplasmaconcentrationofDMHFreachedapeak(43.1261mg/
ml)30minafterdosing,andgraduallydecreasedtoabout35%(14.9626mg/ml)of
themaximumplasmaconcentrationsafter8h.Within30minand8hafterdosing,
itwasnotedthat72%and95%oftheadministereddoseofDMHF,respectively,
1
Synonymfor4-hydroxy-2,5-dimethyl-3(2H)-furanone,DMHF,No.1446.
2
Synonymfor2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone,HEMF,No.1449.
TETRAHYDROFURAN AND FURANONE DERIVATIVES 499
K2
wasabsorbedintoallorgantissuesoftherats(particularlythekidney,heart,and
liver).DMHFand/oritsmetaboliteswereexcretedprimarilyintheurine,withmost
being excreted in the frst 6h after dosing (87% of the total amount of DMHF
excreted). A minor amount (1.34% of the total amount of excreted DMHF) was
eliminated in the faeces within the frst 24h.The authors provided no data as to
whatpercentageoftheadministereddoseofDMHFwasexcretedbytherats(Li
etal.,1990).
In groups of three male and female volunteers, 5994% of an ingested dose
of65.5180.6mgofDMHF(totalincludingfreeandglycosideconjugate),whichis
approximatelyequivalenttoadoseofDMHFof1.13.0mg/kgbw,wasexcretedin
the urine as the glucuronide conjugate within 24h (Roscher et al., 1997).These
datadocumenttherapidabsorptionandeliminationofDMHFand,byimplication,
otherfuranonederivatives,inanimals.
(c) Metabolism
Furanonederivativesarepredictedtoformglucuronicacidconjugates,which
areprimarilyexcretedintheurine.Metabolicdataontheprincipalfuranonederiva-
tiveinthisgroup,DMHF,indicatethatthefuranonesinthisgroupwouldberapidly
conjugatedwithglucuronicacidandexcreted(seeFigure1).Thetetrahydrofurfuryl
alcohol derivatives are anticipated to be metabolized in a manner similar to that
for furfuryl alcohol derivatives. After hydrolysis of the tetrahydrofurfuryl esters,
the resulting primary alcohol is oxidized to the corresponding carboxylic acid,
conjugated, and excreted in the urine (Nomeir et al., 1992). The remaining
tetrahydrofurfuryl alcohol, linalool oxide (No. 1454), is a tertiary alcohol that is
conjugated with glucuronic acid and excreted in the urine (Parke et al., 1974).
The alkyl-substituted tetrahydrofuran derivatives are subjected to ring or side-
chainhydroxylationcatalysedbyhumanCYPtoyieldringorsidechain-substituted
alcohols, which may be conjugated with glucuronic acid and excreted, or further
oxidized, conjugated, and excreted in the urine (White et al., 1979; Guengerich
etal.,1984;Kremers&Beaune,1987;OrtizdeMontellano,1995).
In a study of metabolism in humans, performed using fresh strawberries, a
sourceofDMHF(No.1446)(Roscheretal.,1997),twogroupsofthreevolunteers
(twomalesandonefemale)fastedfor18handsubsequentlyingestedeither2.5kg
offreshstrawberriesfromSpain,or2.53.0kgoffreshstrawberriesfromItalyover
the next 8h.Analysis of the two batches of strawberries for free DMHF and gly-
O
O OH
O
O
OGluc
major urinary
metabolite
Figure 1. Metabolic pathway for DMHF in humans
500 TETRAHYDROFURAN AND FURANONE DERIVATIVES
K2
cosideconjugatecontentrevealedthatSpanishstrawberriescontainedDMHFat
a concentration of 26.2mg/kg (ratio of free:glycoside DMHF, 2:13), while Italian
strawberriescontainedDMHFataconcentrationof60.2mg/kg(ratiooffree:gly-
coside DMHF, 3:2). This corresponds to a total intake of DMHF of 65.5mg for
volunteerswhoingestedSpanishstrawberries(approximatelyequivalentto1.1mg/
kgbw),and150.5180.6mgofDMHFforthosewhoingestedItalianstrawberries
(approximatelyequivalentto2.513.0mg/kgbw).Samplesofurinecollected24h
aftertheinitialingestionofstrawberriesrevealedthatallvolunteersexcreted59
94% of the ingested DMHF as the glucuronic acid conjugate (4-hydroxy-2,5-
dimethyl-3(2H)-furanone b-D-glucuronide). Urinary excretion of the DMHF
glucuronic acid conjugate was greater in females (8194% of the administered
dose)thaninmales(5969%oftheadministereddose),independentofthedose.
NounchangedDMHForglycosidicallyboundforms,DMHFglucosideor6-malonyl
derivative, were detected in the urine of any of the subjects (Roscher et al.,
1997).
In conclusion, furanone derivatives may undergo direct conjugation with glu-
curonic acid and rapid elimination via the urine. The esters of tetrahydrofurfuryl
alcoholarepredictedtoundergorapidhydrolysis,andtheresultingtetrahydrofur-
furylalcoholisexpectedtobeoxidizedtothecorrespondingacidthatisthencon-
jugatedandexcretedintheurine.Afterundergoingringorside-chainhydroxylation,
the 4 alkyl-substituted tetrahydrofurans are conjugated and excreted, or further
oxidized,conjugated,andexcreted.
2.2.2 Toxicological studies
(a) Acute toxicity
Oralmedianlethaldose(LD
50
)valueshavebeenreportedforeightofthetet-
rahydrofuran and furanone derivatives in this group (see Table 3). In rats, LD
50

valuesareintherangeof549mg/kgbwfor2,5-dimethyl-4-methoxy-3(2H)-furanone
(No.1451)to4500mg/kgbwfortetrahydrofurfurylalcohol(No.1443),demonstrat-
ing that the acute toxicity of these compounds when administered orally is low
(Gajewski&Alsdorf,1949;Colaianni,1967;Moreno,1977;Cooper&Good,1979;
Levenstein,1982;Reagan&Becci,1984a,1984b;Burdock&Ford,1990).Inmice,
LD
50
values range from 1000mg/kgbw for 5-isopropenyl-2-methyl-2-vinyltetrahy-
drofuran(No.1455)to>2000mg/kgbwfor2,5-dimethyl-4-methoxy-3(2H)-furanone
(No. 1451), also confrming the low acute toxicity of these compounds in this
species(Moran&Easterday,1980;Bonetti,1983).
(b) Short-term studies of toxicity
The results of short-term studies with representative tetrahydrofuran and
furanonederivativesaresummarizedinTable4andaredescribedbelow.
TETRAHYDROFURAN AND FURANONE DERIVATIVES 501
K2
Table 3. Studies of the acute toxicity of tetrahydrofuran and furanone
derivatives administered orally
No. Flavouringagent Species Sex LD
50
Reference
(mg/kgbw)
1443 Tetrahydrofurfurylalcohol Rat NR 4500 Gajewski&
Alsdorf(1949)
1448 2-Methyltetrahydrofuran-3- Mouse M,F 1860 Moran&
one Easterday
(1980)
1449 4-Hydroxy-2-ethyl-5-methyl- Mouse M,F 1932 Moran&
3(2H)-furanone(HEMF) Easterday
(1980)
1451 2,5-Dimethyl-4-methoxy- Rat NR >0.5ml/kgbw Levenstein
3(2H)-furanone (>549mg/kgbw)
a
(1982)
1452 2,2,-Dimethyl-5-(1- Rat M,F 3900 Cooper&Good
methylpropen-1-yl) (1979)
tetrahydrofuran
1453 2,5-Diethyltetrahydrofuran Rat M,F 3800 Burdock&Ford
(1990)
1453 2,5-Diethyltetrahydrofuran Rat M,F 3754 Reagan&Becci
(1984a)
1454 Linalooloxide Rat NR 1150 Moreno(1977)
1454 Linalooloxide Rat M,F 2210 Colaianni(1967)
1454 Linalooloxide Rat M,F 1924 Reagan&Becci
(1984b)
1455 5-Isopropenyl-2-methyl-2- Mouse NR 10002000 Bonetti(1983)
vinyltetrahydrofuran
F,female;M,male;NR,notreported.
a
Calculatedusingdensityof2,5-dimethyl-4-methoxy-3(2H)-furanone=1.097g/ml.
(i) 2-Hexyl-4-acetoxytetrahydrofuran (No. 1440)
Rats
Ina13-weekfeedingstudy,groupsof1014maleand1014femaleCharles
Riveralbinoratsweregivendietscontaining2-hexyl-4-acetoxytetrahydrofuran(in
gumarabic)ataconcentrationof39,65and78mg/kgfromweeks0to4,5to10,
and 11 to 13.The control group was maintained on a basal diet containing only
thevehicle,gumarabic,fromweeks0to13ofthestudy.Thefoodconsumption,
foodeffciency,andbodyweightofeachratweredeterminedweekly.Duringweek
7ofthestudy,haematologicalexaminationandclinicalchemistrydeterminations
were conducted on seven of the animals in each group. After 90 days, all the
animalswerekilledandnecropsiesperformed.Theweightsoftheliverandkidney
ofeachratweremeasured,whileawiderangeoftissuesandorgansfromseven
of the animals in each group were subjected to histopathological examinations.
Over the 13-week experimental period, the average daily intake of 2-hexyl-4-
acetoxytetrahydrofuran by male and female rats was reported to be 4.45 and
5.32mg/kgbw,respectively.Foodeffciency,foodconsumption,bodyweights,and
502 TETRAHYDROFURAN AND FURANONE DERIVATIVES
K2
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504 TETRAHYDROFURAN AND FURANONE DERIVATIVES
K2
relative weightsof the kidney and liver were not signifcantly different between
control and treated groups. In males, the percentage of haemoglobin and mean
corpuscular concentration of haemoglobin were signifcantly increased during
week7,butonlythepercentageofhaemoglobininmaleswasincreasedatweek
13.Thebiologicalsignifcanceofthisincrease(710%)wasnotdiscussedbythe
authors.Histologicalexaminationofawiderangeoftissuesshowednoevidence
ofanylesionthatcouldbeattributedtoadministrationofthetestsubstance(Rab-
inowicz,1963).
(ii) 2-(3-Phenylpropyl)tetrahydrofuran (No. 1441)
Rats
In a 90-day dietary study, groups of 1016 male and 1016 female Charles
River CD rats were given diets containing 2-(3-phenylpropyl)tetrahydrofuran at a
concentrationprovidingadoseofapproximately42.98and48.58mg/kgbwperday
tomaleandfemalerats,respectively.Acontrolgroup,receivingabasaldiet,also
wasstudied.Theconcentrationofthetestmaterialinthedietwasadjustedduring
thestudytomaintainconstantlevelsofdietaryintake.Haematologicalexamination
andbloodureadeterminationswereperformedon50%oftheanimalsatweek7,
andonalltheanimalsattheendoftheexperimentalperiod.After90days,allthe
animalswerekilled,andsubjectedtoadetailednecropsyandgrosshistopathologi-
calexamination.Clinicalobservationsrecordeddailyandfoodconsumption,food
utilization,andbodyweightsmeasuredweeklyrevealednosignifcantdifferences
betweentreatedandcontrolgroups.Therewasnodifferenceinabsoluteandrela-
tiveweightsoftheliverorkidneybetweentreatedandcontrolanimals.Notoxico-
logicallysignifcanteffectsonhaematologyandclinicalchemistrywerereportedin
anyofthetreatedrats.Grossandhistopathologicalexaminationofawiderange
oftissuesandorgansfromeachanimalshowednoevidenceoftissuealterations
thatcouldbeassociatedwithadministrationof2-(3-phenylpropyl)tetrahydrofuran.
The NOEL for 2-(3-phenylpropyl)tetrahydrofuran was 42.98mg/kgbw per day in
males and 48.58mg/kgbw per day in females, respectively (Posternak et al.,
1969).
(iii) Tetrahydrofurfuryl alcohol (No. 1443)
Rats
Ina7-daydoserange-fndingstudy,groupsoffvemaleandfvefemaleFischer
344 rats (aged 5 weeks) were maintained on diets containing tetrahydrofurfuryl
alcoholataconcentrationof0(control),50,500or2500mg/kgofdiet.Theanimals
wereobservedtwicedailyforclinicalsignsoftoxicity.Bodyweightswererecorded
ondays0,6and7ofthestudy.Onday7,theanimalswerekilledbyexsanguina-
tion through the abdominal aorta under carbon dioxide/oxygen anaesthesia.
The brain, heart, kidneys, liver, spleen, ovaries, and testes were weighed. The
actualconsumptionoftetrahydrofurfurylalcoholwas0,6,62or332mg/kgbwper
dayformalerats,and0,6,62or312mg/kgbwperdayforfemalerats.Therewere
TETRAHYDROFURAN AND FURANONE DERIVATIVES 505
K2
no compound-related clinical fndings reported in any of the rats. Mean body
weights, and relative and absolute organ weights were comparable beween test
and control groups. No compound-related abnormalities were observed upon
grossnecropsytetrahydrofurfurylalcohol(Arts&Lina,2003).
Onthebasisoftheresultsofthis7-dayrange-fndingstudy,groupsoffvemale
and fve female Fischer 344 rats (aged 56 weeks) were given diets containing
tetrahydrofurfurylalcoholataconcentrationof0(control),60,600or3000mg/kg
ofdietfor28days.Thesedietarylevelswerecalculatedbytheauthorstoprovide
average daily intakes of of tetrahydrofurfuryl alcohol of 0, 6, 60 or 300mg/kgbw.
Theanimalswereobservedtwicedailyforclinicalsignsoftoxicity.Measurements
ofbodyweightandfoodconsumptionwererecordedweekly,andwaterintakewas
monitoredduringthefrstandthirdweeksofthestudy.Neurobehavioralobserva-
tionsandmotoractivitytestingwereperformedonallratsafter28daysofexposure
tothetestsubstance.After28days,theanimalsweresacrifcedbyexsanguination
from the abdominal aorta under carbon dioxide/oxygen anaesthesia. Complete
haematology analyses were performed on all of the rats at the end of the study.
Meancorpuscularvolume,meancorpuscularhaemoglobinandmeancorpuscular
haemoglobinconcentrationswerecalculated.Routineanalysesforplasmachem-
istrywereperformed,includingenzymaticactivities,andconcentrationsofprotein,
lipidandvariousinorganicsalts.Themajororgansincluding,butnotonly,theliver,
kidneys, lungs, heart, brain, and sex organs were weighed and necropsied. All
majortissuetypeswerepreserved.Theliver,lungs,andkidneysfromallanimals,
in addition to any tissues showing gross abnormalities, were subjected to micro-
scopic examination. No overt clinical signs of toxicity were noted in any of the
treated rats. The results of neurobehavioral testing and body-weight measure-
ments indicated no signifcant differences between control rats and rats treated
withtetrahydrofurfurylalcohol.Malesatthehighestdose(300mg/kgbwperday)
showedslightlylowerfoodintake,foodconversioneffciencyandwaterintakeby
day16ofthestudy.Nosignifcantchangesinbodyweight,foodconsumption,and
food conversion effciency were noted in females at the highest dose, and no
consistent changes were observed in the water consumption of treated females.
Meancorpuscularvolume(p<0.05)andmeancorpuscularhaemoglobin(p<0.01)
values in males at the highest dose were signifcantly decreased compared to
thoseofcontrols.Whiletherewasnosignifcantdifferenceinthenumberoflym-
phocytesforanimalsinthecontrolandtreatedgroups,asignifcantlydecreased
thrombocyte count (p < 0.01 and p < 0.05 for males and females, respectively)
wasobservedinratsatthehighestdoserelativetocontrols.Malesandfemales
at the highest dose exhibited increased concentrations of plasma cholesterol,
triglycerides,andphospholipids;however,theseconcentrationsonlyreachedsta-
tistical signifcance in females at the highest dose. No compound-related effects
on absolute and relative organ weights, or macroscopic and microscopic effects
were reported in any of the rats. In particular, the authors noted that there were
nogrossormicroscopicchangestothelivertocorroboratetheclinicalchemistry
changes observed in treated rats. The NOEL for tetrahydrofurfuryl alcohol in
FischerF344ratswas60mg/kgbwperday(Arts&Lina,2003).
506 TETRAHYDROFURAN AND FURANONE DERIVATIVES
K2
(iv) 4-Hydroxy-2,5-dimethyl-3(2H)-furanone (DMHF; No. 1446)
Rats
In a 90-day dietary study, groups of 1016 male and 1016 female Charles
River CD rats were given diets containing DMHF at a concentration providing a
dose of approximately 6.15mg/kgbw per day in males or 7.04mg/kgbw per day
infemales.Acontrolgroup,receivingabasaldiet,alsowasstudied.Theconcen-
tration of the test material in the diet was adjusted during the study to maintain
constant levels of dietary intake. Haematological examination and blood urea
determinationswereperformedon50%oftheanimalsatweek7,andonallthe
animals at the end of the experimental period.After 90 days, all of the animals
were killed, and subjected to a detailed necropsy and gross histopathological
examination. Clinical observations recorded daily, and food consumption, food
utilization,andbodyweightsmeasuredweeklyrevealednosignifcantdifferences
betweencontrolgroupsandgroupstreatedwithDMHF.Therewerenosignifcant
differencesinabsoluteandrelativeweightsoftheliverorkidneyincontrolanimals
andinanimalstreatedwithDMHF.Notoxicologicallysignifcanteffectsonhaema-
tology or clinical chemistry parameters were reported in any of the rats treated
with DMHF. Gross and histopathological examination of a wide range of tissues
andorgansfromeachanimalshowednoevidenceoftissuealterationsthatcould
be associated with administration of DMHF. The NOEL for DMHF was 6.15mg/
kgbwperdayinmaleratsandand7.04mg/kgbwperdayinfemalerats(Posternak
etal.,1969).
(v) 2-Methyltetrahydrofuran-3-one (No. 1448)
Rats
Groupsof23maleand23femaleweanlingSprague-Dawleyratsweregiven
diets containing 2-methyltetrahydrofuran-3-one at a concentration providing an
expecteddailyintakeof0or91.4mg/kgbwfor90days.Weeklymeasurementsof
weight and food consumption were obtained for each of the animals during the
13-weekexperimentalperiod.Animalswereobserveddailyforappearance,physi-
ological responses, behaviour, any pharmacological or toxicological responses,
and mortality. During weeks 6 and 13 of the study, urine from eight males and
eightfemalesineachgroupwascollectedfordeterminationofpH,specifcgravity,
microscopicexaminationofsedimentandqualitativeestimatesofconcentrations
ofalbumin,glucose,occultblood,ketones,andbilirubin.Additionally,atweek6of
thestudy,eightmalesandeightfemalesineachgroupwerekilledandsubjected
to haematological examinations (erythrocyte volume fraction, haemoglobin, and
erythrocyte,leukocyteanddifferentialleukocytecounts)andbloodchemicaldeter-
minations (glucose, blood urea nitrogen, serum glutamic-oxaloacetic transami-
nase,serumglutamic-pyruvictransaminaseandserumalkalinephosphatase).The
remaining rats (15 of each sex per group) were necropsied after sacrifce at the
endofthestudy,andsubjectedtothesamehaematologicalandbloodchemistry
determinationsasthoseratskilledatweek6ofthestudy.Allratsappearednormal
throughout the study, and no compound-related physiological effects were
observed. The averaged daily intake of DMHF in male and female rats was
reported to be 91.6 and 91.2mg/kgbw, respectively. There were no signifcant
TETRAHYDROFURAN AND FURANONE DERIVATIVES 507
K2
differencesbetweencontrolandtreatedratsinparametersofgrowth,weightgain,
feed consumption, or feed utilization. Results of urine analysis, blood chemistry,
andhaematologicalexaminationsoftreatedratsrevealednosignifcantdifference
whencomparedwithcontrolrats.Measurementofserumconcentrationsofsodium,
potassium,calcium,andchlorideoftreatedratsatweek13alsorevealedvalues
thatwerenotsignifcantlydifferentfromthoseofcontrols.Grosspathologicaland
histologicalexaminationoftreatedandcontrolratsfailedtorevealanyconsistent
signifcantdifferencebetweenthetwogroups.Noadverseeffectswereobserved
inmaleandfemaleratsgiven2-methyltetrahydrofuran-3-oneatadoseof91.6or
91.2mg/kgbwperday,respectively,for90days(Shellenberger,1970).
(vi) 2-Ethyl-4-hydroxy-5-methyl-3(2H)-furanone (HEMF; No.
1449)
Rats
Groups of 15 male and female weanling Sprague-Dawley rats were given
HEMFataconcentrationcalculatedtoprovideanaverageintakeof1.52mg/kgbw
per day for 93 days. However, it was reported that the actual average intake of
HEMFbymaleandfemaleratswas1.43mg/kgbwperday.Thecontrolgroupwas
given a basal diet containing only the vehicle, acetone, for a period of 93 days.
Daily observation for symptoms and weekly measurement of body weight, food
intake volume, and food utilization revealed no signifcant differences between
treated and control groups. No compound-related effects were observed upon
haematologicalexamination(erythrocyteandleukocytecounts,erythrocytevolume
fraction,orconcentrationofhaemoglobin),bloodchemicaldetermination(glucose,
bloodureanitrogen,glutamic-oxalacetictransaminase,glutamic-pyruvictransami-
nase,andalkalinephosphatase)andurineanalysisduringweeks6and12ofthe
study.Allofthesurvivinganimalswerekilledattheendofthe93-daystudy,and
grossandhistopathologicalexaminationoftheliverandkidneyswereperformed
forallanimals.Inaddition,histopathologicalexaminationof25tissuesandorgans
wasperformedforeightanimalsofeachsexinthecontrolandtestgroups.Abso-
luteandrelativeorganweights(liver,kidneys,adrenals,andspleen)werereported
nottobesignifcantlydifferentbetweentreatedandcontrolgroups.Therewasalso
no evidence of any compound-related tissue alterations observed in rats treated
withHEMF(Cox&Re,1978).
(vii) 4-Hydroxy-5-methyl-3(2H)-furanone (No. 1450)
Rats
Groups of three male and female rats (strain not specifed) were maintained
on diets containing a meat favour cocktail at a concentration of 197, 492, 983,
1966,2949or3932mg/kgofdietfor3weeks.Thecocktailincluded:4-hydroxy-5-
methyl-3(2H)-furanone (No. 1450), 74.1%; 2,3-dimethyl-4-hydroxy-2,5-dihydrofu-
ran-5-one,23.2%;2-acetyl-2-thiazole,1.8%;and2-acetylthiazoline,0.9%.These
dietary concentrations provided average daily intakes of the favour cocktail of
approximately10,25,49,98,147or197mg/kgbw,equivalenttoapproximately7,
18,36,73,109and146mg/kgbwof4-hydroxy-5-methyl-3(2H)-furanone,respec-
508 TETRAHYDROFURAN AND FURANONE DERIVATIVES
K2
tively(Food&DrugAdministration,1993).Groupsofsixmaleandsixfemalerats
were fed a control diet. Based on twice or thrice weekly measurements of body
weight,foodandwaterconsumption,andfoodutilization,noconsistentsignifcant
differenceswereobservedbetweentestandcontrolanimalsoverthe4-weekstudy
period.Haematologicalexamination(erythrocytevolumefractionandtotalleuko-
cyte counts) of all rats at the end of the study revealed a signifcant increase in
erythrocyte volume fraction in males fed diets containing the favour cocktail at
492,983,1966or2949mg/kgofdietandinfemalesfeddietscontainingthefavour
cocktail at 492 or 983mg/kg of diet. Total leukocyte counts of female rats given
diets containing the favour cocktail at 983mg/kg of diet were also signifcantly
greaterthanthoseofthecontrols.Theauthorsconcludedthat,owingtothelack
of a doseresponse relationship in these effects, the fndings must be regarded
asachanceoccurrencethatwasofnobiologicalsignifcance.Uponnecropsyat
studytermination,absoluteandrelativeorganweights(liver,spleen,heart,kidneys,
testes) of male rats were observed not to be signifcantly different between test
andcontrolgroups.Femalesatthehighestdose(favourcocktail,3932mg/kgof
diet)exhibitedrelativeweightsoftheliverthatweresignifcantlyhigherthanthose
ofthecontrols;however,macroscopicexaminationofthetissuesofallanimalsat
postmortem showed no signifcant fndings attributable to the compound tested
(Munday&Kirkby,1971a).
Inafollow-upstudy,groupsofeightmaleandeightfemaleweanlingColworth
Wistar rats were fed diets containing the same meat favour cocktail used in the
previous study (Munday & Kirkby, 1971a) at a concentration of 197, 492, 983,
1966,2949or3932mg/kgofdietfor13weeks.Groupsof16malesandfemales
were maintained on a control diet throughout the duration of the study. These
dietaryconcentrationsprovidedaveragedailyintakesofthemeatfavourcocktail
of approximately 10, 25, 49, 98, 147 or 197mg/kgbw, equivalent to intakes of
4-hydroxy-5-methyl-3(2H)-furanone of approximately 7, 18, 36, 73, 109 and
146mg/kgbwperday,respectively(FoodandDrugAdministration,1993).Clinical
chemistrytestswereperformedat13weeksforbothsexesfedthedietscontaining
the meat favour cocktail at 492, 983 and 2949mg/kg of diet. Haematological
examinationwasperformedat6and13weeksforgroupsofanimalsfedthediets
containing the meat favour cocktail at 197, 1966 and 3932mg/kg of diet. All
animals were subjected to post-mortem examination, measurement of organ
weights,andhistologicalexaminationoforgansweighedattheconclusionofthe
study.Inaddition,groupsofsixmalesandsixfemaleswerefeddietscontaining
themeatfavourcocktailataconcentrationof197,1966or3932mg/kgofdietfor
6weeks,andbiochemicalstudieswerecarriedoutonsamplesofbloodandurine.
Agroupof12malesand12femaleswerefedabasaldietfor6weeksandserved
ascontrols.Attheendofthe6weeks,alltheanimalsweresacrifced,andmea-
surementsoforganweightandmacroscopicandmicroscopicexaminationswere
performed.
Weeklymeasurementofbodyweight,foodandwaterintake,andfoodutiliza-
tion revealed a signifcantly decreased food intake in males and females at
2949mg/kg,andasignifcantlyincreasedwaterintakeinmalesat492mg/kgand
femalesat983mg/kg,whencomparedwithcontrols.Intheabsenceofanydose
responserelationship,theauthorsregardedthesechangesasbeingunrelatedto
TETRAHYDROFURAN AND FURANONE DERIVATIVES 509
K2
theadministrationofthefavourcompound.Measurementofurinerefractiveindex
andurineglutamic-oxalacetictransaminaseactivityforkidneyfunctionandqualita-
tiveurineanalysis(pH,protein,glucose,andblood)inratsfedwithdietscontaining
themeatfavourcocktailat197,1966or3932mg/kgfor6weeks,andinthosefed
with diets containing the meat favour cocktail at 492, 983 or 2949mg/kg for 13
weeks failed to show any adverse effects attributable to the compound adminis-
tered.Clinicalchemistrymeasurementsrevealednormalvaluesthatwerenotsig-
nifcantly different between control groups and those given diets containing the
favour cocktail for 6 or 13 weeks. Results of serum protein electrophoresis
(albumin, globulin, and fbrinogen) revealed no signifcant differences between
animalstreatedfor6or13weeksandthecontrols.Whileratsfedwithdietscon-
tainingthemeatfavourcocktailat492,983or2949mg/kgfor13weeksexhibited
no signifcant changes in haematological parameters when compared with the
controls,ratsfedwithdietscontainingthemeatfavourcocktailat3932mg/kgfor
6 weeks exhibited signifcantly higher erythrocyte volume fraction and leukocyte
counts than the controls.Additionally, rats fed diets containing the meat favour
cocktail at 197mg/kg for 6 weeks showed signifcantly lower erythrocyte volume
fractionsthanthecontrols.Theauthorsconsideredtheseobservationsnottobe
relatedtothefavourcompoundadministered.
For animals treated for 6 weeks, relative weights of the liver were increased
(p<0.05)infemalesatthetwohighestdietaryconcentrations(1966and3932mg/
kg), compared with controls. Other isolated increases in relative organ weights
were found, but none was dose-related. The authors noted that the increase in
relative weight liver observed in treated females was slight, and questioned the
biological signifcance of the effect, given that the increase noted at 1966mg/kg
was not observed at 1966mg/kg in the 13-week study. In addition, there was no
evidenceofgrossandhistopathologicalalterationstotheliveroranyotherorgans
examinedinratsinthe6-weekstudy.
Foranimalstreatedfor13weeks,theabsoluteweightsofthekidneyinmales
and females at 1966 and 3932mg/kg, and the relative weights of the kidney in
malesat1966mg/kgandinfemalesat1966and3932mg/kgweregreaterthan(p
< 0.05) those of the controls.Absolute and relative weights of the liver of males
and females at 197, 1966 or 3932mg/kg were increased; however, according to
theauthors,theincreaseswerenotmarked,andwerenotsupportedbyanyevi-
denceofhistopathologicalchangesintheliver.Likewise,histopathologicalexami-
nation of the remaining organs showed no evidence of alterations that could be
associated with administration of the test material. Therefore, the authors con-
cludedthatnoadverseeffectswereobservedthatcouldbeattributedtothetest
compound(Munday&Kirkby,1971b).
(viii) 4-Acetoxy-2,5-dimethyl-3(2H)-furanone (No. 1456)
Rats
GroupsoffvemaleandfemaleCharlesRiverCrl:CDBRratsweregivendiets
containing 4-acetoxy-2,5-dimethyl-3(2H)-furanone at concentrations estimated to
provideadoseof0or18mg/kgbwperdayfor14days.Animalswereexamined
510 TETRAHYDROFURAN AND FURANONE DERIVATIVES
K2
forviabilitytwiceperday.Bodyweightsandfoodconsumptionwererecordedon
days0,7and14.Agrossnecropsywasperformedoneachoftheanimalsatthe
end of the study; this included examination of the external surface of the body,
carcass, all orifces and the cranial, thoracic and abdominal cavities and their
contents.Kidneyandliverweightswererecordedbeforefxationin10%buffered
formalin.Allgrosslesionswerealsofxedforhistologicalexamination.Duringweek
1, the mean measured dietary intake of 4-acetoxy-2,5-dimethyl-3(2H)-furanone
was 40% higher in males (25.2mg/kgbw per day) and 32% higher in females
(23.8mg/kgbw per day) when compared to the target intake of 18mg/kgbw per
day. By week 2, the average dietary intakes of 4-acetoxy-2,5-dimethyl-3(2H)-
furanonebymalesandfemaleswere17.2and16.7mg/kgbwperday,respectively.
Nostatisticallysignifcantdifferencesinanyoftheparameterstestedwerenoted
intreatedandcontrolanimals.Theauthorsconcludedthatdietaryadministration
of 4-acetoxy-2,5-dimethyl-3(2H)-furanone produced no evidence of toxic effects
undertheconditionsofthisstudy(Drummond,1993).
(c) Long-term studies of toxicity and carcinogenicity
Theresultsoflong-termstudiesoftoxicityandcarcinogenicitywithrepresenta-
tivetetrahydrofuranandfuranonederivativesaresummarizedinTable4andare
describedbelow.
(i) 4-Hydroxy-5-methyl-3(2H)-furanone (No. 1450)
Rat
GroupsoffourmaleandfemaleColworthWistarratswerefeddietscontaining
ameatfavourcocktailataconcentrationof197,492,983,1966,2949or3932mg/
kgofdiet(equivalenttoadoseof4-hydroxy-5-methyl-3(2H)-furanone)ofapproxi-
mately 7, 18, 36, 73, 109 and 146mg/kgbw per day, respectively) for 13 weeks.
Owingtothelackoforganweightchangesattributabletotheadministrationofthe
favourmixtureina13-weekstudydiscussedabove(Munday&Kirkby,1971b),all
treatment groups (24 males and 24 females) were subsequently maintained on
dietscontainingthefavourcocktailatthehighestdietaryconcentration(3932mg/
kg) from week 15 of the study, for an additional 37 weeks (i.e. up to week 52).
Evaluation of fnal mean body weights and haematological parameters revealed
nosignifcantdifferencesbetweentestandcontrolanimalsatweek15andatthe
endofthe1-yearstudy.Nocompound-relatedeffectsongeneralhealthandsur-
vival were noted in any of the treated rats.At necropsy, there was no signifcant
differenceinabsoluteorrelativeorganweights(liver,spleen,heart,kidneys,brain,
adrenals,pituitary,thyroid,andtestes)betweentreatedandcontrolanimals.Gross
andhistopathologicalexaminationofalltreatedandcontrolratsrevealednosig-
nifcantmacroscopicfndingsattheendofthestudy.Uponmicroscopicexamina-
tion, lesions (subcutaneous sarcoma, chloroma, pituitary adenoma, and
parafollicularthyroidadenoma)wereobservedintreatedandcontrolrats.Similar
fndings have been reported in previous studies using control rats of the same
strain; therefore, these lesions were considered not to be related to the favour
cocktailadministered.Theauthorsconcludedthatadministrationofdietscontain-
TETRAHYDROFURAN AND FURANONE DERIVATIVES 511
K2
ing the favour cocktail at a concentration of up to 3932mg/kg (approximately
equivalent to a dose of 4-hydroxy-5-methyl-3(2H)-furanone of 146mg/kgbw per
day)forupto1yearproducednoeffectsonthetype,incidence,ortimeofdevel-
opmentoftumoursinColworthWistarrats(Munday&Kirkby,1973).
(ii) 4-Hydroxy-2,5-dimethyl-3(2H)-furanone (DMHF; No. 1446)
Rat
Groupsof60maleand60femaleSprague-Dawleyratsgivendietscontaining
DMHFatadoseof0(control),100,200or400mg/kgbwperdayfor24months.
Observationforsignsofmortalityorclinicaltoxicitywasconductedtwiceperday.
Measurements of body weight and food consumption were recorded twice per
weekforthefrst16weeksofthestudyandtwicepermonththereafter.Haematol-
ogyandcoagulationparameterswereevaluatedat12,18and24months.Atsac-
rifce,absoluteandrelativeorganweightswererecorded.Completemacroscopic
and histopathological examinations of selected tissues and gross lesions were
performed.
Nosignifcantcompound-relatedeffectswerereportedinanyoftheanimalsat
100and200mg/kgbwperday.Themeanbodyweightsandbody-weightgainsof
malesandfemalesatthehighestdose(400mg/kgbwperday)weresignifcantly
lower than those of control animals at 24 months. Haematological examination
revealedsignifcantdifferencesinabsoluteortotalleukocytecountsbetweenrats
treated with DMHF at a dose of 200 or 400mg/kgbw per day and the controls,
whichtheauthorsattributedtonormalbiologicalvariability.
Themeansurvivalrateformalesinthegroupreceivingthehighestdosewas
signifcantlylower(approximately20%,p<0.05)thanthatofmalesinthecontrol
groupat24months.Theauthorsconcludedthatthisfndingwasattributabletoan
increasedincidenceofadenomasoftheparsdistalisofthepituitary,withsubse-
quent compression of the hypothalamic region within the brains of males at the
highest dose. However, it was noted that the incidence of pituitary adenomas in
malesatthehighestdosewaswithintherangeforhistoricalcontrols,aswellas
within reported ranges in the literature for the age and strain of rat (McComb et
al.,1984).Pituitaryadenomasarecommoninageingandagedrats,andgenerally
appearbetweenage13and24months.Inthisstudy,allofthedecedentanimals
with adenomas of the pars distalis died during month 18 of the study or later. In
addition,Petoanalysis,whichcomparesincidenceoftumoursandtimetotumour
formation,revealednostatisticaldifferencebetweencontrolandtreatedmalesand
females in this study. Therefore, it was concluded that these adenomas were
common,spontaneoustumoursthatwereunrelatedtotheadministrationofDMHF.
Noothersignifcantcompound-relatedhaematological,biochemical,macroscopic,
histopathological,orneoplasticchangeswerereported.TheNOELforDMHFwas
200mg/kgbwperdayonthebasisofdecreasedbody-weightgainsinmalesand
females, and decreased survival rate in males at 400mg/kgbw per day (Kelly &
Bolte,2003).
512 TETRAHYDROFURAN AND FURANONE DERIVATIVES
K2
The incidence of pituitary adenoma of the par distalis is a spontaneous neo-
plasm,whichalsooccursinstrainsofratsotherthanSprague-Dawley(Haseman
et al., 1985, 1998). For example, the incidence of pituitary adenomas of the par
distalis in control male Fischer F344 rats in dietary studies performed by
theNationalToxicologyProgramisintherangeof14to60%,withameanrateof
30%.
(d) Genotoxicity
Theresultsoftestsforgenotoxicityinvitroandinvivoperformedwithrepre-
sentativetetrahydrofuranandfuranonederivativesaresummarizedinTable5and
describedbelow.
(i) In vitro
NoevidencewasfoundforreversemutationintestsinSalmonella typhimurium
strainsTA1535,TA1537,TA1538,TA100,TA98andTA102withtetrahydrofurfuryl
alcohol (1102100mg/plate) (No. 1443), tetrahydrofurfuryl propionate (3600mg/
plate)(No.1445)or2-(3-phenylpropyl)tetrahydrofuran(3600mg/plate)(No.1441)
(Wildetal.,1983;Aeschbacheretal.,1989).
4-Hydroxy-5-methyl-3(2H)-furanone (No. 1450) (1012000mg/plate), DMHF
(No. 1446) (1010000mg/plate), and HEMF (No. 1449) (up to 10000mg/plate)
inducedreversemutationsinstandardandmodifedAmesassays.Positiveresults
wereobtainedforDMHF(No.1446)inS. typhimurium strainsTA100,TA102,TA98
and TA97 at the highest dose tested (4000mg/plate) with or without metabolic
activation(Xingetal.,1988).Incontrast,Gilroyetal.(1978)andHiramotoetal.
(1996b)reportedpositiveresultsforthiscompoundonlyinS. typhimuriumstrain
TA100whentestedatconcentrationsof10000mg/platewithorwithoutmetabolic
activation. Similarly, HEMF (No. 1449) and 4-hydroxy-5-methyl-3(2H)-furanone
(No.1450)producedpositiveresultsinS. typhimuriumstrainTA100withorwithout
metabolicactivation(Hiramotoetal.,1996a;Lietal.,1998).
ThestandardRecassaywithBacillus subtilisH17(rec
+
)andM45(rec
-
)exposed
toDMHF(No.1446)ataconcentrationof20,40,60,80and120mg/discofyielded
adose-dependentDNAdamageresponse(Xingetal.,1988).
(ii) In vivo
Inanassayforgenotoxicityinvivo,groupsoffveICRmiceweregivenanega-
tivecontrol,orDMHF(No.1446)orHEMF(No.1449)ataconcentrationof1000,
2000 or 3000mg/kgbw by oral administration. Blood was drawn at intervals of
15min after administration for up to 120min. For DMHF, the frequency of micro-
nucleatedperipheralreticulocyteswasincreasedatadoseof2000and3000mg/
kgbw, but not at 1000mg/kgbw. For HEMF, the frequency was increased at all
threedoses(Hiramotoetal.,1998).
KunmingmiceinjectedintraperitoneallywithDMHF(No.1446)atadoseof0,
186,232or309mg/kgbwdemonstratedadose-dependentincreaseinerythrocyte
TETRAHYDROFURAN AND FURANONE DERIVATIVES 513
K2
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514 TETRAHYDROFURAN AND FURANONE DERIVATIVES
K2
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TETRAHYDROFURAN AND FURANONE DERIVATIVES 515
K2
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516 TETRAHYDROFURAN AND FURANONE DERIVATIVES
K2
micronucleusformationinthebonemarrow,reachingamaximumincreaseof2.6-
fold(Xingetal.,1988).Also,malemiceinjectedwithDMHFatadoseof0,232,
464or928mg/kgbwexhibitedincreasesinspermatocytechromosomeaberrations
(Xingetal.,1988).InasimilarassayinmaleKunmingmicegivenDMHFatadose
of 200, 400 or 800mg/kgbw by intragastric instillation, a signifcant increase in
sister chromatid exchanges in spermatogonial cells compared to controls was
reportedatallthreedoses(Tianetal.,1992).Positiveresultswerealsoobtained
in an assay for micronucleus formation in male mice given DMHF at a dose of
200, 400 or 800mg/kgbw by intraperitoneal injection (Tian et al., 1992). The
increasesobservedateachdosedidnotestablishacleardoseresponserelation-
ship,althoughincreasesweresignifcantlyhigherthanforthenegativecontrol.
Groups of fve or six male ICR mice were given DMHF at a dose of 0, 500,
1000or1500mg/kgbwbyintraperitonealinjection.Bloodsamplesweredrawnat
24,48and72hafterinjection.Thefrequencyofmicronucleatedperipheraleryth-
rocyteswassignifcantlyincreasedat500mg/kgbw,withthemaximumfrequency
of1.6%beingobtainedat48hafterdosing(Hiramotoetal.,1996b).
Groups of fve or six male ICR mice were given HEMF (No. 1449) at a dose
of500,1000or1500mg/kgbwbyintraperitonealinjectionandsamplesofperiph-
eralbloodweretakenat24,48and72hafterinjection.Allthemiceinthegroup
givenHEMFatadoseof1500mg/kgbwdiedbefore24h.Thefrequencyofmicro-
nucleatedperipheralerythrocyteswassignifcantlyhigherthanthatinthecontrols
ingroupsgivenHEMFatadoseof500or1000mg/kgbw.Themaximumnumber
ofmicronucleatedperipheralerythrocyteswasobservedat48hat1000mg/kgbw
group(0.58%)andat500mg/kgbw(approximately0.3%).Thefrequencyofmicro-
nucleatedperipheralerythrocytesreportedinmicegiventhepositivecontrolsub-
stance,mitomycinC,atadoseof1mg/kgbw,was3.1%(Lietal.,1998).
Insummary,positiveresultswereobtainedinseveralassaysforgenotoxicity
in vivo in mice given DMHF via intraperitoneal injection at doses as low as
196mg/kgbw(Xingetal.,1988).Similarly,positiveresultswerealsoobtainedfor
DMHF administered orally; however, there are conficting data pertaining to the
lowest dose at which DMHF elicits a positive response: 200mg/kgbw according
toTianetal.(1992);2000mg/kgbwaccordingtoHiramotoetal.(1998).
Putative mechanism of genotoxicity of
furanone derivatives
FuranonesinduceDNAdamageinvitrobygeneratingfreeradicalsthatinduce
strand scission. In the presence of metals (e.g. Fe
3+
) and dissolved oxygen, the
enolichydroxylgroup(OH)ofthefuranoneisoxidizedbysingleelectrontransfer
toyieldthecorrespondingcarbon-centeredradicalandareducedmetalion(e.g.
Fe
2+
).Thecarbon-centeredradicalcancoupletomolecularoxygentoproducea
peroxyl radical that may damage DNA. Alternately, the reduced metal ion can
auto-oxidizetoformasuperoxideradicalanion.Thesuperoxideradicalthendis-
mutatesintohydrogenperoxide(H
2
O
2
).Itiswellrecognizedthatreducedmetals
reactwithH
2
O
2
toformahydroxylradical,whichisapowerfuloxidizingagent(see
Figure2).Hydrogenperoxidealsooxidizesglutathioneleadingtodecreasedglu-
TETRAHYDROFURAN AND FURANONE DERIVATIVES 517
K2
tathione S-transferase/oxidized glutathione and an increase in cellular oxidative
stress.
InthecaseofDMHF,experimentalevidenceforthisH
2
O
2
-producingpathway
includesthefollowing:
Fe
3+
orCu
2+
isreadilyreducedtoFe
2+
orCu
+
inthepresenceofDMHF;
DNAstrand-breakingofsupercoiledplasmidDNAintoanopencircularformin
the presence of DMHF is inhibited in the presence of superoxide dismutase
and catalase, enzymes used to detoxicate superoxide to form oxygen and
water;
hydroxylradicalscavengerssuchaspotassiumiodine,sodiumazide,orethanol
alsoinhibitDNAstrand-breaking;
free radical spin-trapping agents (e.g. 5,5-dimethyl-1-pyrroline N-oxide) also
inhibitDNAstrand-breaking;
oxygen radical trapping agents such as 2-mercaptoethanol and cysteine are
alsoinhibitory;
removalofdissolvedoxygenbynitrogenpurgedecreasesDNAstrand-breaking
andH
2
O
2
formation;
additionofmetalchelatingagentsalsoinhibitsDNAstrand-breakingbydeplet-
ingthemetalionsrequiredforthisprocess;
DNAstrand-breakingbyDMHFwasmuchfasterinthepresenceofFe
3+
than
initsabsence;and
electron spin resonance of a solution of DMHF and 5,5-dimethyl-1-pyrroline
N-oxide showed the presence of hydroxyl radicals and bicarbonate radicals
(Hiramotoetal.,1996a,1996b;Yamashitaetal.,1998).
On the basis of these observations, cellular oxidative stress is related to the
dose-dependent oxidation of DMHF and structurally related furanones, yielding
H
2
O
2
and eventually hydroxyl radicals (Hiramoto et al., 1996a, 1996b; Li et al.,
1998;Yamashitaetal.,1998).
TheabilityofDMHFtoinduceoxygenradicalformationandDNAstrandbreaks
isreminiscentofsimilaractivitiesobservedforvitaminC.VitaminC(ascorbicacid)
contains an enediol that is superfcially related to the enol of DMHF. Being both
H O O H
+ F e
+ + +
+ O
2
O O
+ F e
+ +
+ O
2
- .
O
2
- .
s u p e r o x i d e
d i s m u t a s e
H
2
O
2
O H
.
h y d r o x y r a d i c a l ,
D N A s t r a n d b r e a k i n g
Figure 2. Mechanism of oxidation of furanone derivatives in vitro
518 TETRAHYDROFURAN AND FURANONE DERIVATIVES
K2
anenoletherandana,b-unsaturatedketone,DMHFissubjecttohydrolyticring
opening, to yield an enediol. Like DMHF, vitamin C also reduces metal ions and
produces superoxide anions to generate hydroxyl radicals that cleave DNA. As
anticipated,vitaminCexhibitsgenotoxicityintestsystemssimilartothoseinwhich
furanones give positive results. In standardAmes assays, ascorbic acid (vitamin
C)inducesreversemutationsinS. typhimuriumstrainsTA104,TA102,TA100and
TA98atconcentrationsof3521761mg/plate(Ichinotsuboetal.,1981;DAgostini
etal.,2000).IntheE. coli Mutoxitest,positiveresultswereobtainedwhenascorbic
acid at a concentration of 200, 300 or 400mg/plate in the presence of Cu
2+
was
incubatedwithE. colistrainIC203(Martinezetal.,2000).E.coliIC203carriesan
oxyR mutation that effectively removes its ability to turn on the biosynthesis of
H
2
O
2
-protective proteins and makes the strain sensitive to DNA damage under
conditionsofoxidativestress(Blancoetal.,1998).
Increasedfrequenciesofmicronucleusformationwereobservedwhenascorbic
acid(400,500or600mg/ml)wasincubatedwithChinesehamstercells(Milleret
al., 1995).An increase in sister chromatid exchanges was observed in Chinese
hamsterovarycellsinthepresenceofascorbicacidat500mg/mlwithoutmetabolic
activation (Tennant et al., 1987). In a standard assay for micronucleus formation
in mice, ascorbic acid at a dose of 1500mg/kgbw induced a signifcant increase
(Shelbyetal.,1993).
(iii) Conclusion
Furanones are a class of substances present naturally in food and that are
alsoaddedasfavouringagents.Theprincipalfuranoneusedasafavouringagent
is DMHF. In humans, DMHF is rapidly absorbed in the gastrointestinal tract and
conjugatedwithglucuronicacidintheliver.FreeDMHFisnotdetectedintheblood
ofhumanvolunteerstowhomitisadministeredasaconstituentofstrawberries;
its glucuronic acid conjugate is the principal urinary metabolite (Roscher et al.,
1997). Thus, the potential for chemical reaction of DMHF with important cellular
macromolecules,especiallyDNA,appearstobelow.
Genotoxicity with 3-(2H)-furanone derivatives, notably DMHF and 2-ethyl-4-
hydroxy-5-methyl-3-(2H)-furanone,wasobservedinstandardizedbacterial(Gilroy
etal.,1978;Xingetal.,1988;Hiramotoetal.,1996a,1996b;Lietal.,1998)and
mammalian assays (Xing et al., 1988;Tian et al., 1992; Hiramoto et al., 1996b).
A mechanism for genotoxicity involving dose-dependent formation of H
2
O
2
and
oxidized furanones has been extensively studied (Hiramoto et al., 1995, 1996a,
1996b);thesestudiesindicatethat,athighdoses,DNAsingle-strandbreaksresult
fromthereactionofhydroxylradicalswithDNA.
DespitethefactthatDMHFcausesgenotoxicity,itisnotcarcinogenicinrats.
Two studies, one with DMHF and the other with a structurally related furanone,
showed no evidence of carcinogenicity at intakes that are orders of magnitude
greaterthantheintakeoffuranonesaddedasfavouringagents(Munday&Kirkby,
1973;Kelly&Bolte,2003).Furthermore,vitaminC,astructurallysimilarcompound
withagenotoxicitytestproflesimilartothatofDMHF,doesnotdemonstratecar-
cinogenicity (National Research Council, 1996). In a 2-year bioassay, the NOEL
TETRAHYDROFURAN AND FURANONE DERIVATIVES 519
K2
forDMHFwas200mg/kgbwperdayinrodents.Thisintakeisapproximately2000
timeshigherthanthedailypercapitaintake(eatersonly)of0.088mg/kgbwper
dayfromuseofDMHFasafavouringagent.
Afterconsiderationofalltheavailabledata,theCommitteeconcludedthatitis
highly unlikely that DMHF, other furanones or tetrahydrofurans would pose any
signifcant genotoxic risk to humans under the conditions of use as favouring
agents.Similarly,2-ethyl-4-hydroxy-5-methyl-3(2H)-furanonewasconsiderednot
toposeagenotoxicrisk.
(e) Other relevant studies: anti-tumour studies
Japanese-style fermented soy sauce (shoyu) contains signifcant amounts
(230mg/kg)oftheantioxidantHEMF(No.1449),whichhasbeenreportedtohave
anticarcinogenic properties. In order to induce forestomach neoplasia, groups of
2527femaleICRmice(aged9weeks),weregivenbenzo[a]pyreneatadoseof
1.5mgincornoilbygavageonceperweekfor4weeks(Nagaharaetal.,1992).
AfterthefourthdoseofBP,theanimalswerefedanexperimentaldietcontaining
HEMFatadoseof0,25,50or75mg/kgofdietfor120days.Thesedietarycon-
centrationsprovidedanaveragedailyintakeofofHEMFof3.75,7.5or11.25mg/
kgbw(Food&DrugAdministration,1993).Theanimalsweresacrifcedatage211
days.Asignifcantreductionintheincidenceofforestomachneoplasiainducedby
benzo[a]pyrene(p0.05)andthenumberofneoplasmspermousewasobserved
inallgroupsofmicegivendietscontainingHEMF.Inaddition,dietaryadministra-
tionofHEMFproducednoeffectonfoodintakeorbodyweight,indicatingthatthe
anticarcinogeniceffectofHEMFwasnotcausedbycaloricrestriction.Theauthors
suggestedthatHEMFinhibitstumourpromotioninmice(Nagaharaetal.,1992).
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K2
K2
525
PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS AND RELATED
ALDEHYDES AND ESTERS
First draft prepared by
Professor I.G. Sipes
1
and Dr D.G. Hattan
2
1
Department of Pharmacology, College of Medicine, University
of Arizona, Tucson, AZ, USA; and
2
Offce of Food Additives Safety, Center for Food Safety and
Applied Nutrition, Food and Drug Administration,
College Park, MD, USA
Evaluation ............................................................................... 525
Introduction......................................................................... 525
Estimateddailyintake........................................................ 526
Absorption,distribution,metabolismandelimination........ 526
ApplicationoftheProcedurefortheSafety
EvaluationofFlavouringAgents.................................. 536
Considerationofsecondarycomponents.......................... 536
Considerationofcombinedintakesfromuse
asfavouringagents.................................................... 536
Conclusions........................................................................ 536
Relevantbackgroundinformation............................................. 537
Additionalconsiderationsonintake................................... 537
Biologicaldata.................................................................... 537
Biochemicaldata......................................................... 537
Hydrolysis.............................................................. 537
Absorption,distributionandexcretion................... 538
Metabolism............................................................ 541
Toxicologicalstudies.................................................... 546
Acutetoxicity......................................................... 546
Short-termstudiesoftoxicity................................. 547
Long-termstudiesoftoxicityand
carcinogenicity................................................ 548
Genotoxicity........................................................... 548
References............................................................................... 556
1. EVALUATION
1.1 Introduction
TheCommitteeevaluatedagroupof22phenyl-substitutedaliphaticalcohols
and related aldehydes and esters (Table 1) using the Procedure for the Safety
Evaluation of Flavouring Agents (see Figure 1, p 192). The Committee has not
previouslyevaluatedanymemberofthegroup.
Seven of the 22 favouring agents (Nos 1465, 1467, 14721474, 1478 and
1479) have been reported to occur naturally in various foods. They have been
526 PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS
K2
detectedinroastednuts,cookedpotatoes,cheese,wine,fruit,vegetables,coffee,
tea,andcocoa(Nijssenetal.,2003).
1.2 Estimated daily intake
The total annual volume of production of the 22 phenyl-substituted aliphatic
alcoholsandrelatedaldehydesandestersinthisgroupisapproximately1300kg
inEurope(InternationalOrganizationoftheFlavorIndustry,1995)and3000kgin
the USA (NationalAcademy of Sciences, 1970, 1982, 1987; Lucas et al., 1999)
(see Table 2). Approximately 70% of the total annual volume of production in
Europe is accounted for by 2-phenylpropionaldehyde (No. 1467), while approxi-
mately87%ofthetotalannualvolumeofproductionintheUSAisaccountedfor
by2-methyl-3-(p-isopropylphenyl)propionaldehyde(No.1465).Thedailyintakeof
2-phenylpropionaldehyde(No.1467)wascalculatedtobe125and6mg/personin
EuropeandtheUSA,respectively.Thedailyintakeof2-methyl-3-(p-isopropylphe-
nyl)propionaldehyde (No. 1465) was calculated to be 22 and 343mg/person, in
Europe and the USA, respectively. The daily per capita intake values for each
agentarereportedinTable1.
1.3 Absorption, distribution, metabolism and elimination
The esters of phenyl-substituted favouring agents (Nos 1458, 1460, 1461,
1464,1469,1470and1475)willbehydrolysedrapidlybycarboxyesterasestothe
corresponding 2-phenyl substituted alcohol or acid (Heymann, 1980). Before
absorptiontheseesters,aswellas2-phenylpropionaldehydedimethylacetal(No.
1468),arepredictedtoundergohydrolysis(Williams,1959)inthegastrointestinal
tract to yield compounds such as 2-phenylpropionaldehyde, b-methylphenethyl
alcohol, 2-ethyl-3-phenylpropionic acid, 4-phenylbutyric acid, and 2-methyl-4-
phenyl-2-butanol,whichwouldberapidlyabsorbed.
Once absorbed, the phenyl-substituted alcohols, aldehydes and acids may
followmultiplemetabolicpathways.Thealcoholsandaldehydescanbeconverted
tophenyl-substitutedcarboxylicacids.Theseacidscanbeconjugatedwithgluc-
uronicacidandexcretedintheurine.Theycanalsoundergob-oxidationtobenzoic
acidorphenylaceticacidderivatives,whichareconjugatedwithglycineorgluta-
minebeforebeingexcretedintheurine.Phenyl-substitutedalcoholscanalsobe
conjugateddirectlywithglucuronicacidbeforeexcretion(Williams,1959).
2-Oxo-3-phenylpropionic acid (phenylpyruvate and its sodium salt, Nos 1478
and1479)isametaboliteofphenylalanine.Itisprimarilydecarboxylatedtoyield
phenylacetateandisreadilyexcretedintheurine(Nelson&Cox,2000).
a,b-Unsaturated 2-phenylaldehyde derivatives (Nos 14721474) are electro-
philicinnatureandarepredictedtobedetoxifedbyglutathioneconjugation.The
structurallyrelatedsubstance2-phenylpropenal(atropaldehyde)readilyformsglu-
tathione conjugates when incubated with glutathione in vitro (Thompson et al.,
1996).
PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS 527
K2
T
a
b
l
e

1
.

S
u
m
m
a
r
y

o
f

t
h
e

r
e
s
u
l
t
s

o
f

s
a
f
e
t
y

e
v
a
l
u
a
t
i
o
n
s

o
f

p
h
e
n
y
l
-
s
u
b
s
t
i
t
u
t
e
d

a
l
i
p
h
a
t
i
c

a
l
c
o
h
o
l
s

a
n
d

r
e
l
a
t
e
d

a
l
d
e
h
y
d
e
s

a
n
d

e
s
t
e
r
s
a

u
s
e
d

a
s

f
a
v
o
u
r
i
n
g

a
g
e
n
t
s
F
l
a
v
o
u
r
i
n
g

a
g
e
n
t

N
o
.

C
A
S

N
o
.

a
n
d

S
t
e
p

A
3
a
,
b

D
o
e
s

C
o
m
m
e
n
t
s

C
o
n
c
l
u
s
i
o
n

b
a
s
e
d

s
t
r
u
c
t
u
r
e

i
n
t
a
k
e

e
x
c
e
e
d

o
n

c
u
r
r
e
n
t

i
n
t
a
k
e

t
h
e

t
h
r
e
s
h
o
l
d

f
o
r

h
u
m
a
n

i
n
t
a
k
e
?
S
t
r
u
c
t
u
r
a
l

c
l
a
s
s

I
E
t
h
y
l

4
-
p
h
e
n
y
l
b
u
t
y
r
a
t
e

1
4
5
8

1
0
0
3
1
-
9
3
-
3

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

O
O

E
u
r
o
p
e
:

N
D

a
n
d

U
S
A
:

0
.
0
1

-
M
e
t
h
y
l
p
h
e
n
e
t
h
y
l

a
l
c
o
h
o
l

1
4
5
9

1
1
2
3
-
8
5
-
9

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

H
O

E
u
r
o
p
e
:

0
.
1

U
S
A
:

0
.
0
1
2
-
M
e
t
h
y
l
-
4
-
p
h
e
n
y
l
-
2
-
b
u
t
y
l

1
4
6
0

1
0
3
-
0
7
-
1

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
c
e
t
a
t
e

O
O

E
u
r
o
p
e
:

0
.
4

a
n
d

U
S
A
:

0
.
0
4
2
-
M
e
t
h
y
l
-
4
-
p
h
e
n
y
l
-
2
-
b
u
t
y
l

1
4
6
1

1
0
0
3
1
-
7
1
-
7

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

i
s
o
b
u
t
y
r
a
t
e

O
O

E
u
r
o
p
e
:

a
n
d

U
S
A
:

1
528 PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS
K2
T
a
b
l
e

1
.

(
C
o
n
t
d
)
F
l
a
v
o
u
r
i
n
g

a
g
e
n
t

N
o
.

C
A
S

N
o
.

a
n
d

S
t
e
p

A
3
a
,
b

D
o
e
s

C
o
m
m
e
n
t
s

C
o
n
c
l
u
s
i
o
n

b
a
s
e
d

s
t
r
u
c
t
u
r
e

i
n
t
a
k
e

e
x
c
e
e
d

o
n

c
u
r
r
e
n
t

i
n
t
a
k
e

t
h
e

t
h
r
e
s
h
o
l
d

f
o
r

h
u
m
a
n

i
n
t
a
k
e
?
2
-
M
e
t
h
y
l
-
4
-

1
4
6
2

4
0
6
5
4
-
8
2
-
8

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

p
h
e
n
y
l
b
u
t
y
r
a
l
d
e
h
y
d
e

O
H

E
u
r
o
p
e
:

0
.
4

U
S
A
:

0
.
4
3
-
M
e
t
h
y
l
-
2
-

1
4
6
3

2
4
3
9
-
4
4
-
3

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

p
h
e
n
y
l
b
u
t
y
r
a
l
d
e
h
y
d
e

O
H

E
u
r
o
p
e
:

N
D

U
S
A
:

0
.
0
7
M
e
t
h
y
l

4
-
p
h
e
n
y
l
b
u
t
y
r
a
t
e

1
4
6
4

2
0
4
6
-
1
7
-
5

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

O
O

E
u
r
o
p
e
:

N
D

a
n
d

U
S
A
:

0
.
0
1
2
-
M
e
t
h
y
l
-
3
-
(
p
-
i
s
o
p
r
o
p
y
l
p
h
e
n
y
l
)

1
4
6
5

1
0
3
-
9
5
-
7

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

p
r
o
p
i
o
n
a
l
d
e
h
y
d
e

O
H

E
u
r
o
p
e
:

2
2

U
S
A
:

3
4
3
PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS 529
K2
2
-
M
e
t
h
y
l
-
3
-

1
4
6
6

4
1
4
9
6
-
4
3
-
9

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

t
o
l
y
l
p
r
o
p
i
o
n
a
l
d
e
h
y
d
e

(
o
,

m
,

O
H
C
H
3

E
u
r
o
p
e
:

0
.
6

a
n
d

p
)

U
S
A
:

2
7
2
-
P
h
e
n
y
l
p
r
o
p
i
o
n
a
l
d
e
h
y
d
e

1
4
6
7

9
3
-
5
3
-
8

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

O
H

E
u
r
o
p
e
:

1
2
5

U
S
A
:

6
2
-
P
h
e
n
y
l
p
r
o
p
i
o
n
a
l
d
e
h
y
d
e

1
4
6
8

9
0
-
8
7
-
9

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

d
i
m
e
t
h
y
l

a
c
e
t
a
l

O
O

E
u
r
o
p
e
:

a
n
d

U
S
A
:

3
2
-
P
h
e
n
y
l
p
r
o
p
y
l

b
u
t
y
r
a
t
e

1
4
6
9

8
0
8
6
6
-
8
3
-
7

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

O
O

E
u
r
o
p
e
:

0
.
0
0
4

a
n
d

U
S
A
:

0
.
5
2
-
P
h
e
n
y
l
p
r
o
p
y
l

i
s
o
b
u
t
y
r
a
t
e

1
4
7
0

6
5
8
1
3
-
5
3
-
8

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

O
O

E
u
r
o
p
e
:

a
n
d

U
S
A
:

0
.
0
5
530 PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS
K2
T
a
b
l
e

1
.

(
C
o
n
t
d
)
F
l
a
v
o
u
r
i
n
g

a
g
e
n
t

N
o
.

C
A
S

N
o
.

a
n
d

S
t
e
p

A
3
a
,
b

D
o
e
s

C
o
m
m
e
n
t
s

C
o
n
c
l
u
s
i
o
n

b
a
s
e
d

s
t
r
u
c
t
u
r
e

i
n
t
a
k
e

e
x
c
e
e
d

o
n

c
u
r
r
e
n
t

i
n
t
a
k
e

t
h
e

t
h
r
e
s
h
o
l
d

f
o
r

h
u
m
a
n

i
n
t
a
k
e
?

2
-
(
p
-
T
o
l
y
l
)
p
r
o
p
i
o
n
a
l
d
e
h
y
d
e
,

1
4
7
1

9
9
-
7
2
-
9

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

O
H

E
u
r
o
p
e
:

0
.
0
4

U
S
A
:

0
.
0
1
2
-
P
h
e
n
y
l
-
2
-
b
u
t
e
n
a
l

1
4
7
4

4
4
1
1
-
8
9
-
6

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

O
H

E
u
r
o
p
e
:

U
S
A
:

0
.
0
7
E
t
h
y
l

2
-
e
t
h
y
l
-
3
-

1
4
7
5

2
9
8
3
-
3
6
-
0

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n
p
h
e
n
y
l
p
r
o
p
a
n
o
a
t
e

O O

E
u
r
o
p
e
:

N
D

a
n
d

U
S
A
:

0
.
9
2
-
P
h
e
n
y
l
-
4
-
p
e
n
t
e
n
a
l

1
4
7
6

2
4
4
0
1
-
3
6
-
3

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

O
H

E
u
r
o
p
e
:

0
.
0
3

U
S
A
:

0
.
0
4
PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS 531
K2
2
-
M
e
t
h
y
l
-
4
-
p
h
e
n
y
l
-
2
-
b
u
t
a
n
o
l

1
4
7
7

1
0
3
-
0
5
-
9

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

O
H

E
u
r
o
p
e
:

U
S
A
:

0
.
0
1
2
-
O
x
o
-
3
-
p
h
e
n
y
l
p
r
o
p
i
o
n
i
c

1
4
7
8

1
5
6
-
0
6
-
9

N
o

S
e
e

n
o
t
e
s

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
c
i
d

O O
H
O

E
u
r
o
p
e
:

N
D

a
n
d

U
S
A
:

0
.
0
9
S
o
d
i
u
m

2
-
o
x
o
-
3
-

1
4
7
9

1
1
4
-
7
6
-
1

p
h
e
n
y
l
p
r
o
p
i
o
n
a
t
e

O O
N
a
O
S
t
r
u
c
t
u
r
a
l

c
l
a
s
s

I
I
5
-
M
e
t
h
y
l
-
2
-
p
h
e
n
y
l
-
2
-
h
e
x
e
n
a
l

1
4
7
2

2
1
8
3
4
-
9
2
-
4

N
o

S
e
e

n
o
t
e

N
o

s
a
f
e
t
y

c
o
n
c
e
r
n

O
H

E
u
r
o
p
e
:

1
8

U
S
A
:

6
532 PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS
K2
T
a
b
l
e

1
.

(
C
o
n
t
d
)
F
l
a
v
o
u
r
i
n
g

a
g
e
n
t

N
o
.

C
A
S

N
o
.

a
n
d

S
t
e
p

A
3
a
,
b

D
o
e
s

C
o
m
m
e
n
t
s

C
o
n
c
l
u
s
i
o
n

b
a
s
e
d

s
t
r
u
c
t
u
r
e

i
n
t
a
k
e

e
x
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PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS 533
K2
Table 2. Annual volumes of production of phenyl-substituted aliphatic alcohols
and related aldehydes and esters used as favouring agents in Europe and the
USA
Flavouring Mostrecent Intake
b
Intakeof Annual Consumption
agent(No.)

annual mg/day
mg/kgbw alcohol
volumein ratio
e

volume(kg)
a
perday equivalents
naturally
(mg/kgbw
occurring
perday)
c

foods(kg)
d
Ethyl4-phenylbutyrate(1458)
Europe ND ND ND ND
USA 0.05 0.01 0.0001 0.00002 - NA
b-Methylphenethylalcohol(1459)
Europe 0.7 0.10 0.002
USA
f
0.05 0.01 0.0001 - NA
2-Methyl-4-phenyl-2-butylacetate(1460)
Europe 3 0.4 0.01 0.008
USA
f
0.2 0.04 0.0006 0.0005 - NA
2-Methyl-4-phenyl-2-butylisobutyrate(1461)
Europe 12 2 0.03 0.02
USA 8 1 0.02 0.01 - NA
2-Methyl-4-phenylbutyraldehyde(1462)
Europe 3 0.4 0.01
USA 3 0.4 0.01 - NA
3-Methyl-2-phenylbutyraldehyde(1463)
Europe ND ND ND
USA 0.5 0.07 0.001 - NA
Methyl4-phenylbutyrate(1464)
Europe ND ND ND ND
USA 0.05 0.01 0.0001 0.00002 - NA
2-Methyl-3-(p-isopropylphenyl)propionaldehyde(1465)
Europe 153 22 0.4
USA 2604 343 6 1380 0.5
2-Methyl-3-tolylpropionaldehyde(o,m,andp)(1466)
Europe 4 0.6 0.01
USA 204 27 0.4 - NA
2-Phenylpropionaldehyde(1467)
Europe 878 125 2
USA 45 6 0.1 + NA
2-Phenylpropionaldehydedimethylacetal(1468)
Europe 32 5 0.08 0.03
USA 20 3 0.04 0.01 - NA
2-Phenylpropylbutyrate(1469)
Europe 0.03 0.004 0.0001 0.00007
USA 4 0.5 0.01 0.007 - NA
2-Phenylpropylisobutyrate(1470)
Europe 13 2 0.03 0.02
USA
f
0.3 0.05 0.001 0.0007 - NA
2-(p-Tolyl)propionaldehyde(1471)
Europe 0.3 0.04 0.001
USA 0.05 0.01 0.0001 - NA
5-Methyl-2-phenyl-2-hexenal(1472)
Europe 129 18 0.3
USA 42 6 0.1 206 5
534 PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS
K2
Table 2. (Contd)
Flavouring Mostrecent Intake
b
Intakeof Annual Consumption
agent(No.)

annual mg/day
mg/kgbw alcohol
volumein ratio
e

volume(kg)
a
perday equivalents
naturally
(mg/kgbw
occurring
perday)
c

foods(kg)
d
4-Methyl-2-phenyl-2-pentenal(1473)
Europe 3 0.4 0.01
USA 41 5 0.1 + NA
2-Phenyl-2-butenal(1474)
Europe 11 2 0.03
USA 0.5 0.07 0.001 902 1804
Ethyl2-ethyl-3-phenylpropanoate(1475)
Europe ND ND ND ND
USA
g
5 0.9 0.01 0.002 - NA
2-Phenyl-4-pentenal(1476)
Europe 0.2 0.03 0.0005
USA
f
0.2 0.04 0.001 - NA
2-Methyl-4-phenyl-2-butanol(1477)
Europe 25 4 0.06
USA
f
0.05 0.01 0.0001 - NA
2-Oxo-3-phenylpropionicacidand2-oxo-3-phenylpropionicacidsodiumsalt
(1478and1479)
Europe ND ND ND
USA
g
0.5 0.09 0.001 + NA
Total
Europe 1267
USA 2979
NA,notavailable;ND,nointakedatareported;+,reportedtooccurnaturallyinfoods
(Nijssenetal.,2003),butnoquantitativedata;-,notreportedtooccurnaturallyinfoods
a
FromInternationalOrganizationoftheFlavourIndustry(1995)andLucasetal.(1999)or
NationalAcademyofSciences(1970,1982,1987).
b
Intakeexpressedasmg/personperdaywascalculatedasfollows:
[(annualvolume,kg)(110
9
mg/kg)]/[populationsurveycorrectionfactor365days],
wherepopulation(10%,eatersonly)=3210
6
forEuropeand2610
6
fortheUSA.
Thecorrectionfactor=0.6forEuropeand0.8fortheUSA,representingtheassumption
thatonly60%and80%oftheannualvolumeofproductionofthefavour,respectively,
wasreportedinthepoundagesurveys(Lucasetal.,1999;InternationalOrganizationof
theFlavourIndustry,1995;NationalAcademyofSciences,1970,1982,1987).Intake
expressedasmg/kgbwperdaywascalculatedasfollows:
[(mg/personperday)/bodyweight],wherebodyweight=60kg.Slightvariationsmay
occurfromrounding.
c
Calculatedasfollows:(Relativemolecularmassofalcohol/Relativemolecularmassof
ester)Dailypercapitaintake(eatersonly)ester.
d
QuantitativedatafortheUSAreportedbyStofberg&Grundschober(1987).
e
Theconsumptionratioiscalculatedasfollows:
(annualconsumptionviafood,kg)/(mostrecentlyreportedvolumeasafavouringagent,kg).
f
AnnualvolumereportedinpreviousUSAsurveys(NationalAcademyofSciences,1970,
1982,1987).
g
Thevolumecitedistheanticipatedannualvolumeofproduction,whichwasthe
maximumamountoffavouringagentestimatedtobeusedannuallybythemanufacturer
atthetimethematerialwasproposedforuseasafavour.
PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS 535
K2
1.4 Application of the Procedure for the Safety Evaluation of
Flavouring Agents
Step 1. InapplyingtheProcedure,theCommitteeassigned20ofthe22favour-
ingagentsinthisgroup(Nos14581471,14741479)tostructuralclass
I.Theothertwofavouringagents(Nos1472and1473)wereassigned
tostructuralclassII(Crameretal.,1978).
Step 2. Allthefavouringagentsinthisgroupareexpectedtobemetabolizedto
innocuousproducts.TheirevaluationthereforeproceededviatheA-side
ofthedecision-tree.
Step A3. Theestimateddailyintakesofthe20favouringagentsinstructuralclass
IandthetwofavouringagentsinstructuralclassII,arebelowtherespec-
tive thresholds of concern (i.e. 1800mg/person for class I and 540mg/
personforclassII).AccordingtotheProcedure,thesafetyofthese22
favouring agents raises no concern when they are used at estimated
currentintakes.
Theintakeconsiderationsandotherinformationusedtoevaluatethe22phenyl-
substituted aliphatic alcohols and related aldehydes and esters in this group
accordingtotheProcedurearesummarizedinTable1.
1.5 Consideration of secondary components
Onememberofthisgroupoffavouringagents,2-methyl-3-(p-isopropylphenyl)-
propionaldehyde(No.1465),hasaminimumassayvalueof<95%.Informationon
thesafetyofthesecondarycomponentofthiscompoundissummarizedinAnnex
5 (Summary of the safety evaluation of secondary components for favouring
agentswithminimumassayvaluesoflessthan95%).Thesecondarycomponent,
2-methyl-3-(p-isopropylphenyl)propionicacid,isstructurallyrelatedtotheprimary
favouringagentandisexpectedtosharethesamemetabolicfate.Onthisbasis,
the Committee considered that 2-methyl-3-(p-isopropylphenyl)propionaldehyde
doesnotpresentasafetyconcernatestimatedcurrentintakes.
1.6 Consideration of combined intakes from use as favouring agents
Intheeventthatall20agentsinstructuralclassIwereconsumedconcurrently
on a daily basis, the estimated combined intake would not exceed the human
intake threshold for class I (1800mg/person per day). In the event that the two
agents in structural class II were consumed concurrently on a daily basis, the
estimatedcombinedintakewouldnotexceedthehumanintakethresholdforclass
II(540mg/personperday).Overallevaluationofthedataindicatedthatcombined
intakeoftheagentsinthisgroupwouldnotpresentasafetyconcern.
1.7 Conclusions
The Committee concluded that none of the favouring agents in this group of
phenyl-substitutedaliphaticalcoholsandrelatedaldehydesandesterswouldraise
a safety concern at estimated current intakes.Available data on the toxicity and
536 PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS
K2
metabolism of these substances were consistent with the results of the safety
evaluation.
2. RELEVANT BACKGROUND INFORMATION
2.1 Additional considerations on intake
The volumes of production and daily per capita intakes of each agent in this
grouparereportedinTable2.
Phenyl-substituted aliphatic alcohols and related substances have been
detectedinavarietyoffoodsincludingnuts(i.e.nutmeg,roastedflberts,peanuts,
macadamia nuts), cheeses (i.e. blue, provolone), sesame seeds, french-fried
potatoes,porkliver,wine,malt,starfruit,mushrooms,asparagus,okra,coffee,tea,
andcocoa(Nijssenetal.,2003).AsshowninTable2,sevenoftheagentsinthis
grouphavebeenreportedtooccurnaturallyinfoods(Nijssenetal.,2003).Quan-
titativedataonnaturaloccurrenceandconsumptionratioshavebeenreportedfor
threeagentsinthegroup.Dataonannualvolumesofproductionfor2-phenyl-2-
butenal (No. 1474), 5-methyl-2-phenyl-2-hexenal (No. 1472), and 2-methyl-3-(p-
isopropylphenyl)propionaldehyde (No. 1465) demonstrate that their consumption
occurspredominantlyfromtraditionalfoods(i.e.consumptionratio,>1).2-Methyl-
3-(p-isopropylphenyl)propionaldehyde (No. 1465) is present in nutmeg; intake of
this agent from this food is approximately 50% of the intake of 2-methyl-3-(p-
isopropylphenyl)propionaldehyde as an added favouring agent (Stofberg &
Kirschman,1985;Stofberg&Grundschober,1987)(seeTable2).
2.2 Biological data
2.2.1 Biochemical data
(a) Hydrolysis
Phenyl-substituted esters are expected to be hydrolysed rapidly to the corre-
sponding phenyl-substituted alcohols or carboxylic acids by classes of enzymes
knownascarboxylesterases(Heymann,1980).Inmammals,theseenzymesoccur
in most tissues (Heymann, 1980;Anders, 1989), but predominate in the hepato-
cytes(Heymann,1980).Acetalsalsoareknowntohydrolysetotheircorresponding
aldehydes and alcohols under acidic conditions, even in the absence of enzyme
catalysis(Morgareidge,1962).
Methodsusingsimulatedgastricjuiceandintestinalfuidhavebeendeveloped
to study ester hydrolysis in vitro. Aromatic esters such as phenylethyl acetate,
methylphenylacetate,ethylphenylacetate,isopropylphenylacetate,isoamylphen-
ylacetate,andcitronellylphenylacetaterapidlyhydrolyseinsimulatedgastricjuice
and intestinal fuid (Longland et al., 1977). Rapid hydrolysis (t
1/2
1.4 0.1min)
occurredwhenaseriesofstructurallyrelated3-phenylpropenyland3-phenylpro-
pionic acid esters (cinnamyl propionate, propyl cinnamate, butyl cinnamate, 3-
phenylpropyl cinnamate and cinnamyl cinnamate) were incubated with artifcial
intestinalfuidcontainingpancreatin(Buck&Renwick,2000).
PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS 537
K2
Aromaticacetalsalsorapidlyhydrolyseinacidicsimulatedgastricjuice.Incuba-
tion of the aromatic acetal, 2-phenylpropionaldehyde dimethyl acetal (No. 1468)
ataconcentrationof1mmol/linthepresenceofsimulatedgastricjuiceat37C
invitroresultedin97%hydrolysiswithin1h,butincubationwithsimulatedintestinal
fuidfor5hresultedinonly5.1%hydrolysis(Morgareidge,1962).
Analogously,phenyl-substitutedestersandacetalsareanticipatedtobehydro-
lysedtothecorrespondingalcohols,aldehydes,andcarboxylicacids,beforebeing
metabolizedfurtherandexcreted.
(b) Absorption, distribution and excretion
2-Phenylpropanolderivatives(Nos1159,1463,14671474,and1476)inthis
groupareexpectedtoberapidlyabsorbedinthegastrointestinaltractandexcreted
primarilyintheurine.
Fivechinchillarabbitsweregivenasingledoseofb-methylphenethylalcohol
(No.1459)at500mg/kgbwsuspendedinwaterbyoralgavage.Sixtytoeightyper
centoftheadministereddosewasexcretedasglucuronicacidconjugatesinthe
urineat24h(Robinsonetal.,1955).InastudybyGruneberg&Ladgecker(1957),
agroupofsevenrabbitswasgivenb-methylphenethylalcoholorallyatadoseof
100, 250, 500, or 1000mg/kgbw, while a group of four dogs received b-
methylphenethylalcoholorallyatdosesrangingfrom50to200mg/kgbw.Maximum
urinary excretion of glucuronic acid conjugates was reached 3h after dosing in
bothspecies.Pooledsamplesofurineoftherabbitsordogsat24hrevealedthat
50and58%,respectively,oftheadministereddosewasexcretedasunconjugated
or conjugated (glucuronic acid) metabolites. A human given b-methylphenethyl
alcohol orally at a dose of 100mg/kgbw excreted 18.7% of the administered
dose in the urine, mainly as glucuronic acid conjugates, within 24h after dosing
(Gruneberg&Ladgecker,1957).
Three chinchilla rabbits received 2-phenylpropionaldehyde (No. 1467) as a
single dose at 300mg/kgbw by gavage. At 24h, it was found that 52% of the
administered dose was excreted as the glucuronic acid conjugate of the corre-
spondingacid,2-phenylpropionicacid,intheurine(Robinsonetal.,1955).
Intwomengiven5mgof(+/-)-[methyl-
14
C]2-phenylpropionicacidorally,95%
and100%,respectively,oftheadministereddosewasexcretedintheurineasthe
correspondingglucuronicacidconjugatewithin24h.IntwoRhesusmonkeysgiven
(+/-)-[methyl-
14
C]2-phenylpropionicacidatadoseof81mg/kgbwbyintramuscular
injection, 82 and 71% of the administered dose, respectively, was excreted as
glucuronicacidconjugatesinurinecollectedfor24hafterdosing.Inrabbitsgiven
(+/-)-[methyl-
14
C]2-phenylpropionic acid at a dose of 81mg/kgbw, 82% of the
administereddosewasexcreted,predominantlyastheglucuronicacidconjugate
(73%),intheurinewithin24h.Bile-ductcannulatedratsweregiven(+/-)-[methyl-
14
C]2-phenylpropionic acid at a dose of 5, 50, or 500mg/kgbw and bile was col-
lected for 3h after dosing. Independent of dose, approximately 2030% of the
administereddosewasexcretedinthebile,mainlyastheglucuronicacidconju-
gate (75%) with smaller amounts excreted as the unchanged acid (Dixon et al.,
1977).
538 PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS
K2
MaleSprague-DawleyratsgivenR-2-phenylpropionicacidorS-2-phenylpropi-
onicacidatadoseof200mg/kgbwbyintraperitonealinjectionexcretedthecor-
responding glucuronic acid conjugates into the urine within 16h (Li et al., 2002).
MaleSprague-Dawleyratsgiven2-phenylpropionicacidatadoseof80mg/kgbw
perdayfor7daysexhibitedincreasedlevelsofglucuronicacidconjugatesinthe
urine (Lake et al., 1980). Male Japanese white rabbits given 2-phenylpropionic
acid at a dose of 200mg/kgbw as the sodium salt in aqueous solution by an
unspecifed route excreted the corresponding glucuronic acid conjugate in the
urinewithin24hurine(Nakamura&Yamaguchi,1987).
Inamultispeciesstudy,approximately50%ofadoseof150mg/kgbwofR,S-
[methyl-
14
C]2-phenylpropionic acid administered by intraperitoneal injection was
excretedintheurineofmale(n=4)andfemalerats(n=4)(57.511.6and46.6
4.9%,respectively),mice(n=4)(51.35.6%),andtworabbits(46.6and54.9%,
respectively),within48h(valuesquotedaremeanstandarddeviation).Inboth
sexesofallspeciestested,mostoftheadministereddosewaseliminatedwithin
the frst 24h (Fournel & Caldwell, 1986). Male Wistar albino rats were given R-,
S-, or R,S-2-phenylpropionic acid at a dose of 150mg/kgbw by intraperitoneal
injection, and samples of blood, urine, and faeces were obtained. The plasma
half-livesoftheR-,S-,andR,S-isomers(3.00,4.78,and3.83h,respectively)and
theeliminationrates(0.2313,0.1451,and0.1811perhour,respectively)indicate
that the R-(-) isomer is more rapidly cleared than the S-(+) isomer (65.4 versus
43.6ml/h). In rats, the S/R ratio increased from 1 (racemic) to >2 within 8h of
dosing. The stereoselective inversion of R- to S-has been reported to be more
pronouncedinfemaleratsthaninmales.GiventheinversionoftheR-toS-isomer,
itwasnotunexpectedthatthebodyburden,measuredbytheareaunderthecurve
(AUC),wouldbegreaterfortheS-isomer(AUC
08h
=214.5,272.0,and486.5mg/
mlhfortheR-,S-,andR,S-isomers,respectively)(Fournel&Caldwell,1986).
Onceformed,theacyl-glucuronideofR-andS-2-phenylpropionicacidunder-
goesinternalacylmigration.TherateofconversionoftheRformisabouttwofold
greaterthanthatfortheSform.Thusthedegradationhalf-lifefortheconjugated
formofR-2-phenylpropionicacidisapproximatelyone-halfofthatfortheS-isomer
(i.e.1.8versus3.3h)(Akiraetal.,2000).Takentogether,thesepharmacokinetic
datademonstratethatR-,S-,orR,S-2-phenylpropionicacidisrapidlydistributed
andeliminatedinlaboratoryanimals(Fournel&Caldwell,1986;Meffnetal.,1986;
Nakamura&Yamaguchi,1987;Akiraetal.,2000).R-,S-,orR,S-2-Phenylpropionic
acidderivedfromthemetabolismofphenylpropanolanditsesterswouldalsobe
rapidlydistributedandeliminated.
Thestereoselectivedispositionof(+/-)-2-phenylpropionicacidwasstudiedin
groupsofintact,bile-ductcannulated,bile-ductligated,andbothnephrectomized
and bile-duct cannulated rats given a single dose at 20mg/kg bw by intrave-
nousinjection.After5min,plasmaproteinbindingoftheR-(-)stereoisomerwas
slightly higher than that of the S-(+) stereoisomer, which corresponded to a
slightlyincreasedtissuedistributionoftheS-(+)isomer.After1h,theenantiomeric
excess of the S-(+) isomer in the plasma was 60%, suggesting selective loss of
theR-(-)-isomer.After6h,bile-ductcannulatedratsexcretedagreaterproportion
of the administered dose of (+/-)-2-phenylpropionic acid in the bile (51% as the
PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS 539
K2
glucuronicacidconjugate)thanintheurine(40%,comprising8%asfreeand32%
as conjugated acid).The total amount of S-(-) isomer excreted into the bile and
urineaccountedfor63%oftheadministereddose,suggestingR-toS-isomeriza-
tion.At any time-point, the total elimination (i.e. biliary and urinary excretion) of
freeandconjugatedR-(-)isomerwas<50%.Thecombinationofalowerrateof
excretion and lower plasma concentrations for the R-(-) isomer provide further
evidenceofR-toS-isomerization(Yamaguchi&Nakamura,1985).
Nonsteroidal anti-infammatory drugs (NSAIDs) such as ibuprofen, 2-(4-
isobutylphenyl)propionicacid,arederivativesof2-phenylpropionicacid.Thephar-
macokineticactivityofNSAIDs,especiallyibuprofen,hasbeendirectlyrelatedto
chiral inversion and the subsequent yield of the pharmacologically more active
stereoisomer(Leeetal.,1985).Therefore,extensivestudies,bothmetabolicand
toxicological,inhumanshavebeenperformedforthischemicalclass.Noattempt
hasbeenmadetocompletelyreviewallthesestudieshere.However,theresults
ofthemostrecentandthoroughstudiesareincludedtoproviderelevantdatafor
humans.
Six men and six women were each given 400mg (a dose of approximately
5.7mg/kgbw)of(+/-)-ibuprofenandpharmacokineticparametersweremonitored
for 10h after dosing. Maximum serum concentrations (C
max
) of both enantiomers
were achieved 3h after dosing, with the S-(+) isomer showing a slightly higher
valuethantheR-(-)isomer.AsignifcantlyhigherAUCfortheS-(+)isomercorre-
spondedtoamorerapideliminationhalf-lifefortheR-(-)isomer.Stereoselective
proteinbindingshowedthatthefractionoftheunboundS-(+)isomerissignifcantly
greaterthantheunboundR-(-)isomer(S/R=2/1).Basedonthestereochemical
compositionof(+/-)-ibuprofenanditsmetabolitesintheurine,approximately68%
oftheR-(-)isomerundergoeschiralinversion,withasignifcantlyhigherclearance
rateviainversionthanbyotherroutes(Tanetal.,2002).
Inasimilarstudy,sixhealthyhumanvolunteersweregiven400mgofibuprofen
intheformoftwotabletsontwoseparateoccasionsandpharmacokineticparam-
etersweremonitoredfor6hafteradministration.Maximumserumconcentrations
were achieved within the frst 2h and, as in the above study, the S-isomer dem-
onstrated a signifcantly higher serum concentration than the R-isomer. Both the
AUCandtheserumhalf-lifeweresignifcantlyhigherfortheS-isomerthantheR-
isomer.TherateofeliminationmeasuredfortheR-isomerwassignifcantlygreater
thanthatoftheS-isomer(Surietal.,1997).
Ina1-monthstudy,threegroupsofhumanvolunteers,young(onemale,seven
females),elderly(eightmales,sixfemales),andelderlywithrenalimpairment(four
males,ninefemales),weregiven800mgofracemicibuprofenthreetimesperday
(2400mg/day). S-D
4
-Ibuprofen (10mg) was administered with the frst and last
doses of the study. The half-life of S-D
4
-Ibuprofen was signifcantly longer (p <
0.05)intheelderlyvolunteerswithrenalimpairment.TheinversionofR-ibuprofen
to S-ibuprofen was comparable in all three groups.After the frst dose, the S/R
ratio for all groups was 2.1 and after the last dose the S/R ratios were 2.8, 2.4,
and 2.4 for young, elderly, and elderly with renal impairment, respectively. The
groupofyoungpeopleshowedsignifcantly(p<0.05)lowerlevelsofunboundS-
ibuprofenthanthegroupsofelderlypeopleandelderlypeoplewithrenalimpair-
540 PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS
K2
mentatthefrstandlastdosestested.Asintheabovestudy,incomparisonwith
the S-enantiomer, greater proportions of the R-enantiomer were found bound to
serum protein. After the frst and last dose tested, the body burden for the S-
enantiomer was signifcantly (p < 0.0001) greater than that of the R-enantiomer,
whichwasassociatedwithamorerapidclearanceoftheR-enantiomer.Repeated
dosingincreasedtherateofinversion of R-toS-,buthadnoeffectonthefraction
inverted (Rudy et al., 1995). On the basis of these data, the pharmacokinetic
behaviourof2-phenylpropionicacidderivativesaresimilarinratsandhumans.
Thefour3-phenylpropyl(Nos1465,1466,1475,and1478)derivativesinthis
groupareanticipatedtoshowpharmacokineticbehavioursimilartothatofother,
previously evaluated, 3-phenylpropyl derivatives (Annex 1, reference 149). Like-
wise, the six 4-phenylbutyl derivatives (Nos 1458, 14601462, 1464, and 1477)
areanticipatedtoshowapatternofabsorption,distribution,andeliminationsimilar
tothatof2-phenylethylderivatives(Annex1,reference160).
(c) Metabolism
Afterhydrolysis,theresultingalcoholsandaldehydesinthisgroupoffavouring
agentsareoxidizedtoyieldthecorrespondingcarboxylicacids.Inthecaseof2-
phenylpropylderivatives,thecarboxylicacidissubsequentlyconjugatedwithgluc-
uronic acid and excreted primarily in the urine. Derivatives of 3-phenylpropionic
acidand4-phenylbutyricacidmayalsoundergob-oxidationandcleavagetoyield
thecorrespondingderivativesofbenzoicacid(Annex1,reference149)andphen-
ylaceticacid(Annex1,reference160).Theseacidsareconjugatedpredominantly
withglycineorglutamine,respectively,andexcretedintheurine(seeFigure1).
Groups of three rabbits were given b-methylphenethyl alcohol (No. 1459)
at a dose of 500mg/kgbw, 2-phenylpropionaldehyde (No. 1467) at a dose of
300mg/kgbw, or (+/-)-, (+)-, or (-)-2-phenylpropionic acid at a dose of
300mg/kgbwbyoraladministration,andurinewascollectedfor24h.Inallcases,
theprincipalmetabolite(>52%)wasidentifedastheglucuronicacidconjugateof
2-phenylpropionic acid (Robinson et al., 1955). After administration of p-
methylphenethyl alcohol at a dose of 544mg/kgbw, approximately 70% of the
administereddosewasexcretedintheurineastheglucuronicacidconjugateof
theacidmetabolite.Asmallerquantity(12%)oftheadministereddosewasexcreted
astheglucuronicacidconjugateoftheparentalcohol(Williams,1959).
A comprehensive study has been performed on the effect of species, dose,
and mode of administration on the metabolism of (+/-)-2-phenylpropionic acid
(Dixon et al., 1977). For doses in the range of 5 to 500mg/kgbw, the principal
metaboliteinman(5mg/kgbw,oral),Rhesusmonkeys(81mg/kgbw,administered
byintramuscularinjection),rabbits(81mg/kgbw,administeredorally),cats(81mg/
kgbw, administered by intraperitoneal injection), and rats (5, 50, 500mg/kgbw,
administeredbyintraperitonealinjection)wastheglucuronicacidconjugateofthe
2-phenylpropionic acid administered. Only in the cat were appreciable quantities
oftheglycine(9%)andtaurine(13%)metaboliteformidentifed.
Inafollow-upstudywiththedifferentstereochemicalforms(R,S,andR,S)of
2-phenylpropionicacid(Fournel&Caldwell,1986),itwasshownthatchiralinver-
PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS 541
K2
sionoccursduringthemetabolismof2-phenylpropionicacidinanimals(seeFigure
2).Thisprocessisindependentofglucuronidationoftheacid.Groupsofrats(four
ofeachsex),mice(fourmales),andrabbits(twofemales)weregivenR,S-[methyl-
14
C]2-phenylpropionic acid at a dose of 150mg/kgbw intraperitoneally, and urine
was collected for 24h.The female rats excreted slightly more of the free (7.4%)
andconjugated(37.4%)2-phenylpropionicacidthanthemalerats(3and30.1%,
respectively). The rabbits showed a metabolic profle similar to that of the rats,
whilethemiceexcretedmoreoftheunconjugatedacid(13.4%freeversus29.1%
conjugated). Measurement of the amounts of urinary R-(-) and S-(+) stereoiso-
mers showed that male and female rats and rabbits exhibited stereoselective
inversion of the R-(-) to the S-(+), with greater stereoselectivity occurring during
the 2448h after dosing.Although, chiral inversion was reported not to occur in
miceduringmetabolism,micedemonstratedaslightpreferenceforglucuronidation
oftheS-(+)isomer(Fournel&Caldwell,1986).Theresultsobtainedinratswere
confrmedinanotherstudyinwhichratsweregiven(+/-)-2-phenylpropionicacid
R
CO
2
H
CO
2
Glu
R= -CH
2
OH or -CHO
OH
O OH
O OR'
R'= Gly or Gln
H
O
H
O SG
GSH
Figure 1. Metabolism of phenyl-substituted aliphatic alcohols and related
aldehydes and esters
Gln,glutamine;Gly,glycine;Glu,glutamicacid;GSH,glutathione
542 PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS
K2
at a dose of 20mg/kgbw by intravenous injection. The enantiomeric enrichment
of the S-(+) isomer appearing in the bile and urine within 6h was approximately
63%(Yamaguchi&Nakamura,1985).
Inthesamestudy(Fournel&Caldwell,1986),theinfuenceofdoseandroute
of administration on the stereochemical course of metabolism was examined.
WhengroupsoffourmaleratsweregivenR-(-)-2-phenylpropionicacidatadose
of30,150,or300mg/kgbw,thestereoisomerratiomeasuredintheurineat24h
wasapproximatelyR/S=7/3atthetwohigherdoses,butonly11/9atthelowest
dose.Inallcases,inversiontotheS-(+)formwasgreateronday2thanonday
1ofdosing.S-(+)-Phenylpropionicacid,administeredatthesamedoses,showed
amuchslowerrateofconversiontotheR-(-)form.Finally,theisomericcomposi-
tionintheurineofratsgiventheracemicform(R,S),eitherorallyorbyintraperi-
tonealinjection,wasindependentoftherouteofadministration.Theauthorsnoted
that both enantiomers undergo glucuronidation and that there is some stereose-
lectivityforglucuronidationoftheS-(+)isomerintheratandthemouse.Foribu-
profen, a derivative of 2-phenylpropionic acid, glucuronidation of the S-form was
favouredinhumans(Leeetal.,1985).
Inastudyusingdeuteriumlabelled[
2
H
5
](+/-)-4-isobutyl-2-phenylpropionicacid
1

(i.e. ibuprofen) in rats it was shown that chiral inversion involves the loss of the
hydrogen at the chiral position. However, hydrogen loss was not identifed as a
ratelimitingstep.Theauthorsproposedthatchiralinversionoccursviaenzyme-
catalysedstereoselectiveformationofacoenzymeAesteroftheacidandsubse-
quentformationofaplanarenolatetautomer(Saninsetal.,1991).
In the pharmacokinetic study discussed above (section 2.2.1(b); Tan et al.,
2002),metabolitesintheurineinhumanswereanalysed24hafteradministration
of(+/-)-ibuprofenasanoraldoseatapproximately5.7mg/kgbw.Oftheadminis-
tereddose,74%wasexcretedastheS-(+)isomer(unchangedacid,11%;oxidized
positionsontheisobutylsidechain,63%).Adifferentstereochemicalpreference
for the S-(+) isomer occurred for each metabolite. The S/R ratio of 7/1 was
recorded for the glucuronic acid conjugate of (+/-)-ibuprofen, 4.8/1 for the 2-
hydroxylated metabolite, and 3.4/1 for the 4-carboxy metabolite. No signifcant
differenceinmetabolismwasobservedbetweenmalesandfemales.
Inconclusion,estersofb-methylphenethylalcoholarehydrolysedtothecom-
ponent alcohol, and then subsequently oxidized to the corresponding aldehyde
andcarboxylicacid.Theresulting2-phenylpropanionicacidisthenconjugatedwith
glucuronicacidandexcretedprimarilyintheurine.Ithasbeenshownthatstere-
oselectivechiralinversion(R-toS-)occursduringthemetabolismof2-phenylpro-
pionicacidinvivo.
Ifthealkylside-chaincontainsa,b-unsaturationasin2-phenylalkenylaldehyde
derivatives(Nos14721474),theelectrophilica,b-unsaturatedalkeneissuscep-
C O
2
H
1

(+/-)-4-isobutyl-2-phenylpropionicacid
PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS 543
K2
tibletonucleophilicattackbyglutathioneorotherendogenousthiols.Forexample,
2-phenylpropenal(No.1467),ametaboliteoftheanti-epilepticdrugfelbamate,has
been shown to undergo glutathione conjugation to yield mercapturic acid conju-
gates in rodents and humans (Thompson et al., 1997). When six adult male
Sprague-Dawleyratsweregivenfelbamate
2
atadoseof800mg/kgbwbygavage,
2-phenylpropenalmercapturicacidconjugatewasidentifedintheurineat18h.In
the same study, in one healthy male human volunteer given a tablet containing
600mgoffelbamate(adoseof9mg/kgbwdeterminedbystudyauthors),theurine
O N H
2
O H
2
N
O O
2

felbamate
CH
3
O
OH
R-2-phenylpropionic acid
CH
3
O
OH
S-2-phenylpropionic acid
O
OH
RS-2-phenylpropionic acid
CH
3
CH
3
O
OGlu
CH
3
O
OGlu
major urinary metabolite
S/R ratio increases over time
Figure 2. Metabolism of RS-2-phenylpropionic acid in mammals
544 PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS
K2
at8hcontainedreduced
3
andoxidized
4
mercapturicacidconjugatesof2-phenyl-
propenal(Thompsonetal.,1997).Similarly,thea,b-unsaturatedderivativesof2-
phenylalkenealdehyde(Nos14721474)mayconjugatewithglutathione,before
beingreadilyconvertedintomercapturicacidconjugatesandeliminated.
Experiments in vitro provide additional evidence for glutathione conjugation.
2-Phenylpropenalreadilyformsglutathioneconjugateswhenincubatedinvitrowith
freeglutathioneatpH8.0(Thompsonetal.,1996).Thehalf-lifefor2-phenylpro-
penalisapproximately1min(Thompsonetal.,1996).Thehalf-lifeofformationof
theglutathioneconjugateof2-phenylpropenalwasdeterminedindividuallyinthe
presence of three isoforms of human glutathione transferase (GST), GSTM1-1,
GSTP1-1,orGSTA1-1,andintheabsenceofGST.Theformationoftheglutathi-
oneconjugatewasmoreeffcientlycatalysedbyGSTM1-1thanGSTP1-1,followed
byGSTA1-1(half-livesofformation(t
1/2
)were11.31.2,17.70.4,and26.4
2.4,respectivelyandk
cat
/K
m
valueswere0.2750.035,0.1640.005,and0.042
0.005mmol/l
-1
s
-1
,respectively)(Dieckhausetal.,2001).Invitro,2-phenylprope-
nalinhibitedthecatalyticactivityofGSTM1-1andGSTP1-1,withtheinhibitionof
thelatterbeingreversibleinthepresenceofexcessglutathione(Diekhausetal.,
2001).
Onthebasisofthesedata,twometabolicoptionswereanticipatedtoexistfor
themetabolicdetoxicationof2-phenylalkenylaldehydes(e.g.Nos1472,1473,and
1476). In one pathway, the aldehyde is oxidized to the corresponding carboxylic
acid,andthensubsequentlyconjugatedwithglucuronicacidandexcreted.Inthe
alternative pathway, the alkene function may react with glutathione to yield mer-
capturicacidconjugatesthatarereadilyexcretedprimarilyintheurine.
Derivativesof3-phenylpropanal(Nos1465and1466)andarelatedester(No.
1475) undergo b-oxidation and cleavage to yield benzoic acid and are subse-
quentlyconvertedtohippuricacidandexcretedintheurine.Metabolicdatarelated
to the evaluation of 3-phenylpropyl derivatives was reviewed previously by the
Committee (Annex 1, reference 149). Dogs given the sodium salt of 2-methyl-3-
phenylpropionic acid at a dose of approximately 500mg/kgbw excreted 77% of
theadministereddoseashippuricacidandbenzoicacidintheurine(Kay&Raper,
1924). Sterically-hindered acids, 2-ethyl- and 2-propyl-3-phenylpropionic acids,
wereshownnotundergob-oxidationindogsgivenanunspecifedoraldose,but
insteadwereexcretedunchangedintheurine(Carter,1941).
O H
S C O
2
H
N H O C H
3
3
reducedmercapturicacidconjugateof2-phenylpropenal
OH
S CO
2
H
NHOCH
3
O
4

oxidizedmercapturicacidconjugateof2-phenylpropenal
PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS 545
K2
The 3-phenylpropanoic acid derivatives, 2-oxo-3-phenylpropionic acid (Nos
1478and1479),areendogenousinhumans,beingformedbytheoxidativedeami-
nation of phenylalanine (Nelson & Cox, 2000). Extensive scientifc studies have
been performed on this substance as it is a major catabolite of phenylalanine in
patients with phenylketonuria. Excess phenylalanine undergoes transamination
with pyruvate to form alanine and phenylpyruvate, which can accumulate in the
blood, tissues, and urine in patients with phenylketonuria (Nelson & Cox, 2000).
Atlowlevels,endogenous2-oxo-3-phenylpropionicacidwasfoundtobeprimarily
decarboxylatedtoyieldphenylacetate,andreadilyexcretedintheurine.Aminor
metabolite, the reduction product phenylacetate, was also excreted in the urine
(Nelson&Cox,2000).
Afterhydrolysisof4-phenylbutyrateesters(Nos1458and1464),andoxidation
of 2-methyl-4-phenylbutyraldehyde (No. 1462), the resulting carboxylic acids
undergob-oxidationandcleavagetoyieldphenylaceticacid,beforebeingexcreted
primarilyastheglutamineconjugateinhumansandaglycineconjugateinrodents
(Annex 1, reference 160). Of the doses of the sodium salt of 2-methyl-4-phenyl-
butyric acid of 480 and 600mg/kgbw administered hypodermically to two cats,
approximately42%wasexcretedintheurineastheglycineconjugateofphenyl-
aceticacid(Kay&Raper,1924).Indogs,4-phenylbutyricacidhasbeenshownto
undergo b-oxidation, resulting in urinary excretion of phenylacetic acid and its
glycineconjugate(Raper&Wayne,1928).
Theremainingthree4-phenylbutylderivatives(Nos1460,1461,and1477)are
tertiaryalcoholsanddonotundergob-oxidation,butrather,conjugatedirectlywith
glucuronicacidandareexcretedintheurine.2-Phenylpropan-2-ol,astructurally
related tertiary alcohol at a dose of 500mg/kgbw given orally to three rabbits
resultedinalmostcomplete(6098%)excretionoftheglucuronicacidconjugate
oftheunchangedalcoholwithin24h(Robinsonetal.,1955).
In conclusion, the 2-phenylpropyl or propenyl derivatives in this group are
readily hydrolysed and oxidized to the corresponding carboxylic acids, before
beingsubsequentlyconjugatedwithglucuronicacidandexcreted.a,b-Unsaturated
aldehyde derivatives may be conjugated with glutathione and excreted as mer-
capturic acids, while 3-phenylpropyl derivatives, are expected to oxidize to the
corresponding3-phenylpropionicderivatives.Subsequently,the3-phenylpropionic
derivativesaresubjectedtob-oxidationandcleavage,toyieldbenzoicacid.The
resulting benzoic acid is conjugated with glycine and excreted as hippuric acid.
Finally,4-phenylbutylderivativesundergohydrolysisfollowedbyfunctionalgroup
oxidation (b-oxidation) and cleavage to phenylacetic acid, which is conjugated
primarilywithglutamineandexcretedintheurine.Conversely,4-phenylbutylderiv-
atives occurring as tertiary alcohols are instead conjugated with glucuronic acid,
beforeeliminationintheurine.
2.2.2 Toxicological studies
(a) Acute toxicity
Oralmedianlethaldose(LD
50
)valueshavebeenreportedfor9ofthe21sub-
stances in this group and are summarized in Table 3. Eight of the phenyl-
546 PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS
K2
T
a
b
l
e

3
.

S
t
u
d
i
e
s

o
f

t
h
e

a
c
u
t
e

t
o
x
i
c
i
t
y

o
f

p
h
e
n
y
l
-
s
u
b
s
t
i
t
u
t
e
d

a
l
i
p
h
a
t
i
c

a
l
c
o
h
o
l
s

a
n
d

r
e
l
a
t
e
d

a
l
d
e
h
y
d
e
s

a
n
d

e
s
t
e
r
s

a
d
m
i
n
i
s
t
e
r
e
d

o
r
a
l
l
y
N
o
.

F
l
a
v
o
u
r
i
n
g

a
g
e
n
t

S
p
e
c
i
e
s

S
e
x

L
D
5
0

R
e
f
e
r
e
n
c
e

(
m
g
/
k
g

b
w
)
1
4
5
9

b
-
M
e
t
h
y
l
p
h
e
n
e
t
h
y
l

a
l
c
o
h
o
l

R
a
t

N
R

3
0
0

M
c
G
e
e

(
1
9
7
4
)
1
4
6
0

2
-
M
e
t
h
y
l
-
4
-
p
h
e
n
y
l
-
2
-
b
u
t
y
l

a
c
e
t
a
t
e

R
a
t

N
R

8
0
0

L
e
v
e
n
s
t
e
i
n

(
1
9
7
5
)
1
4
6
5

2
-
M
e
t
h
y
l
-
3
-
(
p
-
i
s
o
p
r
o
p
y
l
p
h
e
n
y
l
)
p
r
o
p
i
o
n
a
l
d
e
h
y
d
e

R
a
t

M
,

8
1
0

J
e
n
n
e
r

e
t

a
l
.

(
1
9
6
4
)
1
4
6
6

2
-
M
e
t
h
y
l
-
3
-
t
o
l
y
l
p
r
o
p
i
o
n
a
l
d
e
h
y
d
e

R
a
t

N
R

1
0
0

M
o
r
e
n
o

(
1
9
7
7
)
1
4
6
7

2
-
P
h
e
n
y
l
p
r
o
p
i
o
n
a
l
d
e
h
y
d
e

R
a
t

M
,

8
0
0

J
e
n
n
e
r

e
t

a
l
.

(
1
9
6
4
)
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(
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(
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,

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,

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.
PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS 547
K2
substitutedalcoholsandrelatedaldehydesandestersusedasfavouringagents
(Nos1459,1460,14651468,1471,and1477)haveoralLD
50
valuesintherange
of 1850 to 5000mg/kgbw in rats (Jenner et al., 1964; Br & Griepentrog, 1967;
Wong & Weir, 1971; Moreno, 1973, 1975, 1977; Shellanski & Moldovan, 1973;
McGee, 1974; Levenstein, 1975). In mice, oral LD
50
values of >41000mg/kgbw
were reported for ethyl 2-ethyl-3-phenylpropanoate (No. 1475) (Pellmont, 1969),
demonstrating that the oral acute toxicities of phenyl-substituted alcohols and
relatedaldehydesandestersinmicearealsolow.
(b) Short-term studies of toxicity
The results of short-term studies of toxicity with phenyl-substituted aliphatic
alcoholsandrelatedaldehydesandestersusedasfavouringagentsaresumma-
rizedinTable4.
(i) b-Methylphenethyl alcohol (No. 1459)
Rats
Groupsof15maleand15femaleweanlingWistarratswerefeddietsdesigned
to provide b-methylphenethyl alcohol at a dose of 0 (control), 10, 40, or 160mg/
kgbwperday,for13weeks.Animalswereobservedthroughoutthestudyforsigns
oftoxicityandoverallcondition,andweightwasmeasuredatthebeginningofthe
studyandtwiceperweekthereafter.Samplesofblood,forevaluationofhaema-
tological and clinical chemistry parameters, and urine were collected at week 6
and at study termination. At the end of the treatment period, all animals were
necropsiedandorganswereexaminedgrosslyforanyabnormalities.Samplesof
organ tissue for microscopic examination were obtained from the control group
and the group receiving the highest dose. In comparison with the controls, body
weights of males were unaffected by treatment, while treated females showed
statisticallysignifcant,butnon-dose-dependentreductionsatweek3,whichper-
sistedfortheremainderoftheperiod.Inmales,foodintakewasslightlydecreased
at every dose compared with that in the control group; however, the decrease
wasstatisticallysignifcant(p<0.01)onlyat40mg/kgbwperday.Foodintakein
females was signifcantly decreased at 160mg/kgbw per day, which was initially
reportedatweek4andateveryweekthereafter.Withtheexceptionofsignifcant
decreasesobservedatthelowestdose,waterintakeintreatedmaleswascom-
parabletothatofthecontrols,whileinfemalesatthelowestandhighestdoses,
non-dose-related,statisticallysignifcantincreases(p<0.01)werereported.Hae-
matologicalanalysisperformedatstudyterminationrevealedastatisticallysignif-
cantdecrease(p<0.05)inerythrocytecountsinmalesatthehighestdose.Urine
analysis conducted at 13 weeks revealed an increased incidence of trace pro-
teinuriainfemalesatthelowestdosecomparedwiththecontrolgroup.Signifcant
clinical chemistry results consisted of elevated concentrations of protein and
reducedconcentrationsofalbumin,identifedinmalesatthelowestandintermedi-
ate doses and in females at the lowest dose, respectively. Males at the highest
dose showed signifcantly increased absolute and relative weights of the liver.A
similarincreasewasreportedintherelativeweightsoftheliverinfemalesatthe
548 PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS
K2
T
a
b
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e

4
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b
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h
i
g
h
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r
.
PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS 549
K2
highestdose.Inmales,dose-dependentincreaseswerereportedinrelativeweights
ofthekidney,reachingstatisticalsignifcanceinthegroupsreceivingtheintermedi-
ate and highest doses, while absolute weights of the kidney were signifcantly
increased only at the highest dose. Additionally, a non-dose-related increase in
relative weight of the brain was reported in all treated females. Relative weights
of the full and empty caecum and of the adrenals were signifcantly increased
in comparison to those of controls in females at the highest and lowest doses,
respectively.Signifcantlyelevatedrelativeweightsoftheheartandpituitarywere
reported in males at the intermediate dose. Overall, variations in the relative
weights of the brain, heart, pituitary, caecum, and adrenals were not associated
withcorrespondingchangesinabsoluteweights,and,withtheexceptionofasingle
occurrence of a renal mesenchymal tumour reported in a female at the highest
dose,noneofthechangesinorganweightswereidentifedinthepresenceofany
histopathologicalfndings.Additionalrenalvariationsobservedintheratexhibiting
thetumourincludedseverenephrosis,chronicinfammatorycellinfltrationofthe
kidney, and hyperplasia of the renal pelvis and bladder. A higher incidence of
alveolar thickening and peribronchial cuffng were reported in treated males and
females,respectively,comparedwithcontrols.However,nodoseresponserela-
tionshipwaspresented.Onthebasisoftherenaleffectsobservedinmalesatthe
twohigherintakes,theno-observed-effect-level(NOEL)was10mg/kgbwperday
(Gauntetal.,1982).
(ii) 2-Phenylpropionaldehyde (No. 1467)
Rats
Groupsof15maleand15femaleCFEratsweregiven2-phenylpropionalde-
hyde at a dose of 0 (control), 10, 50, or 500mg /kg bw in corn oil by gavage,
7daysperweekfor15weeks.Additionalgroupsof10(fveofeachsex)and15
(ten males, fve females) rats were given 2-phenylproionaldehyde at the same
dosesfor6and2weeks,respectively.Bodyweightandwaterandfeedconsump-
tionweremeasuredeachweekfor14weeks.Urineanalyseswereconductedon
samples taken during the last week of treatment. At study termination, blood
samplesweretakenforhaematologicalandserumanalyses,andratswerekilled
andnecropsied.Tissuesampleswereobtainedformicroscopicexaminations.All
animalswereingoodconditionandwerereportedtosurviveuntiltheendofthe
studyperiod.Inonemale,asubcutaneousswellingintheneckwasidentifedat
week 6, which was determined by examination to be a cyst.Although the mean
body weights of male rats after 14 weeks tended to be lower at the two higher
doses, the difference was not statistically signifcant when compared with the
controls.Nodifferencesinmeanbodyweightswereobservedbetweenthecontrols
and any treated females. In both sexes at the highest dose, a non-statistically
signifcantincreaseinmeanfeedconsumption,andasignifcantelevationinwater
intakewerereported.Haematologicalevaluationsshowedadecreaseof57%in
theconcentrationofhaemoglobininmalesatthehighestdoseat6weeks,aswell
asat15weeksinbothsexesatthehighestdoseandinfemalesattheintermedi-
ate dose. Slight polycythaemia was noted in males at the intermediate dose at
week15.Thevariation,however,wasnotconfrmedinmalesatthehighestdose
550 PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS
K2
orinanytreatedfemales.Signifcantlyincreasedreticulocytecountswerereported
infemalesatthehighestdoseatweek15.Resultsobtainedfromserumandurine
analysesweresimilarintreatedandcontrolanimals.Atnecropsy,pulmonaryleu-
kocyteinfltrationwasreported,suggestiveofamildrespiratoryinfection(affected
groups not specifed). Statistically signifcant relative organ weight changes (p <
0.05),particularlyintheliver,occurredmainlyinanimalsatthehighestdose.At2
weeks, relative weights of the liver were increased in males at the intermediate
andhighestdosesandinfemalesatthehighestdose.At6weeks,relativeweights
oftheliverwereincreasedformalesatthehighestdoseandfemalesattheinter-
mediateandhighestdoses.At15weeks,relativeweightsoftheliverwereincreased
ateverydoseinmalesandatthehighestdoseinfemales.However,theauthors
questioned the relevance of the increased weights of the liver at the lowest and
intermediate doses, suggesting that they were likely to refect the slightly lower
body weights observed in the two groups relative to those of the controls.At 2
weeks, increased weights of the kidney in males at the intermediate dose and
increasedweightofthepituitaryinfemalesatthehighestdosewerereported.At
6weeks,malesatthelowestandhighestdosesexhibitedsignifcantlyincreased
relative weights of the heart.Additionally, males at the highest dose had signif-
cantlyincreasedrelativeweightsofthekidney,stomach,smallintestineandpitui-
tary,andfemalesattheintermediateandhighestdoseshadsignifcantlyincreased
relative weights of the brain and stomach, respectively.At the highest dose, sig-
nifcantincreasesinrelativeweightsofthekidneyandstomachwereobservedin
bothsexes,whereasincreasesinrelativeweightsofthepituitaryandheartwere
limitedtomalesandfemales,respectively.Withtheexceptionofasmallgranuloma
reported in the liver of one male at the highest dose at 15 weeks, the increased
organweightswerenotaccompaniedbyanyhistologicalchangesrelatedtotreat-
ment.Onthebasisoftheseresults,theNOELfor2-phenylpropionaldehydewas
10mg/kgbwperdayinrats,butbecauseofthemarginalchangesinliverweights
the authors stated that the NOEL may be closer to 50mg/kgbw per day (Pelling
etal.,1976).
(iii) 2-Methyl-3-tolylpropionaldehyde (No. 1466)
Rats
Groups of 1016 male and 1016 female Charles River CD rats were fed a
dietdesignedtoprovide2-methyl-3-tolylpropionaldehydeatadoseof1.17(males)
or1.38(females)mg/kgbwperdayfor90days.Bodyweightandfeedconsump-
tionwererecordedweeklyandeffciencyoffeedutilizationwascalculated.Hae-
matologicalevaluationswereconductedonhalftheanimalsatweek7andonall
theanimalsatstudytermination.Atnecropsy,kidneyandliverweightsweremeas-
ured and gross and histological examinations were conducted on a variety of
organs.Comparedwithcontrols,novariationsinbodyweight,feedconsumption,
haematology or clinical chemistry values, organ weights, or pathology were
observed(Posternaketal.,1969).
PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS 551
K2
5

(iv) 2-Methyl-4-phenylbutyraldehyde (No. 1462)

Rats
Groups of 1016 male and 1016 female Charles River CD rats were fed a
dietdesignedtoprovide2-methy-4-phenylbutyraldehydeatadoseof0.563(males)
or0.656(females)mg/kgbwperdayfor90days.Bodyweightandfeedconsump-
tionwererecordedweeklyandeffciencyoffeedutilizationwascalculated.Hae-
matological evaluations were conducted on half the animals at week 7 and all
theanimalsatstudytermination.Atnecropsy,weightsofthekidneyandliverwere
measured,andgrossandhistologicalexaminationswereconductedonavariety
of organs. No effects on body weight, feed consumption, haematology, clinical
chemistry,organweightorpathologywerereported.Slightlydepressedconcentra-
tionsofbloodureawereobservedinmalesandfemales,buttheauthorsdidnot
considerthistobeofanytoxicologicalsignifcance(Posternaketal.,1969).
(c) Long-term studies of toxicity and carcinogenicity
Noinformationwasavailable
(d) Genotoxicity
(i) In vitro
Testingforgenotoxicityinvitrohasbeenperformedforfour(Nos1459,1467,
1468, and 1470) representative members of this group of phenyl-substituted ali-
phaticalcoholsandrelatedaldehydesandestersusedasfavouringagents(see
Table5).
In standard assays for mutagenicity in Salmonella typhimurium, b-methyl-
phenethylalcohol(No.1459),2-phenylpropionaldehyde(No.1467),2-phenylpro-
pionaldehyde dimethyl acetal (No. 1468) and 2-phenylpropyl isobutyrate (No.
1470)werenotmutagenicinS. typhimuriumstrainsTA98,TA100,TA1535,TA1537,
andTA1538whentestedatconcentrationsofupto3600mg/plate,withandwithout
metabolicactivation(Wildetal.,1983).
Derivativesofcinnamyl
5
arestructurallyrelateda,b-unsaturatedphenyl-substi-
tutedalcohols,aldehydesandcarboxylicacids.TheCommitteepreviouslyevalu-
ated a group of cinnamyl alcohol and related favouring agents and found them
tobeofnosafetyconcern(Annex1,reference150).Cinnamaldehyde(transand
unspecifed stereochemistry), cinnamyl alcohol (trans and unspecifed
cinnamyl alcohol
OH
cinnamaldehyde
H
O
cinnamic acid
OH
O
552 PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS
K2
T
a
b
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5
.

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PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS 553
K2
stereochemistry), cinnamic acid, a-methylcinnamaldehyde, cinnamyl acetate,
benzylcinnamate,cyclohexylcinnamate,a-amylcinnamaldehyde,a-hexylcinnam-
aldehyde, and p-methoxy-a-methylcinnamaldehyde predominantly were inactive
in S. typhimurium, including strains TA92, TA94, TA97, TA98, TA100, TA102,
TA104,TA1535,TA1537,TA1538,andTA2637.Theassayswereperformedusing
concentrations ranging up to the level of cytotoxicity, both in the absence and
presence of metabolic activation (S9 fraction) obtained from the livers ofAroclor
1254 or methylcholanthrene-induced Sprague-Dawley rats or Syrian hamsters
(Dunkel&Simon,1980;Ederetal.,1980;Florinetal.,1980;Lijinsky&Andrews,
1980;Lutzet al.,1980;Ederetal.,1982a,1982b;Kasamakietal.,1982;Lutzet
al.,1982;Privaletal.,1982;Sekizawa&Shibamoto,1982;Neudeckeretal.,1983;
Wild et al., 1983; Ishidate et al., 1984; Huang et al., 1985; Marnett et al., 1985;
Mortelmansetal.,1986;Fujita&Sasaki,1987;Tennantetal.,1987;Katoetal.,
1989;Ederetal.,1991;Dillonetal.,1992;Azizan&Blevins,1995).Thereaderis
referredtotheevaluationmadebytheCommitteeatitsffty-ffthmeeting(Annex
1, reference 149) of other reported studies of genotoxicity with cinnamyl com-
poundsandusedtosupporttheevaluationofthea,b-unsaturatedphenyl-substi-
tutedalcohols,aldehydes,andcarboxylicacids.
(ii) In vivo
The potential of 2-phenylpropionaldehyde (No. 1467) and 2-phenylpropional-
dehydedimethylacetal(No.1468)toinducesex-linkedrecessivelethalmutations
inadultDrosophila melanogasterwerestudiedintheBasctest.Thefrequencyof
mutationwasunaffectedwhensolutionsof2-phenylpropionaldehydeand2-phen-
ylpropionaldehydedimethylacetalataconcentrationof10and5mmol/l,respec-
tively (1341 and 901mg/ml, respectively) were fed to the fies for 3 days (Wild
etal.,1983).
Inatestformicronucleusformation,groupsoffourmaleandfourfemaleNMRI
micegiven2-phenylpropionaldehydeasasingleintraperitonealdoseat134,402,
or670mg/kgbwor2-phenylpropionaldehydedimethylacetalasasingleintraperi-
tonealdoseat360,630,or900mg/kgbw,demonstratednoincreaseinmicronucle-
ated erythrocytes in samples of bone marrow obtained 30h after administration
(Wildetal.,1983).
Dataongenotoxicityafteradministrationofcinnamaldehydeinvivosupportthe
conclusionsfor2-phenylpropionaldehydeaswellasforthethreea,b-unsaturated
aldehydes(Nos14721274).Anincreaseinthefrequencyofsex-linkedrecessive
lethalmutationswasreportedwhenD. melanogasterwasinjectedwithcinnamal-
dehydeataconcentrationof20000mg/l.However,noincreaseinthefrequency
ofmutationsoccurredwhenD. melanogasterwerefedcinnamaldehydeatacon-
centration of 800mg/kg of diet for 3 days. Reciprocal translocations were not
observedineitherassay(Woodruffetal.,1985).Inmammaliantestsystems,there
was no evidence for an increase in unscheduled DNA synthesis in hepatocytes
inratsormicegivencinnamaldehydeatadoseof1000mg/kgbwbyoralgavage
(Mirsalisetal.,1989).Intheassayformicronucleusformationinrodents,thefre-
quencyofformationofmicronucleiwasnotincreasedwhenratsormiceweregiven
cinnamaldehydeatadoseof1700mg/kgbwor1100mg/kgbw,respectively,byoral
554 PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS
K2
gavage(Meretoetal.,1994),orwhenmiceweregivencinnamaldehydeatadose
of500mg/kgbwbyintraperitonealinjection(Hayashietal.,1984,1988).
In one study (Mereto et al., 1994) an increase in micronucleated cells was
reportedinratandmousehepatocytes,andinrat(butnotinmouse)forestomach
cellsafteroralgavagedosingwithcinnamaldehydeatadoseofupto1100(rats)
or1700(mice)mg/kgbw.Noincreaseintheformationofmicronucleiinthefore-
stomachwereobservedinratsormiceatdosesof850mg/kgbw.Theincreased
frequencyofmicronucleusformationwasabsentinhepatocytesfromratsgivena
dose of 500mg/kgbw, but present in hepatocytes from mice given a dose of
850mg/kgbw. No DNA fragmentation was observed in the rat hepatocytes or
gastricmucosacells.AnincreaseintheincidenceandsizeofGST-positivefociin
hepatocytes of rats pre-treated with N-nitrosodiethylamine and then given cinna-
maldehydeatadoseof500mg/kgbwperdaybyoralgavagefor14days(Mereto
etal.,1994).
The positive fndings with cinnamaldehyde in the rat forestomach and in the
liverofbothratsandmiceinvivo areinconsistentwithnegativeresultsobserved
inthestandardassayinbonemarrowandareobservedatdosesthatfarexceed
those resulting from intake of cinnamaldehyde in food. It has been reported
thatcinnamaldehydegivenatoraldosesof500mg/kgbwresultsinthedepletion
of hepatocellular glutathione (Swales & Caldwell, 1991, 1992, 1993). Therefore,
increasesinmicronucleusformationwerereportedatdoses(1100and1700mg/
kgbw) that appear to affect cellular defence mechanisms (i.e. glutathione deple-
tion). On the basis of the fact the micronucleus formation is dose-dependent, it
appearsthatinductionofmicronucleiisathresholdphenomenonwhichoccursat
intakesthatareordersofmagnitudegreaterthanintakeofcinnamaldehydeasa
favouringsubstance.Also,itislikelythatthebolusdosesresultingfromgavage
administrationproducemuchgreaterexposuresofboththeforestomachandliver
than does administration by dietary admixture. The author (Mereto et al., 1994)
acknowledgedthesefactsandconcludedthatthedatadidnotjustifytheconclu-
sion that cinnamaldehyde was clastogenic.As a result of the apparent threshold
forinductionofmicronucleiandthelackofactivityintheremainderofthestudies
in vivo, the results obtained with bolus, highest-dose exposures occurring in the
liverandforestomacharenotconsideredtoberelevanttothesafetyofcinnamal-
dehydeusedasafavouringagent.
(iii) Conclusion
The testing of these representative 2-phenylpropanol derivatives in bacterial
testsystemsinvitro(Amesassay)andinmammaliansystems(testformicronu-
cleusformation)invivoshowednoevidenceofgenotoxicpotential.Theseresults
arefurthersupportedbythelackofpositivefndingsintheBasctest.Byanalogy
tostructurallyrelatedcinnamylderivatives,thea,b-unsaturatedphenyl-substituted
aliphaticalcoholsandrelatedaldehydesandesters(Nos14721474,and1476)
presentnoevidenceofgenotoxicityinbacterialtestsystems(Amesassay)invitro
andinmammaliansystemsinvivo(testsforunscheduledDNAsynthesisandfor
micronucleusformation).
PHENYL-SUBSTITUTED ALIPHATIC ALCOHOLS 555
K2
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K2
L1
561
GLYCYRRHIZINIC ACID
First draft prepared by
Gary Williams
1
, Catherine Leclercq
2
and Dr A.G.A.C. Knaap
3
1
Environmental Pathology and Toxicology, New York Medical College,
Valhalla, USA; and
2
National Research Institute on Food and Nutrition (INRAN), Rome, Italy
3
Center for Substances and Risk Assessment, National Institute of Public
Health and the Environment, Bilthoven, Netherlands
Explanation............................................................................... 561
Biologicaldata.......................................................................... 563
Biochemicalaspects.......................................................... 563
Absorption,distribution,andexcretion........................ 563
Absorption............................................................. 563
Distribution............................................................. 566
Metabolism............................................................ 567
Excretion................................................................ 569
Pharmacodynamics(mechanismofaction)................ 572
Toxicologicalstudies.......................................................... 577
Acutetoxicity................................................................ 577
Short-termstudiesoftoxicity....................................... 578
Long-termstudiesoftoxicityandcarcinogenicity....... 581
Genotoxicity................................................................. 583
Reproductivetoxicity.................................................... 587
Specialstudies............................................................. 588
Observationsinhumans.................................................... 589
Casereports................................................................ 589
Single-dosestudies..................................................... 590
Multiple-dosestudies................................................... 591
Studiesinpregnantwomen......................................... 597
Intake........................................................................................ 598
Presenceandconcentrationofglycyrrhizinicacidin
foodsandbeverages...................................... 598
Glycyrrhizinicacidasanaturalconstituent.......... 598
Glycyrrhizinicacidasafavouringagent.............. 599
Assessmentofintake......................................................... 600
Comments ............................................................................... 602
Evaluation ............................................................................... 605
References............................................................................... 605
1. ExPLANATIoN
TheCommitteewasaskedtocommentonthesafetyofglycyrrhizinicacidand
itsmonoammoniumsaltasanaturalconstituentofliquorice(USA,licorice)and
initsuseasafavouringsubstanceinvariousfoodproducts.Inthecallfordata,
thetermglycyrrhizicacidwasusedratherthanthealternativetermglycyrrhizinic
562 GLYCYRRHIZINIC ACID
L1
acid. The Committee agreed to use the latter term. Glycyrrhizinic acid and its
monoammoniumsalthavenotbeenevaluatedpreviouslybytheCommittee.
Glycyrrhizinic acid is a naturally occurring triterpenoid saponin found in the
extractsofrootsandrhizomesfromGlycyrrhiza glabra,theliquoriceplant.Dried
extracts of the roots of the liquorice plant, which may contain between 4% and
25%glycyrrhizinicacid,arepresentinliquoriceconfectionery,liquoriceherbalteas
andinsomehealthproducts. Glycyrrhizinicacidandthemonoammoniumsaltare
both used as favouring agents. It should be noted that in the literature some
authorshaveusedthetermglycyrrhizininterchangeablywithglycyrrhizinicacid;
however,thisisnottechnicallycorrect.Glycyrrhizinisthetermhistoricallyusedto
describethecrudeacidextractoftheliquoriceplant.
Glycyrrhizinic acid has been determined to be generally recognized as safe
(GRAS)intheUnitedStatesofAmerica(USA)(FederalRegister,1985)andhas
beenevaluatedbytheNordicCouncilofMinisters(Strmeretal.,1993a)andby
theScientifcCommitteeonFood(ScientifcCommitteeonFood,1991,2003).
This monograph reviews the safety of glycyrrhizinic acid (and its ammonium
salt)fromalldietarysourceswithaviewtoestablishinganacceptabledailyintake
(ADI).Dataonglycyrrhizin(acrudeliquoriceextractcontainingglycyrrhizinicacid),
varioussaltsofglycyrrhizinicacid(existsinbothaandbstereoisomers),andon
thehydrolysisproductofglycyrrhizinicacid,glycyrrheticacid(whichexistsasboth
a and b stereoisomers, with the b-isomer being the metabolite of glycyrrhizinic
acid),wereevaluated.
Figure 1. Structural formula of glycyrrhizinic acid
O
O
C
O
HO
HO
OH
O
OH
OH
OH
HO
O
C
O
H
3
C
H
CH
3
CH
3
CH
3
CH
3
CH
3
H
H
3
C
H
C
O
O
OH
GLYCYRRHIZINIC ACID 563
L1
2. BIoLoGICAL DATA
2.1 Biochemical aspects
It is important to clarify the term glycyrrhizin. In the literature, some authors
haveusedthisterminterchangeablywithglycyrrhizinicacid;however,thisisnot
technicallycorrect.Glycyrrhizinisthetermusedhistoricallytodescribethecrude
acid extract of liquorice isolated by the Houseman (1922) gravimetric method
(describedinBell,1980).Glycyrrhizinicacid(showninFigure1)isacomponent
ofglycyrrhizinandisaglycosidethatoccursnaturallyintheliquoricerootasthe
calcium, potassium, or ammonium salt (Mitchell, 1956; Morris & Muller, 1965;
Wangetal.,2000;EuropeanFlavourandFragranceAssociation,2001).Moreover,
the terms glycyrrhizinic acid and 18b-glycyrrhizinic acid are interchangeable.
Similarly, the term glycyrrhetic acid is synonymous with 18b-glycyrrhetic acid.
Currentanalyticaltechniquesdeterminetheglycyrrhizinicacidcontentofliquorice
and its products; past analytical methods (i.e. Houseman gravimetric method)
determined glycyrrhizin content. Glycyrrhizinic acid is composed of glycyrrhetic
acid and two molecules of glucuronic acid. Glycyrrhetic acid, also commonly
referred to as glycyrrhetinic acid, can be released upon cleavage with
glucuronidase.
2.1.1. Absorption, distribution, and excretion
(a) Absorption
Glycyrrhizinicacid,whetherinthefreeformorastheammoniumsalt,ispoorly
absorbedfromthegastrointestinaltract.Inthegastrointestinaltract,glycyrrhizinic
acidishydrolysed,mainlybytheactivityofintestinalmicrofora,toglycyrrheticacid
(thefreeaglyconeofglycyrrhizinicacid),asubstancethatisreadilyabsorbed.
The results of studies in humans (Carlat et al., 1959; Guillaume et al., 1999)
and rats (Parke et al., 1963; Iveson et al., 1971; Ichikawa et al., 1986; Takeda
et al., 1996) indicated that glycyrrhizin and monoammonium glycyrrhizinate are
hydrolysed in the gastrointestinal tract to glycyrrhetic acid, which is almost com-
pletelyabsorbedintothebody.Atlowdoses(i.e.<10mg/kgbw),uptakeofglycyr-
rhetic acid by the liver after absorption results in low whole-body exposures and
rapid elimination via the bile (Terwasawa et al., 1986; Yamamura et al., 1992;
Ishidaetal.,1994;Krhenbhletal.,1994).
The times at which maximum plasma concentrations of glycyrrhetic acid are
achievedafteroralingestionofglycyrrhizinicacidarereportedtobeintherange
of1216and812hinratsandhumans,respectively.Dosesinexcessof25mg/
kgbwmaysaturatethecapacityofintestinalmicroforatohydrolyseglycyrrhizinic
acid to glycyrrhetic acid, thereby limiting C
max
values and producing a high-dose
absorptionplateau.Inhumans,absorptionofglycyrrheticacidfromthegutisvirtu-
allycomplete,regardlessofwhetheritisformedfromthehydrolysisofglycyrrhi-
zinic acid or is initially present as either the glycoside or the aglycone in a food
matrix(e.g.liquorice)(Strmeretal.,1993b;Mensingaetal.,1998).
Six healthy volunteers were given capsules containing 0.5, 1.0, or 1.5g of
18b-glycyrrhetic acid (corresponding to a dose of approximately 8.3, 16.7, or
564 GLYCYRRHIZINIC ACID
L1
25mg/kgbw, respectively) (Krhenbhl et al., 1994). Blood samples were taken
immediatelybeforeadministration,every30minuntil8hafterdosing,andat9,10,
12, 14, 24, and 48h after administration.Analytical results showed that the bio-
availabilityof18b-glycyrrheticacidwasconstantovertherangeofdosestested.
Maximumbloodconcentrationsreachedafteradministrationof0.5,1.0,and1.5g
were 4.5, 7.0, and 9.0mg/l, respectively. At the lowest dose, the elimination of
18b-glycyrrhetic acid from the blood ftted a single compartment model, but was
biphasicatdosesof1.0and1.5g.Theeliminationhalf-lifeforthefrstphasewas
approximately 2h.The half-life for the second phase was 11.5 and 38.7h at 1.0
and 1.5g, respectively. On the basis of the slow second elimination phase and
highlipidsolubility,theauthorssuggestedthatextensivetissuedistributionof18b-
glycyrrheticacidoccurredatthehigherdoses.
Thepharmacokineticprofleofglycyrrhizinwasexaminedinthreehealthyvol-
unteers(Yamamuraetal.,1992).Afteroraladministrationof100mgofglycyrrhizin
(corresponding to a dose of approximately 1.67mg/kg bw), glycyrrhetic acid was
detectedinplasmaatamaximumconcentrationof200mg/ml;noglycyrrhizinwas
foundinplasma.Afterintravenousinjection,however,onlyglycyrrhizinwasdetected
in plasma, suggesting that it is not hydrolysed to glycyrrhetic acid in the blood.
Eliminationofglycyrrhizinfromplasmawasreportedtobebiphasicandoccurred
with a half-life of between 2.7 and 4.8h. It was also reported in this study that
glycyrrhizinwasnothydrolysedinstomachjuices.Theauthorsconcludedthatthe
appearance of the major metabolite of glycyrrhizin (i.e. glycyrrhetic acid) in the
plasmaafteroraladministrationofglycyrrhizinwasduetohydrolysisbyintestinal
bacteriaandthatlittlehydrolysisoccurredafterabsorption.
In 15 healthy volunteers fed liquorice candy (amount not specifed) for 24
weeks, peak plasma concentrations of glycyrrhetic acid were reported to range
from0to480mg/l(Hughes&Cowles,1977).Additionally,theauthorsreportedthat
maximum plasma concentrations of glycyrrhetic acid reached 84mg/ml in four
healthy volunteers who ingested liquorice candy (amount not specifed) for 2
days.
Serumconcentrationsofglycyrrheticacidweremeasuredinfvehealthymale
volunteers given a single oral dose of liquorice containing 133mg of glycyrrhizin
(corresponding to a dose of glycyrrhetic acid of approximately 2.22mg/kgbw)
(Terwasawa et al., 1986). The maximum serum concentration was reported to
be 30mg/l, which was reached approximately 24h after administration. The
peak serum concentration of glycyrrhetic glycosides was reportedly reached
within4h.
Therateofabsorptionofglycyrrheticacidinhumanvolunteerswhoconsumed
anextractoftheGlycyrrhizaplantwasreportedtobeapproximately75%ofthat
associated with the consumption of pure glycyrrhizinic acid (Cantelli-Forti et al.,
1994).
In an investigation of the bioavailability of glycyrrhetic acid after ingestion of
liquorice,sixhealthyvolunteers(fvefemalesandonemale)weregivenaliquorice
confection containing approximately 200mg of glycyrrhizinic acid (approximately
3.3mg/kgbw)(Gunnarsdttir&Jhannesson,1997).Meanplasmaconcentrations
GLYCYRRHIZINIC ACID 565
L1
ofglycyrrheticacidrosetoapeakofapproximately570mg/mlat10hafteringes-
tion. The area under the curve of concentrationtime (AUC) was approximately
7569mgh/l,whilethemeanC
max
andT
max
wereapproximately794mg/land13h,
respectively. The authors suggested that the biotransformation of glycyrrhizinic
acidtoglycyrrheticacidoccursinthelargeintestineinhumans.
Inanotherstudyofthebioavailabilityofglycyrrheticacidinhumans(Mensinga
etal.,1998),eightmenandeightwomenweregiveneither:(a)130mgofglycyr-
rhetic acid in aqueous suspension; (b) 225mg of glycyrrhizinic acid in aqueous
solution;(c)225mgofglycyrrhizinicacidin150gofsweetliquorice;or(d)225mg
ofglycyrrhizinicacidinsaltedliquorice.Samplesofplasmawerecollectedforup
to56hafterconsumption.Plasmaconcentrationsofglycyrrheticacidpeakedafter
3and810hfortreatments(a)and(bd),respectively.Intra-individualdifferences
between the treatments were reported to be small, while large inter-individual of
differencesoffour-tofvefoldoccurredwithrespecttoC
max
andAUCvalues,e.g.
fortreatment(c),C
max
andAUCvalueswerereportedtorangefrom0.4to1.6mg/l
andfrom4.8to23.1mg/lh,respectively.
Egashiraetal.(2003)investigatedthepharmacokineticsofglycyrrhizin(actu-
allyglycyrrhizinicacid)andglycyrrheticacidinmaleWistarratsgivensingleand
multiple doses by various routes of exposure. Groups of three rats were given
monoammoniumglycyrrhizinate(asaltofglycyrrhizinicacid)byintravenousinjec-
tion,intraperitonealinjectionorbyoralintubationinsalineasasinglebolusdose
of10mg/kgbw.Anothergroupofratsreceived20dailydoses(eitherintravenously
ororally)of2mg/kgbw.Ineachcase,samplesofplasmaandbilewerecollected
at various time-points and analysed for the presence of glycyrrhizin (actually
glycyrrhizinic acid) and glycyrrhetic acid.Administration of single doses by intra-
venousorintraperitonealinjectionresultedincurvesfortheplasmaconcentration
ofglycyrrhizinicacidthatcloselyresembledatwo-compartmentmodel.Administra-
tionofsingleoraldosesresultedinmuchmoregradualabsorptionofglycyrrhizinic
acid,withpeakplasmaconcentrationsof70mg/lbeingachievedwithin1012hof
administration.Approximately 60% of the oral dose administered was absorbed
within 24h. This value appears to include the absorption of glycyrrhetic acid in
addition to any glycyrrhizinic acid. After administration of single does by intra-
venous,intraperitoneal,ororalroutes,theAUCvaluesat24hforglycyrrheticacid
werereportedtobe1.155,1.167,and1.004mgh/ml,respectively.Theappear-
ance and concentrations of glycyrrhetic acid were similar for all these routes of
exposure.Biliaryconcentrationsofglycyrrhizinicacidandglycyrrheticacidpeaked
within2hofdosing(200250mg/ml)anddeclinedrapidlywithin8hofdosing.This
fndingessentiallywasthereverseoftheobservationsinplasma.
Afterdosingat2mg/kgbwperdayfor20days,plasmaconcentrationsofglycyr-
rhizinicacidwere210mg/mland0.20.7mg/mlfortheintravenousandoralroutes
of exposure, respectively. Concentrations of glycyrrhetic acid after multiple intra-
venousandoraldosesrangedfrom6to50mg/landfrom5to60mg/l,respectively.
Equilibriumconcentrationsofglycyrrhizinicacidandglycyrrheticacidwereachieved
within 2 days, with no accumulation of either compound over the 20-day course
oftreatment(Egashiraetal.,2003).
566 GLYCYRRHIZINIC ACID
L1
Wistarratsweregivenradiolabelledglycyrrheticacidatadoseof20mg/kgbw
bytwodifferentroutes(Ivesonetal.,1971).Afteroralgavage,absorptionfromthe
stomachwasreportedtorangefrom27to47%within6h,whileafterintraduodenal
infusion, absorption was reported to range from 83 to 95%. When bile collected
from the rats was re-administered by intraduodenal infusion to rats that had not
been dosed, 31% of the radiolabel given to the frst rat was reabsorbed and
excretedinthebile.
Takeda et al. (1996) reported that in male Wistar and Sprague-Dawley rats
treaedorallywithglycyrrhizin(i.e.glycyrrhizinicacid),glycyrrhizinwashydrolysed
byintestinalbacteriatoglycyrrheticacid,whichwascompletelyabsorbedfromthe
gastrointestinaltract.Intheabsenceofintestinalbacteria,astestedusinggerm-
freerats,itwasreportedthatglycyrrhizinwasnotabsorbedand,therefore,didnot
appearintheplasma.Absorptionofglycyrrheticacidfromtheintestineoftherats
that were not germ-free was considered by the authors to have been complete,
becausecomparablecumulativeplasmaconcentrationsofglycyrrheticacidwere
obtained both after intravenous injection of glycyrrhetic acid at 5.7mg/kgbw and
oraladministrationofglycyrrhizinat10mg/kgbw(anamountequimolarto5.7mg
ofglycyrrheticacid).
Wangetal.(1995)investigatedthegastrointestinalabsorptionofpureglycyr-
rhizinandglycyrrhizinasaconstituentofGlycyrrhizaextract.NormalmaleWistar
ratsandratswithbilefstulaweregivenglycyrrhizinatadoseof200mg/kgbwor
an equivalent dose of glycyrrhiza extract in saline (corresponding to a dose of
glycyrrhizin of 200mg/kgbw). The absorption of glycyrrhizin in the Glycyrrhiza
extractwasreportedtobereducedwhencomparedwiththatofpureglycyrrhizin.
The excretion of glycyrrhizin in the bile after administration of the Glycyrrhiza
extractwasreportedtobeapproximately1.1%oftheadministereddoseafter30h,
slightlylessthanthatofpureglycyrrhizin.
Ichikawaetal.(1986)testedtheintestinalabsorptionofglycyrrhizinicacidby
injecting bile containing glycyrrhizinic acid into the duodenum of portal vein-
cannulated rats. Blood samples were collected for up to 2h after administration.
Absorption from the gut was reported to be apparent as revealed by the steady
increase in concentrations of glycyrrhizinic acid in the samples of portal plasma
over time. The authors estimated that approximately 80% of the glycyrrhizin
excretedinthebilewasreabsorbedfromthegastrointestinaltract.
(b) Distribution
Theresultsofstudiesinrats,andinferencesthatcanbedrawnfromtheresults
of studies in humans, indicate that both glycyrrhizinic acid and its hydrolysis
productglycyrrheticacidarelargelyconfnedtotheplasmaandarenottakenup
in bodily tissues to a signifcant extent. In plasma, glycyrrhizinic acid and glycyr-
rheticacidareboundtoserumalbumin.Somehepaticuptakeofglycyrrhizininthe
bloodhasbeenreportedtooccur,possiblythroughtheactionsoforganicanion-
transportingpolypeptidesinbothratsandhumans(Ismairetal.,2003).
Inastudyinratsgivenasingleintravenousdoseofglycyrrheticacidat2,5,
or 12mg/kgbw, analysis of the brain, lung, heart, liver, kidney, stomach, small
GLYCYRRHIZINIC ACID 567
L1
intestine,pancreas,spleen,muscle,skin,adiposetissue,plasma,andlymphfuid
showedthatdistributionwaspoor(Ishidaetal.,1989).Theauthorsalsoreported
poor uptake of glycyrrhetic acid into erythrocytes. Ishida et al. (1989) concluded
thattheeliminationofglycyrrheticacidintheplasmawasgenerallybiexponential
innatureanddose-dependent.
Ishidaetal.(1988)reportedthatglycyrrheticacidbindstoplasmaalbuminin
vitroat>96%inratsand>99.9%inhumans,atconcentrationsof<0.5mg/mland
<5mg/ml, respectively. Ishida et al. (1994) later reported that glycyrrhizin was
activelytakenupbytheisolatedratliverataratethatexceededitsexcretioninto
thebile.
(c) Metabolism
Glycyrrhizinwasreportednottobehydrolysedtoasignifcantdegreebyhuman
stomachjuicesinvitro,norwasithydrolysedinhumanplasmainvivo;however,
glycyrrhizin appears in the blood as glycyrrhetic acid after oral administration in
humans(Yamamuraetal.,1992).Theappearanceofglycyrrheticacidintheblood
afteradministrationofglycyrrhizin(i.e.glycyrrhizinicacid)isexplainedbythefact
that the intestinal microorganisms in several mammalian species, including
humans,arecapableofhydrolysingglycyrrhizin(glycyrrhizinicacid)toglycyrrhetic
acid (Hattori et al., 1983; Hattori et al., 1985; Akao et al., 1987; Akao, 1997a;
Shibataetal.,2000;Akao,2001).
Hattori et al. (1983) studied the metabolism of glycyrrhizinic acid by human
intestinal bacteria isolated from faeces. An intestinal bacterial mixture was pre-
pared and incubated with either monoammonium glycyrrhizinate (glycyrrhizinic
acid) or glycyrrhetic acid for 24 and 48h, respectively. Three metabolites were
reportedandidentifedfromglycyrrhizinicacid:glycyrrheticacid,3-epi-glycyrrhetic
acid,and3-dehydro-glycyrrheticacid.Onemetabolite,3-epi-glycyrrheticacid,was
identifedfromglycyrrheticacidandtheparentcompoundwasalsorecovered.3-
Dehydro-glycyrrheticacidwasrecoveredfromtheculturemedium.Thismetabolite
wasincubatedwiththebacterialmixtureandbecametransformedtoglycyrrhetic
acid and 3-epi-glycyrrhetic acid, indicating that it may be an intermediate in the
formation of 3-epi-glycyrrhetic from either glycyrrhetic acid or glycyrrhizinic acid.
Figure2illustratesthemetabolicpathwayofglycyrrhizinicacidinthepresenceof
humanintestinalfora.
In a later study, Hattori et al. (1985) examined which microbes of the human
intestinal fora were responsible for the metabolism of glycyrrhizinic acid. Their
resultsindicatedthatPeptostreptococcus intermedius andClostridium perfringens
togetherhydrolysedglycyrrhizinicacidtoglycyrrheticacid.Ruminococcus sp.and
Clostridium innocuum were isolated from fresh human faeces and identifed as
activebacteriaintheconversionof3-dehydro-glycyrrhizinicacidtoeitherglycyr-
rhizinic acid or 3-epi-glycyrrhizinic acid. The authors reported that a fair number
of bacteria studied from the human intestinal fora showed b-glucuronidase
activity and, thus, had the ability to metabolize glycyrrhizinic acid to glycyrrhetic
acid. However, only a moderate number of bacteria showed the activity required
to reduce 3-dehydro-glycyrrhizinic acid to either glycyrrhizinic acid or 3-epi-
glycyrrhizinicacid.
568 GLYCYRRHIZINIC ACID
L1
In a more recent study byAkao (1997b), the b-glucuronidase reported to be
responsibleforthehydrolysisofglycyrrhetylmono-glucuronide,anintermediatein
thehydrolysisofglycyrrhizintoglycyrrheticacid,wasreportedtohavebeeniso-
latedfromhumanintestinalbacteria(Eubacteriumsp.).
Imaietal.(1999)reportedthatitisthehydrolysisofdipotassiumglycyrrhizinate
to glycyrrhetinyl monoglucuronide that is the rate-limiting step in the metabolic
pathway,andisfollowedbytherapidhydrolysisofglycyrrhetinylmonoglucuronide
toglycyrrheticacid.
Itwasreportedthatglycyrrhizinismetabolizedbyhumanintestinalbacteriato
18b-glycyrrheticacidthroughtwopossiblepathways(Kimetal.,1999;Kimetal.,
2000).Themainpathway,whichiscatalysedbyEubacteriumL-8b-glucuronidase,
proceedstotheformationofthemajorproduct,18b-glycyrrheticacid.Thesecond
pathway,catalysedbyStreptococcusLJ-22b-glucuronidase,resultsintheproduc-
tion of the minor product, 18b-glycyrrhetic acid-3-O-b-D-glucuronide (GAMG),
whichissubsequentlyhydrolysedto18b-glycyrrheticacidbyEubacteriumL-8b-
glucuronidase.According to the authors, GAMG and 18b-glycyrrhetic acid have
H O
O
C O O H
18b-glycyrrhetic acid
O
C O O H
O
3-dehydro-18b-glycyrrhetic acid
H O
O
C O O H
3-epi-18b-glycyrrhetic acid
glycyrrhizinic acid
Figure 2. Metabolic pathway of glycyrrhizinic acid
FromHattorietal.(1983)
GLYCYRRHIZINIC ACID 569
L1
potent anti-platelet aggregation activity in vitro. Conversely, 18b-glycyrrhetic acid
was reported to have the most potent cytotoxic activity against tumour cell lines
andN-nitrosamines(Malagolietal.,1998;Kimetal.,2000;Martinezetal.,2000),
aswellasinhibitoryactivityagainstgrowthofHelicobacter pyloriandinfectionwith
rotavirus(Kimetal.,2000).
Okamura et al. (2003) reported that glycyrrhizin, incubated aerobically or
anaerobically for 48h with fresh faeces from male Wistar rats, was metabolized
mainly to glycyrrhetic acid. Under anaerobic conditions, trace amounts of 3-a-
hydroxyglygyheticacidand3-dehydroglycyrrheticacidwereformed.Underaerobic
conditions, the amount of 3-dehydroglycyrrhetic acid was found to increase con-
stantly throughout the incubation period, so that after 48h concentrations of this
metabolitewereequaltothoseofglycyrrheticaciditself(i.e.about35mmol/l).
Akao et al. (1990a, 1990b) reported on a reversible conversion of 18b-
glycyrrhetic acid to 3-oxo-18b-glycyrrhetic acid. Akao et al. (1990b) studied the
hydroxylationof18b-glycyrrheticacid,3-oxo-18b-glycyrrheticacid,and3-epi-18b-
glycyrrheticacidbyratlivermicrosomes.Theresultsshowedthatinthepresence
of nicotinamide adenine dinucleotide phosphate (oxidized form) (NADP
+
), 18b-
glycyrrhetic acid was converted to 3-oxo-18b-glycyrrhetic acid; however, in the
presenceofnicotinamideadeninedinucleotidephosphate(reducedform)(NADPH)
the same compound was converted to 22a-hydroxy-18b-glycyrrhetic acid and
24-hydroxy-18b-glycyrrheticacid.Similarly,3-oxo-18b-glycyrrheticacidand3-epi-
18b-glycyrrheticacidwereconvertedto22a-and24-hydroxyderivatives.
Kawakamietal.(1993)reportedthatglycyrrhetyl-3-O-glucuronide,glycyrrhetic
acid-3-O-hydrogensulfate,andaglucuronideconjugateofglycyrrheticacidwere
biliarymetabolitesofglycyrrheticacidinrats.
Humansubjectsgiven2.28or2.50gofglycyrrheticacidorallyfailedtoshow
anyoftheunchangedcompoundintheurine,althoughanunidentifedmetabolite
waspresent(VanKatwijk&HuisintVeld,1954).
(d) Excretion
In both humans and rats, glycyrrhizinic acid and glycyrrhetic acid formed by
microbe-mediatedhydrolysisareexcretedmainlyinthefaeces.Inaddition,asig-
nifcant proportion of glycyrrhetic acid absorbed after hydrolysis is subjected to
enterohepaticcirculationandexcretionintothebile.
Inapreviouslydescribedstudy(Krhenbhletal.,1994)inwhichsixhealthy
volunteers were given capsules containing 0.5, 1.0, or 1.5g of 18b-glycyrrhetic
acid(adoseofapproximately8.3,16.7,and25mg/kgbw,respectively),elimination
ofglycyrrheticacidfromthebloodfttedasinglecompartmentmodelatthelowest
dose,butwasbiphasicat1.0and1.5g.Theeliminationhalf-lifewas approximately
2hforthefrstphase.Thehalf-lifeofthesecondphasewas11.5and38.7hat1.0
and 1.5g, respectively. On the basis of the slow second elimination phase and
high lipid solubility, the authors suggested that extensive tissue distribution of
glycyrrheticacidoccurredatthehigherdose.
570 GLYCYRRHIZINIC ACID
L1
After oral administration of 100mg of glycyrrhizin (a dose of approximately
1.67mg/kg bw), elimination of glycyrrhetic acid from plasma was reported to be
biphasicandoccurredwithahalf-lifeof2.74.8h.Italsowasreportedinthisstudy
that glycyrrhizinic acid was not hydrolysed in stomach juices. The authors con-
cluded that the appearance of the major metabolite of glycyrrhizinic acid in the
plasma after oral administration was caused by hydrolysis by intestinal bacteria
andthatlittlehydrolysisoccurredafterabsorption(Yamamuraetal.,1992).
Carlat et al. (1959) reported that orally administered radiolabelled monoam-
monium glycyrrhizinate was hydrolysed to form glycyrrhetic acid and excreted
unchangedinthefaecesinhumans.
Theurinaryexcretionof18b-glycyrrheticacidwasexaminedintwovolunteers
(ageandsexnotreported)whoreceivedDutchconfectioneryliquoriceatadose
of 500g (consumed within 4h), or at a daily dose of 40g for a period of 5 days
(Guillaumeetal.,1999).Allurinewascollectedfromthesubjectsduringthedays
ofliquoriceconsumption,aswellasarbitrarilyondays6,8,and10ofthestudy.
Itwasreportedthat18b-glycyrrheticacidwasfrstdetectedintheurineat8and
9hafterthefrstoraladministrationof500and40gofliquorice,respectively.The
authors attributed this delay to the hydrolysis of glycyrrhizin to 18b-glycyrrhetic
acidbyintestinalbacteria,anditssubsequentabsorptionfromthegut.Additionally,
itwasreportedthat urinary18b-glycyrrheticacidcouldbe detectedat up to53h
afterconsumptionof500gofliquorice,andupto72haftercessationofconsump-
tionofliquoriceat40g/dayfor5days.Theauthorsattributedtheextendedexcre-
tiontothephenomenonofenterohepaticcycling.
Tostudybiliaryexcretionandenterohepaticcyclingofglycyrrhizin(i.e.glycyr-
rhizinicacid),fastedratswereintravenouslyinjectedwithglycyrrhizinatadoseof
100mg/kgbw(Ichikawaetal.,1986).Biliaryfstulasandurinarybladdercannulas
were placed in some of the rats. Blood samples were taken between 0.5 and
360minaftertheadministrationofglycyrrhizin.Infstulatedandcannulatedanimals,
bileandurinesamplesweredrawnforupto60hafteradministration.Samplesof
faeceswerealsotaken.Approximately70%oftheadministereddosewasexcreted
in the bile within 12h, and the compound was reported not to affect bile fow.
Glycyrrhizinicacidwasreportedlynotdetectedinthebileorurineofratsafter48h.
In rats without fstulas, the total cumulative excretion in the faeces was 5.5% of
theinjecteddose.Itwasreportedthattherewasnodetectableexcretionofglycyr-
rhizinicacidinthefaecesoffstulatedrats.Theauthorsconcludedthathighcon-
centrationsoftheadministereddoseofglycyrrhizin(glycyrrhizinicacid)remained
intheliver,andwereslowlyexcretedintothebile;however,thistransportprocess
was considered to be saturable.Approximately 80% of the glycyrrhizinic acid in
thebilewasreportedtobereabsorbedfromthegastrointestinaltractviaentero-
hepaticrecycling.
Ishida et al. (1992) gave monoammonium glycyrrhizinate (glycyrrhizinic acid)
in5%glucosesolutionasasingleintravenousinjectionatadoseof5,10,20,or
50mg/kgbw to male Wistar rats (number not specifed). Blood samples were
collected at 1, 5, 10, 30, 60, and 120min after dosing of all animals.Additional
blood samples were collected at 3h after dosing for the group receiving 10mg/
kgbw,3and5hafterdosingforthegroupreceiving20mg/kgbwand3,5,8,and
GLYCYRRHIZINIC ACID 571
L1
12hafterdosingforthegroupreceiving50mg/kgbw.Theeliminationofglycyrrhi-
zinate from the plasma was reported to be biexponential for all doses, while the
volumeofdistributionwassignifcantlygreaterforthetwohighestdoses(20and
50mg/kgbw)comparedwiththeotherdoses;however,therewasnoevidenceof
bioaccumulationevenatthehighestdose.Theauthorsreportedthattheplasma
distributionofglycyrrhizinatewasdose-dependent.Inanotherstudy,similarresults
wereobtainedwhenmaleSprague-Dawleyratswereinjectedwithasinglebolus
intravenousdoseofglycyrrhizinat20,50,or100mg/kgbw(Tsaietal.,1992).In
this study, blood samples collected for up to 24h after dosing were reported to
showabiphasic,dose-dependentdecreaseinplasmaconcentrationsofglycyrrhi-
zinatalldoses.Inanotherbioavailabilitystudy,italsowasreportedthatasimilar
biphasicresponseoccurredintheplasmainthreestrainsofratsafterintravenous
administrationofglycyrrheticacidat10mg/kgbw(Takedaetal.,1996).
Todeterminethepharmacokineticsofglycyrrhizinicacidandglycyrrheticacid
inbile,groupsofsixfastedmaleSprague-Dawleyratsweregivendistilledwater
(vehicle),glycyrrhizinicacidat480mg/kgbw,orliquoriceextractat6278mg/kgbw
by oral gavage (Cantelli-Forti et al., 1997).Additional groups of rats were intra-
venously injected with glycyrrhizinic acid at 2.5mg/kgbw or with liquorice extract
at32.7mg/kgbwtoavoidgastrointestinalabsorptionofthetestsubstances.Inboth
experiments, after administration of liquorice extract a signifcant reduction was
reportedinthebioavailability(7.4timesless)andclearancerate(20%slower)of
glycyrrhizinicacidcomparedwithpureglycyrrhizinicacid.
In male Wistar rats given monoammonium glycyrrhizinate in a 5% glucose
solution as a single intravenous dose at 5, 10, 20, or 50mg/kgbw (Ishida et al.
(1992),theprimaryrouteofexcretionwasreportedtobeviathebile.Therateof
totalandrenalclearanceinanimalswassignifcantlydecreasedat20and50mg/
kgbw, indicating that the elimination of monoammonium glycyrrhizinate is dose-
dependent;however,therewasnoevidenceofbioaccumulation.Thiswasalsothe
caseinmaleSprague-Dawleyratsgivenasingleintravenousdoseofglycyrrhizin
at20,50,or100mg/kgbw(Tsaietal.,1992).Bloodsampleswerecollectedforup
to24hafterdosing.Theauthorsreporteddose-dependentdecreasesintotalclear-
anceandvolumeofdistributionofglycyrrhizinintherats.
Albino rats were given radiolabelledb-glycyrrhetic acid in arachis oil orally at
adoseof25mg/kgbw(Parkeetal.,1963).After23days,70%oftheadministered
dosewasreportedtoberecoveredinthebile,1320%inthefaeces,and23%
intheurine.Additionally,intraperitonealinjectionofthesamedoseofb-glycyrrhetic
acidwasreportedtoresultinvirtually100%ofthedosebeingexcretedinthebile
within12h,withonlytraceamountsappearingintheurine.
In Wistar rats, 18b-glycyrrhetic acid, administered either by oral gavage or
intraperitoneal injection, at a dose of 20mg/kgbw, was reported to be excreted
largelyinthefaeces(approximately95%)withasmallproportion(approximately
1.7%)appearingintheurine(Ivesonetal.,1971).Biliaryexcretionwasreported
to account for 75% of the administered dose within 7h after intraperitoneal
injection. Biliary metabolites were identifed as the glycyrrhetic acid conjugates
glycyrrhetyl-3-O-glucuronide, glycyrrhetic acid 3-O-hydrogen sulfate, and glycyr-
rhetyl-3-O-glucuronide;however,onlyglycyrrheticacidwasreportedtoberecov-
572 GLYCYRRHIZINIC ACID
L1
ered from the faeces after oral administration, suggesting that hydrolysis of the
biliarymetabolitesbyintestinalmicroforaoccurred.
Metabolitesexcretedinthebileofratsgivenglycyrrheticacidbyintravenous
administration have been identifed as glycyrrhetyl-3-O-glucuronide, glycyrrhetic
acid 3-O-hydrogen sulfate, and a glucuronide conjugate of glycyrrhetic acid
(Kawakamietal.,1993).Thesearesimilartometabolitesidentifedafteroraland
intraperitonealadministration(Ivesonetal.,1971).Thefrstappearanceofmetabo-
litesinthebilewasreportedtooccurafter3hand,uponimmediateintraduodenal
infusiontoasecondrat,appeared intheplasmaoftherecipientratafter4h.The
totalpercentageofglycyrrheticacidor metabolitesexcretedinthebilewasapprox-
imately 17% in the frst rat and <2% in the second rat (Kawakami et al., 1993).
The authors estimated that the contribution of enterohepatic circulation to the
plasmaconcentrationsofglycyrrheticacidwas<3.5%.Incontrast,Ploegeretal.
(2000a)reportedthatenterohepaticrecyclingwasresponsiblefortheslowterminal
clearanceofglycyrrheticacid.Kawakamietal.(1993)comparedtheplasmacon-
centrationtimecourseforglycyrrheticacidinbileduct-cannulatedandcontrolrats
after intravenous administration of glycyrrhetic acid and reported no signifcant
differences; however, the duration of this study was only 3h and Ploeger et al.
(2000a)reportedthattheinfuenceoftheenterohepaticrecyclingdoesnotbecome
evidentuntil5hafterdosing.Inanotherstudy,Ploegeretal.(2000b)reportedthat
enterohepaticrecyclingofmetabolitesofglycyrrheticacidhasasignifcanteffect
onthekineticsofglycyrrheticacidinhumansandrats.
2.1.2. Pharmacodynamics (mechanism of action)
Glycyrrhizinic acid (glycyrrhizin), liquorice, and glycyrrhetic acid have been
reported to have anti-infammatory, anti-arthritic, antioxidant, and hypolipidaemic
properties (Elmadjian et al., 1956; Finney et al., 1958; Evdokimova & Kamilov,
1967; Whitehouse et al., 1967; Mezenova, 1984; Sitohy et al., 1991; Strmer et
al.,1993b;Fuhrmanetal.,1997;Mauricioetal.,1997),aswellashepatoprotective
andimmunoprotectiveeffectsinhumansandanimals(Kisoetal.,1984;Yamamura
et al., 1997; Okamoto & Kanda, 1999; Paolini et al., 1999; Shiga et al., 1999;
Wen-Yuetal.,1999;Al-Qarawietal.,2001;Daietal.,2001;Horigomeetal.,2001;
Okamotoetal.,2001;Utsunomiyaetal.,2001;Jeong&Kim,2002).
Absorbed glycyrrhetic acid (aglycone of glycyrrhizinic acid) has also been
reported to produce effects similar to those of the adrenal steroid aldosterone
(Groen et al., 1951; Groen et al., 1952; Kraus, 1957; Epstein et al., 1977a;
Chandler,1985;Strmeretal.,1993b;Bijlsmaetal.,1996;Ploegeretal.,2000a,
2000b).Theprecisemechanismofactionofglycyrrheticacidinvolvestheinhibition
of 11b-hydroxysteroid dehydrogenase (11b-HSD), which is an enzyme that con-
vertscortisoltocortisone(Epsteinetal.,1978;Stewartetal.,1987,Stewartetal.,
1988;Monderetal.,1989;MacKenzieetal.,1990a;Kageyama,1992;Kageyama
etal.,1992a;Edwardsetal.,1993;Morris,1993).
Theauthorsofseveralstudieson11b-HSDhavereportedthatthismicrosomal
enzyme catalyses both the conversion of cortisol to cortisone and the reverse
reaction, and consists of two enzymes, 11b-HSD1 (11-oxo-reductase) and 11b-
HSD2(Monderetal.,1989;Stewartetal.,1990;Stewartetal.,1994;Stewart&
GLYCYRRHIZINIC ACID 573
L1
Mason, 1995). It was reported that 11b-HSD2 catalyses the oxidation of cortisol
tocortisoneandshowsrelativelyhighactivityinthekidney,while11b-HSD1cataly-
ses the conversion of cortisone to cortisol and is relatively inactive in the kidney
butisactiveintheliver(Monderetal.,1989;Latifetal.,1990;Stewartetal.,1990;
Edwardsetal.,1993).Monderetal.(1989)reportedthatglycyrrheticacidinhibited
11b-HSD2butnot11b-HSD1.Inhibitionof11b-HSD2byglycyrrheticacidwithout
inhibition of 11b-HSD1 contributes further to the accumulation of cortisol in vivo
(Monderetal.,1989;Mantero&Boscaro,1992;Edwardsetal.,1993).
Cortisolhasmineralocorticoidactivityreportedlyequivalenttothatofaldoste-
rone,butnormallydoesnotexpressthisactivityinthetissueswherealdosterone
acts(i.e.thekidney)becauseitisconvertedtoinactivecortisoneby11b-HSD2.The
inhibitionof11b-HSD2by glycyrrheticacidleadstohightissueconcentrationsof
cortisol,resultingininappropriatemineralocorticoidactivity(i.e.sodiumandwater
retention and loss of potassium), as has been reported to occur in humans and
animals(Hassanetal.,1954;Revers,1946; Macabiesetal.,1963a,1963b;Cotterill
&Cunliffe,1973;Epsteinetal.,1978;Valentinoetal.,1987;Morris,1993,Bernardi
et al., 1994; Funder, 1995; Stewart & Mason, 1995;Armanini et al., 1996; Rossi
etal.,1999;Wuetal.,1999;Horigomeetal.,2001).Inaddition,theresultsoftwo
studiesinrats(Hanafusaetal.,2002;Tanahashietal.,2002)indicatethatglycyrrhi-
zinicacid,inadditiontoinhibiting11b-HSDafterhydrolysistoglycyrrheticacid,sup-
pressesexpressionof11b-HSDmRNAandproteinthroughindirecteffectsinvolving
aglucocorticoidfeedbackmechanism.Typically,theeffectdescribedaboveoccurs
intheabsenceofanygrossorhistologicalabnormalities(Fujimura,1974).
Investigationintoaphysiologicalmechanismforcertaineffectsreportedtobe
caused by liquorice was initiated following a study by Revers (1946), in which
liquorice extract had been successfully used in the treatment of 45 patients with
gastriculcer.Inthisstudy,itwasreportedthatliquoriceextractat10g/day,admin-
isteredorallyinthreesub-doses,reducedintestinaltonusandmobility,anddimin-
ishedorcuredgastriculcers;however,liquoriceextractwasalsoreportedtocause
oedemaofthefaceandlegsinapproximately20%ofthepatients.Reductionof
the liquorice dose to 3g/day was reported to result in a decrease in the number
ofcasesandseverityofoedemainthepatients.
Insubsequentinvestigations,ithasbeenreportedthatconsumptionofglycyr-
rhizinicacidandliquoriceorliquoriceextractscaninducesodiumandwaterreten-
tion,andpotassiumexcretioninhealthyindividuals(Molhuysenetal.,1950;Card
etal.,1953;Chandler,1985;Heikensetal.,1995).Theseeffectshavebeencom-
pared with those of both aldosterone and deoxycorticosterone, which are miner-
alocorticoidhormonessecretedbytheadrenalcortex(Groenetal., 1951;Groen
etal., 1952;Kraus,1957).Theprincipalbiologicalactionofaldosteroneand,toa
lesserextent,deoxycorticosteroneistheregulationofelectrolyteandwaterbalance
in the body by promoting the retention of sodium and water, and excretion of
potassiumbythekidney.Theregulationofconcentrationsofelectrolytesiscritical
inmaintainingnormalbloodpressure,muscleaction,andpHofbodyfuids(Fox,
1984;Gornall,1986).
In patients with a defective mechanism for the release of aldosterone (e.g.
Addison disease), or in those who have had their adrenals removed because of
574 GLYCYRRHIZINIC ACID
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disease, the administration of glycyrrhizinic acid (glycyrrhizin) or the ingestion of
liquorice has been reported to lessen the symptoms associated with electrolyte
imbalance,despitetheinabilityofthesepatientstosecretealdosterone(Groenet
al., 1951; Groen et al., 1952; Card et al., 1953; Pelser et al., 1953; Cotterill &
Cunliffe,1973).Forthisreason,liquoriceandliquoriceextractshavebeenreported
tohavemineralocorticoid-likeactivity(Epsteinetal.,1978;Morris,1993;Strmer
et al., 1993b; Funder, 1995; Heikens et al., 1995; Sweetman, 2002). In healthy
individualswhoconsumelargequantitiesofproductscontainingglycyrrhizinicacid,
such as liquorice, these compounds have been reported to result in sodium and
waterretention, andpotassiumloss,leadingtohypertension,compensatoryreduc-
tionsinplasmaconcentrationsofaldosteroneandrenin,andothereffectsrelated
toelectrolyteimbalance(Epsteinetal.,1977a,1977b;Beretta-Piccolietal.,1985;
MacKenzieetal.,1990a;Fareseetal.,1991;Morris,1993;Strmeretal.,1993b;
Bernardi et al. 1994; Kerlan et al., 1994; Funder, 1995; Olukoga & Donaldson,
2000; Brouwers & van der Meulen, 2001). The term apparent mineralocorticoid
excess(AME)hasbeenusedtodescribetheelectrolyteabnormalitiesinvolvedin
araremonogenicformofearlyonsethypertension(Edwardsetal.,1993;Stewart
&Mason,1995)andhasbeenappliedtotheeffectsonelectrolytebalancecaused
by consumption of glycyrrhizinic acid (glycyrrhizin) (Morris, 1993; Olukoga &
Donaldson,2000).
In individuals who consume liquorice and who show evidence of electrolyte
imbalance,ithasbeenreportedthatconcentrationsofaldosteronearedecreased,
suggestingthatglycyrrhizinorglycyrrheticacidmayhavedirectmineralocorticoid
activity;however,datafromstudiesexaminingthebindingofglycyrrhizinorglycyr-
rheticacidtomineralocorticoidreceptors(MR)invitrodemonstratethattheaffnity
ofthesecompoundsforMRisapproximately10000timeslessthanthatofaldo-
sterone(Molhuysenetal.,1950;Ulmannetal.,1975;Armaninietal.,1983,Arma-
ninietal.,1985;Tamayaetal.,1986;Takedaetal.,1987;Baker&Fanestil,1991).
Datafromstudiesinvivoindicatethattheconcentrationsofglycyrrheticacidinthe
bloodavailableforbindingtoMR,incombinationwiththeloweraffnity forMRof
glycyrrhetic acid compared with that of aldosterone, are not suffcient to explain
AME attributable to ingestion of liquorice (Monder et al., 1989; Edwards, 1991);
however,severalinvestigatorshavereportedthaturinaryconcentrationsofcortisol
andtheratioofserumcortisoltocortisoneareincreased,whileplasmaandurinary
concentrationsofcortisonearedecreasedbyconsumptionofliquoricecontaining
glycyrrhizinicacid(Epsteinetal.,1978;MacKenzieetal.,1990a;Kageyama,1992;
Kageyamaetal.,1992a;Krhenbhletal.,1994).Cortisolhasbeenreportedto
have mineralocorticoid activity and an affnity for MR that is equivalent to that of
aldosterone,whilecortisonehasnomineralocorticoidactivityanddoesnotbindto
MR (Edwards & Stewart, 1990; Strmer et al., 1993b). On the basis of these
observations, several investigators have suggested that inducedAME occurs in
responsetoincreasedrenalconcentrationsofcortisoland,morespecifcally,asa
resultofinhibitionoftheenzyme11b-HSD2,whichconvertsactivecortisoltoinac-
tive cortisone (Epstein et al., 1978; Stewart et al., 1987; Funder et al., 1988;
Monderetal.,1989;MacKenzieetal.,1990a;Kageyama,1992;Kageyamaetal.,
1992a; Edwards et al., 1993; Morris, 1993; Funder, 1995; Heikens et al., 1995;
Sweetman,2002).
GLYCYRRHIZINIC ACID 575
L1
Undernormalphysiologicalconditions,theconcentrationofcortisolintheblood
is1001000timeshigherthanthatofaldosterone,whiletheaffnityofcortisolfor
MRisequivalenttothatofaldosterone(Edwards&Stewart,1990;Strmeretal.,
1993b);however,cortisolispreventedfrombindingtoMR,particularlyintissues
withhighmineralocorticoidactivity(e.g.kidney),becauseitisconvertedintoinac-
tivecortisoneby11b-HSD2(Edwards&Stewart,1990;Morris,1993).Glycyrrhetic
acid inhibits 11b-HSD2, resulting in elevated renal concentrations of cortisol and
inappropriatemineralocorticoideffects(Edwardsetal.,1993;Morris,1993;Strmer
etal.,1993b).Evidencetosupportthismechanismcomesfromseveralsources.
Data from studies of metabolism in vitro using kidney microsomal preparations
have confrmed that glycyrrhetic acid, but not glycyrrhizinic acid (glycyrrhizin),
inhibits human renal 11b-HSD2 (Monder et al., 1989; Stewart et al., 1994). In
addition,glycyrrhizinappearsinthebloodasglycyrrheticacidafteroraladministra-
tionand,inrats,oralglycyrrhizinalsohasbeenreportedtoproduceinhibitionof
11b-HSD2 (Valentino et al., 1987; Wu et al., 1999). Glycyrrhizin and glycyrrhetic
acidhavealsobeenreportedtodecrease thelevelsofmRNAthatcodefor11b-
HSD2 in several tissues (Whorwood et al., 1993; Wu et al., 1999). Furthermore,
the effects of glycyrrhizin and, therefore, glycyrrhetic acid do not occur in the
absenceofcortisol(i.e.followingadrenalectomy)(Kageyamaetal.,1992b).Finally,
theeffectsofliquoricehavebeenreportedtobethesameasthoseseeninindi-
viduals known to have a defciency of 11b-HSD2 (Stewart et al., 1987; Stewart
et al., 1988; Monder et al., 1989; Kageyama, 1992; Kageyama et al., 1992a;
Morris,1993).
The time frame for changes in concentrations in cortisol and cortisone in
humansafteroraladministrationof18b-glycyrrheticacidwasstudiedbyKrhen-
bhl et al. (1994). In this study, single oral doses of 0.5, 1.0, or 1.5g of 18b-g
lycyrrhetic acid were provided to six healthy males (approximately 8.3, 16.7, or
25mg/kgbw, respectively). No adverse effects occurred in any subject and no
changesinbloodpressureorpulsewerereportedtooccur.Plasmaconcentrations
of cortisol were reportedly not affected at any dose. Plasma concentrations of
cortisone was reported to be decreased at each dose compared with those of a
controlgroup.Thedecreasesinplasmaconcentrationsofcortisonewerereported
to be signifcantly different at 0.5 and 1.0g at 2.57.5h after administration, and
at1.5gat2.510hafteradministration.Asaresultofthedecreaseinconcentra-
tionsofcortisone,theratioofplasmacortisoltoplasmacortisonewas increased,
indicating competitive inhibition of 11b-HSD2. Based on the pharmacokinetic
parametersof18b-glycyrrheticacidalsoexaminedinthisstudy,theauthorscon-
cludedthatthedevelopmentofhypertensionandhypokalaemiainhumanswould
require continuous intake of substantial amounts of glycyrrhizinic acid; however,
signifcant variation in the ability of liquorice to induce AME has been reported
(Strmeretal.,1993b).
Teelucksinghetal.(1990)investigatedhoweffectivelyglucocorticoidreceptors
in human skin could be targeted upon inhibition of cortisol metabolism. Twenty-
threehealthyvolunteerswithnopreviousexposuretoexogenouscorticosteroids
wereexposeddermallytoacombinationofglycyrrheticacid(20mg/ml)andhydro-
cortisone(0.1,0.3,1,3,or10mg/ml),whichwasadministeredinavolumeof10ml
to77mmsites.Whenhydrocortisonewasadministeredatconcentrationsof1,
576 GLYCYRRHIZINIC ACID
L1
3, or 10mg/ml, a signifcant potentiation of the cutaneous vasoconstrictor effect
was reported. In a companion experiment in vitro, the skin of nude mice was
homogenized and incubated with glycyrrhetic acid (0.01, 1, or 100mmol/l). The
activityof11b-HSDinthisskinwasreportedtobesignifcantlyinhibitedinadose-
dependentmanneratallconcentrationsofglycyrrheticacid.
Inadditiontotheinhibitoryeffectofliquoricecomponentson11b-HSD2,their
effects on other enzymes also contribute toAME in some individuals (Kumagai
et al., 1966; Tamura, 1975; Sakamoto & Wakabayashi, 1988; Latif et al., 1990;
Morrisetal.,1990;Morris,1993).Glycyrrheticacidhasbeenreportedtoinhibit5b-
reductaseand3b-hydroxysteroiddehydrogenase(13b-HSD)enzymesintheliver,
resulting in an accumulation of aldosterone and 5a-dihydroaldosterone, both of
whicharepotentmineralocorticoids(Tamura,1975;Latifetal.,1990).Inrats,both
glycyrrhizin and glycyrrhetic acid, administered orally at a dose of 45mg/kgbw
perdayfor20days,werereportedtoinhibitenzymesinLeydigcellsinvivoand
also inhibited enzymes in ovarian microsomal preparations (Sakamoto &
Wakabayashi,1988).Theauthorsofthisstudysuggestedthattheresultingaccu-
mulation of steroids could provide the necessary precursors for conversion to
othercorticosteroids(i.e.cortisol).
Beyond effects on enzyme and biochemical systems, it has recently been
reported that male Sprague-Dawley rats given glycyrrhizin at a dose of 200mg/
kgbw per day for 0.52 weeks showed upregulation of the aquaporin-2 and -3
waterchannelsintherenalinnerandoutermedulla,asanalysedbyWesternblot
(Kangetal.,2003).Creatinineclearancewasreportedtobeunchanged.Upregula-
tion of aquaporin-2 and -3 results in decreased fow of urine and excretion of
sodium.Intraperitonealtreatmentoftheratswithspironolactone(amineralocorti-
coidreceptorblocker)atadoseof20mg/kgbwperdaywasfoundtoreversethe
effectsofglycyrrhizin.Theauthorssuggestedthattheirresultsmayindicatethat
waterandsaltretentionassociatedwithhighratesofglycyrrhizinconsumptionmay
in part be related to upregulation of aquaporins through the action of cortisol on
MRs.
Morrisetal.(1990)havesuggestedthatinhibitionof5b-steroidreductasemay
occurinusersofchewingtobacco.Thiswasbasedontheinhibitionof5b-steroid
reductaseincytosolicpreparationsfromratliverbysalivatakenfromindividuals
whohadchewed1goftobaccofor5min.Theinhibitoryeffectwasequivalentto
thatreportedtooccurinvitroafterexposureto1mmolofglycyrrheticacid.
Paolinietal.(1999)reportedthatoralintakeofliquoricerootextract(3138or
6276mg/kgbw) or its natural constituent, glycyrrhizin, (240 or 480mg/kgbw) in
Sprague-Dawley rats affected their levels of liver monooxygenases. Although a
singledosewasreportednottohaveaneffect,dailydosesgivenonfourconsecu-
tive days reportedly induced cytochrome P450 (CYP) CYP3A, CYP1A2 and, to
varying extents, CYP2B1-linked enzymes.Also, a lesser increase in activity was
reported for testosterone 6b-(CYP3A1/2, CYP1A1/2), 7a-(CYP1A1/2, CYP2A1),
16a-(CYP2B1,CYP2C11),2a-(CYP2C11)and2b-(CYP3A1,CYP1A1)-dependent
oxidasesandandrost-4-ene-3,17-dione-(CYP3A1/2)-supportedmonooxygenases.
Theauthorsstatedthatthehighestreportedconsumptionofliquoricerootextract
inhumansisaboutfourtimesgreaterthanthehighestdose(6276mg/kgbw)used
inthisstudy.
GLYCYRRHIZINIC ACID 577
L1
RadixGlycyrrhiza uralensisFisch(GRZ),alsoknownasChineseliquorice,is
a component of Glycyrrhizae radix that has been traditionally used as an anti-
infammatory, anti-ulcer, detoxifcation, and liver-protective agent (Wen-Yu et al.,
1999;WHO,1999).GroupsoftenmaleKunmingmiceweregivenGRZatadose
of10g/kgbwperdayorglycyrrheticacidatadoseof50mg/kgbwperdayorally
for 7 days (Wen-Yu et al., 1999). The control animals were given an equivalent
volumeofnormalsaline.MicetreatedwithGRZorglycyrrheticacidwerereported
tohavesignifcantlyincreasedlevelsofthehepaticenzymesCYP450,aminopyrine
N-demethylase,7-ethoxycumarinO-deethylase,andarylhydrocarbonhydroxylase
comparedwithcontrols.TheauthorsattributedthehepatoprotectiveeffectofGRZ
toitsCYP450-inducingeffect,whichrendersxenobioticslessharmfulviametabo-
lismanddetoxifcation.
2.2 Toxicological studies
2.2.1. Acute toxicity
Themedianlethaldose(LD
50
)valuesforglycyrrhizinicacidandrelatedcom-
poundsadministeredbyvariousroutesinavarietyofanimalspeciesarepresented
in Table 1. Notably, no verifable LD
50
value was identifed for glycyrrhizinic acid
itself.
TheoralLD
50
valuesfortheammonium,diammonium,potassium,monopotas-
sium,anddipotassiumsaltsofglycyrrhizinicacidwerereportedtobeintherange
of 122012700mg/kgbw in mice (Klosa, 1957; Fujimura, 1974; Fujimura &
Okamoto,1974).Additionally,nodeathswerereportedinmicegivenglycyrrhetic
Table 1. Acute toxicity of compounds related to glycyrrhizinic acid
Compound Species Route LD
50
Reference
(mg/kgbw)
Glycyrrhizin Mouse Oral >2000 Sasakietal.(2002)
Guinea-pig Subcutaneous 1000 Tocco(1924)
Dog Intravenous 500 Tocco(1924)
Glycyrrheticacid Mouse Intraperitoneal 308 Finneyetal.(1958)
Ammonium Mouse Oral 12700 Fujimura(1974)
glycyrrhizinate Mouse Intraperitoneal 1050 Fujimura(1974)
Monoammonium Mouse Intraperitoneal 1070 Fujimura(1974)
glycyrrhizinate
Diammonium Mouse Oral 9600 Fujimura&Okamoto(1974)
glycyrrhizinate Mouse Intraperitoneal 1250 Fujimura&Okamoto(1974)
Potassium Mouse Oral 2400 Fujimura(1974)
glycyrrhizinate Mouse Intraperitoneal 1260 Fujimura(1974)
Monopotassium Mouse Oral 1220 Klosa(1957)
glycyrrhizinate Mouse Intravenous 412 Klosa(1957)
Mouse Intramuscular 695 Klosa(1957)
Mouse Subcutaneous 697 Klosa(1957)
Dipotassium Mouse Oral 8100 Fujimura&Okamoto(1974)
glycyrrhizinate Mouse Intraperitoneal 1400 Fujimura&Okamoto(1974)
578 GLYCYRRHIZINIC ACID
L1
acid at a maximum dose of 610mg/kgbw by oral administration (Finney et al.,
1958). No effects were reported on the nervous system, cardiovascular system,
respiratory system, or the gastrointestinal tract of cats given a single dose of
glycyrrhetic acid at 125mg/kgbw by intraperitoneal administration (Finney et al.,
1958).
IngroupsoffouradultfemaleSprague-Dawleyrats,intraperitonealadministra-
tion of single doses of glycyrrhizin, 18a- or 18b-glycyrrhetic acid at 0.5, 1, or
1.5g/kgbwwasreportedtobewelltoleratedandtoproducenosignifcantlesions
uponexaminationbyelectrocardiogram(ECG).Conversely,theadministrationof
asingleintraperitonealdoseof18a-glycyrrheticacidat2g/kgbw,ortwodosesof
18a-glycyrrhetic acid at 1g/kgbw within 1h, was reported to result in the death
ofallfourrats,whichtheauthorsattributedtoanatrio-ventricularblock.Uponhis-
topathological examination, these rats were reported to exhibit oedema of the
brain, cerebellum and lung, and haematic stasis of the kidney, adrenal glands,
spleen and liver. Focal changes in the papillary muscles of the heart also were
reported,includingoedema,myolisis,andcelldistortion(Rossietal.,1999).
2.2.2. Short-term studies of toxicity
Severalshort-termstudieshavereportednotoxicologicallysignifcantadverse
effectsinmiceandratstreatedorallywithglycyrrhizinicacidat442mg/kgbwper
day(Quaschningetal.,2001),withglycyrrhizinat533mg/kgbwperday(Macabies
etal.,1963a),orwithglycyrrheticacidat1500mg/kgbwperday(Linko&Vasama,
1958;Horigomeetal.,2001).
After repeated administration (30 times) of ammonium glycyrrhizinate at a
maximumdailydoseof7or28mg/kgbwbygavageintothestomachsofmiceand
rats (study duration unknown), there were no signs of intoxication, essential
changes in the haematological and integral parameters, shifts in the activity of
serumenzymes,ormorphologicalchangesincellstructuresoftheinternalorgans.
A second dose of ammonium glycyrrhizinate at 28mg/kgbw given during the
courseoftreatment,reportedlyledtochangesintheactivityofsomeenzymesin
the brain, and the development of parenchymatous dystrophy of the liver, which
wasreportedtodeveloptoacidophilicnecrosis,attendedwithsignsofregenera-
tion(Antovetal.,1997).
Thirty-eight favouring agents, including liquorice extract, were evaluated for
possible immunotoxicity by studying their effect on humoral and cell-mediated
immunity.In135femaleCD-1orB6C3F
1
micegivenliquoriceextractatadoseof
0, 1250, 2500, or 5000mg/kgbw per day by gavage for 5 days, liquorice extract
wasreportedtoproduceachangeinspleenweightandspleen:bodyweightratio,
buttheseeffectswereconsideredbytheauthorstobeincidentalchanges,asthe
organweightdifferencewasnotdose-dependentandnootherimmunomodulation
wasreported(Vollmuthetal.,1989).
Thepossibleuseofglycyrrheticacidinthetreatmentofautoimmunediseases
was investigated in groups of seven to eight male MRL/Fas 1pr mice given
drinking-watercontainingglycyrrheticacidatadoseof5mg/kgbwperdayfor21
or170days.Thecontrolgroupreceivedthevehicle(0.3%tragacanthgum)only.
GLYCYRRHIZINIC ACID 579
L1
After 21 days of treatment, two mice per group were killed and their organs ex-
tractedforexamination.Nosignifcantdifferencesintheweightsoftheliver,kidney,
thymus, and spleen were reported between animals in the groups treated with
glycyrrheticacidandthecontrolgroup.Bodyweightsofmicetreatedwithglycyr-
rheticacidandmiceinthecontrolgroupwerealsoreportednottobesignifcantly
different at the end of the 170-day study. A gradual increase in urinary protein
concentrations ( 150mg/ml) was reported in the control mice from days 22170
ofthestudy.Incontrast,urinaryproteinconcentrationsinmicetreatedwithglycyr-
rheticacidwerereportedtoreach150mg/mlaftermorethan90daysofmonitoring.
Fromdays1850ofthestudy,urinaryproteinconcentrationsinmicetreatedwith
glycyrrheticacidwerereportedtobesignifcantlylowerthanthoseofthecontrols.
Treatment with glycyrrhetic acid was reported to signifcantly decrease serum
concentrationsofIgGinthemiceafter21daysoftreatment,butnotserumcon-
centrations of anti-rheumatoid factor and anti-cardiolipin antibodies.Although no
signifcant changes in the serum concentrations of corticosterone were reported
inmicetreatedwithglycyrrheticacid,thesignifcantdecreaseinserumconcentra-
tions of dehydrocorticosterone resulted in a signifcant suppression of the mean
ration of dehydrocorticosterone to corticosterone, which the authors attributed to
systemic11b-HSD2activity.Concentrationsofcorticosteroneanddehydrocortico-
steroneintheliverofmicetreatedwithglycyrrheticacidwerereportedtobesig-
nifcantly higher than those of controls; however, there were no changes in the
rationofdehydrocorticosteronetocorticosteroneintheliver.Nosignifcantchanges
intheconcentrationsofthesesteroidswerereportedinthekidneys,thymus,and
spleenofmicetreatedwithglycyrrheticacidorofmiceinthecontrolgroup.11b-
HSD activity was reported to be signifcantly inhibited in the liver and kidneys of
micetreatedwithglycyrrheticacidcomparedwithcontrols;however,nosignifcant
difference in 11b-HSD activity in the thymus and spleen was reported between
mice treated with glycyrrhetic acid and mice in the control groups.According to
the authors, glycyrrhetic acid inhibits 11b-HSD activity and subsequently modu-
latescorticosteroidmetabolismin1prmice,resultinginthesuppressionofurinary
protein excretion and serum concentrations of IgG. Therefore, the authors con-
cludedthatcontinuoustreatmentwithglycyrrheticacidwouldretardthedevelop-
mentofautoimmunediseasesin1prmice(Horigomeetal.,2001).
GroupsofmaleWistar-Kyotoratsweregivendrinking-watercontainingglycyr-
rhizinicacidataconcentrationof3000mg/lformorethan21days(approximately
442mg/kgbw per day). Additonal groups were given placebo, spironolactone
(5.8mg/kgbwperday),oreplerenone(182mg/kgbwperday)bygavageondays
8 to 21. No signifcant differences in body weight and heart rate were reported
for any of the treatment groups. However, signifcantly increased systolic blood
pressure, endothelin-1 (ET-1) protein concentrations, and ET-1-induced vascular
contractions were reported for the group receiving placebo + glycyrrhizinic acid
compared with the other groups. Conversely, concentrations of nitrate in aortic
tissueandnitrousoxide-mediatedendothelialfunctionwerereportedtobesignif-
cantlyreducedinthegroupreceivingplacebo+glycyrrhizinicacidcomparedwith
theothergroups.Theauthorsreportedthatalltheeffectsattributedtoglycyrrhizinic
acid (i.e. observed in the group receiving placebo + glycyrrhizinic acid) were
restoredornormalizedbytreatmentwitheitheroneofthetwoaldosteronereceptor
antagonists (i.e. spironolactone and eplerenone). The authors attributed the
580 GLYCYRRHIZINIC ACID
L1
observed glycyrrhizinic acid-induced hypertension in rats to decreased bioavail-
abilityofnitrousoxideandactivationoftheET-1vascularsystem,andsuggested
the possible use of aldosterone receptor antagonists in the treatment of cardio-
vascular diseases associated with reduced 11b-HSD activity (Quaschning et al.,
2001).
The possible correlation between high intake of glycyrrhizin and myolysis of
the papillary muscles has been studied in groups of 40 adult female Sprague-
Dawleyratsgivendistilledwater(control),18a-or18b-glycyrrheticacidat15mg/
kgbwperday,orglycyrrhizinat30mg/kgbwperdaybyoraladministrationfor30
days.Tenratspergroupwerekilledatdays0,15,and30ofthestudyinorderto
monitor the possible biochemical or histological effects of the treatment. Mild
myolysisoftheheartpapillarymuscleswasreportedinratstreatedwithglycyrrhi-
zin or 18a-glycyrrhetic acid at day 30 of the study, and no regression of effects
was reported 30 days after cessation of treatment (i.e. day 60). Treatment with
18a-glycyrrheticacidwasalsoreportedtoproducetubularcalculi(associatedwith
signifcantly enhanced excretion of calcium ions) and a slight expansion of the
bronchus-associated lymphoid tissue of the lungs at day 15. Similar increases
in excretion of calcium ions were also reported in rats given glycyrrhizin or 18b-
glycyrrheticacid,butsuchincreaseswerereportedtoreachstatisticalsignifcance
onlyonday30ofthestudy.Increasedexcretionofcalciumionswasreportedto
persistintheratsevenaftercessationoftreatment,particularlyinratstreatedwith
18a-glycyrrhetic acid.According to the authors, the biochemical and histological
effects observed are not, in themselves, considered to be serious side-effects;
however, they recommended that consumption of products containing liquorice
(i.e. liquorice as a confectionery or drug) should be avoided by individuals with
hyperaldosteronism, heart disease, or those being treated with diuretics (Rossi
etal.,1999).
Ina50-daystudy,ratsfedliquoriceextractatadoseof10g/kgbwperdayor
ammoniatedglycyrrhizinatadoseof1g/kgbwperdaywerereportedtoshowa
progressive increase in blood pressure and growth depression. Relative weight
increases and severe lesions were reported in the kidneys, adrenal glands, and
heart.Thesurvivalrate,after50days,wasreportedtobe36%forratsreceiving
liquorice extract and 77% for those receiving ammoniated glycyrrhizinate, com-
paredwith100%forcontrols(Girerdetal.,1958).
Ina165-daystudy,ratsweregivenglycyrrhizinorallyatadoseof160mg/day
(approximately533mg/kgbwperday)atcertainintervals.A25%increaseinblood
pressurewasreported,whichreturnedtonormalvalueswhentreatmentwasdis-
continued (Macabies et al., 1963a). The possible hypertensive action of several
glycyrrhizin-related substances was also examined and similar results reported
(Macabiesetal., 1963b).
Gordon (1974) studied the effect of ammonium glycyrrhizinate on blood
pressure, electrolytes, and corticosterone. Within 23 weeks, increases in blood
pressurewerereportedinSprague-Dawleyratsfedammoniumglycyrrhizinateat
a dose of 1 or 2g/kgbw per day. These effects were reported not to occur in
Osborne-Mendelratsduringa20-weekstudy;however,furtherdetailsofthisstudy
werenotavailable.
GLYCYRRHIZINIC ACID 581
L1
Ina90-daystudy,ratswerefeddietscontainingdiammoniumanddipotassium
glycyrrhizinate at 0.1 and 0.5% (approximately 50 and 250mg/kgbw per day,
respectively). Male rats given the highest dose (250mg/kgbw per day) were
reportedtoexhibitaslowerrateofbody-weightgain,butnogrossorhistological
abnormalitieswerereportedintheorgans(Fujimura&Okamoto,1974).
A possible neurobehavioural infuence was studied in male Sprague-Dawley
ratsgivendietscontainingammoniatedglycyrrhizinat0,2,3,or4%(approximately
0,1.23,1.87,and2.55g/kgbwperday,respectively).Thestudyconsistedofthree
experimentalgroupsof40rats.Withineachexperimentalgroup,fourgroupsof10
rats were fed the respective diets. The frst experimental group, which was fed
thetestdietfor4months,wasusedtoassessgrowth,physiologicalstate,motor
functionandexploratory behaviour.Thesecondgroup,alsofedthetestdietfor4
months,wasusedtoassessbasiclearningskills,inadditiontogrowthandphysi-
ological parameters.The last experimental group, fed the test diet for 6 months,
wasusedtoassessshock-inducedbehaviourandoperantconditioningbehaviour.
Duringtheexposureperiod,growthwasreportedtobesignifcantlyreducedinthe
group receiving the highest dose (2.55g/kgbw per day) in the frst experimental
group;however,growthwasnotreducedineitheroftheotherexperimentalgroups
receivingthisdiet(containing4%ammoniatedglycyrrhizin).Asimilarpatternwas
seenforfoodconsumption,whichwasreportedtobesignifcantlyreducedat4%
inthesecondexperimentalgrouptwo,butnotineitherthefrstorthirdexperimen-
tal groups. Blood pressure and heart rate measurements in the frst and second
experimentalgroupswerereportedtobesignifcantlydifferentfromcontrolvalues
atvarioustimesduringthestudyandshowedanoverallhypertensiveandbrady-
cardic effect. No signifcant differences in blood pressure or heart rate were
reportedinratsreceivingdietscontaining2%ammoniatedglycyrrhizin.Atterminal
necropsy, signifcant trends towards dose-related increases in heart weight were
reportedinthefrstandsecondexperimentalgroups,inkidneyweightsinallthree
experimental groups, and increased brain and decreased adrenal weights in the
secondexperimentalgroup;however,differencesinorganweightsfortheindivid-
ualexposuregroupswithinexperimentalgroupswerenotreported.Testsformotor
function,exploratorybehaviour,andoperantperformancewerereportednottobe
signifcantly different from control values in any group treated with ammoniated
glycyrrhizin.Overall,avoidancebehaviourwasreportedtosurpasscontrollevels,
whilenoeffectswerereportedonresponseinhibitionlearning,retention,orshock
sensitivity.Theauthorssuggestedthattheeffectsofammoniatedglycyrrhizinon
thepituitaryadrenalaxis,whicharemanifestedphysiologicallyaseffectsonheart
rateandbloodpressure,mayhavebeenrelatedtothepositiveeffectsonavoid-
ancebehaviour(Sobotkaetal.,1981).
Linko&Vasama(1958)reportedanincreaseinbodyweightandexcretionof
potassiuminratsgivendietscontainingglycyrrheticacidatadoseof1500mg/
kgbwperdayfor8,10,or14days.
2.2.3. Long-term studies of toxicity and carcinogenicity
Astudyofcarcinogenicitywiththedisodiumsaltofglycyrrhizinicacidhasbeen
conductedinB6C3F
1
mice.Groupsof50malemiceweregivendrinking-water
582 GLYCYRRHIZINIC ACID
L1
containing0.04,0.08,or0.15%disodiumglycyrrhizinate(approximately 71,166,
and 229mg/kgbw per day, respectively) for up to 96 weeks. Groups of 50 or
morefemalemiceweregivengivendrinking-watercontaining0.08,0.15,or0.3%
disodium glycyrrhizinate (approximately 117, 217, and 407mg/kgbw per day,
respectively)forupto96weeks.Thehighestdoseforeachsexwasthemaximum
tolerateddose(MTD)determinedinapreviousstudyofsubacutetoxicity.Afterthe
treatmentperiod,miceweremaintainedonabasaldietwithnodisodiumglycyr-
rhizinateinthedrinkingwaterforanadditional14weeks.Therewasadose-related
reductionintheamountofwaterconsumedbythetreatedanimalscomparedwith
that in the control animals (signifcance not stated); however, no dose-related
increasewasreportedintheincidenceoftumoursorinthespecifcdistributionof
benign and malignant neoplasms in mice treated with disodium glycyrrhizinate
compared with controls. The most common neoplasms reported were liver cell
tumoursandlymphoidleukaemiainmaleandfemalemice,respectively.Noother
observations(e.g.haematology)werereported(Kobukeetal.,1985).
In femaleA/J mice treated orally, a water extract of liquorice was reported to
signifcantly inhibit the formation of lung and forestomach tumours induced by
benzo[a]pyrene and N-nitrosodiethylamine (Wang & Mukhtar, 1992; Wang et al.,
1992).Theliquoriceconstituent,glycyrrhizin,alsowasreportedtoprotectagainst
theinitiationofskintumoursby7,12-dimethylbenz[a]anthracene(DMBA)infemale
Sencarmice(Wang&Mukhtar,1992).
Glycyrrhizinic acid (glycyrrhizin) has also been reported to inhibit the occur-
rence of hepatocellular carcinoma induced by diethylnitrosamine (DEN). Groups
of50micereceivedDENatadoseof75100mg/kgbwperweekbyintraperitoneal
injectionfor6weekspluseither2mgofglycyrrhizinadministeredthreetimesper
week intramuscularly, or the same volume of saline.Additional control groups of
30 mice received glycyrrhizin or saline without DEN. Mice were killed every 5
weeks after the last injection of DEN, up to 25 weeks. The average number of
tumours(adenomasandhepatocellularcarcinomas)at25weekswasreportedto
be0.71and1.64ingroupstreatedwithglycyrrhizin+DENandDENonly,respec-
tively.Inthegrouptreatedwithglycyrrhizin+DEN,theincidenceofhepatocellular
carcinomawasreportedtobelowerandthenumberofapoptoticcellswasreported
tobehigherthanthatinthegrouptreatedwithDENonly(Shiotaetal.,1997).
In an investigation of the reported chemopreventative effects of liquorice,
groups of 16 male Fischer rats were given diets containing 0.38, 1.5, or 3%
liquorice root extract (corresponding to a dose of liquorice root of approximately
190,750,or1500mg/kgbwperday,respectively)for3months.Halfoftheanimals
in each group were killed after 1 month of treatment.A control group of 20 rats
received only the basal diet for the same treatment periods. Analyses of liver
enzymesat1and3monthswerereportedtoshowincreasedactivityofglutathione
transferase, catalase, and protein kinase C. The increases were not statistically
signifcantinallcases,butwerereportedtoshowatrendtowardsincreaseacross
thetreatedgroups.Levelsofornithinedecarboxylase,whichindicatepromotional
activity,werereportedtobedecreased.Nosignifcantchangesinfoodconsump-
tion,bodyweight,haematological,clinicalchemistry,organweight,orhistological
parameterswerereportedatanydose.Theauthorssuggestedthattheincreases
inprotectiveenzymeswithoutsubsequentincreasesinso-calledriskenzymesmay
GLYCYRRHIZINIC ACID 583
L1
contributedirectlyorindirectlytothereportedchemoprotectiveactivityofliquorice
(Webbetal.,1992).
2.2.4. Genotoxicity
(a) In vitro
Severalglycyrrhizinatesaltsandliquoriceextractsand/orvariouscomponents
ofliquoricecontainingglycyrrhizinicacidhavebeentestedinanumberofshort-
termtestsofmutagenicity/genotoxicity.Theresultsofthesetestsaresummarized
inTable2.
Inmammaliancells,chromosomeaberrationswerereportedinChinesehamster
lungfbroblaststreatedwithglycyrrhizinorsodiumglycyrrhizinate,butnotinhuman
embryoniclungcellstreatedwithammoniatedglycyrrhizin(LittonBionetics,1972;
Ishidateetal.,1984;Ishidate,1988).Glycyrrhizinicacidtrisodiumsaltwasreported
to produce negative results in tests for sister chromatid exchange and micro-
nucleusformationinChinesehamstercellculturesandhumanfbroblasticcelllines
(Sasakietal.,1980).Liquoriceextractswerereportedtoproducenegativeresults
intheassayforunscheduledDNAsynthesisassayinrathepatocytes,butreport-
edlyproducedpositiveresultsinmouselymphomacells(Hecketal.,1989).
(b) In vivo
The results of tests on glycyrrhizinic acid salts in vivo are presented inTable
3.Ammoniatedglycyrrhizinatewasreportedtogivenegativeresultsintheassay
forchromosomeaberrationinratbonemarrow(LittonBionetics,1972),andnega-
tiveresultsinassaysfordominantlethalmutationinmice(Sheuetal.,1986)and
host-mediatedassaysinwhichS. typhimuriumbacterialstrainswereisolatedfrom
theorgansofhostanimalsandevaluatedformutageniceffects(LittonBionetics,
1972);however,mixedresultswerereportedforassayfordominantlethalmutation
in rats (Litton Bionetics, 1972; Jorgenson et al., 1977; Sheu et al., 1986). The
single positive result reported was based on a statistically signifcant increase in
the number of dead implants, which was reportedly due to a high frequency of
dead implants in fve out of 33 pregnant females mated in the frst week after
treatment. No effect on the frequency of dead implants was reported in females
matedduringthesecondweekaftertreatment.
In a study in male ddY mice (Sasaki et al., 2002), the comet (alkaline single
cell gel electrophoresis) assay was used to investigate DNA damage in eight
organs (i.e. stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone
marrow)aftertreatmentwithasingleoral(gavage)doseofglycyrrhizinat2000mg/
kgbw.Theauthorsreportednegativeresults(i.e.noDNAdamageatthelevelof
thesinglecell)foreachoftheorgansstudied.
In addition to the studies with ammonium glycyrrhizinate, both disodium and
trisodiumglycyrrhizinatewerereportedtogivenegativeresultsinatestformicro-
nucleusformationinmaleddYmicewhenadministeredbyintraperitonealinjection
atdosesof140mg/kgbw(Hayashietal.,1988).
584 GLYCYRRHIZINIC ACID
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GLYCYRRHIZINIC ACID 587
L1
Overall,althoughsomefndingswerepositive,theavailabledatafromstudies
conductedinvitroandinvivoindicatethatglycyrrhizinicacidanditsrelatedsalts
arenotgenotoxic.
2.2.5. Reproductive toxicity
Nobirthdefectsoreffectsonmaternalorfetalsurvivalwerereportedinmice,
rats,hamsters,orrabbitsgivenammoniumglycyrrhizinateatadoseof1g/kgbw
per day by gavage (Food and Drug Research Laboratories, 1972; Mantovani et
al.,1988).Anothersalt,disodiumglycyrrhizinate,wasalsoreportedtobenon-tera-
togenicinratsgivenadoseof601480mg/kgbwperdayduringthefrst20days
ofgestation(Itamietal.,1985).
In albino CD-1 mice or Wistar albino rats, monoammonium glycyrrhizinate at
adoseof0,27,90,300,or1000mg/kgbwadministeredbyoralintubationondays
615 of gestation was reported not to produce teratogenic effects or result in
decreasedmaternalorfetalsurvival.Administrationofmonoammoniumglycyrrhi-
zinateatthesamedosestoSyriangoldenhamstersondays610ofgestationor
toDutch-beltedrabbitsondays618ofgestationwasalsoreportednottoproduce
teratogeniceffectsorresultindecreasedmaternalorfetalsurvival.Theexamina-
tions in this study consisted of a caesarean section to determine the number of
implantationsites,resorptionsites,andliveanddeadfetuses.Theurogenitaltract
ofeachdamwasexaminedandthefetuseswerealsoexaminedforanyabnormali-
ties(FoodandDrugResearchLaboratories,1972).
A dose-related increase in embryotoxicity and minor anomalies has been
reportedinratsgivenammoniumglycyrrhizinatebyoraladministration.Groupsof
1620 pregnant rats were given drinking-water containing ammonium glycyrrhiz-
inateatadoseof0,21,239,or680mg/kgbwperdayondays717ofgestation.
The authors reported that the effects in the fetuses were not signifcantly dose-
related,withtheexceptionofthenumberofvariantsofthesternum.Sternalvaria-
tions were reported to show a similar increase in the groups receiving the
intermediatedose(239mg/kgbwperday)andthehighestdose(680mg/kgbwper
day).The incidence of haemorrhages, haematomas, and ectopic (i.e. displaced)
orhypoplastic(i.e.incompletedevelopment)kidneyswasreportedtoshowasig-
nifcantincreaseinthegroupreceivingthelowestdose(21mg/kgbwperday)and
the highest dose compared with that in the control group, while the incidence in
groupreceivingtheintermediatedosewasreportedtoshownosignifcantchange.
Asignifcantincrease(p<0.01)inrenalvariantswasreportedinthegroupreceiv-
ingthelowestdose;asignifcantincrease(p<0.05)ininternalhaemorrhageswas
reportedonlyinthegroupreceivingtheintermediatedose.Noabnormalitieswere
reportedinthedamsexceptforasignifcantincreaseinwaterintakeinthegroups
receivingtheintermediateandhighestdoses.Theconcentrationsofpotassiumin
thedamswerereportedtoremainnormalatalldoses.Theauthorsalsoreported
asignifcantincreaseinresorptionsinthegroupstreatedwithammoniumglycyr-
rhizinate,butthisoccurredatamoresensitivelevel(p<0.03)thanthatnormally
used in this type of study (p < 0.05). The sternal variations are
consideredtorepresentfetotoxicityandconsequentlythestudyshowednodose-
relatedteratogenicity(Mantovanietal.,1988).
588 GLYCYRRHIZINIC ACID
L1
PregnantWistarratsweregivendietscontaining0,0.08,0.4,or2.0%disodium
glycyrrhizinate(approximately0,60,290,and1480mg/kgbwperday,respectively)
duringthefrst20daysofgestation.Fetuseswereexaminedonday20ofgesta-
tionforexternal,skeletal,orinternalmalformations,bodyweight,placentalweight,
mortality, and sex ratio. No signifcant differences between animals treated with
disodium glycyrrhizinate and control animals were reported. Postnatal develop-
mentoftheoffspringtreatedwithdisodiumglycyrrhizinatewasnodifferenttothat
of control animals. The only signifcant difference between animals treated with
disodium glycyrrhizinate and control animals reported by the authors was in the
post-partumbody-weightgainofthedamsreceivingthehighestdose(2.0%diso-
diumglycyrrhizinate).Atthetwohigherdoses(0.4and2.0%disodiumglycyrrhiz-
inate), a lower body-weight gain was reported up to 3 weeks after delivery. No
explanationwasgivenforthisobservation.Theauthorsconcludedthatdisodium
glycyrrhizinatewasnotteratogenictoratsfedatthelevelsindicated(Itamietal.,
1985).
2.2.6. Special studies
Inadditiontoconductingstudiestoevaluatethemetabolismandpharmacoki-
netics of glycyrrhizinic acid and glycyrrhetic acid in rats (Ploeger et al., 2000a,
2000b), Ploeger (2000) and co-workers (2000a, 2000b) have developed a
physiologically-based pharmacokinetic-pharmacodynamic model to characterize
theprobabilityofhumansdevelopingpseudohyperaldosteronismasaresultofthe
consumptionofglycyrrhizinicacid,particularlyfromliquoriceandproductscontain-
ingliquorice.Themodelwascalibratedonthebasisofdatafromtheliteratureon
plasma concentrations of glycyrrhizinic and glycyrrhetic acid after oral intake of
glycyrrhizinicacid/glycyrrhizin,occurrenceofpseudohyperaldosteronismatvarious
plasma concentrations of glycyrrhizinic and glycyrrhetic acid, and variables such
astherateofbowelmovements,stomachemptying,rateofhydrolysisofglycyr-
rhizinicacidtoglycyrrheticacid,andenterohepaticcirculationofglycyrrheticacid
werealsoincluded.Withthesevariables,themodelwasabletoprovideestimates
of the effects of intake of glycyrrhizinic acid, with hydrolysis to glycyrrhetic acid,
ontheactivityof11b-HSD2throughthepredictionoftheratioofcortisol/cortisone
in urine samples at 24h (Heilmann et al., 1999). Much of these data were also
validated against data obtained from independently conducted clinical trials in
volunteersreceivingmultipleoraldosesofglycyrrhizinicorglycyrrheticacid.
Fromanalysisoftheirmodel,Ploeger(2000)andPloegeretal.(2001a,2001b)
determined that the response (likelihood of developing pseudohyperaldosteron-
ism)tointakeofglycyrrhizinicacidwasmainlyafactorofthemotilityofthegas-
trointestinal tract (slower motility resulted in greater systemic exposures to both
glycyrrhizinic and glycyrrhetic acids), variances in the absorption rate constant,
variability in the plasma concentrations of glycyrrhetic acid that produced 50%
inhibitionof11b-HSD2,andvariabilityintheinitialorbaselineactivityofthe11b-
HSD2enzymeinanygivenindividual(Muneetal.,1995;Ferrarietal.,2001).By
studyingtheeffectsofthesevariablesinaMonteCarlosimulation,Ploeger(2000)
calculatedanestimateofthelikelihoodofdevelopmentofdisturbancesoftheratio
of cortisol to corticosterone, and of biochemical and clinical manifestations of
pseudohyperaldosteronismwithincreasingintakeofglycyrrhizinicacid.Thephar-
GLYCYRRHIZINIC ACID 589
L1
macodynamicportionofthemodelwasinitializedwiththefollowingassumptions
obtained from the scientifc literature: (a) at the no-observed-effect level (NOEL)
for glycyrrhizin of 2.0mg/kgbw per day reported in a randomized double-blind
studyinwhichgroupsof39healthyfemalevolunteersweretreatedwithglycyrrhi-
zinic acid for up to 8 weeks, that the plasma concentration of glycyrrhetic acid
never exceeded 800mg/l (Bijlsma et al., 1996); and (b) the range of potential
responsestoglycyrrhizinicacidwasassumedtohavebeencapturedinthestudies
byBijlsmaetal.(1996)andvanGelderenetal.(2000).
Ploeger(2000)calculatedthatatanintakeofglycyrrhizinicacidof100mg/day,
about2mg/kgbwperdayortheNOELvaluereportedfromtheBijlsmaetal.(1996)
and van Gelderen et al. (2000) 8-week clinical study, approximately 18% of the
exposed population would have glycyrrhizinic acid at concentrations of greater
than 800mg/l. Disturbances of the ration of cortisol to corticosterone and of the
appearance of clinical manifestations of pseudohyperaldosteronism were
predicted to occur in 26% and in 0.04% of the exposed population, respectively.
Theseestimateswereconsideredapplicabletotheconsumptionofglycyrrhizinic
acid from all dietary sources combined. Overall, the physiologically-based phar-
macokinetic-pharmacodynamic model developed by Ploeger (2000) appeared to
agreewellwiththeavailableclinicalstudydataaswellaswiththecasereportsof
effects of glycyrrhizin at intakes of <100mg/day in sensitive members of the
population.
2.3 observations in humans
2.3.1. Case reports
Intheliterature,therehavebeenmanycasereportsofreportedintoxications
related to excessive consumption of liquorice (Revers, 1946; Molhuysen et al.,
1950; Groen et al., 1951; Groen et al., 1952; Pelser et al., 1953; Louis & Conn,
1956;Mollaretetal.,1960;Jennyetal.,1961;Salassaetal.,1962;Chodkiewicz
etal.,1963;Grossetal.,1966;Connetal.,1968;Koster&David,1968;LeFebvre
&Marc-Aurele,1968;Pelner,1969;Chamberlain,1970;Tourtellotte&Hirst,1970;
Robinsonetal.,1971;Cotterill&Cunliffe,1973;Wash&Bernard,1975;Epsteinet
al., 1976; Bannister et al., 1977; Cumming, 1977; Epstein et al., 1977a, 1977b;
Epsteinetal.,1978;Mouradetal.,1979;Takedaetal.,1979;Werneretal.,1979;
Blachley & Knochel, 1980; Cumming et al., 1980; Lai et al., 1980; Ibsen, 1981;
Sundaram&Swaminathan,1981;Simpson&Currie,1982;Yokoyamaetal.,1982;
Corsietal.,1983;Cuginietal.,1983;Heidemann&Kreuzfelder,1983;Joseph&
Kelemen,1984;Nielsen&Pedersen,1984;Pietteetal.,1984;Beretta-Piccoliet
al.,1985;Morietal.,1985;Commeauetal.,1986;Stewartetal.,1987;Acharet
al.,1989;Scalietal.,1990;Bcker&Breithardt,1991;Fareseetal.,1991;Chu-
bachietal.,1992;Hayashietal.,1992,Shintanietal.,1992;Rosseel&Schoors,
1993;Bernardietal.,1994;Brayley&Jones,1994;Kerlanetal.,1994;Hayashiet
al.,1995;Heikensetal.,1995;Barrellaetal.,1997;Chamberlain&Abolnik,1997;
DeKlerk et al., 1997; Kageyama et al., 1997; Blakey, 1998; Dellow et al., 1998;
Doeker&Andler,1999;Erikssonetal.,1999;Famularoetal.,1999;Ishikawaet
al., 1999; Nishioka & Seguchi, 1999; Dobbins & Saul, 2000; Negro et al., 2000;
Olukoga & Donaldson, 2000; Russo et al., 2000; Brouwers & van der Meulen,
590 GLYCYRRHIZINIC ACID
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2001).Effectsreportedinthesecasesincludedretentionofserumsodium,deple-
tionofserumpotassium,oedema,hypertension,andmyopathy.Theseeffectswere
reportedtobeintensifedinindividualstakingmedication,especiallydiuretics.
InWesternEurope,peoplewhoconsumed13lofalcoholicbeveragescon-
taining liquorice (levels not specifed) daily were reported to develop muscular
weakness,paralysis,tetany,andhypokalaemia(Mollaretetal.,1960;Jennyetal.,
1961; Chodkiewicz et al., 1963). According to the authors, the symptoms sug-
gestedprimaryaldosteronism(i.e.electrolyteimbalanceduetoexcessivesecre-
tionofaldosterone),butsuchsymptomshavealsobeenreportedtobetheresult
oftheingestionoflargequantitiesofglycyrrhizin(Jennyetal., 1961).
The case reports document that exposures to glycyrrhizinic acid at <100mg/
daymaybeassociatedwiththedevelopmentofeffectscharacteristicofpseudo-
hyperaldosteronism,includingincreasedbloodpressure.Inthesecases,thereare
often other factors, either genetic or lifestyle, that increase the sensitivity of the
individualstotheeffectsofglycyrrheticacidon11b-HSD2.Forexample,Russoet
al.(2000)reportedtwocasesofhypertensionencephalopathyassociatedwithhigh
blood pressure induced by prolonged daily ingestion of low doses of liquorice
(lengthofexposurenotspecifed).Onecasewasreportedtoinvolveamanaged
42 years who consumed 50g of liquorice per day (equated by the authors to an
intake of glycyrrhizinic acid of 100mg/day), while the other case involved a man
aged46yearswhoingested40gofliquoriceperday(equatedbytheauthorsto
an intake of glycyrrhizinic acid of 80mg/day). Both patients were reported to
present with classic symptoms of hypermineralocorticoidism (i.e. hypertension,
hypokalaemia and suppressed renin-aldosterone system) in addition to hyper-
tensionencephalopathy.Thepatientswereadvisedtodiscontinuetheirintakeof
liquorice, whereupon rapid improvements in their symptoms were reported. The
authors suggested that individual variations in susceptibility to glycyrrhizinic acid
exist, such that those who have lower enzyme activity could develop severe
inducedhypermineralocorticoidismoningestionoflowdosesofglycyrrhizinicacid.
Similarly,Rosseel&Schoors(1993)reportedonthecaseofamanaged55years
whodevelopedhighbloodpressure,lowplasmareninactivity,andhypokalaemia,
despitenormalconcentrationsofplasmaaldosterone,afterconsumptionofchewing
gum containing glycyrrhizinic acid at approximately 50mg/day. The symptoms
resolvedafterdiscontinuationofconsumptionofchewinggum.
2.3.2 Single-dose studies
Ithasbeenreportedthatsingleoraldosesof18b-glycyrrheticacidat0.5,1.0,
or 1.5g (approximately 8.3, 16.67, and 25.0mg/kgbw, respectively) given to six
healthymalesdidnottoresultinanyadverseeffectsorchangesinbloodpressure
or pulse (Krhenbhl et al., 1994). Plasma concentrations of cortisol were not
affected at any dose. Plasma concentrations of cortisone were reported to be
decreasedateachdosecomparedwiththoseofacontrolgroup.Thedecreases
in plasma concentrations of cortisone were statistically signifcant in the groups
receiving0.5and1.0gbetween2.5and7.5hafteradministration,andinthegroup
receiving1.5gbetween2.5and10hafteradministration.
GLYCYRRHIZINIC ACID 591
L1
Amanaged33yearsdiagnosedwithviralmeningitiswasadmittedtothehos-
pitalandtreatedwithapreparationcalledStrongerNeo-MinophagenC(SNMC),
adrugconsistingofglycine,cysteineandglycyrrhizin,whereuponthepatientwas
reportedtodevelopadrug-inducedallergichepatitis(Akashietal.,1988).Onthe
basisoftheresultsofanSNMCpatchtest,theauthorsconcludedthattheallergic
reaction was induced by the SNMC treatment; however, they were not able to
determinewhichingredientofSNMCmayhavepotentiatedthecondition.
An National Cancer Institute-sponsored phase-I clinical trial with glycyrrhetic
acidinpatientswithapriorhistoryofbreastcancerwasconductedbyVogeletal.
(1992). Ten healthy female volunteers who had been previously diagnosed with
breastcancerreceivedglycyrrheticacidasasingleoraldoseat100to500mg/m
2

(approximately2.70to13.51mg/kgbw)andwerereportedtoshownoevidenceof
signifcanttoxicity.Nochanges(comparedwithbaseline)werereportedinsystolic
ordiastolicbloodpressure,serumsodium,orplasmareninconcentrations.None
ofthesubjectsreportedanyadverseeffectsandglycyrrheticacidwasreportedto
bewelltolerateduptoadoseof500mg/m
2
.
2.3.3 Multiple-dose studies
In a randomized double-blind treatment study conducted in the Netherlands,
39 healthy female volunteers (aged 1840 years; body weights, 5090kg) were
given capsules containing glycyrrhizinic acid at a dose of 0, 1, 2, or 4mg/kgbw
perdayadministeredorallyfor8weeks(Bijlsmaetal.,1996;vanGelderenetal.,
2000).Therewere10womeninthecontrolgroup,9inthegroupreceiving1mg/
kgbw per day, 9 in the group receiving 2mg/kgbw per day, and 11 in the group
receiving4mg/kgbwperday.Thestudyusedfemalevolunteersbecauseinapilot
studywithhigherdoseswomenappearedtobemoresensitivethanmentoglycyr-
rhizinic acid (van Vloten et al., 1989). Women were eliminated from the study
if their blood pressure rose above 95mmHg (12.7kPa) diastolic or 150mmHg
(20kPa)systolic,oriftheyexhibitedperipheraloedemaoradecreaseintheserum
concentrationofpotassiumtobelow3.0mmol/l.Onepersonfromthegroupreceiv-
ing the highest dose (4mg/kgbw per day) was withdrawn from the study after 6
weeksduetoconcentrationdiffcultiesandgeneraldiscomfort.Uponexamination,
thebloodpressureofthissubjectwasreportedtobeslightlyelevated(+7mmHg
(+0.9kPa)systolicanddiastolic),aswasthebodyweight(by3kg)comparedwith
baseline levels. Once consumption was discontinued, these parameters were
reportedtohavereturnedtonormalvalues.Duringthecourseofthestudy,there
was no change in body weight in any group, indictating no retention of water.
Plasmareninactivitydecreasedsignifcantlyonlyatthehighestdose.Theserum
concentration of aldosterone in the group receiving the highest dose was also
signifcantlylowerthanthatofthecontrolgroupafter2,4,6,and8weeksoftreat-
ment,butnotafter10weeks.Therewasnodifferencesbetweentheconcentrations
ofaldosteronereportedinthegroupsreceivingadoseof1or2mg/kgbwperday
and those reported in the control group. At 4mg/kgbw per day, but not at 1 or
2mg/kgbwperday,anapparentincreaseintheconcentrationofatrialnatriuretic
peptide was found, which decreased after cessation of treatment.At the highest
dose, blood pressure was unchanged, but was relatively higher than that in the
592 GLYCYRRHIZINIC ACID
L1
controlgroup(diastolic,72.9mmHg(9.7kPa)comparedwith67.8mmHg(9.0kPa)
inthecontrolgroup;andsystolic,125mmHg(16.7kPa)comparedwith120mmHg
(16.0kPa) in the control group), owing to a reduction in blood pressure in the
controlgroupoverthecourseofthestudy.Duringweeks24,asignifcantdecrease
inplasmaconcentrationsofpotassiuminthegroupreceivingadoseof4mg/kgbw
perday,relativetothecontrolgroup,wasreported;however,concentrationsgradu-
allyreturnedtobaselinevaluesthroughouttheremainderoftheexperiment.The
NOEL was 2mg/kgbw per day, albeit based on evaluation of a small number of
individuals.
Inanolderclinicalstudywithpureglycyrrhizinicacid(VanVlotenetal.,1989),
groupsofeightmaleandeightfemalehumanvolunteersconsumedglycyrrhizinic
acidatdosesof400or800mg/dayfor24weeks.Duringthestudyeffectsrelated
topseudohyperaldosteronismwerereportedlyobserved,withfemalesshowinga
greatersensitivitytotheeffect.
Revers(1946)reportedanimprovementinthehealingofgastriculcersin45
casesinwhichpatientsweretreatedwithliquoriceextract;however,oedemawas
reportedin20%ofthepatientswhoweregivenoneteaspoonofapreparationof
100gofextractin50gofwaterthreetimesperday(equivalenttoatotalof10g
liquoriceextractperdayorapproximately850mgofglycyrrhizinicacid,assuming
thatliquoriceextractcontains8.5%glycyrrhizinicacid).Theseeffectswerereported
toincludeinitialswellingoftheface,followedbyenlargedlegsandhands.Often,
theoedemawasreportedtobeaccompaniedbyviolentheadache,dizziness,pain
in the upper abdomen, tightness of the chest, a marked increase in weight, and
slighthypertension.Haematologyandcardiaceffectswerenotstudied.Thetotal
dose, when reduced to 3g liquorice extract per day (approximately 255mg of
glycyrrhizinic acid, assuming liquorice extract contains 8.5% glycyrrhizinic acid),
was reported not to produce the oedematous effects; however, the author com-
mentedthat,onoccasion,oedemaalsooccurredattheselowerdoses.Inaddition
tothisreportedincidenceofperipheralandfacialoedema,ararecaseofpulmo-
nary oedema has been reported, for which excessive consumption of liquorice
(approximately3.6gofglycyrrhizinicacid)wastheonlyreportedassociatedevent
(structural abnormalities of the heart were ruled out) (Chamberlain & Abolnik,
1997).
Inaseriesofclinicaltrials,Smorenberg-Schoorl&Vree(1963)evaluatedthe
effectsoftheconsumptionofsuccusliquoritiae,reportedlycontaining26%glycyr-
rhizinicacid,onbody-weightgain,waterretention,oedemaandbloodpressurein
17individualsoveranunspecifedperiodoftime.Theindividualsreportedlycon-
sumed 1560mg of glycyrrhizinic acid per day. In six of the subjects, intake of
780mg/day was reported to be associated with less pronounced body-weight
increases,andinfourofthesesix,withincreasedbloodpressure.Oneofthe17
subjectsappearedtobeespeciallysensitivetoglycyrrhizinicacidsinceanincrease
in blood pressure was reported at an intake of 130mg/day over an unspecifed
periodoftime.
Epsteinetal.(1977a)measuredthelevelsofelectrolyteandtherenin-angio-
tensin-aldosterone axis in four women (aged 3855 years) withdrawn from con-
sumptionofliquorice.Thewomenwerehospitalizedforchronictoxicitycausedby
GLYCYRRHIZINIC ACID 593
L1
liquorice after 6 months to 5 years of ingesting 25200g of liquorice candy per
day.Serumpotassium,serumaldosterone,andurinaryaldosteronewerereported
to be below normal concentrations. It was reported that the renin-angiotensin-
aldosteroneaxiswassuppressedandplasmareninactivitywasdecreasedinall
four subjects. All levels returned to the normal range after electrolyte treatment
andabstinencefromliquoriceingestion.
In a clinical study, 14 healthy individuals ingested 100 or 200g of liquorice
candy(equatedbytheauthorstoapproximately700or1400mgofglycyrrhizinic
acid,respectively)for14weeks(Epsteinetal.,1977b).Consumptionofliquorice
wasstoppedprematurelyinsixwomenowingtothedevelopmentofhypokalaemia
and/or oedema of the extremities (the dose received by the subjects was not
specifed). Other reported side-effects included headaches, lethargy, and weight
gain.Itwasreportedthatplasmaconcentrationsofpotassiumdropped,therenin-
angiotensin axis was suppressed, plasma renin activity decreased, and urinary
and plasma concentrations of aldosterone decreased in subjects continuing the
treatment.Theseeffectswerereversibleuponcessationofliquoriceingestion.No
clear distinction was made between the effects seen at the high and low doses;
however,theauthorsdidnotethatthereportedeffectshadamorerapidonsetat
thehigherdose.Onemalesubjectwasreportedtoadapttothelevelsofliquorice
given and his clinical symptoms (oedema and weight gain) returned to normal
throughoutcontinuedingestionofliquorice.Theauthorssuggestedthatthismay
beduetoanadaptivealterationofabsorptionordegradationoftheliquorice.
Thepossibleeffectofliquoriceingestiononthepituitary-adrenalfunctionof13
healthysubjects(aged1846years)wasdeterminedbyEpsteinetal. (1978).Eight
subjects(fvemen,threewomen)ingested100gofliquoricedailyandfvesubjects
(all women) ingested 200g of liquorice daily for 14 weeks. The authors deter-
mined the dosage to be equivalent to 7001400mg of glycyrrhetic acid per day;
however,inapreviouspaperbytheseauthors(Epsteinetal.,1977b),theintake
ofliquoricewasdeterminedtobeequivalentto7001400mgofglycyrrhizinicacid
perday.Glycyrrhizinicacidiscomprisedofonemoleculeofglycyrrheticacidand
twomoleculesofglucuronicacid.Itwasassumed,therefore,thattheauthorswere
actuallyreferringtoglycyrrhizinicacidandnotglycyrrheticacid.Thisassumption
was confrmed by personal communication with one of the co-authors, Dr E.A.
Espiner.Thedailyintakeofglycyrrhizinicacidwasestimatedtobe11.723.3mg/
kgbw per day for a 60kg individual.The same authors also measured excretion
ofcortisolinfourwomenwithhypokalaemichypertensionattributedtoeitherlong-
term ingestion of liquorice or aldosterone-secreting adenomas. Normal subjects
were reported to show a signifcant increase in mean urinary concentration of
cortisol during the ingestion period and into the subsequent 1-week observation
period.Plasmacortisolandadrenocorticotropichormone(ACTH)valueswerenot
changed.Thewomenreportedlysufferingfromhypokalaemic(i.e.reducedserum
potassium) hypertension due to toxicity caused by liquorice also showed a sub-
stantial elevation in urinary cortisol excretion above the normal. This effect was
not seen in the women with primary aldosteronism (i.e. alteration of electrolyte
metabolismduetoexcessivesecretionofaldosterone).
In a 4-week clinical study, groups of three male and three female volunteers
were given a dried aqueous liquorice extract containing glycyrrhizin at 108, 217,
594 GLYCYRRHIZINIC ACID
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380or814mg/day(approximatelyequivalenttoadoseofglycyrrhizinof1.8,3.6,
6.2,or13mg/kgbwperday,respectively)(Bernardietal.,1994).Anthropometric
measurements,renalfunction,serumelectrolyte(sodiumandpotassiumconcen-
trations), blood glucose, renin-aldosterone axis and erythrocyte volume fraction
wereallassessedaspartofthisstudy.Noeffectswerereportedatthetwolowest
dosesofglycyrrhizin(1.8and3.6mg/kgbwperday),whileatthetwohigherdoses
(6.2and13mg/kgbwperday),itwasreportedthatthreesubjectswithdrewfrom
thestudyowingtoside-effectsincludingheadaches,oedema,andhypertension.
Two of the subjects were female and their complaints, headaches and oedema,
werereportedtobedirectlyassociatedwithpremenstrualsymptoms.Ofthesetwo
subjects, one female receiving glycyrrhizinic acid at 13mg/kgbw per day, who
withdrewfromthestudyduetosymptomsofborderlinehypertension,hypokalae-
mia, and peripheral oedema, was also reported to be taking an estroprogestinic
drug(i.e.anoralcontraceptive)atthesametime.Thethirdsubject,amale,had
ahistoryofhypertensionthatwasaggravatedbythestudyprotocol.Noneofthe
otherparticipantsexhibitedanysignifcantsymptoms.
Insixhealthymalevolunteers,ingestionof7gofliquorice(containing0.5gof
glycyrrhizinicacid;adoseofglycyrrhizinicacidofapproximately8.33mg/kgbwper
day) daily for 7 days was reported to result in a signifcant decrease in serum
concentrationoftestosteroneandasignifcantincreasein17b-hydroxyprogester-
one,whileserumandrostenedionedidnotchange(Armaninietal.,1999).Onthe
basisoftheseresults,theauthorsconcludedthattheenzyme17,20-lyase,which
isresponsiblefortheconversionof17-hydroxyprogesteronetoandrostenedione,
wasinhibitedbyglycyrrhizinicacid.
Hayashietal.(1995)reportedtwocasesofhypokalaemicmyopathyinpatients
withahistoryofingestionofglycyrrhizinorliquorice.Thefrstpatient,amanaged
62years,wasreportedtohaveconsumed273mgofglycyrrhizinperdayforthe
treatment of liver dysfunction for a period of 2 months (glycyrrhizin at a dose of
approximately 4.6mg/kgbw per day).The second patient in this study, a woman
aged27years,wasreportedtohavetakenavarietyofChineseherbsforweight
reductionandconstipationfor3years;however,theamountofliquoriceorglycyr-
rhizin consumed was not reported. In addition to myopathy, both patients were
reportedtoexperiencesevereweaknessintheirupperandlowerextremities.They
alsowerereportedtohaveserumandurinechemistryvaluesindicativeofminer-
alocorticoid excess, which the authors attributed to the intake of glycyrrhizin.
Muscle biopsies performed in the patients were reported to show varying fbre
diameters, necrosis, phagocytosis, and occasional vacuolated fbres in the male
patient,withnovacuolations,butsmallmyofbrillardegenerationinsomemuscle
fbres in the female patient. The authors concluded that the muscle changes
observedinthefrstpatientrefectedaseverelyaffectedstage,andthoseinthe
secondpatientwereindicativeofamoderatelyaffectedstage.Upondiscontinua-
tion of consumption of glycyrrhizin and oral treatment with potassium chloride, it
was reported that both patients were discharged from the hospital, having made
afullrecoveryoftheirmusclestrengths.
InastudyonthepotentialinteractionofliquoriceandCYP3A-substratedrugs,
10healthymenweregiven1gofliquoriceorplacebobyoraladministrationtwice
GLYCYRRHIZINIC ACID 595
L1
daily(approximatelyequivalentto33mg/kgbwperday)for7daysplus7.5mgof
midazolamonday8.Pre-treatmentwithplaceboorliquoricewasreportednotto
result in signifcantly different effects on the pharmacokinetic and pharmacody-
namic parameters of midazolam. On the basis of the results obtained for mid-
azolam, which is a known substrate of CYP3A enzymes, the authors suggested
that liquorice does not induce CYP3A activities in humans, and that therefore
liquoricewouldnotbeexpectedtointeractwithCYP3A-substratedrugsinhumans
(Shonetal.,2001).
Healthyvolunteersweregiveneither225mgofglycyrrhizinperday,or0.1mg
of 9a-fuorocortisol per day, or 225mg of glycyrrhizin plus 1.5mg of dexametha-
sone per day, for 7 days. The glycyrrhizinic acid content of the glycyrrhizin was
not given.The authors reported that treatment with glycyrrhizin only signifcantly
suppressedplasmareninactivity,urinaryaldosteroneandserumpotassiumcon-
centrations,whileserumsodium,urinarypotassium,urinarycortisolexcretionand
plasmaandurinarycortisoneconcentrationsweresignifcantlyincreased.Similar
resultswerealsoreportedaftertreatmentwith9a-fuorocortisol.Themineralocor-
ticoid effects of glycyrrhizin were reported to be signifcantly decreased after co-
treatmentwithglycyrrhizinplusdexamethasone(Kageyamaetal.,1992a).
In a similar study, fve male and fve female healthy volunteers were given
glycyrrhetic acid at a dose of 500mg/day (equivalent to approximately 200g of
confectionery per day); however, the glycyrrhizinic acid equivalent of the glycyr-
rhetic acid was not given. Concentrations of urinary cortisol, urinary potassium,
plasma sodium were reported to be signifcantly elevated with treatment, while
plasmaandurinarycortisone,plasmapotassium,plasmareninactivityandaldo-
sterone were reported to be signifcantly decreased with treatment. Plasma con-
centrationsofcortisolwerereportedtohavedecreased,butnotsignifcantly.Also,
oraladministrationof250mgofglycyrrheticacidonthefrstdayoftreatmentwas
reportedtodecreaseplasmacortisoneandincreasetherationofplasmacortisol
tocortisonewithin40min(MacKenzieetal.,1990b).
Blood pressure and endocrine functions were studied in 83 male users of
chewing tobacco and 22 age-matched controls. Users of chewing tobacco were
reported to have signifcantly higher mean blood pressure (133/78mmHg
(17.7/10.4kPa) compared with 128/73mmHg (17.1/9.7kPa), lower plasma renin
activity, and lower ratios of spot urinary tetrahydrocortisone to tetrahydrocortisol.
Additionally,infvenormalvolunteersgivenglycyrrhizinicacidatadoseof200mg/
day(reportedlyequivalenttotwopouchesofliquorice-containingchewingtobacco
perday)for1week,signifcantreductionswerereportedinplasmareninactivity,
ratiosofurinarytetrahydrocortisonetotetrahydrocortisol,andurinaryexcretionof
aldosterone.Theauthorsconcludedthattheuseofonetotwopouchesofchewing
tobaccomayproducemineralocorticoideffects.Thisstudydidnotprovidedetails
ofthemethodologyorthelevelsofuseofchewingtobacco.Inaddition,individual
andstatisticaldatawerenotreported.Furthermore,noadversehealtheffectswere
reportedinanyofthevolunteers(Guthrie,1992).
The dose and timeresponse relationships between liquorice consumption
and rise in blood pressure, as well as the variability in response to glycyrrhetic
596 GLYCYRRHIZINIC ACID
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acidbetweenindividualswereinvestigatedinhealthy,Caucasianvolunteersfrom
Iceland and Sweden. In the frst study, 10 volunteers (one male, nine females)
received200gofsweetliquoricedailyfor2weeks(equatedtoanintakeofglycyr-
rhetic acid of 540mg/day by the authors, which corresponds to approximately
9mg/kgbw per day in a 60kg individual). In the second study, 30 volunteers (11
males,19females)weregiven100gofsweetliquoricedailyfor4weeks(equated
toanintakeofglycyrrheticacidof270mg/daybytheauthors,whichcorresponds
toapproximately4.5mg/kgbwperdayina60kgindividual).Athirdgroupofvol-
unteers(12males,12females)wasgiven50gofsweetliquoricedailyfor4weeks
(equatedtoanintakeofglycyrrheticacidof75mg/daybytheauthors,whichcor-
responds to approximately 0.83mg/kgbw per day in a 60kg individual). Blood
pressure was measured using a standard mercury sphygmomanometer in all of
the subjects, twice during each visit, as well as before and after the period of
liquoriceconsumption.Allthevolunteersweregivenphysicalexaminationsatthe
beginning of the study, at the end of the period of liquorice consumption, and at
theendofthestudy.Inthefrststudy,meanvaluesforsystolicanddiastolicblood
pressureswerereportedtobesignifcantlyhigherthanbaselineafter2weeksof
consumption. Plasma concentrations of potassium were also reported to be sig-
nifcantly decreased in these subjects after 2 weeks, while no signifcant change
inheartratewasreported.Inthesecondstudy,itwasreportedthatsystolicblood
pressurewassignifcantlyincreasedafter2and4weeksofliquoriceconsumption.
Signifcantlydecreasedplasmaconcentrationsofpotassiumwerealsoreportedin
this group after 2 weeks of liquorice consumption, but no signifcant changes in
diastolic blood pressure and heart rate were reported. Although a signifcantly
increased systolic blood pressure was reported at 2 weeks in the third study, no
signifcantchangesinsystolicbloodpressureat4weeks,diastolicbloodpressure,
heart rate and serum concentrations of potassium were reported in any of the
volunteers. Additionally, it was reported that the individual response to liquorice
followedthenormaldistributioncurve,suchthattherewasaverysmallinterindi-
vidual variance in the rise of blood pressure with consumption of liquorice. The
authorsconcludedthatthephysiologicaleffectsofliquoricearedependentonthe
dose, but not on the length of exposure to liquorice, such that prolongation of
consumptionfrom2to4weeksdoesnotinfuencetheresponse,asthemaximum
bloodpressurelevelisreachedafterthefrst2weeksofconsumption(Sigurjns-
dttiretal.,2001).
In summary, the available clinical studies have reported mild clinical effects
consisting of hypokalaemia, reduced plasma renin activity, and reduced urinary
concentrationsofaldosteroneinindividualsconsumingglycyrrhizinicacidatadose
ofapproximately12mg/kgbwperdayintheformofsoftliquoricecandy.Noclinical
effectshavebeenreportedtobeassociatedwithintakesofglycyrrhizinicacidof
6.2mg/kgbwperday(Bernardietal., 1994).
Theclinicaleffectsreportedtobeproducedbyglycyrrhizinorglycyrrhizinicacid
maybedescribedasphysiologicalinnature,mirroring,forallpracticalpurposes,
the effects produced by excessive doses of the endogenous adrenal cortical
hormone,aldosterone,whichisinvolvedinregulatingelectrolytebalance.Asnoted
above, these effects consist of hypokalaemia and disturbances in renal function
resulting in sodium retention and oedema; however, these effects were reported
GLYCYRRHIZINIC ACID 597
L1
to be reversible upon cessation of administration of glycyrrhizinic acid (Revers,
1946; Molhuysen et al., 1950; Conn et al., 1968; Chamberlain, 1970; Epstein et
al.,1977a,1977b;Epsteinetal.,1978;Laietal.,1980;Sundaram&Swaminathan,
1981;Yokoyamaetal.,1982;Heidemann&Kreuzfelder,1983;Joseph&Kelemen,
1984; Beretta-Piccoli et al., 1985; Achar et al., 1989; Mackenzie et al., 1990b;
Kageyamaetal.,1992a;Morris,1993;Strmeretal.,1993b;Kerlanetal.,1994;
Hayashietal.,1995;Heikensetal.,1995;Olukoga&Donaldson,2000;Russoet
al.,2000;vanGelderenetal.,2000;Brouwers&vanderMeulen,2001;Sigurjns-
dttiretal.,2001).Thereversibilityoftheeffectsarguesfurtherthatthechanges
refect a physiological response, rather than a phenomenon of toxicity (WHO,
1987), and is consistent with homeostatic adjustments known to occur following
releaseofaldosterone(Guyton,1987).
2.3.4 Studies in pregnant women
Three studies were identifed that evaluated the effects of glycyrrhizinic acid,
derivedfromconsumptionofliquorice,onpregnancyoutcomes(Colleyetal.,1982;
Strandbergetal.,2001;Strandbergetal.,2002).Oneofthesestudies(Colleyet
al., 1982), in 313 mothers who consumed liquorice as part of cough medication,
didnotcontrolforanyconfoundingvariablesandisthereforeuninterpretablewith
respecttoevaluatingtheeffectsofglycyrrhizinicacidonpregnancyoutcome.
Thepotentialeffectsofliquoriceconsumptiononbirthweightandgestational
agewerestudiedin1049Finnishwomenwhohadcompletedstudyquestionnaires
betweenMarchandNovemberof1998.Intakeofglycyrrhizinwascalculatedbased
onthequantityandfrequencyofliquoriceconsumptionreportedonthequestion-
naire, together with the data on glycyrrhizin content of liquorice confectionery
obtainedfrommanufacturersandtheNationalFoodAdministrationreportof1993
(Blomberg & Hallikainen, 1993). Maternity records provided data on the type of
delivery,birthweightandmaternalbloodpressure,aswellasthebasisforestimat-
inggestationalage.Thecalculatedmeanintakeofglycyrrhizinwasreportedtobe
363mg/weekamongconsumers(adoseofglycyrrhizinofapproximately0.87mg/
kgbwperday),with46and2%ofthemothersreportingregularweeklyanddaily
consumption of liquorice, respectively. Average values for gestational age were
reported to be 40.1, 40, and 39.7 weeks for low (<250mg/week), medium (250
499mg/week) and high (500mg/week) maternal intakes of glycyrrhizin, respec-
tively.Therewerenosignifcantdifferencesinmeanbirthweightsreportedbetween
the groups with a low, intermediate and high intake of glycyrrhizin. Regression
analysesperformedwerereportedtoresultinnosignifcantrelationshipbetween
birthweightandintakeofglycyrrhizin;however,asignifcantreductioningestational
age(by2.52days)wasreportedtobeassociatedwithamaternalintakeofglycyr-
rhizinof500mg/week(approximately1.2mg/kgbwperday).Theeffectofglycyr-
rhizinconsumptionongestationalagewasreportedtoremainstatisticallysignifcant
despiteadjustmentsforparity,systolicbloodpressure,smoking,coffeeconsump-
tion, and exclusion of augmented or induced births.Additionally, the cumulative
frequencyplotofgestationalagewasreportedlyshiftedtotheleftamongwomen
withahighintakeofglycyrrhizin,comparedwiththerestofthesample.According
totheauthors,possiblemechanismsforthereducedgestationalagemayinclude
598 GLYCYRRHIZINIC ACID
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inhibition of cortisol or prostaglandin metabolism by glycyrrhizin. However, the
authorscautionedthattheresultsobtainedinthisstudymayhavebeenconfounded
by factors associated with liquorice consumption, and they recommended that
furtherstudiesbeundertakentoclarifythismatter(Strandbergetal.,2001).
Inasubsequentstudy,Strandbergetal.(2002)evaluatedtheriskofpre-term
birth (gestational age, <37 weeks) and high rates of consumption of liquorice
(glycyrrhizin,glycyrrhizinicacid)inasampleof95Finnishwomen.Onehundred
andsevenwomenwhogavebirthtoinfantsofnormalgestationalageatthesame
hospital served as controls. Intake of glycyrrhizin was evaluated on the basis of
questionnaire data and was grouped into three categories: <250mg/week, 250
499mg/week, and 500mg/week. The authors compared the risk for pre-term
(gestational age, <37 weeks) and early pre-term (gestational age, <34 weeks)
deliveryinthewomenwithahighconsumptionofliquorice(i.e.500mgofglycyr-
rhizin per week) with that for women with a low (<250mg/week) and moderate
(250499mg/week)consumptioncombined.Noseparateanalysesofthelow-and
moderate-consumptioncategorieswereconducted.Comparedwiththelowercon-
sumptioncategoriescombined,intakeofglycyrrhizinatratesinexcessof500mg/
weekwasreportedtobeassociatedwithanincreasedriskforbothpre-term(age-
adjusted odds ratio (OR), 2.28; 95% CI, 1.015.14) and early pre-term delivery
(OR,3.07;95%CI,1.178.05).Theassociationwithpre-termdeliverywasslightly
weakened(OR,2.15;95%CI,0.834.95)upontheinclusionofconfoundingvari-
ablessuchassmokingandparityintheanalysis.Theassociationwithearlypre-
term delivery was unchanged by the addition of confounding variables in the
analysis.Theauthorsnotedthatonelimitationofthestudywastheretrospective
natureofthequestionnaire.Thisweakeffectonpre-termbirthweightisbiologically
plausibleinlightoftheknowneffectsofglycyrrhizin(glycyrrheticacid)onconcen-
trationsofcortisolandprostaglandinmetabolism.
3. INTAKE
Exposuretoglycyrrhizinicacidmayoccurthroughtheconsumptionofliquorice
confectioneryandofhealthproductsinwhichliquoriceisused,mainlyforitslaxa-
tive and anti-fatulence properties. It may also occur by sucking/chewing dried
Glycyrrhiza roots. Moreover, glycyrrhizinic acid is present in beverages, chewing
gum, tooth paste, and tobacco in which either the purifed acid or its ammonium
salt or the dried crude root extract of Glycyrrhiza glabra have been incorporated
asafavouring.
3.1 Presence and concentration of glycyrrhizinic acid in foods
and beverages
(a) Glycyrrhizinic acid as a natural constituent
Concentrationsofglycyrrhizinicacidhavebeendeterminedinliquoriceconfec-
tionery products available on the market in the USA, Germany, Belgium and the
UK(Strmeretal.1993b).Similarrangesofconcentrationsofglycyrrhizinicacid
(2907900mg/kg) were found in these different markets, with few products con-
GLYCYRRHIZINIC ACID 599
L1
taining >3500mg of glycyrrhizinic acid per kg. The mean content of 17 liquorice
products from the UK was 2000mg/kg (Spinks & Fenwick, 1990). In the Nether-
lands, the glycyrrhizinic acid content of liquorice distributed on the Dutch market
bythefourmajormanufacturers(excludingspecialtiesinsmallpackaging)varied
from 27000 to 115000mg/kg in block liquorice and from 700 to 2300mg/kg in
liquorice,withcheapproductscontaininglessglycyrrhizinicacid.Onthebasisof
blockliquoricepurchasesandmarketsharesofthedifferentliquoricevarieties,the
meancontentofliquoriceproductsmarketedintheNetherlandswasassumedto
be 900mg/kg (TNO, 1995). On the other hand, Maas (2000) reported analytical
dataonglycyrrhizinicacidin19samplesofliquoricemarketedintheNetherlands;
valuesrangedfrom300to5100mg/kg,withameanof1700mg/kg.
In most exposure assessments for glycyrrhizic acid, liquorice is typically
assumed to contain glycyrrhizic acid at 2000mg/kg, i.e. the mean content in
liquoriceintheUK(Spinks&Fenwick,1990;Strmeretal.,1993b;vanGelderen
etal.,2000;CommissionoftheEuropeanCommunities2003).
Certainhealthproducts,suchasliquoriceherbaltea(dried)andthroatpearls,
may contain glycyrrhizinic acid at much higher concentrations 20000 and
47000mg/kg,respectively(Spinks&Fenwick,1990).
In10brandsofherbalteasforwhichliquoriceplantmaterialwasstatedasan
ingredient, the concentration of glycyrrhizinic acid in the prepared beverage was
foundtovaryfrom2to450mg/l(average,126mg/l).Asimilarrangeofconcentra-
tionswasfoundinherbalalcoholicbeverages(Maas,2000).
(b) Glycyrrhizinic acid as a favouring agent
Theupperuselevels(95thpercentileofreportedusage)forammoniumglycyr-
rhizinate and glycyrrhizinic acid used as chemically defned favouring agents in
foodshavebeenprovidedtotheScientifcCommitteeforFoodbytheEuropean
FlavourandFragranceAssociationforvariouscategories(EuropeanFlavourand
FragranceAssociation, 2001, 2003). upper use levels of glycyrrhizinic acid were
375mg/kg in dairy products and edible ices, 200mg/kg in bakery wares, 135
550mg/kg in alcoholic beverages, 50mg/kg in non-alcoholic soft beverages,
25mg/kg in meat and meat products, 20mg/kg in fsh and fsh products, and
10mg/kg in composite foods (including casseroles, meat pies, mincemeat). The
upperuselevelsvariedfrom400to5000mg/kginconfectionery,fondant,sweets
andchewinggum.
Maximumreporteduselevelsoftheammoniumsaltofglycyrrhizinicacidfrom
theFlavorandExtractManufacturersAssociation(1994)werelistedinthelatest
versionofFenarolishandbook(Burdock,2002):non-alcoholicbeverages,51mg/
kg; alcoholic beverages, 59mg/kg; baked goods, 61mg/kg; gelatin/puddings,
79mg/kg;frozendairyproducts,91mg/kg;frostingconfectionery,625mg/kg;hard
candies,676mg/kg;softcandies,1511mg/kg;andchewinggum,2278mg/kg.
Glycyrrhizinic acid is used as a favouring agent in the USA at the following
maximumpermittedlevels:bakedfoods,500mg/kg;alcoholicbeverages,1000mg/
kg;non-alcoholicbeverages,1500mg/kg;chewinggum,11000mg/kg;hardcandy,
600 GLYCYRRHIZINIC ACID
L1
160000mg/kg;herbsandseasonings,1500mg/kg;plantproteinproducts,1500mg/
kg;softcandy,31000mg/kg;vitaminormineraldietarysupplements,5000mg/kg;
allotherfoodsexceptsugarsubstitutes,1000mg/kg(CodeofFederalRegulations,
2003).
3.2 Assessment of intake
Recent cases of intoxication attributable to glycyrrhizinic acid reported in the
Opinion of the Scientifc Committee for Food referred to prolonged consumption
of large quantities of either liquorice confectionery (50g/day), herbal tea (3l per
day)andchewinggums(two16gpacksperday)(ScientifcCommitteeforFood,
2003).Thesemaythereforebeconsideredimportantpotentialsourcesofglycyr-
rhizinicacidinsomesectionsofthepopulation.
Refnedassessmentsofexposuretoglycyrrhizinicacidhavebeenperformed
bydifferentauthorsconsideringonlytheconsumptionofliquorice.Inthefollowing
calculations, it was assumed that liquorice contains glycyrrhizinic acid at
2000mg/kg.
An exposure assessment was conducted using food consumption data from
the 1995 Australian National Nutrition Survey (Australian Bureau of Statistics,
1999) and 1997 New Zealand National Nutrition Survey (Russell et al., 1999).
Liquorice consumption data was assessed though individual food recall for 24h.
Individualbodyweightsofrespondentswereusedtoexpressexposureperkgof
bodyweight.
In the survey from Australia, the estimated consumption of liquorice for all
respondents(13858subjects)was0.3g/day,correspondingtoanaveragedietary
exposuretoglycyrrhizinicacidequalto0.6mg/day(0.01mg/kgbwperday).Esti-
mated consumption of liquorice for consumers only (108 subjects) was 40.2mg/
dayatthemeanand162.8g/dayatthe95thpercentile,correspondingtodietary
exposuretoglycyrrhizinicacidof80.4mg/day(1.4mg/kgbwperday)atthemean
and325.5mg/day(5.6mg/kgbwperday)atthe95thpercentile.
InthesurveyfromNewZealand,theestimatedconsumptionofliquoriceforall
respondents(4636subjects)was0.25g/day,correspondingtoanaveragedietary
exposure to glycyrrhizinic acid of 0.5mg/day (0.01mg/kgbw per day). Estimated
meanconsumptionofliquoriceforconsumersonly(30subjects)was38.2mg/day,
correspondingtodietaryexposuretoglycyrrhizinicacidof76mg/day(1.1mg/kgbw
perday).The95thpercentileforconsumersonlyisnotreportedhereduetothe
smallsamplesize.
Owing to the short duration of the surveys fromAustralia and New Zealand,
levels of exposure in consumers with a high intake are probably overestimated,
whilethepercentageofconsumersisunderestimated.
In Nordic countries (Denmark, Finland, Iceland, Norway and Sweden) gross
consumption of liquorice was estimated at 1 to 2.5kg/person per year. These
fgurescorrespondtoabout2.76.8gliquorice/personperdayandthereforetoa
meanintakeofglycyrrhizinicacidof6to15mg/personperday.
GLYCYRRHIZINIC ACID 601
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The consumption of different types of liquorice was also estimated using the
data of the Second Dutch National Food Consumption Survey (TNO, 1995). In
order to provide estimates comparable to those of other countries, exposure to
glycyrrhizinic acid was assessed considering a mean content of 2000mg/kg of
liquorice, although the use of a different value (900mg/kg) was proposed in the
TNOreport.Thestudyreferredto6218personsaged192years.Foodconsump-
tiondatawereobtainedthrougha2-dayrecord.
Theresultsshowedthatdailyconsumptionofliquoriceproducts(inallrespon-
dents)was2gatthemeanand10gatthe95thpercentile.Highestvalueswere
found in children and teenagers due to the high percentage of consumers of
liquorice in these age classes (35%). In children aged 710 years, mean intake
was5gandintakeatthe95thpercentilewas20g.
Amongliquoriceusersonly(15%ofthewholesample),thedailyconsumption
of liquorice products was 13g at the mean and 50g at the 95th percentile. The
highestvaluesforconsumptioninconsumersonlywerefoundinwomenandmen
aged1619and1922years.Intheseagegroups,meanintakerangedfrom17
to26g/dayandintakeatthe95thpercentilerangedfrom54to122g/day.Intake
inchildrenliquoriceusersonlyaged710yearswas8gand7gatthemeanand
25gand16gatthe95thpercentileinmalesandfemalesrespectively.
Based on these data, estimated dietary intake of glycyrrhizinic acid for all
respondentswastherefore4mg/dayand20mg/dayattheaverageand95thper-
centilerespectively,i.e.0.07and0.3mg/kgbwassumingabodyweightof60kg.
Inchildrenaged710years,intakewouldbe10mg/dayatthemeanand40mg/day
atthe95thpercentile.
Amongliquoriceusersonly,estimateddietaryintakewas26and100mg/day
(mean and 95th percentile). In women and men aged 1619 and 1922 years,
meanintakerangedfrom34to52mg/dayandintakeatthe95thpercentileranged
from108to244g/day.Inchildrenaged710years,meanestimatedintakeranged
from to 14 to 16mg/day and intake at the 95th percentile ranged from 32 to
50mg/day.
The consumption of liquorice confectionery and the corresponding intake of
glycyrrhizinic acid were also estimated through ad hoc statistical analysis of the
foodconsumptiondataoftheINRAN-RM-2001foodsurveyconductedinasample
of Italian secondary school students. The study referred to 233 adolescents of
mean age 17 1.1 and mean body weight 64.1 13.2 (Leclercq et al., 2004).
Foodintakewasassessedonthebasisof1224-hdietaryrecords(4consecutive
days in three different periods of the year). In the whole sample the estimated
consumptionofliquoricewas0.41.4g/dayatthemeanand2.5g/dayatthe95th
percentile. Estimated consumption of liquorice for consumers only (39 subjects,
17%ofthewholesample)was,onaverage,2.42.8g/daywithamaximumintake
of13.5g/day.Therefore,inthewholesampletheestimateddietaryintakeofglycyr-
rhizinic acid was 0.8mg/day at the mean and 5mg/day at the 95th percentile.
Amongconsumersonly,estimateddietaryintakewas4.8mg/day,withamaximum
of27mg/day.
In summary, the available data on consumption of liquorice confectionery in
Australia, New Zealand and, in some European countries, suggest that mean
602 GLYCYRRHIZINIC ACID
L1
intake in the whole population is in the range of 0.01 to 0.1mg/kgbw per day.
Where data were available, the percentage of consumers of liquorice in Europe
was reported to be in the range of 1520%, with higher rates in children and
teenagers (35%). However, these percentages are probably underestimated
owing to the short duration of most surveys, which would not allow identifcation
ofoccasionalconsumers.
Inthesesurveys,intakebyconsumersonlywasestimatedtobeintherange
of 550mg/day at the mean and to reach 100300mg/day at the 95th
percentile.
Thepresentassessmentofintakewasperformedconsideringameancontent
ofglycyrrizinicacidof2000mg/kgofliquorice.Higherlevelsofexposure(upfour
times higher) can be expected in consumers with a high intake who are loyal to
liquorice products which contain higher concentrations of glycyrrhizinic acid
(7900mg/kgofliquorice).
Norefnedassessmentofintakehasbeenprovidedorpublishedinrelationto
glycyrrhizinicacidintakeviaconsumptionofherbalteacontainingliquorice.Herbal
teasareavailableasdriedproducts(teabags)forhomepreparationorasready-
for-useproductswhicharepackagedintobottlesasregularsoftdrinksandusually
consumedcool.
Itisclaimedthatthepopularityofherbalteashasincreasedsignifcantlyduring
thepastdecadesinwesterncountries(Manteiga,1997).However,noquantitative
estimates of consumption were made available to the Committee at its present
meeting. High levels of consumption of ready-for-use herbal teas were therefore
assumedtobesimilartothatofothersoftdrinks.Sincethehighestconsumption
(perkgbw)ofsoftdrinksisknowntobefoundinchildrenandteenagers,estimates
ofintakeareprovidedinthisagegroupinordertoprovideaconservativeestimate.
Dataretrievedfromafoodconsumptionstudybasedon14-dayindividualrecords
collectedinatotalof948teenagersfromfvecitiesintheEuropeanUnion(Dublin,
Ghent,Helsinki,PotsdamandRome)wereused.Meanagerangedfrom13to16
yearsandmeanbodyweightrangedfrom53to64kg,accordingtothecity.Mean
intakeofregularcarbonateddrinks(excludingdietproducts)was136ml/dayinthe
totalpopulation(Lambeetal.,2000).Thehighlevelofconsumption(97.5thper-
centile of the total population) varied from 371ml/day in Rome to 995ml/day in
Ghent (Institute of European Food Studies, 1998). These levels of consumption
are referred to average intake in 14 days and therefore may be considered as
representativeofthelong-termintakeinconsumerswithahighintake.Exposure
in consumers with a high intake would range from 46mg to 125mg assuming a
mean concentration of of glycyrrhizinic acid of 126mg/l in herbal teas. The esti-
matedexposureinconsumersloyaltothebrandsofherbalteawithahighercon-
centrationofglycyrrhizinicacid(450mg/l)couldreach167448mg.
4. CoMMENTS
The absorption, distribution, biotransformation and excretion of glycyrrhizinic
acidand/oritsmonoammoniumsalthavebeeninvestigatedinratsandhumans.
GLYCYRRHIZINIC ACID 603
L1
In both species, glycyrrhizinic acid, whether in the free form or as the monoam-
moniumsalt,ispoorlyabsorbedfromthegastrointestinaltract.Inthegastrointes-
tinal tract, glycyrrhizinic acid is hydrolysed, mainly by the activity of intestinal
microfora, to 18b-glycyrrhetic acid (the aglycone of glycyrrhizinic acid), a sub-
stancethatisreadilyabsorbed.18b-Glycyrrheticacidissubjecttoenterohepatic
circulationandcanbefurthermetabolizedbyintestinalbacteriato3-dehydro-18b-
glycyrrhetic acid and 3-epi-18b-glycyrrhetic acid. The time at which maximum
plasma concentrations of glycyrrhetic acid are achieved after oral ingestion of
glycyrrhizinicacidisreportedtobeintherangeof1216and812hinratsand
humans,respectively.Dosesinexcessof25mg/kgbwmaysaturatethecapacity
of intestinal microfora to hydrolyse glycyrrhizinic acid to glycyrrhetic acid. In
humans,absorptionofglycyrrheticacidfromthegutisvirtuallycomplete,regard-
lessofwhetheritisformedfromthehydrolysisofglycyrrhizinicacidorisinitially
presentaseithertheglycosideortheaglyconeinafoodmatrix(e.g.liquorice).In
humans, at a dose of 0.5g the half-life was approximately 2h, while at doses of
1.0and2.0g,asecond,slowerphaseofeliminationoccurred.
Theresultsofstudiesinrats,andinferencesthatcanbedrawnfromtheresults
of studies in humans, indicate that both glycyrrhizinic acid and its hydrolysis
productglycyrrheticacidarelargelyconfnedtotheplasma.Inplasma,glycyrrhi-
zinicacidandglycyrrheticacidareboundtoserumalbuminandarenottakenup
inbodytissuestoasignifcantextent.
Absorbedglycyrrheticacidhasbeenreportedtoproduceeffectsthataresimilar
to those of the adrenal steroid aldosterone. The mechanism of action of glycyr-
rhetic acid involves inhibition of the type-2 11b-HSD, an enzyme that converts
cortisol to cortisone. As a result, levels of cortisol, which has mineralocorticoid
activityreportedlyequivalenttothatofaldosterone,increase.Thehighrenalcon-
centrationsofcortisolcauseretentionofsodiumandexcretionofpotassium.This
electrolyteimbalancehasbeenreferredtoasapparentmineralocorticoidexcess
orpseudohyperaldosteronism.
TheoralLD
50
valuesforglycyrrhizinicacidandvarioussaltsinmice,guinea-
pigs and dogs were reported to be in the range of 308 to 12700mg/kgbw. The
toxicity of glycyrrhizinic acid and/or its monoammonium salt has been evaluated
inanumberofshort-termstudiesinratsandmice.Athighdoses,effectsreported
includedthoserelatedtoapparentmineralocorticoidexcessorpseudohyperaldo-
steronism. Mild myolysis of the heart papillary muscles was reported in female
Sprague-Dawley rats treated with glycyrrhizin (crude extract) at 30mg/kgbw per
dayorwith18a-or18b-glycyrrheticacidat15mg/kgbwperdayfor30days(note:
glycyrrhizinicacidisnotmetabolizedto18a-glycyrrheticacid).
Inastudyofcarcinogenicity,B6C3F
1
miceweretreatedfor96weekswiththe
disodium salt of glycyrrhizinic acid at a dose of 229mg/kgbw per day in males
and407mg/kgbwperdayandobservedforanadditional14weeks.Therewasa
dose-related reduction in the amount of water consumed by the treated animals
when compared with the control animals (statistical signifcance not stated);
however,nodose-relatedincreasewasreportedintheincidenceoftumoursorin
thespecifcdistributionofbenignandmalignantneoplasmsintreatedmicecom-
paredwithcontrols.
604 GLYCYRRHIZINIC ACID
L1
Oral administration of glycyrrhizin, like glycyrrhizinic acid, has been reported
to inhibit the development of chemical-induced neoplasms in several models in
miceandrats.
The available data indicated that glycyrrhizinic acid and its salts do not have
carcinogenicactivity.
Several glycyrrhizinic acid salts and liquorice extracts and/or various compo-
nentsofliquoricecontainingglycyrrhizinicacidhavebeeninvestigatedinanumber
oftestsformutagenicityand/orgenotoxicity.Overall,althoughsomepositivefnd-
ings were reported, the available data indicated that glycyrrhizinic acid and its
relatedsaltsarenotgenotoxicinvitroorinvivo.
Ammonium and disodium salts of glycyrrhizinic acid at doses of 1.5g/kgbw
perdayhavebeenevaluatedinseveralstudiesofdevelopmentaltoxicityinmice,
rats,hamstersandrabbits.Inoneofthesestudies,embryotoxicitywasobserved,
but overall the data indicated that glycyrrhizinic acid and its salts are not
teratogenic.
Therehavebeenmanycasereportsofeffectsrelatedtoexcessiveconsump-
tionofliquorice(i.e.equivalenttoanintakeofglycyrrhizinicacidof>200mg/day).
Theseincludedretentionofserumsodium,depletionofserumpotassium,oedema,
hypertension,andmyopathy.Thecasereportsalsodocumentedthatconsumption
ofproductscontainingliquoriceatlevelsthatwouldresultinintakesofglycyrrhi-
zinic acid of <100mg/day could be associated with the development of effects
characteristic of pseudohyperaldosteronism, including increased blood pressure.
Thebasisforsusceptibilityinsuchcaseswasnotknown,althoughseveralexpla-
nationsarepossible.
Theavailableclinicalstudieshavebeenreportedtodemonstratemildclinical
effects, consisting of hypokalemia, reduced plasma renin activity, and reduced
urinaryconcentrationsofaldosterone.
Inarandomizeddouble-blindstudy,39healthyfemalevolunteersweregiven
glycyrrhizinic acid at a dose of 0, 1, 2, or 4mg/kgbw per day for 8 weeks. No
adverseeffectswereobservedinthegroupsreceivingadoseof1or2mg/kgbw
perday.Inthegroupreceivingadoseof4mg/kgbwperday,decreasesinplasma
reninactivityandserumconcentrationsofaldosteronewerefound.Therewasan
apparentincreaseintheconcentrationofatrialnatriureticpeptide,whichreturned
to normal after discontinuation of exposure, but there was no increase in blood
pressure.However,meanbloodpressurewasgreateratthehighestdosethanin
thecontrols,owingtoareductioninthebloodpressureofthelatteroverthecourse
ofthestudy.
A physiologically-based pharmacokinetic-pharmacodynamic model has been
developedtocharacterizetheprobabilityofhumansdevelopingpseudohyperaldo-
steronism as a result of the consumption of glycyrrhizinic acid. On the basis of
modelling,itwascalculatedthatatanintakeofglycyrrhizinicacidof100mg/day
(about2mg/kgbwperday),approximately18%oftheexposedpopulationwould
haveglycyrrhizinicacidatconcentrationsof>800mg/l.Also,itwaspredictedthat
disturbances of the ratio of cortisol to corticosterone would occur in 26% of the
GLYCYRRHIZINIC ACID 605
L1
exposedpopulation,andthatclinicalmanifestationsofpseudohyperaldosteronism
wouldappearin0.04%ofexposedpersons(95%CI,0.000463.0%).
Intake
Exposuretoglycyrrhizinicacidthroughconsumptionofliquoriceconfectionery
was assessed on the basis of a number of food surveys lasting 114 days.
Assuming a mean content of 2000mg of glycyrrhizinic acid per kg of liquorice
confectionery,theexposuresforconsumersonlyinthesesurveyswerecalculated
tobeintherangeof5to50mg/dayatthemeanandreached100to300mg/day
atthe95thpercentile.
On the basis of a mean content of 126mg of glycyrrhizinic acid per litre of
herbalteacontainingliquorice,highlevelsofexposuremaybeexpectedinregular
consumersofthesebeverages.
5. EVALUATIoN
The most signifcant effect of glycyrrhizinic acid, after hydrolysis in the gut to
glycyrrheticacidandsubsequentabsorption,isinhibitionofthetype-211b-HSD,
withaconsequentincreaseinconcentrationsofcortisol,whichleadstoincreased
mineralocorticoid activity with retention of sodium and water and symptoms of
apparentmineralocorticoidexcess.Thisphysiologicalactionofglycyrrhizinicacid
(glycyrrhetic acid) is reversible, but when sustained can lead to elevated blood
pressure.
TheCommitteeconcludedthatthesafetyevaluationofglycyrrizinicacidshould
bebasedonthedatafromhumans.Itwasobservedthatthereisasensitivesubset
ofthepopulationwhoappeartoshowsignsofpseudohyperaldosteronismatlower
exposures than those which produce effects in the general population, but the
available data did not allow the Committee to adequately characterize this sub-
group,andhencethedatacouldnotbeusedtoassignanADI.Theavailabledata
suggestthatanintakeof100mg/daywouldbeunlikelytocauseadverseeffects
inthemajorityofadults.TheCommitteerecognizedthat,incertainhighlysuscep-
tibleindividuals,physiologicaleffectscouldoccuratintakessomewhatbelowthis
fgure.Thedataindicatethatconsumerswithahighintakeofliquoriceconfection-
eryorherbalteacontainingliquoricemayhaveanintakeofglycyrrhizinicacidof
>100mg/day.
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L1
621
ANNEX 1
REPORTS AND OTHER DOCUMENTS RESULTING FROM PREVIOUS MEETINGS OF
THE JOINT FAO/WHO EXPERT COMMITTEE ON FOOD ADDITIVES
1. General principles governing the use of food additives (First report of the Joint
FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Report
Series,No.15,1957;WHOTechnicalReportSeries,No.129,1957(outofprint).
2. Proceduresforthetestingofintentionalfoodadditivestoestablishtheirsafety
foruse(SecondreportoftheJointFAO/WHOExpertCommitteeonFoodAdditives).
FAONutritionMeetingsReportSeries,No.17,1958;WHOTechnicalReportSeries,
No.144,1958(outofprint).
3. Specifcationsforidentityandpurityoffoodadditives(antimicrobialpreserva-
tives and antioxidants) (Third report of the Joint FAO/WHO Expert Committee on
Food Additives). These specifcations were subsequently revised and published as
Specifcations for identity and purity of food additives, Vol. I. Antimicrobial
preservatives and antioxidants, Rome, Food and Agriculture Organization of the
UnitedNations,1962(outofprint).
4. Specifcations for identity and purity of food additives (food colours) (Fourth
reportoftheJointFAO/WHOExpertCommitteeonFoodAdditives).Thesespecifca-
tionsweresubsequentlyrevisedandpublishedasSpecifcationsforidentityandpurity
of food additives, Vol. II. Food colours, Rome, Food andAgriculture Organization of
theUnitedNations,1963(outofprint).
5. Evaluationofthecarcinogenichazardsoffoodadditives(FifthreportoftheJoint
FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Report
Series,No.29,1961;WHOTechnicalReportSeries,No.220,1961(outofprint).
6. Evaluationofthetoxicityofanumberofantimicrobialsandantioxidants(Sixth
report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition
MeetingsReportSeries,No.31,1962;WHOTechnicalReportSeries,No.228,1962
(outofprint).
7. Specifcationsfortheidentityandpurityoffoodadditivesandtheirtoxicological
evaluation: emulsifers, stabilizers, bleaching and maturing agents (Seventh
report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition
MeetingsSeries,No.35,1964;WHOTechnicalReportSeries,No.281,1964(outof
print).
8. Specifcationsfortheidentityandpurityoffoodadditivesandtheirtoxicological
evaluation:foodcoloursandsomeantimicrobialsandantioxidants(Eighthreport
oftheJointFAO/WHOExpertCommitteeonFoodAdditives).FAONutritionMeetings
Series,No.38,1965;WHOTechnicalReportSeries,No.309,1965(outofprint).
9. Specifcationsforidentityandpurityandtoxicologicalevaluationofsomeanti-
microbialsandantioxidants.FAONutritionMeetingsReportSeries,No.38A,1965;
WHO/FoodAdd/24.65(outofprint).
10. Specifcationsforidentityandpurityandtoxicologicalevaluationoffoodcolours.
FAONutritionMeetingsReportSeries,No.38B,1966;WHO/FoodAdd/66.25.
11. Specifcationsfortheidentityandpurityoffoodadditivesandtheirtoxicological
evaluation: some antimicrobials, antioxidants, emulsifers, stabilizers, four
treatment agents, acids, and bases (Ninth report of the Joint FAO/WHO Expert
Committee on FoodAdditives). FAO Nutrition Meetings Series, No. 40, 1966; WHO
TechnicalReportSeries,No.339,1966(outofprint).
12. Toxicological evaluation of some antimicrobials, antioxidants, emulsifers, sta-
bilizers,fourtreatmentagents,acids,andbases.FAONutritionMeetingsReport
Series,No.40A,B,C;WHO/FoodAdd/67.29.
L1
13. Specifcationsfortheidentityandpurityoffoodadditivesandtheirtoxicological
evaluation:someemulsifersandstabilizersandcertainothersubstances(Tenth
report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition
MeetingsSeries,No.43,1967;WHOTechnicalReportSeries,No.373,1967.
14. Specifcationsfortheidentityandpurityoffoodadditivesandtheirtoxicological
evaluation: some favouring substances and non nutritive sweetening agents
(Eleventh report of the Joint FAO/WHO Expert Committee on FoodAdditives). FAO
Nutrition Meetings Series, No. 44, 1968; WHO Technical Report Series, No. 383,
1968.
15. Toxicologicalevaluationofsomefavouringsubstancesandnonnutritivesweet-
ening agents. FAO Nutrition Meetings Report Series, No. 44A, 1968; WHO/Food
Add/68.33.
16. Specifcationsandcriteriaforidentityandpurityofsomefavouringsubstances
and non-nutritive sweetening agents. FAO Nutrition Meetings Report Series, No.
44B,1969;WHO/FoodAdd/69.31.
17. Specifcationsfortheidentityandpurityoffoodadditivesandtheirtoxicological
evaluation:someantibiotics(TwelfthreportoftheJointFAO/WHOExpertCommit-
teeonFoodAdditives).FAONutritionMeetingsSeries,No.45,1969;WHOTechnical
ReportSeries,No.430,1969.
18. Specifcationsfortheidentityandpurityofsomeantibiotics.FAONutritionMeet-
ingsSeries,No.45A,1969;WHO/FoodAdd/69.34.
19. Specifcationsfortheidentityandpurityoffoodadditivesandtheirtoxicological
evaluation: some food colours, emulsifers, stabilizers, anticaking agents, and
certainothersubstances(ThirteenthreportoftheJointFAO/WHOExpertCommittee
on Food Additives). FAO Nutrition Meetings Series, No. 46, 1970; WHO Technical
ReportSeries,No.445,1970.
20. Toxicologicalevaluationofsomefoodcolours,emulsifers,stabilizers,anticak-
ing agents, and certain other substances. FAO Nutrition Meetings Report Series,
No.46A,1970;WHO/FoodAdd/70.36.
21. Specifcations for the identity and purity of some food colours, emulsifers,
stabilizers, anticaking agents, and certain other food additives. FAO Nutrition
MeetingsReportSeries,No.46B,1970;WHO/FoodAdd/70.37.
22. Evaluation of food additives: specifcations for the identity and purity of food
additives and their toxicological evaluation: some extraction solvents and
certain other substances; and a review of the technological effcacy of some
antimicrobial agents. (Fourteenth report of the Joint FAO/WHO Expert Committee
on Food Additives). FAO Nutrition Meetings Series, No. 48, 1971; WHO Technical
ReportSeries,No.462,1971.
23. Toxicological evaluation of some extraction solvents and certain other sub-
stances. FAO Nutrition Meetings Report Series, No. 48A, 1971; WHO/Food
Add/70.39.
24. Specifcationsfortheidentityandpurityofsomeextractionsolventsandcertain
othersubstances.FAONutritionMeetingsReportSeries,No.48B,1971;WHO/Food
Add/70.40.
25. Areviewofthetechnologicaleffcacyofsomeantimicrobialagents.FAONutrition
MeetingsReportSeries,No.48C,1971;WHO/FoodAdd/70.41.
26. Evaluation of food additives: some enzymes, modifed starches, and certain
othersubstances:Toxicologicalevaluationsandspecifcationsandareviewof
thetechnologicaleffcacyofsomeantioxidants(FifteenthreportoftheJointFAO/
WHOExpertCommitteeonFoodAdditives).FAONutritionMeetingsSeries,No.50,
1972;WHOTechnicalReportSeries,No.488,1972.
622 ANNEX 1
L1
27. Toxicologicalevaluationofsomeenzymes,modifedstarches,andcertainother
substances.FAONutritionMeetingsReportSeries,No.50A,1972;WHOFoodAddi-
tivesSeries,No.1,1972.
28. Specifcations for the identity and purity of some enzymes and certain other
substances.FAONutritionMeetingsReportSeries,No.50B,1972;WHOFoodAddi-
tivesSeries,No.2,1972.
29. Areviewofthetechnologicaleffcacyofsomeantioxidantsandsynergists.FAO
NutritionMeetingsReportSeries,No.50C,1972;WHOFoodAdditivesSeries,No.3,
1972.
30. Evaluation of certain food additives and the contaminants mercury, lead, and
cadmium(SixteenthreportoftheJointFAO/WHOExpertCommitteeonFoodAddi-
tives). FAO Nutrition Meetings Series, No. 51, 1972; WHO Technical Report Series,
No.505,1972,andcorrigendum.
31. Evaluationofmercury,lead,cadmiumandthefoodadditivesamaranth,diethyl-
pyrocarbamate,andoctylgallate.FAONutritionMeetingsReportSeries,No.51A,
1972;WHOFoodAdditivesSeries,No.4,1972.
32. Toxicologicalevaluationofcertainfoodadditiveswithareviewofgeneralprin-
ciples and of specifcations (Seventeenth report of the Joint FAO/WHO Expert
Committee on FoodAdditives). FAO Nutrition Meetings Series, No. 53, 1974; WHO
TechnicalReportSeries,No.539,1974,andcorrigendum(outofprint).
33. Toxicological evaluation of some food additives including anticaking agents,
antimicrobials, antioxidants, emulsifers, and thickening agents. FAO Nutrition
MeetingsReportSeries,No.53A,1974;WHOFoodAdditivesSeries,No.5,1974.
34. Specifcations for identity and purity of thickening agents, anticaking agents,
antimicrobials, antioxidants and emulsifers. FAO Food and Nutrition Paper,
No.4,1978.
35. Evaluationofcertainfoodadditives(EighteenthreportoftheJointFAO/WHOExpert
Committee on FoodAdditives). FAO Nutrition Meetings Series, No. 54, 1974; WHO
TechnicalReportSeries,No.557,1974,andcorrigendum.
36. Toxicological evaluation of some food colours, enzymes, favour enhancers,
thickeningagents,andcertainotherfoodadditives.FAONutritionMeetingsReport
Series,No.54A,1975;WHOFoodAdditivesSeries,No.6,1975.
37. Specifcations for the identity and purity of some food colours, enhancers,
thickening agents, and certain food additives. FAO Nutrition Meetings Report
Series,No.54B,1975;WHOFoodAdditivesSeries,No.7,1975.
38. Evaluation of certain food additives: some food colours, thickening agents,
smokecondensates,andcertainothersubstances.(NineteenthreportoftheJoint
FAO/WHOExpertCommitteeonFoodAdditives).FAONutritionMeetingsSeries,No.
55,1975;WHOTechnicalReportSeries,No.576,1975.
39. Toxicological evaluation of some food colours, thickening agents, and certain
othersubstances.FAONutritionMeetingsReportSeries,No.55A,1975;WHOFood
AdditivesSeries,No.8,1975.
40. Specifcationsfortheidentityandpurityofcertainfoodadditives.FAONutrition
MeetingsReportSeries,No.55B,1976;WHOFoodAdditivesSeries,No.9,1976.
41. Evaluationofcertainfoodadditives(TwentiethreportoftheJointFAO/WHOExpert
CommitteeonFoodAdditives).FAOFoodandNutritionMeetingsSeries,No.1,1976;
WHOTechnicalReportSeries,No.599,1976.
42. Toxicological evaluation of certain food additives. WHO Food Additives Series,
No.10,1976.
43. Specifcationsfortheidentityandpurityofsomefoodadditives.FAOFoodand
NutritionSeries,No.1B,1977;WHOFoodAdditivesSeries,No.11,1977.
ANNEX 1 623
L1
44. Evaluation of certain food additives (Twenty-frst report of the Joint FAO/WHO
Expert Committee on Food Additives). WHO Technical Report Series, No. 617,
1978.
45. Summary of toxicological data of certain food additives. WHO Food Additives
Series,No.12,1977.
46. Specifcationsforidentityandpurityofsomefoodadditives,includingantioxi-
dant,foodcolours,thickeners,andothers.FAONutritionMeetingsReportSeries,
No.57,1977.
47. Evaluation of certain food additives and contaminants (Twenty-second report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report
Series,No.631,1978.
48. Summary of toxicological data of certain food additives and contaminants.
WHOFoodAdditivesSeries,No.13,1978.
49. Specifcationsfortheidentityandpurityofcertainfoodadditives.FAOFoodand
NutritionPaper,No.7,1978.
50. Evaluation of certain food additives (Twenty-third report of the Joint FAO/WHO
ExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,No.648,1980,
andcorrigenda.
51. Toxicological evaluation of certain food additives. WHO Food Additives Series,
No.14,1980.
52. Specifcations for identity and purity of food colours, favouring agents, and
otherfoodadditives.FAOFoodandNutritionPaper,No.12,1979.
53. Evaluation of certain food additives (Twenty-fourth report of the Joint FAO/WHO
Expert Committee on Food Additives). WHO Technical Report Series, No. 653,
1980.
54. Toxicological evaluation of certain food additives. WHO Food Additives Series,
No.15,1980.
55. Specifcations for identity and purity of food additives (sweetening agents,
emulsifying agents, and other food additives). FAO Food and Nutrition Paper,
No.17,1980.
56. Evaluation of certain food additives (Twenty-ffth report of the Joint FAO/WHO
Expert Committee on Food Additives). WHO Technical Report Series, No. 669,
1981.
57. Toxicological evaluation of certain food additives. WHO Food Additives Series,
No.16,1981.
58. Specifcationsforidentityandpurityoffoodadditives(carriersolvents,emulsi-
fers and stabilizers, enzyme preparations, favouring agents, food colours,
sweeteningagents,andotherfoodadditives).FAOFoodandNutritionPaper,No.
19,1981.
59. Evaluationofcertainfoodadditivesandcontaminants(Twenty-sixthreportofthe
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.683,1982.
60. Toxicological evaluation of certain food additives. WHO Food Additives Series,
No.17,1982.
61. Specifcationsfortheidentityandpurityofcertainfoodadditives.FAOFoodand
NutritionPaper,No.25,1982.
62. Evaluationofcertainfoodadditivesandcontaminants(Twenty-seventhreportof
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report
Series,No.696,1983,andcorrigenda.
63. Toxicologicalevaluationofcertainfoodadditivesandcontaminants.WHOFood
AdditivesSeries,No.18,1983.
64. Specifcationsfortheidentityandpurityofcertainfoodadditives.FAOFoodand
NutritionPaper,No.28,1983.
624 ANNEX 1
L1
65. Guide to specifcations, general notices, general methods, identifcation tests,
test solutions, and other reference materials. FAO Food and Nutrition Paper,
No.5,Rev.1,1983.
66. Evaluation of certain food additives and contaminants (Twenty-eighth report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report
Series,No.710,1984,andcorrigendum.
67. Toxicologicalevaluationofcertainfoodadditivesandcontaminants.WHOFood
AdditivesSeries,No.19,1984.
68. Specifcationsfortheidentityandpurityoffoodcolours.FAOFoodandNutrition
Paper,No.31/1,1984.
69. Specifcationsfortheidentityandpurityoffoodadditives.FAOFoodandNutrition
Paper,No.31/2,1984.
70. Evaluationofcertainfoodadditivesandcontaminants(Twenty-ninthreportofthe
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.733,1986,andcorrigendum.
71. Specifcationsfortheidentityandpurityofcertainfoodadditives.FAOFoodand
NutritionPaper,No.34,1986.
72. Toxicologicalevaluationofcertainfoodadditivesandcontaminants.WHOFood
AdditivesSeries,No.20.CambridgeUniversityPress,1987.
73. Evaluationofcertainfoodadditivesandcontaminants(ThirtiethreportoftheJoint
FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series,
No.751,1987.
74. Toxicologicalevaluationofcertainfoodadditivesandcontaminants.WHOFood
AdditivesSeries,No.21.CambridgeUniversityPress,1987.
75. Specifcationsfortheidentityandpurityofcertainfoodadditives.FAOFoodand
NutritionPaper,No.37,1986.
76. Principlesforthesafetyassessmentoffoodadditivesandcontaminantsinfood.
WHOEnvironmentalHealthCriteria,No.70.Geneva,WorldHealthOrganization,1987
(outofprint).Thefulltextisavailableelectronicallyatwww.who.int/pcs.
77. Evaluation of certain food additives and contaminants (Thirty-frst report of the
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.759,1987andcorrigendum.
78. Toxicological evaluation of certain food additives. WHO Food Additives Series,
No.22.CambridgeUniversityPress,1988.
79. Specifcationsfortheidentityandpurityofcertainfoodadditives.FAOFoodand
NutritionPaper,No.38,1988.
80. Evaluationofcertainveterinarydrugresiduesinfood(Thirty-secondreportofthe
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.763,1988.
81. Toxicologicalevaluationofcertainveterinarydrugresiduesinfood.WHOFood
AdditivesSeries,No.23.CambridgeUniversityPress,1988.
82. Residuesofsomeveterinarydrugsinanimalsandfoods.FAOFoodandNutrition
Paper,No.41,1988.
83. Evaluation of certain food additives and contaminants (Thirty-third report of the
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.776,1989.
84. Toxicologicalevaluationofcertainfoodadditivesandcontaminants.WHOFood
AdditivesSeries,No.24.CambridgeUniversityPress,1989.
85. Evaluationofcertainveterinarydrugresiduesinfood(Thirty-fourthreportofthe
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.788,1989.
86. Toxicologicalevaluationofcertainveterinarydrugresiduesinfood.WHOFood
AdditivesSeries,No.25,1990.
ANNEX 1 625
L1
87. Residuesofsomeveterinarydrugsinanimalsandfoods.FAOFoodandNutrition
Paper,No.41/2,1990.
88. Evaluation of certain food additives and contaminants (Thirty-ffth report of the
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.789,1990,andcorrigenda.
89. Toxicologicalevaluationofcertainfoodadditivesandcontaminants.WHOFood
AdditivesSeries,No.26,1990.
90. Specifcations for identity and purity of certain food additives. FAO Food and
NutritionPaper,No.49,1990.
91. Evaluation of certain veterinary drug residues in food (Thirty-sixth report of the
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.799,1990.
92. Toxicologicalevaluationofcertainveterinarydrugresiduesinfood.WHOFood
AdditivesSeries,No.27,1991.
93. Residuesofsomeveterinarydrugsinanimalsandfoods.FAOFoodandNutrition
Paper,No.41/3,1991.
94. Evaluation of certain food additives and contaminants (Thirty-seventh report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report
Series,No.806,1991,andcorrigenda.
95. Toxicologicalevaluationofcertainfoodadditivesandcontaminants.WHOFood
AdditivesSeries,No.28,1991.
96. Compendium of food additive specifcations (Joint FAO/WHO Expert Committee
onFoodAdditives(JECFA)).Combinedspecifcationsfrom1stthroughthe37thmeet-
ings, 19561990. Rome, Food and Agricultural Organization of the United Nations,
1992(2volumes).
97. Evaluationofcertainveterinarydrugresiduesinfood(Thirty-eighthreportofthe
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.815,1991.
98. Toxicologicalevaluationofcertainveterinaryresiduesinfood.WHOFoodAddi-
tivesSeries,No.29,1991.
99. Residuesofsomeveterinarydrugsinanimalsandfoods.FAOFoodandNutrition
Paper,No.41/4,1991.
100. GuidetospecifcationsGeneralnotices,generalanalyticaltechniques,iden-
tifcation tests, test solutions, and other reference materials. FAO Food and
NutritionPaper,No.5,Ref.2,1991.
101. Evaluation of certain food additives and naturally occurring toxicants (Thirty-
ninth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO
TechnicalReportSeriesNo.828,1992.
102. Toxicological evaluation of certain food additives and naturally occurring toxi-
cants.WHOFoodAdditiveSeries,No.30,1993.
103. Compendiumoffoodadditivespecifcations:addendum1.FAOFoodandNutri-
tionPaper,No.52,1992.
104. Evaluationofcertainveterinarydrugresiduesinfood(FortiethreportoftheJoint
FAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,No.
832,1993.
105. Toxicologicalevaluationofcertainveterinarydrugresiduesinfood.WHOFood
AdditivesSeries,No.31,1993.
106. Residuesofsomeveterinarydrugsinanimalsandfood.FAOFoodandNutrition
Paper,No.41/5,1993.
107. Evaluation of certain food additives and contaminants (Forty-frst report of the
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.837,1993.
626 ANNEX 1
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108. Toxicologicalevaluationofcertainfoodadditivesandcontaminants.WHOFood
AdditivesSeries,No.32,1993.
109. Compendiumoffoodadditivespecifcations:addendum2.FAOFoodandNutri-
tionPaper,No.52,Add.2,1993.
110. Evaluationofcertainveterinarydrugresiduesinfood(Forty-secondreportofthe
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.851,1995.
111. Toxicologicalevaluationofcertainveterinarydrugresiduesinfood.WHOFood
AdditivesSeries,No.33,1994.
112. Residuesofsomeveterinarydrugsinanimalsandfoods.FAOFoodandNutrition
Paper,No.41/6,1994.
113. Evaluation of certain veterinary drug residues in food (Forty-third report of the
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.855,1995,andcorrigendum.
114. Toxicologicalevaluationofcertainveterinarydrugresiduesinfood.WHOFood
AdditivesSeries,No.34,1995.
115. Residuesofsomeveterinarydrugsinanimalsandfoods.FAOFoodandNutrition
Paper,No.41/7,1995.
116. Evaluationofcertainfoodadditivesandcontaminants(Forty-fourthreportofthe
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.859,1995.
117. Toxicologicalevaluationofcertainfoodadditivesandcontaminants.WHOFood
AdditivesSeries,No.35,1996.
118. Compendiumoffoodadditivespecifcations:addendum3.FAOFoodandNutri-
tionPaper,No.52,Add.3,1995.
119. Evaluationofcertainveterinarydrugresiduesinfood(Forty-ffthreportoftheJoint
FAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,No.
864,1996.
120. Toxicologicalevaluationofcertainveterinarydrugresiduesinfood.WHOFood
AdditivesSeries,No.36,1996.
121. Residuesofsomeveterinarydrugsinanimalsandfoods.FAOFoodandNutrition
Paper,No.41/8,1996.
122. Evaluation of certain food additives and contaminants (Forty-sixth report of the
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.868,1997.
123. Toxicological evaluation of certain food additives. WHO Food Additives Series,
No.37,1996.
124. Compendiumoffoodadditivespecifcations:addendum4.FAOFoodandNutri-
tionPaper,No.52,Add.4,1996.
125. Evaluationofcertainveterinarydrugresiduesinfood(Forty-seventhreportofthe
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.876,1998.
126. Toxicologicalevaluationofcertainveterinarydrugresiduesinfood.WHOFood
AdditivesSeries,No.38,1996.
127. Residuesofsomeveterinarydrugsinanimalsandfoods.FAOFoodandNutrition
Paper,No.41/9,1997.
128. Evaluationofcertainveterinarydrugresiduesinfood(Forty-eighthreportofthe
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.879,1998.
129. Toxicologicalevaluationofcertainveterinarydrugresiduesinfood.WHOFood
AdditivesSeries,No.39,1997.
130. Residuesofsomeveterinarydrugsinanimalsandfoods.FAOFoodandNutrition
Paper,No.41/10,1998.
ANNEX 1 627
L1
131. Evaluation of certain food additives and contaminants (Forty-ninth report of the
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.884,1999.
132. Safety evaluation of certain food additives and contaminants. WHO FoodAddi-
tivesSeries,No.40,1998.
133. Compendiumoffoodadditivespecifcations:addendum5.FAOFoodandNutri-
tionPaper,No.52,Add.5,1997.
134. Evaluationofcertainveterinarydrugresiduesinfood(FiftiethreportoftheJoint
FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series,
No.888,1999.
135. Toxicologicalevaluationofcertainveterinarydrugresiduesinfood.WHOFood
AdditivesSeries,No.41,1998.
136. Residuesofsomeveterinarydrugsinanimalsandfoods.FAOFoodandNutrition
Paper,No.41/11,1999.
137. Evaluationofcertainfoodadditives(Fifty-frstreportoftheJointFAO/WHOExpert
CommitteeonFoodAdditives).WHOTechnicalReportSeries,No.891,2000.
138. Safety evaluation of certain food additives. WHO FoodAdditives Series, No. 42,
1999.
139. Compendiumoffoodadditivespecifcations:addendum6.FAOFoodandNutri-
tionPaper,No.52,Add.6,1998.
140. Evaluationofcertainveterinarydrugresiduesinfood(Fifty-secondreportofthe
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.893,2000.
141. Toxicologicalevaluationofcertainveterinarydrugresiduesinfood.WHOFood
AdditivesSeries,No.43,2000.
142. Residuesofsomeveterinarydrugsinanimalsandfoods.FAOFoodandNutrition
Paper,No.41/12,2000.
143. Evaluation of certain food additives and contaminants (Fifty-third report of the
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.896,2000.
144. Safety evaluation of certain food additives and contaminants. WHO FoodAddi-
tivesSeries,No.44,2000.
145. Compendiumoffoodadditivespecifcations:addendum7.FAOFoodandNutri-
tionPaper,No.52,Add.7,1999.
146. Evaluation of certain veterinary drug residues in food (Fifty-fourth report of the
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.900,2001
147. Toxicologicalevaluationofcertainveterinarydrugresiduesinfood.WHOFood
AdditivesSeries,No.45,2000.
148. Residuesofsomeveterinarydrugsinanimalsandfoods.FAOFoodandNutrition
Paper,No.41/13,2000.
149. Evaluationofcertainfoodadditivesandcontaminants(Fifty-ffthreportoftheJoint
FAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeriesNo.
901,2001.
150. Safety evaluation of certain food additives and contaminants. WHO FoodAddi-
tivesSeries,No.46,2001.
151. Compendiumoffoodadditivespecifcations:addendum8.FAOFoodandNutri-
tionPaper,No.52,Add.8,2000.
152. Evaluationofcertainmycotoxinsinfood(Fifty-sixthreportoftheJointFAO/WHO
ExpertCommitteeonFoodAdditives).WHOTechnicalReportSeriesNo.906,2002.
153. Safety evaluation of certain mycotoxins in food. WHO Food Additives Series,
No.47/FAOFoodandNutritionPaper74,2001.
628 ANNEX 1
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154. Evaluationofcertainfoodadditivesandcontaminants(Fifty-seventhreportofthe
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.909,2002.
155. Safety evaluation of certain food additives and contaminants. WHO FoodAddi-
tivesSeries,No.48,2002.
156. Compendiumoffoodadditivespecifcations:addendum9.FAOFoodandNutri-
tionPaper,No.52,Add.9,2001.
157. Evaluation of certain veterinary drug residues in food (Fifty-eighth report of the
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.911,2002.
158. Toxicologicalevaluationofcertainveterinarydrugresiduesinfood.WHOFood
AdditivesSeries,No.49,2002.
159. Residuesofsomeveterinarydrugsinanimalsandfoods.FAOFoodandNutrition
Paper,No.41/14,2002.
160. Evaluation of certain food additives and contaminants (Fifty-ninth report of the
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.913,2002.
161. Safety evaluation of certain food additives and contaminants. WHO FoodAddi-
tivesSeries,No.50,2003.
162. Compendiumoffoodadditivespecifcations:addendum10.FAOFoodandNutri-
tionPaperNo.52,Add.10,2002.
163. Evaluationofcertainveterinarydrugresiduesinfood(SixtiethreportoftheJoint
FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series,
No.918,2003.
164. Toxicologicalevaluationofcertainveterinarydrugresiduesinfood.WHOFood
AdditivesSeries,No.51,2003.
165. Residuesofsomeveterinarydrugsinanimalsandfoods.FAOFoodandNutrition
Paper,No.41/15,2003.
166. Evaluationofcertainfoodadditivesandcontaminants(Sixty-frstreportoftheJoint
FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series,
No.922,2004.
167. Safety evaluation of certain food additives and contaminants. WHO FoodAddi-
tivesSeries,No.52,2004.
168. Compendiumoffoodadditivespecifcations:addendum11.FAOFoodandNutri-
tionPaper,No.52,Add.11,2003.
169. Evaluationofcertainveterinarydrugresiduesinfood(Sixty-secondreportofthe
JointFAO/WHOExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,
No.925,2004.
170. Residuesofsomeveterinarydrugsinanimalsandfoods.FAOFoodandNutrition
Paper,No.41/16,2004.
171. Toxicologicalevaluationofcertainveterinarydrugresiduesinfood.WHOFood
AdditivesSeries,No.53,2005.
172. Compendiumoffoodadditivespecifcations:addendum12.FAOFoodandNutri-
tionPaper,No.52,Add.12,2004.
173. Evaluation of certain food additives (Sixty-third report of the Joint FAO/WHO
Expert Committee on Food Additives). WHO Technical Report Series, No. 928,
2005.
174. Evaluationofcertainfoodcontaminants(Sixty-fourthreportoftheJointFAO/WHO
ExpertCommitteeonFoodAdditives).WHOTechnicalReportSeries,No.930,2005
(inprint).
ANNEX 1 629
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ANNEX 2
ABBREVIATIONS USED IN THE MONOGRAPHS
ADH alcoholdehydrogenase
ADI acceptabledailyintake
ALDH aldehydedehydrogenase
ATP adenosinetriphosphate
AUC areaunderthecurve
bw bodyweight
cDNA complementaryDNA
C
max
maximumplasmaconcentration
CI confdenceinterval
coA coenzymeA
CYP cytochromeP450
DEN diethylnitrosamine
DMBA 7,12-dimethylbenz[a]anthracene
DMSO dimethylsulfoxide
DNA deoxyribonucleicacid
ECG electrocardiogram
ELISA enzyme-linkedimmunosorbentassay
F female
FAO FoodandAgricultureOrganizationoftheUnitedNations
GEMS/Food Global Environment Monitoring System Food Contamination
MonitoringandAssessmentProgramme
GLP goodlaboratorypractice
GSH glutathione
GST glutathioneS-transferase
HDL high-densitylipoprotein
1
HNMR Protonnuclearmagneticresonancespectroscopy
IDL intermediate-densitylipoprotein
Ig immunoglobulin
IPCS InternationalProgrammeonChemicalSafety
LD
50
medianlethaldose
LDL low-densitylipoprotein
L1
631
L1
M male
MTD maximumtolerateddose
mRNA messengerRNA
NAD nicotinamideadeninedinucleotide
NADH nicotinamideadeninedinucleotide,reduced
NADPH nicotinamideadeninedinucleotidephosphate,reduced
NCE normochromaticerythrocyte
NOEL no-observed-effectlevel
NSAID nonsteroidalanti-infammatorydrug
PARP poly-ADP-ribosepolymerase
PCE polychromaticerythrocyte
QA qualityassurance
S9 9000gmicrosomalfractionofratliver
SCE sisterchromatidexchange
t
1/2
half-life
TOS totalorganicsolids
TPA 12-O-tetradecanoylphorbol-13-acetate
UDPGT UDP-glucuronosyltransferase
UK UnitedKingdom
USA UnitedStatesofAmerica
VLDL very-low-densitylipoprotein
V
max
maximalvelocity
WHO WorldHealthOrganization
w/w weightforweight
632 ANNEX 2
L1
ANNEX 3
JOINT FAO/WHO EXPERT COMMITTEE ON FOOD ADDITIVES
GENEVA, 817 JUNE 2004
Members
Professor J.R. Bend, Professor and Chair, Department of Pharmacology & Toxicology,
Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario,
Canada
DrD.G.Hattan,SeniorToxicologist,OffceofFoodAdditivesSafety,CenterforFoodSafety
andAppliedNutrition,FoodandDrugAdministration,CollegePark,MD,USA
DrY.Kawamura,NationalInstituteofHealthSciences,Setagaya,Tokyo,Japan
DrA.G.A.C.Knaap,CenterforSubstancesandRiskAssessment,NationalInstituteofPublic
HealthandtheEnvironment(RIVM),Bilthoven,TheNetherlands
DrP.M.Kuznesof,OffceofFoodAdditiveSafety,CenterforFoodSafetyandAppliedNutri-
tion,FoodandDrugAdministration,CollegePark,MD,USA
Dr J.C. Larsen, Senior Consultant, Division of Toxicology and Risk Assessment, Danish
InstituteofFoodandVeterinaryResearch,Sborg,Denmark
Mrs I. Meyland, Senior ScientifcAdviser, Danish Food and Veterinary Research, Mrkhj,
Sborg,Denmark
Dr M.V. Rao, Director, Central Laboratories Unit, United Arab Emirates University, Al Ain,
UnitedArabEmirates
Dr J. Schlatter, Food Toxicology Section, Swiss Federal Offce of Public Health, Zurich,
Switzerland
DrM.C.deFigueiredoToledo,ProfessorofFoodToxicology,StateUniversityofCampinas,
FacultyofFoodEngineeringUnicamp,CampinasSP,Brazil
MrsE.Vavasour,FoodDirectorate,HealthCanada,Ottawa,Ontario,Canada(Rapporteur)
DrP.Verger,UnitDirectorINRA1204,Foodriskanalysismethodologies,NationalInstitute
forAgriculturalResearch/NationalInstituteforAgricultureParis-Grignon,Paris,France
Professor R. Walker, Emeritus Professor of Food Science, School of Biomedical and Life
Sciences,UniversityofSurrey,Guildford,Surrey,England
Secretariat
Dr P.J.Abbott, Food StandardsAustralia New Zealand (FSANZ), Canberra,ACT,Australia
(WHO Temporary Adviser)
Dr M.C. Archer, Department of Nutritional Sciences, Faculty of Medicine, University of
Toronto,Toronto,Canada(WHO Temporary Adviser)
DrMa.P.V.Azanza,DepartmentofFoodScienceandNutrition,CollegeofHomeEconomics,
UPDiliman,QuezonCity,Philippines(FAO Consultant)
DrD.Benford,FoodStandardsAgency,London,England(WHO Temporary Adviser)
633
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Dr R. Cantrill, American Oil Chemists Society (AOCS), Champaign, IL, USA (FAO
Consultant)
MsM.L.Costarrica,FoodQualityLiaisonGroup,FoodQualityandStandardsService,Food
and Nutrition Division, Food andAgriculture Organization of the United Nations (FAO),
Rome,Italy(FAO Staff Member)
Dr M. Das, Food Toxicology Laboratory, Industrial Toxicology Research Centre, Mahatma
GandhiMarg,Lucknow,India(WHO Temporary Adviser)
DrM.DiNovi,OffceofFoodAdditiveSafety,CenterforFoodSafetyandAppliedNutrition,
FoodandDrugAdministration,CollegePark,MD,USA(WHO Temporary Adviser)
ProfessorY.El-Samragy,FoodScienceDepartment,AinShamsUniversity,HeliopoilsWest,
Cairo,Egypt(FAO Consultant)
Dr A.B. Hanley, Leatherhead Food International, Leatherhead, Surrey, England (FAO
Consultant)
ProfessorH.Ishiwata,SeitokuUniversity,Chiba,Japan(FAO Consultant)
ProfessorF.Kayama,DivisionofEnvironmentalMedicine,CenterforCommunityMedicine,
JichiMedicalSchool,Tochigi,Japan(WHO Temporary Adviser)
Professor R. Kroes, Institute for Risk Assessment Sciences, Utrecht University, Soest,
Netherlands(WHO Temporary Adviser)
DrS.Lawrie,FoodStandardsAgency,London,England(FAO Consultant)
DrC.Leclercq,NationalResearchInstituteforFoodandNutrition(INRAN),Rome,Italy(FAO
Consultant)
DrM.Luetzow,FoodandAgricultureOrganizationoftheUnitedNations,Rome,Italy(FAO
Joint Secretary)
DrA. Mattia, Division of Biotechnology and GRAS Notice Review, Offce of FoodAdditive
Safety, Center for Food Safety and Applied Nutrition, Food and Drug Administration,
CollegePark,MD,USA(WHO Temporary Adviser)
DrG.Moy,FoodSafetyProgramme,WorldHealthOrganization,Geneva,Switzerland(WHO
Staff Member)
DrI.C.Munro,CanToxHealthSciencesInternational,Mississauga,Ontario,Canada(WHO
Temporary Adviser)
DrA.Nishikawa,SectionChief,DivisionofPathology,NationalInstituteofHealthSciences,
Kamiyoga,Setagaya-ku,Tokyo,Japan(WHO Temporary Adviser)
Dr Z. Olempska-Beer, Food and DrugAdministration, Center for Food Safety andApplied
Nutrition,OffceofFoodAdditiveSafety,CollegePark,MD,USA(FAO Consultant)
Dr S. Page, International Programme on Chemical Safety, World Health Organization,
Geneva,Switzerland(WHO Staff Member)
MrsM.E.J.Pronk,CenterforSubstancesandRiskAssessment,NationalInstituteofPublic
HealthandtheEnvironment(RIVM),Bilthoven,Netherlands(WHO Temporary Adviser)
Professor A.G. Renwick, Clinical Pharmacology Group, University of Southampton,
Southampton,England(WHO Temporary Adviser)
DrS.K.Saxena,Delhi,India(FAO Consultant)
Professor I.G. Sipes, Professor and Head, Department of Pharmacology, College of Medi-
cine,UniversityofArizona,Tucson,AZ,USA(WHO Temporary Adviser)
DrJ.Smith, PrinceEdwardIslandFoodTechnologyCentre,Charlottetown,PE,Canada(FAO
Consultant)
634 ANNEX 3
L1
ProfessorI.Stankovic,InstituteofBromatology,FacultyofPharmaacy,Belgrade(Kumodraz),
SerbiaandMontenegro(FAO Consultant)
DrA.Tritscher, WHO Joint Secretary, International Programme on Chemical Safety, World
HealthOrganization,Geneva,Switzerland(WHO Joint Secretary)
MsA.deVeer,DeputyDirectoroftheDepartmentofFoodandVeterinaryAffairs,Chairman
of the Codex Committee on FoodAdditives and Contaminants, Ministry ofAgriculture,
Nature Management and Fisheries, The Hague, The Netherlands (WHO Temporary
Adviser)
MrsH.Wallin,NationalFoodAgency,Helsinki,Finland(FAO Consultant)
DrD.B.Whitehouse,Consultant,Bowdon,Cheshire,England(FAO Consultant)
Professor G. Williams, Professor of Pathology and Director, Environmental Pathology and
Toxicology,NewYorkMedicalCollege,Valhalla,NY,USA(WHO Temporary Adviser)
ANNEX 3 635
L1
ANNEX 4
ACCEPTABLE DAILY INTAKES AND OTHER TOXICOLOGICAL INFORMATION,
AND INFORMATION ON SPECIFICATIONS
Food additives and ingredients evaluated toxicologically
Foodadditive Specifcations
a
Acceptabledailyintake(ADI)inmg/kgbwand
othertoxicologicalrecommendations
Benzoylperoxide R Treatmentofwheywithbenzoylperoxideata
maximumconcentrationof100mg/kgdoes
notposeasafetyconcern
a-Cyclodextrin a-Cyclodextrindoesnotposeasafety
concernattheproposeduselevelsand
resultingpredictedconsumptionasfood
ingredientandfoodadditive
ThepreviouslyestablishedADInotspecifed
foruseasacarrierandstabilizerfor
favours,colours,andsweeteners,asa
water-solubilizerforfattyacidsandcertain
vitamins,asafavourmodiferinsoyamilk,
andasanabsorbentinconfectionerywas
maintained
Hexoseoxidase N Notspecifed
b
fromChondrus
crispus expressed
inHansenula
polymorpha
Luteinfrom N 02(groupADIforluteinandzeaxanthin)
c
Tagetes erecta L.
Peroxyacid Theperoxycompoundsinthesesolutions
antimicrobial (hydrogenperoxide,peroxyaceticacidand
solutions peroxyoctanoicacid)wouldbreakdowninto
containing1- aceticacidandoctanoicacid,andsmall
hydroxyethylidene- residualquantitiesoftheseacidsonfoods
1,1-diphosphonic atthetimeofconsumptionwouldnotpose
acid(HEDP) asafetyconcern.HEDPdoesnotposea
safetyconcernatthelevelsofresiduethat
Containing HEDP areexpectedtoremainonfoodsatthetime
and three or more consumption.
of the following
components:
peroxacetic acid,
acetic acid,
hydrogen peroxide,
octanoic acid and
peroxyoctanoic
acid
L1
637
638 ANNEX 4
L1
Food additives and ingredients evaluated toxicologically(Contd)
Foodadditive Specifcations
a
Acceptabledailyintake(ADI)inmg/kgbwand
othertoxicologicalrecommendations
Aceticacid R
1-Hydroxyethylidene- N
1,1-diphosphonic
acid(HEDP)
Hydrogenperoxide R
Octanoicacid(as N
foodadditive)
Steviolglycosides N,T 02(temporary)
d-Tagatose Notspecifed
b
Xylanasefrom N Notspecifed
b
Bacillus subtilis
expressedin
Bacillus subtilis
Xylanase(resistant N Notspecifed
b
toxylanase
inhibitor)from
Bacillus subtilis
containinga
modifedxylanase
genefrom
Bacillus subtilis
Zeaxanthin N 02(groupADIforluteinandzeaxanthin)
c
a
N,newspecifcationsprepared;R,existingspecifcationsrevised;T,tentative
specifcations.
b
ADInotspecifedisusedtorefertoafoodsubstanceofverylowtoxicitywhich,onthe
basisoftheavailabledata(chemical,biochemical,toxicologicalandother)andthetotal
dietaryintakeofthesubstancearisingfromitsuseatthelevelsnecessarytoachieve
thedesiredeffectsandfromitsacceptablebackgroundlevelsinfood,doesnot,inthe
opinionoftheCommittee,representahazardtohealth.Forthatreason,andforthe
reasonsstatedintheindividualevaluations,theestablishmentofanADIexpressedin
numericalformisnotdeemednecessary.Anadditivemeetingthiscriterionmustbeused
withintheboundsofgoodmanufacturingpractice,i.e.itshouldbetechnologically
effcaciousandshouldbeusedatthelowestlevelnecessarytoachievethiseffect,it
shouldnotconcealfoodofinferiorqualityoradulteratedfood,anditshouldnotcreatea
nutritionalimbalance.
c
ThisgroupADIdoesnotapplytootherxanthophyll-containingextractswithaluteinor
zeaxanthincontentlowerthanthatcitedinthespecifcations.
ANNEX 4 639
L1
Food additives considered for specifcations only
Foodadditive Specifcations
a
Aluminiumlakesofcolouringmattersgeneralspecifcations R
Aluminiumpowder R
Hydroxypropylcellulose R
Hydroxypropylmethylcellulose R
Ironoxides R
Magnesiumsulfate
b
N,T
Polyvinylalcohol R
Titaniumdioxide R
Zeaxanthin-richextractfromTagetes erectaL. N,T
a
R,existingspecifcationsrevised;R,existingspecifcationsrevised;T,tentative
specifcations.
b
Magnesiumsulfatewasnotevaluatedatthepresentmeetingbecausetheintendeduse
anduselevelswerenotidentifed.
Revision of heavy metals limits for food additives
INS Foodadditive Limits,notmorethan
(mg/kg)
As Pb Cd Hg
523 Aluminiumammoniumsulfate 3
510 Ammoniumchloride 2
503 (ii) Ammoniumhydrogencarbonate 2
927 a Azodicarbonamide 2
901 Beeswax 2
210 Benzoicacid 2
Benzylalcohol 2
Butan-1,3-diol 2
Butan-1-ol 2
Butan-2-ol 2
Butylp-hydroxybenzoate 2
263 Calciumacetate 2
213 Calciumbenzoate 2
170 Calciumcarbonate 3 3
509 Calciumchloride 2
952 Calciumcyclamate 1
341 (ii) Calciumhydrogenphosphate 3 4
516 Calciumsulfate 2
902 Candelillawax 2
1503 Castoroil 2
925 Chlorine 2 1
Citraxanthin 2
459 Cyclodextrin,b- 1
Cyclohexane 2
Dammargum 2
Diethyltartrate 2
Diethyleneglycolmonoethylether 2
242 Dimethyldicarbonate 2
640 ANNEX 4
L1
Revision of heavy metals limits for food additives (Contd)
INS Foodadditive Limits,notmorethan
(mg/kg)
As Pb Cd Hg
Diphenyl 2
Ediblegelatin 1 1.5 0.5 0.15
Ferricammoniumcitrate 2
422 Glycerol 2
Glyceroldiacetate 2
Heptanes 2
239 Hexamethylenetetramine 2
Isoamylacetate 2
Isobutanol 2
Isopropylacetate 2
270 Lacticacid 2
Lightpetroleum 2
1105 Lysozymehydrochloride 2
504 (i) Magnesiumcarbonate 2
511 Magnesiumchloride 2
343 (ii) Magnesiumhydrogenphosphate 3 4
329 Magnesiumlactate 2
Methanol 2
905 Mineraloil(highviscosity) 1
Monoglyceridecitrate 2
234 Nisin 1
Norhydroguaiareticacid 2
451 (ii) Pentapotassiumtriphosphate 3 4
231 Phenylphenol,o- 2
1202 Polyvinylpolypyrrolidone,insoluble 2
1201 Polyvinylpyrrolidone 2
261 Potassiumacetate 2
212 Potassiumbenzoate 2
924 a Potassiumbromate 2
508 Potassiumchloride 2
501 (ii) Potassiumdihydrogenphosphate 3 4
917 Potassiumiodate 2
252 Potassiumnitrate 2
249 Potassiumnitrite 2
337 PotassiumsodiumL(+)tartrate 2
515 (i) Potassiumsulfate 2
Propan-1-ol 2
1520 Propyleneglycol 2
211 Sodiumbenzoate 2
466 Sodiumcarboxymethylcellulose 2
952 Sodiumcyclamate 1
262 (ii) Sodiumdiacetate 2
251 Sodiumnitrate 2
250 Sodiumnitrite 2
232 Sodiumo-phenylphenol 2
Sodiumpercarbonate 2
Sodiumthiocyanate 2
200 Sorbicacid 2
ANNEX 4 641
L1
Revision of heavy metals limits for food additives (Contd)
INS Foodadditive Limits,notmorethan
(mg/kg)
As Pb Cd Hg
955 Sucralose 1
181 Tannicacid 2
Tartaricacid,DL- 2
Toluene 2
1518 Triacetin 2
Trichlorotrifuoroethane,1,1,2- 2
927 b Urea 2
Flavouring agents evaluated by the Procedure for the Safety Evaluation of
Flavouring Agents
A. Pyridine, pyrrole and quinoline derivatives
Flavouringagent No. Specifcations
a
Conclusionbasedon
currentintake
Indole 1301 N Nosafetyconcern
6-Methylquinoline 1302 N Nosafetyconcern
Isoquinoline 1303 N Nosafetyconcern
Skatole 1304 N Nosafetyconcern
1-Ethyl-2-acetylpyrrole 1305 N Nosafetyconcern
1-Methyl-2-acetylpyrrole 1306 N Nosafetyconcern
Methyl2-pyrrolylketone 1307 N Nosafetyconcern
2-Pyridinemethanethiol 1308 N Nosafetyconcern
2-Acetylpyridine 1309 N Nosafetyconcern
N-Furfurylpyrrole 1310 N Nosafetyconcern
2-(2-Methylpropyl)pyridine 1311 N Nosafetyconcern
3-(2-Methylpropyl)pyridine 1312 N Nosafetyconcern
2-Pentylpyridine 1313 N Nosafetyconcern
Pyrrole 1314 N Nosafetyconcern
3-Ethylpyridine 1315 N Nosafetyconcern
3-Acetylpyridine 1316 N Nosafetyconcern
2,6-Dimethylpyridine 1317 N Nosafetyconcern
5-Ethyl-2-methylpyridine 1318 N Nosafetyconcern
2-Propionylpyrrole 1319 N Nosafetyconcern
Methylnicotinate 1320 N Nosafetyconcern
2-(3-Phenylpropyl)pyridine 1321 N Nosafetyconcern
2-Propylpyridine 1322 N Nosafetyconcern
a
N,newspecifcationsprepared.
642 ANNEX 4
L1
B. Aliphatic and alicyclic hydrocarbons
Flavouringagent No. Specifcations
a
Conclusionbasedon
currentintake
Camphene 1323 N Nosafetyconcern
b-Caryophyllene 1324 N Nosafetyconcern
d-Limonene 1326 N,T ADInotspecifed
b
Myrcene 1327 N Nosafetyconcern
a-Phellandrene 1328 N Nosafetyconcern
a-Pinene 1329 N Nosafetyconcern
b-Pinene 1330 N Nosafetyconcern
Terpinolene 1331 N Nosafetyconcern
Bisabolene 1336 N Nosafetyconcern
Valencene 1337 N Nosafetyconcern
3,7-Dimethyl-1,3,6-octatriene 1338 N Nosafetyconcern
p-Mentha-1,3-diene 1339 N Nosafetyconcern
p-Mentha-1,4-diene 1340 N Nosafetyconcern
1,3,5-Undecatriene 1341 N Nosafetyconcern
d-3-Carene 1342 N Nosafetyconcern
Farnesene(aandb) 1343 N Nosafetyconcern
1-Methyl-1,3-cyclohexadiene 1344 N Nosafetyconcern
b-Bourbonene 1345 N Nosafetyconcern
Cadinene(mixtureofisomers) 1346 N Nosafetyconcern
Guaiene 1347 N Nosafetyconcern
a
N,newspecifcationsprepared.
b
AnADInotspecifedwasestablishedford-limonenebytheCommitteeatitsforty-frst
meeting(Annex1,reference107),whichwasmaintainedatthepresentmeeting.
C. Aromatic hydrocarbons
Flavouringagent No. Specifcations
a
Conclusionbasedon
currentintake
p-Cymene 1325 N Nosafetyconcern
Biphenyl 1332 N Nosafetyconcern
p,a-Dimethylstyrene 1333 N Nosafetyconcern
4-Methylbiphenyl 1334 N Nosafetyconcern
1-Methylnaphthalene 1335 N Nosafetyconcern
a
N,newspecifcationsprepared.
ANNEX 4 643
L1
D. Aliphatic, linear a,b-unsaturated aldehydes, acids and related alcohols,
acetals and esters
Flavouringagent No. Specifcations
a
Conclusionbasedon
currentintake
Butyl2-decenoate 1348 N Nosafetyconcern
2-Decenal 1349 N Nosafetyconcern
2-Dodecenal 1350 N Nosafetyconcern
Ethylacrylate 1351 N Nosafetyconcern
Ethyl2-nonynoate 1352 N Nosafetyconcern
2-Hexenal 1353 N Nosafetyconcern
2-Hexen-1-ol 1354 N Nosafetyconcern
2-(E)Hexen-1-ylacetate 1355 N Nosafetyconcern
Methyl2-nonynoate 1356 N Nosafetyconcern
Methyl2-octynoate 1357 N Nosafetyconcern
Methyl2-undecynoate 1358 N Nosafetyconcern
2-Tridecenal 1359 N Nosafetyconcern
trans-2-Heptenal 1360 N Nosafetyconcern
trans-2-Hexenoicacid 1361 N Nosafetyconcern
2-Nonenal 1362 N Nosafetyconcern
2-Octenal 1363 N Nosafetyconcern
2-Pentenal 1364 N Nosafetyconcern
trans-2-Nonen-1-ol 1365 N Nosafetyconcern
2-Undecenal 1366 N Nosafetyconcern
trans-2-Octen-1-ylacetate 1367 N Nosafetyconcern
trans-2-Octen-1-ylbutanoate 1368 N Nosafetyconcern
cis-2-Nonen-1-ol 1369 N Nosafetyconcern
(E)-2-Octen-1-ol 1370 N Nosafetyconcern
(E)-2-Butenoicacid 1371 N Nosafetyconcern
(E)-2-Decenoicacid 1372 N Nosafetyconcern
(E)-2-Heptenoicacid 1373 N Nosafetyconcern
(Z)-2-Hexen-1-ol 1374 N Nosafetyconcern
trans-2-Hexenylbutyrate 1375 N Nosafetyconcern
(E)-2-Hexenylformate 1376 N Nosafetyconcern
trans-2-Hexenylisovalerate 1377 N Nosafetyconcern
trans-2-Hexenylpropionate 1378 N Nosafetyconcern
trans-2-Hexenylpentanoate 1379 N Nosafetyconcern
(E)-2-Nonenoicacid 1380 N Nosafetyconcern
(E)-2-Hexenylhexanoate 1381 N Nosafetyconcern
(Z)-3-&(E)-2-Hexenylpropionate 1382 N Nosafetyconcern
(E)-2-Hexenaldiethylacetal 1383 N Nosafetyconcern
2-Undecen-1-ol 1384 N Nosafetyconcern
a
N,newspecifcationsprepared
644 ANNEX 4
L1
E. Monocyclic and bicyclic secondary alcohols, ketones and related esters
Flavouringagent No. Specifcations
a
Conclusionbasedon
currentintake
Borneol 1385 N Nosafetyconcern
Isoborneol 1386 N Nosafetyconcern
Bornylacetate 1387 N Nosafetyconcern
Isobornylacetate 1388 N Nosafetyconcern
Bornylformate 1389 N Nosafetyconcern
Isobornylformate 1390 N Nosafetyconcern
Isobornylpropionate 1391 N Nosafetyconcern
Bornylvalerate 1392 N Nosafetyconcern
Bornylisovalerate(endo-) 1393 N Nosafetyconcern
Isobornylisovalerate 1394 N Nosafetyconcern
d-Camphor 1395 N Nosafetyconcern
d-Fenchone 1396 N Nosafetyconcern
Fenchylalcohol 1397 N Nosafetyconcern
Nootkatone 1398 N Nosafetyconcern
1,3,3-Trimethyl-2-norbornanylacetate 1399 N Nosafetyconcern
Methyljasmonate 1400 N Nosafetyconcern
Cycloheptadeca-9-en-1-one 1401 N Nosafetyconcern
3-Methyl-1-cyclopentadecanone 1402 N Nosafetyconcern
2(10)-Pinen-3-ol 1403 N Nosafetyconcern
Verbenol 1404 N Nosafetyconcern
7-Methyl-4,4a,5,6-tetrahydro-2(3H)- 1405 N Nosafetyconcern
naphthalenone
3-Methyl-2-(n-pentanyl)-2- 1406 N Nosafetyconcern
cyclopenten-1-one
Dihydronootkatone 1407 N Nosafetyconcern
3-L-Menthoxypropane-1,2-diol 1408 N Nosafetyconcern
b-Ionylacetate 1409 N Nosafetyconcern
a-Isomethylionylacetate 1410 N Nosafetyconcern
3-(l-Menthoxy)-2-methylpropane- 1411 N Nosafetyconcern
1,2-diol
Bornylbutyrate 1412 N Nosafetyconcern
D,L-Menthol(+/-)-propyleneglycol 1413 N Nosafetyconcern
carbonate
L-Monomenthylglutarate 1414 N Nosafetyconcern
L-Menthylmethylether 1415 N Nosafetyconcern
p-Menthane-3,8-diol 1416 N Nosafetyconcern
a
N,newspecifcationsprepared
ANNEX 4 645
L1
F. Amino acids and related substances
Flavouringagent No. Specifcations
a
Conclusionbasedon
currentintake
b-Alanine 1418 N Nosafetyconcern
l-Cysteine 1419 N Nosafetyconcern
b
l-Glutamicacid 1420 N Nosafetyconcern
b,c
Glycine 1421 N Nosafetyconcern
b
dl-Isoleucine 1422 N Nosafetyconcern
l-Leucine 1423 N Nosafetyconcern
b
dl-Methionine 1424 N Nosafetyconcern
l-Proline 1425 N Nosafetyconcern
b
dl-Valine 1426 N Nosafetyconcern
dl-(3-Amino-3-carboxypropyl) 1427 N Nosafetyconcern
dimethylsufoniumchloride
l-Phenylalanine 1428 N Nosafetyconcern
b
l-Asparticacid 1429 N Nosafetyconcern
b
l-Glutamine 1430 N Nosafetyconcern
b,c
l-Histidine 1431 N Nosafetyconcern
b
dl-Phenylalanine 1432 N Nosafetyconcern
l-Tyrosine 1434 N Nosafetyconcern
b
Taurine 1435 N Nosafetyconcern
dl-Alanine 1437 N Nosafetyconcern
l-Arginine 1438 N Nosafetyconcern
b
l-Lysine 1439 N Nosafetyconcern
b
a
N,newspecifcationsprepared.
b
NotevaluatedusingtheProcedurefortheSafetyEvaluationofFlavouringAgents.The
substanceisamacronutrientandnormalcomponentofproteinand,assuch,human
exposurethroughfoodisordersofmagnitudehigherthantheanticipatedlevelof
exposurefromuseasfavouringagent.
c
ThegroupADInotspecifedestablishedbytheCommitteeatitsthirty-frstmeetingfor
L-glutamicacidanditsammonium,calcium,magnesium,monosodiumandpotassium
saltswasmaintained.
646 ANNEX 4
L1
G. Tetrahydrofuran and furanone derivatives
Flavouringagent No. Specifcations
a
Conclusionbasedon
currentintake
2-Hexyl-4-acetoxytetrahydrofuran 1440 N Nosafetyconcern
2-(3-Phenylpropyl)tetrahydrofuran 1441 N Nosafetyconcern
Tetrahydrofurfurylacetate 1442 N Nosafetyconcern
Tetrahydrofurfurylalcohol 1443 N Nosafetyconcern
Tetrahydrofurfurylbutyrate 1444 N Nosafetyconcern
Tetrahydrofurfurylpropionate 1445 N Nosafetyconcern
4-Hydroxy-2,5-dimethyl-3(2H)- 1446 N Nosafetyconcern
furanone
Tetrahydrofurfurylcinnamate 1447 N Nosafetyconcern
2-Methyltetrahydrofuran-3-one 1448 N Nosafetyconcern
2-Ethyl-4-hydroxy-5-methyl-3(2H)- 1449 N Nosafetyconcern
furanone
4-Hydroxy-5-methyl-3(2H)- 1450 N Nosafetyconcern
furanone
2,5-Dimethyl-4-methoxy-3(2H)- 1451 N Nosafetyconcern
furanone
2,2-Dimethyl-5-(1-methylpropen- 1452 N Nosafetyconcern
1-yl)tetrahydrofuran
2,5-Diethyltetrahydrofuran 1453 N Nosafetyconcern
cis,trans-2-Methyl-2-vinyl-5-(2- 1454 N Nosafetyconcern
hydroxy-2-propyl)
tetrahydrofuran(linalooloxide)
5-Isopropenyl-2-methyl-2- 1455 N Nosafetyconcern
vinyltetrahydrofuran(cisand
transmixture)
4-Acetoxy-2,5-dimethyl-3(2H) 1456 N Nosafetyconcern
furanone
(+/-)-2-(5-Methyl-5-vinyl- 1457 N Nosafetyconcern
tetrahydrofuran-2-yl)
propionaldehyde
a
N,newspecifcationsprepared
ANNEX 4 647
L1
H. Phenyl-substituted aliphatic alcohols and related aldehydes and esters
Flavouringagent No. Specifcations
a
Conclusionbasedon
currentintake
Ethyl4-phenylbutyrate 1458 N Nosafetyconcern
b-Methylphenethylalcohol 1459 N Nosafetyconcern
2-Methyl-4-phenyl-2-butylacetate 1460 N Nosafetyconcern
2-Methyl-4-phenyl-2-butylisobutyrate 1461 N Nosafetyconcern
2-Methyl-4-phenylbutyraldehyde 1462 N Nosafetyconcern
3-Methyl-2-phenylbutyraldehyde 1463 N Nosafetyconcern
Methyl4-Phenylbutyrate 1464 N Nosafetyconcern
2-Methyl-3-(p-isopropylphenyl) 1465 N Nosafetyconcern
propionaldehyde
2-Methyl-3-tolylpropionaldehyde 1466 N Nosafetyconcern
(mixedo-,m-,p-)
2-Phenylpropionaldehyde 1467 N Nosafetyconcern
2-Phenylpropionaldehydedimethyl 1468 N Nosafetyconcern
acetal
2-Phenylpropylbutyrate 1469 N Nosafetyconcern
2-Phenylpropylisobutyrate 1470 N Nosafetyconcern
2-(p-Tolyl)propionaldehyde 1471 N Nosafetyconcern
5-Methyl-2-phenyl-2-hexenal 1472 N Nosafetyconcern
4-Methyl-2-phenyl-2-pentenal 1473 N Nosafetyconcern
2-Phenyl-2-butenal 1474 N Nosafetyconcern
Ethyl2-ethyl-3-phenylpropanoate 1475 N Nosafetyconcern
2-Phenyl-4-pentenal 1476 N Nosafetyconcern
2-Methyl-4-phenyl-2-butanol 1477 N Nosafetyconcern
2-Oxo-3-phenylpropionicacid 1478 N Nosafetyconcern
Sodium2-oxo-3-phenylpropionate 1479 N,T
a
N,newspecifcationsprepared;T,tentativespecifcations.
648 ANNEX 4
L1
Flavouring agents considered for specifcations only
No. Flavouringagent Specifcations
a
53 Citronellylformate R
55 Nerylformate R
68 Rhodinylbutyrate R
399 Methyl-b-ionone R
471 2,8-Dithianon-4-ene-4-carboxaldehyde R
504 S-Methylbenzothioate R
557 1-Mercapto-2-propanone R
570 Propenylpropyldisulfde R
605 1,3-Nonanediolacetate(mixedesters) R
615 Butylethylmalonate R
628 Ethylaconitate(mixedesters) R
631.2 Sodiumsaltof3-methyl-2-oxobutanoicacid S
b
632.2 Sodiumsaltof3-methyl-2-oxopentanoicacid S
b
633.2 Sodiumsaltof4-methyl-2-oxopentanoicacid S
b
919 Glycerylmonooleate R
1203 Ammoniumisovalerate R
1218 4-Ethyloctanoicacid R
1263 Isoeugenylphenylacetate R
1273 Ethyl5-hexenoate R
1291 3-Mercapto-2-methylpentan-1-ol(racemic) R
1296 spiro[2,4-Dithia-1-methyl-8-oxabicyclo(3.3.0)octane-3,3- R
(1-oxa-2-methyl)-cyclopentane]
a
R,existingspecifcationsrevised;S,existingspecifcationsweremaintained;T,the
existing,new,orrevisedspecifcationsaretentativeandnewinformationisrequired.
b
Specifcationswillbewithdrawnatthenextmeetingatwhichfavouringagentsare
discussedifnoinformationbecomesavailablebythattime.
Evaluation of a natural constituent of food
Constituent Toxicologicalrecommendations
Glycyrrhizinicacid Availabledatasuggestthatanintakeof100mg/daywouldbe
unlikelytocauseadverseeffectsinthemajorityofadults.
Incertainhighlysusceptibleindividuals,physiologicaleffects
couldoccuratexposurelevelssomewhatbelowthisfgure.
Theintakedataindicatethatconsumerswithahighintake
ofliquoriceconfectioneryorherbalteacontainingliquorice
maybeexposedtoglycyrrhizinicacidatmorethan
100mg/day.
L1
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9
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%
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e

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n
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m

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e
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n
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y

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e

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C
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8
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9
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C
1
5
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h
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C
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5
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1
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1
6

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e
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n

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(
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o
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y
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r
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n
s

1
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)
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b
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(
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)
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s
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o
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h
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1
5
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T
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D
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(
%
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1
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V
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9
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r

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o
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f

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3
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m
e
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y
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3
,
6
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o
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a
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r
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e
n
e
.

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s
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s

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e
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d

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o

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y
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r
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a
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s
,

w
h
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h

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n
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e

m
y
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e
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e

(
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o
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7
)
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y
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e
n
e

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s

e
v
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d

b
y

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C
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s

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s
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h
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n

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E
L
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L

o
f

2
5
0

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g
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1
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-
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k

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(
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h
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t

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s
.
1
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3
9

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M
e
n
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h
a
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1
,

8
9
%

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%

1
,
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a
n
d

T
h
e

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m
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1
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(
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1
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1
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h
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1
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e

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l
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N
o
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t

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e
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(
3
)
.
1
3
4
1

1
,
3
,
5
-
U
n
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e
c
a
t
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e

9
4
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4
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e
c
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u
s
l
y

b
y

t
h
e

(
s
u
m

o
f

U
n
d
e
c
a
t
r
i
e
n
e

C
o
m
m
i
t
t
e
e
.

I
t

i
s

e
x
p
e
c
t
e
d

t
o

s
h
a
r
e

t
h
e

s
a
m
e

m
e
t
a
b
o
l
i
c

f
a
t
e

a
s

i
s
o
m
e
r
s
)

(
Z
,
Z
,
E
)

t
h
e

p
r
i
m
a
r
y

c
o
m
p
o
u
n
d

1
,
3
,
5
-
u
n
d
e
c
a
t
r
i
e
n
e

a
n
d

t
h
e

o
t
h
e
r

a
c
y
c
l
i
c

h
y
d
r
o
c
a
r
b
o
n
s

i
n

t
h
i
s

g
r
o
u
p

o
f

f
a
v
o
u
r
i
n
g

a
g
e
n
t
s
,

w
h
i
c
h

a
r
e

o
x
i
d
i
z
e
d

t
o

o
x
y
g
e
n
a
t
e
d

m
e
t
a
b
o
l
i
t
e
s

a
n
d

e
x
c
r
e
t
e
d

i
n

t
h
e

u
r
i
n
e
.

T
h
e

C
o
m
m
i
t
t
e
e

c
o
n
c
l
u
d
e
d

t
h
a
t

t
h
i
s

s
u
b
s
t
a
n
c
e

w
a
s

o
f

n
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.
650 ANNEX 5
1
3
4
2

d
-
3
-
C
a
r
e
n
e

9
2
%

3
%

b
-
P
i
n
e
n
e
;

b
-
P
i
n
e
n
e

(
N
o
.

1
3
3
0
)

w
a
s

e
v
a
l
u
a
t
e
d

b
y

t
h
e

C
o
m
m
i
t
t
e
e

a
t

i
t
s

2
%

l
i
m
o
n
e
n
e
;

p
r
e
s
e
n
t

m
e
e
t
i
n
g
,

w
h
e
n

i
t

w
a
s

c
o
n
c
l
u
d
e
d

t
h
a
t

t
h
i
s

s
u
b
s
t
a
n
c
e

w
a
s

2
%

m
y
r
c
e
n
e
;

o
f

n
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.

1
%

p
-
c
y
m
e
n
e

d
-
L
i
m
o
n
e
n
e

(
N
o
.

1
3
2
6
)

w
a
s

e
v
a
l
u
a
t
e
d

b
y

t
h
e

C
o
m
m
i
t
t
e
e

a
t

i
t
s

p
r
e
s
e
n
t

m
e
e
t
i
n
g
.

B
a
s
e
d

o
n

t
h
e

A
D
I

n
o
t

s
p
e
c
i
f
e
d

t
h
a
t

w
a
s

e
s
t
a
b
l
i
s
h
e
d

f
o
r

d
-
l
i
m
o
n
e
n
e

a
t

t
h
e

f
o
r
t
y
-
f
r
s
t

m
e
e
t
i
n
g

o
f

t
h
e

C
o
m
m
i
t
t
e
e
,

t
h
e

C
o
m
m
i
t
t
e
e

c
o
n
c
l
u
d
e
d

t
h
a
t

t
h
i
s

s
u
b
s
t
a
n
c
e

w
a
s

o
f

n
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.

M
y
r
c
e
n
e

(
N
o
.

1
3
2
7
)

w
a
s

e
v
a
l
u
a
t
e
d

b
y

t
h
e

C
o
m
m
i
t
t
e
e

a
t

i
t
s

p
r
e
s
e
n
t

m
e
e
t
i
n
g
.

T
h
e

L
O
E
L
/
N
O
E
L

f
o
r

m
y
r
c
e
n
e

w
a
s

2
5
0

m
g
/
k
g

b
w

p
e
r

d
a
y

i
n

1
3
-
w
e
e
k

s
t
u
d
i
e
s

i
n

r
a
t
s

a
n
d

m
i
c
e

t
r
e
a
t
e
d

b
y

g
a
v
a
g
e

(
1
,

2
)
.

T
h
e

C
o
m
m
i
t
t
e
e

c
o
n
c
l
u
d
e
d

t
h
a
t

t
h
i
s

s
u
b
s
t
a
n
c
e

w
a
s

o
f

n
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.

p
-
C
y
m
e
n
e

(
N
o
.

1
3
2
5
)

w
a
s

e
v
a
l
u
a
t
e
d

b
y

t
h
e

C
o
m
m
i
t
t
e
e

a
t

i
t
s

p
r
e
s
e
n
t

m
e
e
t
i
n
g
,

w
h
e
n

i
t

w
a
s

c
o
n
c
l
u
d
e
d

t
h
a
t

t
h
i
s

s
u
b
s
t
a
n
c
e

w
a
s

o
f

n
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.
1
3
4
3

F
a
r
n
e
s
e
n
e

6
7
%

2
1
%

b
i
s
a
b
o
l
e
n
e

B
i
s
a
b
o
l
e
n
e

(
N
o
.

1
3
3
6
)

w
a
s

e
v
a
l
u
a
t
e
d

b
y

t
h
e

C
o
m
m
i
t
t
e
e

a
t

i
t
s

(
a

a
n
d

b
)

(
s
u
m

o
f

(
s
u
m

o
f

i
s
o
m
e
r
s
)
;

p
r
e
s
e
n
t

m
e
e
t
i
n
g
,

w
h
e
n

i
t

w
a
s

c
o
n
c
l
u
d
e
d

t
h
a
t

t
h
i
s

s
u
b
s
t
a
n
c
e

w
a
s

i
s
o
m
e
r
s
)

1
0
%

o
t
h
e
r

i
s
o
m
e
r
s

o
f

n
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.

o
f

f
a
r
n
e
s
e
n
e

a
n
d

O
t
h
e
r

i
s
o
m
e
r
s
o
f

f
a
r
n
e
s
e
n
e

a
r
e

e
x
p
e
c
t
e
d

t
o

s
h
a
r
e

t
h
e

s
a
m
e

o
t
h
e
r

C
1
5
H
2
4

t
e
r
p
e
n
e

m
e
t
a
b
o
l
i
c

f
a
t
e

a
s

t
h
e

p
r
i
m
a
r
y

c
o
m
p
o
u
n
d
s

a
n
d

b
-
f
a
r
n
e
s
e
n
e

a
n
d

h
y
d
r
o
c
a
r
b
o
n
s

(
e
.
g
.

t
h
e

s
t
r
u
c
t
u
r
a
l
l
y

r
e
l
a
t
e
d

a
c
y
l
i
c

h
y
d
r
o
c
a
r
b
o
n
s

v
a
l
e
n
c
e
n
e
,

i
n

t
h
i
s

g
r
o
u
p

o
f

f
a
v
o
u
r
i
n
g

a
g
e
n
t
s
,

w
h
i
c
h

i
n
c
l
u
d
e

m
y
r
c
e
n
e

(
N
o
.

b
o
u
r
b
o
n
e
n
e
,

1
3
2
7
)
.

M
y
r
c
e
n
e

w
a
s

e
v
a
l
u
a
t
e
d

b
y

t
h
e

C
o
m
m
i
t
t
e
e

a
t

i
t
s

p
r
e
s
e
n
t

c
a
d
i
n
e
n
e
,

g
u
a
i
e
n
e
)

m
e
e
t
i
n
g
.

T
h
e

L
O
E
L
/
N
O
E
L

f
o
r

m
y
r
c
e
n
e

w
a
s

2
5
0

m
g
/
k
g

b
w

p
e
r

d
a
y

i
n

1
3
-
w
e
e
k

s
t
u
d
i
e
s

i
n

r
a
t
s

a
n
d

m
i
c
e

t
r
e
a
t
e
d

b
y

g
a
v
a
g
e

(
1
,

2
)
.

T
h
e

C
o
m
m
i
t
t
e
e

c
o
n
c
l
u
d
e
d

t
h
a
t

t
h
i
s

s
u
b
s
t
a
n
c
e

w
a
s

o
f

n
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.

T
h
e

C
1
5
H
2
4

t
e
r
p
e
n
e

h
y
d
r
o
c
a
r
b
o
n
s

f
o
u
n
d

a
s

s
e
c
o
n
d
a
r
y

c
o
m
p
o
n
e
n
t
s

o
f

f
a
r
n
e
s
e
n
e

i
n
c
l
u
d
e

v
a
l
e
n
c
e
n
e

(
N
o
.

1
3
3
7
)
,

b
o
u
r
b
o
n
e
n
e

(
N
o
.

1
3
4
5
)
,

c
a
d
i
n
e
n
e

(
N
o
.

1
3
4
6
)
,

a
n
d

g
u
a
i
e
n
e

(
N
o
.

1
3
4
7
)
.

T
h
e

C
o
m
m
i
t
t
e

e
v
a
l
u
a
t
e
d

a
l
l

t
h
e
s
e

a
g
e
n
t
s

a
t

i
t
s

p
r
e
s
e
n
t

m
e
e
t
i
n
g

a
n
d

c
o
n
c
l
u
d
e
d

t
h
a
t

t
h
e
y

w
e
r
e

o
f

n
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.
ANNEX 5 651
L1
S
u
m
m
a
r
y

(
c
o
n
t
d
)
N
o
.

N
a
m
e

M
i
n
i
m
u
m

S
e
c
o
n
d
a
r
y

c
o
m
p
o
n
e
n
t
s

C
o
m
m
e
n
t
s

o
n

s
e
c
o
n
d
a
r
y

c
o
m
p
o
n
e
n
t
s

a
s
s
a
y

v
a
l
u
e

(
%
)
D
.

A
l
i
p
h
a
t
i
c
,

l
i
n
e
a
r

a
,
b
-
u
n
s
a
t
u
r
a
t
e
d

a
l
d
e
h
y
d
e
s
,

a
c
i
d
s

a
n
d

r
e
l
a
t
e
d

a
l
c
o
h
o
l
s
,

a
c
e
t
a
l
s

a
n
d

e
s
t
e
r
s
1
3
4
9

2
-
D
e
c
e
n
a
l

9
2
%

4
%

2
-
d
e
c
e
n
o
i
c

(
E
)
-
2
-
D
e
c
e
n
o
i
c

(
N
o
.

1
3
7
2
)

a
c
i
d

i
s

s
u
b
s
t
r
a
t
e

f
o
r

t
h
e

f
a
t
t
y

a
c
i
d

(
s
u
m

o
f

E

&

a
c
i
d

c
y
c
l
e

a
n
d

i
s

m
e
t
a
b
o
l
i
z
e
d

a
n
d

e
x
c
r
e
t
e
d

p
r
i
m
a
r
i
l
y

a
s

c
a
r
b
o
n

i
s
o
m
e
r
s
)

d
i
o
x
i
d
e

a
n
d

w
a
t
e
r

(
4
)
.

T
h
e

r
e
l
a
t
e
d

m
a
t
e
r
i
a
l
,

2
,
4
-
d
e
c
a
d
i
e
n
a
l
,

w
h
i
c
h

i
s

o
x
i
d
i
z
e
d

t
o

2
,
4
-
d
e
c
a
d
i
e
n
o
i
c

a
c
i
d
,

e
x
h
i
b
i
t
e
d

N
O
E
L
s

o
f

1
0
0

a
n
d

2
0
0

m
g
/
k
g

b
w

p
e
r

d
a
y

f
o
r

m
a
l
e

a
n
d

f
e
m
a
l
e

m
i
c
e
,

r
e
s
p
e
c
t
i
v
e
l
y
,

i
n

9
0
-
d
a
y

f
e
e
d
i
n
g

s
t
u
d
y

(
5
)
.

N
O
E
L
s

o
f

1
0
0

a
n
d

3
3
.
9

m
g
/
k
g

b
w

p
e
r

d
a
y

w
e
r
e

r
e
p
o
r
t
e
d

f
o
r

r
a
t
s

i
n

t
w
o

s
e
p
a
r
a
t
e

9
0
-
d
a
y

s
t
u
d
i
e
s

(
5
,

6
)
.

T
h
e

C
o
m
m
i
t
t
e
e

c
o
n
c
l
u
d
e
d

t
h
a
t

t
h
i
s

s
u
b
s
t
a
n
c
e

w
a
s

o
f

n
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.
1
3
5
0

2
-
D
o
d
e
c
e
n
a
l

9
3
%

4
%

2
-
d
o
d
e
c
e
n
o
i
c

2
-
D
o
d
e
c
e
n
o
i
c

a
c
i
d

i
s

s
t
r
u
c
t
u
r
a
l
l
y

r
e
l
a
t
e
d

t
o

t
h
e

p
r
i
m
a
r
y

m
a
t
e
r
i
a
l

(
s
u
m

o
f

&

a
c
i
d

a
n
d

i
s

e
x
p
e
c
t
e
d

t
o

b
e

m
e
t
a
b
o
l
i
z
e
d

i
n

t
h
e

s
a
m
e

w
a
y
.

I
t

i
s

i
s
o
m
e
r
s
)

s
u
b
s
t
r
a
t
e

f
o
r

t
h
e

f
a
t
t
y

a
c
i
d

c
y
c
l
e
,

a
n
d

i
s

m
e
t
a
b
o
l
i
z
e
d

a
n
d

e
x
c
r
e
t
e
d

p
r
i
m
a
r
i
l
y

a
s

c
a
r
b
o
n

d
i
o
x
i
d
e

a
n
d

w
a
t
e
r

(
4
)
,

a
n
d

t
h
u
s

d
o
e
s

n
o
t

p
r
e
s
e
n
t

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.
1
3
5
3

2
-
H
e
x
e
n
a
l

9
2
%

4
%

2
-
h
e
x
e
n
o
i
c

(
E
)
-
2
-
H
e
x
e
n
o
i
c

a
c
i
d

(
N
o
.

1
3
6
1
)

i
s

s
u
b
s
t
r
a
t
e

f
o
r

t
h
e

f
a
t
t
y

a
c
i
d

(
s
u
m

o
f

&

a
c
i
d

c
y
c
l
e

a
n
d

i
s

m
e
t
a
b
o
l
i
z
e
d

a
n
d

e
x
c
r
e
t
e
d

p
r
i
m
a
r
i
l
y

a
s

c
a
r
b
o
n

i
s
o
m
e
r
s
)

d
i
o
x
i
d
e

a
n
d

w
a
t
e
r

(
4
)
.

9
8
-
d
a
y

s
t
u
d
y

w
i
t
h

t
h
e

s
t
r
u
c
t
u
r
a
l
l
y

r
e
l
a
t
e
d

m
a
t
e
r
i
a
l

2
,
4
-
h
e
x
a
d
i
e
n
a
l
,

w
h
i
c
h

o
x
i
d
i
z
e
s

t
o

2
,
4
-
h
e
x
a
d
i
e
n
o
i
c

a
c
i
d
,

e
x
h
i
b
i
t
e
d

N
O
E
L
s

o
f

1
5

a
n
d

6
0

m
g
/
k
g

b
w

f
o
r

m
a
l
e

a
n
d

f
e
m
a
l
e

r
a
t
s
,

r
e
s
p
e
c
t
i
v
e
l
y

(
7
)
.

T
h
e

C
o
m
m
i
t
t
e
e

c
o
n
c
l
u
d
e
d

t
h
a
t

t
h
i
s

s
u
b
s
t
a
n
c
e

w
a
s

o
f

n
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.
1
3
5
9

2
-
T
r
i
d
e
c
e
n
a
l

9
2
%

4
%

2
-
t
r
i
d
e
c
e
n
o
i
c

2
-
T
r
i
d
e
c
e
n
o
i
c

a
c
i
d

i
s

s
t
r
u
c
t
u
r
a
l
l
y

r
e
l
a
t
e
d

t
o

t
h
e

p
r
i
m
a
r
y

m
a
t
e
r
i
a
l

a
n
d

(
s
u
m

o
f

&

a
c
i
d

i
s

e
x
p
e
c
t
e
d

t
o

b
e

m
e
t
a
b
o
l
i
z
e
d

i
n

t
h
e

s
a
m
e

w
a
y
.

I
t

i
s

s
u
b
s
t
r
a
t
e

i
s
o
m
e
r
s
)

f
o
r

t
h
e

f
a
t
t
y

a
c
i
d

c
y
c
l
e
,

a
n
d

i
s

m
e
t
a
b
o
l
i
z
e
d

a
n
d

e
x
c
r
e
t
e
d

p
r
i
m
a
r
i
l
y

a
s

c
a
r
b
o
n

d
i
o
x
i
d
e

a
n
d

w
a
t
e
r

(
4
)
,

a
n
d

t
h
u
s

d
o
e
s

n
o
t

p
r
e
s
e
n
t

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.
652 ANNEX 4
L1
1
3
6
2

2
-
N
o
n
e
n
a
l

9
2
%

4
%

2
-
N
o
n
e
n
o
i
c

(
E
)
-
2
-
N
o
n
e
n
o
i
c

a
c
i
d

(
N
o
.

1
3
8
0
)

i
s

s
u
b
s
t
r
a
t
e

f
o
r

t
h
e

f
a
t
t
y

a
c
i
d

(
s
u
m

o
f

&

a
c
i
d

c
y
c
l
e
,

a
n
d

i
s

m
e
t
a
b
o
l
i
z
e
d

a
n
d

e
x
c
r
e
t
e
d

p
r
i
m
a
r
i
l
y

a
s

c
a
r
b
o
n

i
s
o
m
e
r
s
)

d
i
o
x
i
d
e

a
n
d

w
a
t
e
r

(
4
)
,

a
n
d

t
h
u
s

d
o
e
s

n
o
t

p
r
e
s
e
n
t

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.
1
3
6
3

2
-
O
c
t
e
n
a
l

9
2
%

4
%

2
-
o
c
t
e
n
o
i
c

a
c
i
d

2
-
O
c
t
e
n
o
i
c

a
c
i
d

i
s

s
t
r
u
c
t
u
r
a
l
l
y

r
e
l
a
t
e
d

t
o

o
t
h
e
r

a
,
b
-
u
n
s
a
t
u
r
a
t
e
d

(
s
u
m

o
f

&

a
n
d

e
t
h
y
l

o
c
t
a
n
o
a
t
e

a
c
i
d
s

a
n
d

i
s

e
x
p
e
c
t
e
d

t
o

b
e

m
e
t
a
b
o
l
i
z
e
d

i
n

t
h
e

s
a
m
e

w
a
y
.

i
s
o
m
e
r
s
)

I
t

i
s

s
u
b
s
t
r
a
t
e

f
o
r

t
h
e

f
a
t
t
y

a
c
i
d

c
y
c
l
e
,

a
n
d

i
s

m
e
t
a
b
o
l
i
z
e
d

a
n
d

e
x
c
r
e
t
e
d

p
r
i
m
a
r
i
l
y

a
s

c
a
r
b
o
n

d
i
o
x
i
d
e

a
n
d

w
a
t
e
r

(
4
)
,

a
n
d

t
h
u
s

d
o
e
s

n
o
t

p
r
e
s
e
n
t

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.

E
t
h
y
l

o
c
t
a
n
o
a
t
e

(
N
o
.

3
3
)

h
a
s

b
e
e
n

e
v
a
l
u
a
t
e
d

b
y

t
h
e

C
o
m
m
i
t
t
e
e
,

w
h
i
c
h

c
o
n
c
l
u
d
e
d

t
h
a
t

i
t

w
a
s

o
f

n
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.
1
3
7
4

(
Z
)
-
2
-
H
e
x
e
n
-
1
-
o
l

9
2
%

3
-
5
%

(
E
)
-
2
-
h
e
x
e
n
-
1
-
o
l

B
o
t
h

t
h
e

a
n
d

i
s
o
m
e
r
s

a
r
e

o
x
i
d
i
z
e
d

i
n

v
i
v
o
,

f
r
s
t

t
o

t
h
e

c
o
r
r
e
s
p
o
n
d
i
n
g

a
l
d
e
h
y
d
e

a
n
d

t
h
e
n

t
o

t
h
e

a
c
i
d

(
8

1
0
)
.

T
h
e
y

t
h
e
n

e
n
t
e
r

t
h
e

f
a
t
t
y

a
c
i
d

c
y
c
l
e

w
h
e
r
e

t
h
e
y

a
r
e

c
o
m
p
l
e
t
e
l
y

m
e
t
a
b
o
l
i
z
e
d

a
n
d

e
x
c
r
e
t
e
d

(
4
)
.

N
O
E
L

o
f

1
2
0

m
g
/
k
g

b
w

p
e
r

d
a
y

w
a
s

i
d
e
n
t
i
f
e
d

i
n

9
8
-
d
a
y

s
t
u
d
y

i
n

r
a
t
s

g
i
v
e
n

d
r
i
n
k
i
n
g
-
w
a
t
e
r

c
o
n
t
a
i
n
i
n
g

t
h
e

s
t
r
u
c
t
u
r
a
l
l
y

r
e
l
a
t
e
d

m
a
t
e
r
i
a
l

c
i
s
-
3
-
h
e
x
e
n
-
1
-
o
l

(
1
1
)
.

T
h
e

C
o
m
m
i
t
t
e
e

c
o
n
c
l
u
d
e
d

t
h
a
t

t
h
e
s
e

s
u
b
s
t
a
n
c
e
s

w
e
r
e

o
f

n
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.
1
3
7
9

t
r
a
n
s
-
2
-
H
e
x
e
n
y
l

9
3
%

3
%

p
r
o
p
a
n
o
i
c

a
c
i
d
;

P
r
o
p
a
n
o
i
c

a
c
i
d

(
N
o
.

8
4
)

h
a
s

b
e
e
n

e
v
a
l
u
a
t
e
d

b
y

t
h
e

C
o
m
m
i
t
t
e
e
,

p
e
n
t
a
n
o
a
t
e

3
%

2
-
h
e
x
e
n
o
l

w
h
i
c
h

c
o
n
c
l
u
d
e
d

t
h
a
t

i
t

w
a
s

o
f

n
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.

F
o
r

2
-
h
e
x
e
n
o
l

s
e
e

N
o
.

1
3
7
4

a
b
o
v
e
.
1
3
8
1

(
E
)
-
2
-
H
e
x
e
n
y
l

9
3
%

3
%

h
e
x
a
n
o
i
c

a
c
i
d
;

H
e
x
a
n
o
i
c

a
c
i
d

(
N
o
.

9
3
)

h
a
s

b
e
e
n

e
v
a
l
u
a
t
e
d

b
y

t
h
e

C
o
m
m
i
t
t
e
e
,

h
e
x
a
n
o
a
t
e

3
%

2
-
h
e
x
e
n
o
l

w
h
i
c
h

c
o
n
c
l
u
d
e
d

t
h
a
t

i
t

w
a
s

o
f

n
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.

F
o
r

2
-
h
e
x
e
n
o
l

s
e
e

N
o
.

1
3
7
4

a
b
o
v
e
.
ANNEX 5 653
L1
S
u
m
m
a
r
y

(
c
o
n
t
d
)
N
o
.

N
a
m
e

M
i
n
i
m
u
m

S
e
c
o
n
d
a
r
y

c
o
m
p
o
n
e
n
t
s

C
o
m
m
e
n
t
s

o
n

s
e
c
o
n
d
a
r
y

c
o
m
p
o
n
e
n
t
s

a
s
s
a
y

v
a
l
u
e

(
%
)
E
.

M
o
n
o
c
y
c
l
i
c

a
n
d

b
i
c
y
c
l
i
c

s
e
c
o
n
d
a
r
y

a
l
c
o
h
o
l
s

a
n
d

k
e
t
o
n
e
s
1
3
8
6

I
s
o
b
o
r
n
e
o
l

9
2
%

5
%

b
o
r
n
e
o
l

B
o
r
n
e
o
l

(
N
o
.

1
3
8
5
)

w
a
s

e
v
a
l
u
a
t
e
d

b
y

t
h
e

C
o
m
m
i
t
t
e

a
t

i
t
s

p
r
e
s
e
n
t

m
e
e
t
i
n
g
.

N
O
E
L
s

o
f

5
2
6

a
n
d

>
1
3
0
0

m
g
/
k
g

b
w

p
e
r

d
a
y

w
e
r
e

r
e
p
o
r
t
e
d

i
n

3
1
-

a
n
d

9
0
-
d
a
y

s
t
u
d
i
e
s

i
n

d
o
g
s
,

r
e
s
p
e
c
t
i
v
e
l
y

(
1
2
)
.

I
n

a
d
d
i
t
i
o
n
,

N
O
E
L
s

o
f

1
5

a
n
d

9
0

m
g
/
k
g

b
w

p
e
r

d
a
y

w
e
r
e

i
d
e
n
t
i
f
e
d

f
o
r

m
a
l
e
s

a
n
d

f
e
m
a
l
e
s
,

r
e
s
p
e
c
t
i
v
e
l
y
,

f
o
r

t
h
e

r
e
l
a
t
e
d

m
a
t
e
r
i
a
l

i
s
o
b
o
r
n
y
l

a
c
e
t
a
t
e

(
N
o
.

1
3
8
8
)

i
n

9
0
-
d
a
y

s
t
u
d
y

i
n

r
a
t
s

(
1
3
)
.

T
h
e

C
o
m
m
i
t
t
e
e

t
h
u
s

c
o
n
c
l
u
d
e
d

t
h
a
t

b
o
r
n
e
o
l

d
o
e
s

n
o
t

p
r
e
s
e
n
t

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.
1
3
9
8

N
o
o
t
k
a
t
o
n
e

9
3
%

3
%

d
i
h
y
d
r
o
n
o
o
t
k
a
t
o
n
e

D
i
h
y
d
r
o
n
o
o
t
k
a
t
o
n
e

(
N
o
.

1
4
0
7
)

i
s

m
e
t
a
b
o
l
i
z
e
d

p
r
i
m
a
r
i
l
y

t
h
r
o
u
g
h
t

t
h
e

e
p
o
x
i
d
a
t
i
o
n

a
n
d

h
y
d
r
a
t
i
o
n

o
f

t
h
e

i
s
o
p
r
e
n
y
l

s
i
d
e
-
c
h
a
i
n

t
o

f
o
r
m

t
h
e

c
o
r
r
e
s
p
o
n
d
i
n
g

1
3
,
1
4
-
d
i
o
l

(
1
4
)
.

T
o

m
i
n
o
r

e
x
t
e
n
t
,

r
e
d
u
c
t
i
o
n

o
f

t
h
e

k
e
t
o
n
e

t
o

t
h
e

s
e
c
o
n
d
a
r
y

a
l
c
o
h
o
l

f
o
l
l
o
w
e
d

b
y

c
o
n
j
u
g
a
t
i
o
n

w
i
t
h

g
l
u
c
u
r
o
n
i
c

a
c
i
d

m
a
y

o
c
c
u
r

a
s

w
i
t
h

t
h
e

a
l
i
p
h
a
t
i
c

a
n
d

m
o
n
o
c
y
c
l
i
c

s
e
c
o
n
d
a
r
y

k
e
t
o
n
e
s

(
1
5

1
7
)

(
W
i
l
l
i
a
m
s
,

1
9
5
9
;

L
i
n
g
t
o
n

&

B
e
v
a
n
,

1
9
9
4
;

T
o
p
p
i
n
g

e
t

a
l
.
,

1
9
9
4
)
.

T
h
e

C
o
m
m
i
t
t
e
e

t
h
u
s

c
o
n
c
l
u
d
e
d

t
h
a
t

d
i
h
y
d
r
o
n
o
o
t
k
a
t
o
n
e

d
o
e
s

n
o
t

p
r
e
s
e
n
t

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.
1
4
0
7

D
i
h
y
d
r
o
n
o
o
t
k
a
t
o
n
e

9
0
%

6
%

n
o
o
t
k
a
t
o
n
e

N
o
o
t
k
a
t
o
n
e

(
N
o
.

1
3
9
8
)

i
s

m
e
t
a
b
o
l
i
z
e
d

p
r
i
m
a
r
i
l
y

t
h
r
o
u
g
h
t

t
h
e

e
p
o
x
i
d
a
t
i
o
n

a
n
d

h
y
d
r
a
t
i
o
n

o
f

t
h
e

i
s
o
p
r
e
n
y
l

s
i
d
e
-
c
h
a
i
n

t
o

f
o
r
m

t
h
e

c
o
r
r
e
s
p
o
n
d
i
n
g

1
3
,
1
4
-
d
i
o
l

(
1
4
)

(
A
s
a
k
a
w
a

e
t

a
l
.
,

1
9
8
6
)
.

T
o

m
i
n
o
r

e
x
t
e
n
t
,

r
e
d
u
c
t
i
o
n

o
f

t
h
e

k
e
t
o
n
e

t
o

t
h
e

s
e
c
o
n
d
a
r
y

a
l
c
o
h
o
l

f
o
l
l
o
w
e
d

b
y

c
o
n
j
u
g
a
t
i
o
n

w
i
t
h

g
l
u
c
u
r
o
n
i
c

a
c
i
d

m
a
y

o
c
c
u
r

a
s

w
i
t
h

t
h
e

a
l
i
p
h
a
t
i
c

a
n
d

m
o
n
o
c
y
c
l
i
c

s
e
c
o
n
d
a
r
y

k
e
t
o
n
e
s

(
1
5

1
7
)
.

T
h
e

C
o
m
m
i
t
t
e
e

t
h
u
s

c
o
n
c
l
u
d
e
d

t
h
a
t

n
o
o
t
k
a
t
o
n
e

d
o
e
s

n
o
t

p
r
e
s
e
n
t

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.
654 ANNEX 5
L1
1
4
0
9

b
-
I
o
n
y
l

a
c
e
t
a
t
e

9
2
%

3
%

a
c
e
t
i
c

a
c
i
d
;

A
c
e
t
i
c

a
c
i
d

(
N
o
.

8
1
)

h
a
s

b
e
e
n

e
v
a
l
u
a
t
e
d

b
y

t
h
e

C
o
m
m
i
t
t
e
e
,

w
h
i
c
h

2
%

b
-
i
o
n
o
l

c
o
n
c
l
u
d
e
d

t
h
a
t

i
t

w
a
s

o
f

n
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.

b
-
I
o
n
o
l

(
N
o
.

3
9
2
)

h
a
s

b
e
e
n

e
v
a
l
u
a
t
e
d

b
y

t
h
e

C
o
m
m
i
t
t
e
e
,

w
h
i
c
h

c
o
n
c
l
u
d
e
d

t
h
a
t

i
t

w
a
s

o
f

n
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.
1
4
1
3

D
,
L
-
M
e
n
t
h
o
l

8
7
%

1
2
%

d
,
l
-
M
e
n
t
h
o
l

T
h
e

s
e
c
o
n
d
a
r
y

c
o
m
p
o
n
e
n
t

i
s

s
t
r
u
c
t
u
r
a
l
l
y

r
e
l
a
t
e
d

t
o

t
h
e

p
r
i
m
a
r
y

(
+
/
-
)
-
p
r
o
p
y
l
e
n
e

2
-
p
r
o
p
y
l
e
n
e

g
l
y
c
o
l

m
a
t
e
r
i
a
l

a
n
d

i
s

e
x
p
e
c
t
e
d

t
o

b
e

m
e
t
a
b
o
l
i
z
e
d

i
n

t
h
e

s
a
m
e

w
a
y
.

g
l
y
c
o
l

c
a
r
b
o
n
a
t
e

c
a
r
b
o
n
a
t
e

B
o
t
h

a
r
e

h
y
d
r
o
l
y
s
e
d

i
n

t
h
e

l
i
v
e
r
,

p
r
o
d
u
c
i
n
g

m
e
n
t
h
o
l

a
n
d

p
r
o
p
y
l
e
n
e

g
l
y
c
o
l
.

M
e
n
t
h
o
l

(
N
o
.

4
2
7
)

a
n
d

p
r
o
p
y
l
e
n
e

g
l
y
c
o
l

(
N
o
.

9
2
5
)

h
a
v
e

b
o
t
h

b
e
e
n

p
r
e
v
i
o
u
s
l
y

e
v
a
l
u
a
t
e
d

b
y

t
h
e

C
o
m
m
i
t
t
e
e

a
n
d

c
o
n
s
i
d
e
r
e
d

n
o
t

t
o

b
e

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.
1
4
1
4

L
-
M
o
n
o
m
e
n
t
h
y
l

7
2
%

2
2

2
4
%

d
i
m
e
n
t
h
y
l

T
h
e

m
e
t
a
b
o
l
i
s
m

o
f

d
i
m
e
n
t
h
y
l

g
l
u
t
a
r
a
t
e

i
s

e
x
p
e
c
t
e
d

t
o

f
o
l
l
o
w

t
h
e

g
l
u
t
a
r
a
t
e

g
l
u
t
a
r
a
t
e
;

2
%

s
a
m
e

p
a
t
h
w
a
y

a
s

t
h
a
t

f
o
r

m
o
n
o
m
e
n
t
h
y
l

g
l
u
t
a
r
a
t
e
.

T
h
e

e
s
t
e
r

g
l
u
t
a
r
i
c

a
c
i
d

f
u
n
c
t
i
o
n
s

a
r
e

h
y
d
r
o
l
y
s
e
d

i
n

v
i
v
o

y
i
e
l
d
i
n
g

m
e
n
t
h
o
l

a
n
d

g
l
u
t
a
r
i
c

a
c
i
d

(
1
8
,

1
9
)
,

a
n
d

t
h
u
s

d
o
e
s

n
o
t

p
r
e
s
e
n
t

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.

M
e
n
t
h
o
l

(
N
o
.

4
2
7
)

h
a
s

b
e
e
n

e
v
a
l
u
a
t
e
d

b
y

t
h
e

C
o
m
m
i
t
t
e
e
,

w
h
i
c
h

c
o
n
c
l
u
d
e
d

t
h
a
t

i
t

w
a
s

o
f

n
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.

G
l
u
t
a
r
i
c

a
c
i
d

h
a
s

n
o
t

b
e
e
n

e
v
a
l
u
a
t
e
d

p
r
e
v
i
o
u
s
l
y

b
y

t
h
e

C
o
m
m
i
t
t
e
e
,

b
u
t

i
s

e
n
d
o
g
e
n
o
u
s

i
n

h
u
m
a
n
s

a
n
d

i
s

s
t
r
u
c
t
u
r
a
l
l
y

r
e
l
a
t
e
d

t
o

v
a
l
e
r
i
c

a
c
i
d

(
N
o
.

9
0
)
,

w
h
i
c
h

h
a
s

b
e
e
n

e
v
a
l
u
a
t
e
d

p
r
e
v
i
o
u
s
l
y

b
y

t
h
e

C
o
m
m
i
t
t
e
e
,

a
n
d

t
h
u
s

d
o
e
s

n
o
t

p
r
e
s
e
n
t

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.
G
.

T
e
t
r
a
h
y
d
r
o
f
u
r
a
n

a
n
d

f
u
r
a
n
o
n
e

d
e
r
i
v
a
t
i
v
e
s
1
4
5
6

4
-
A
c
e
t
o
x
y
-
2
,
5
-
d
i
m
e
t
h
y
l
-

8
5
%

8
%

4
-
h
y
d
r
o
x
y
-
2
,
5
-

4
-
H
y
d
r
o
x
y
-
2
,
5
-
d
i
m
e
t
h
y
l
-
3
(
2
H
)
-
f
u
r
a
n
o
n
e

(
N
o
.

1
4
4
6
)

i
s

e
x
p
e
c
t
e
d

3
(
2
H
)
f
u
r
a
n
o
n
e

d
i
m
e
t
h
y
l
-
3
(
2
H
)
-

t
o

s
h
a
r
e

t
h
e

s
a
m
e

m
e
t
a
b
o
l
i
c

f
a
t
e

a
s

t
h
e

p
r
i
m
a
r
y

m
a
t
e
r
i
a
l
,

i
.
e
.

f
u
r
a
n
o
n
e

c
o
n
j
u
g
a
t
i
o
n

w
i
t
h

g
l
u
c
u
r
o
n
i
c

a
c
i
d

a
n
d

e
x
c
r
e
t
i
o
n

i
n

t
h
e

u
r
i
n
e

(
2
0
)
.

2
-
y
e
a
r

s
t
u
d
y

w
i
t
h

t
h
i
s

m
a
t
e
r
i
a
l

r
e
p
o
r
t
e
d

N
O
E
L

o
f

2
0
0

m
g
/
k
g

b
w

p
e
r

d
a
y

i
n

b
o
t
h

m
a
l
e

a
n
d

f
e
m
a
l
e

r
a
t
s

(
2
1
)
.

T
h
e

C
o
m
m
i
t
t
e
e

c
o
n
c
l
u
d
e
d

t
h
a
t

4
-
h
y
d
r
o
x
y
-
2
,
5
-
d
i
m
e
t
h
y
l
-
3
(
2
H
)
-
f
u
r
a
n
o
n
e

d
o
e
s

n
o
t

p
r
e
s
e
n
t

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.
ANNEX 5 655
L1
S
u
m
m
a
r
y

(
c
o
n
t
d
)
N
o
.

N
a
m
e

M
i
n
i
m
u
m

S
e
c
o
n
d
a
r
y

c
o
m
p
o
n
e
n
t
s

C
o
m
m
e
n
t
s

o
n

s
e
c
o
n
d
a
r
y

c
o
m
p
o
n
e
n
t
s

a
s
s
a
y

v
a
l
u
e

(
%
)
1
4
5
7

(
+
/
-
)
-
2
-
(
5
-
M
e
t
h
y
l
-

9
0
%

6
%

6
-
h
y
d
r
o
x
y
-
2
,
6

r
e
l
a
t
e
d

s
u
b
s
t
a
n
c
e
,

h
y
d
r
o
x
y
c
i
t
r
o
n
e
l
l
a
l

(
N
o
.

6
1
1
)

w
a
s

e
v
a
l
u
a
t
e
d

5
-
v
i
n
y
l
-

(
s
u
m

o
f

d
i
m
e
t
h
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l

2
,
7
-

b
y

t
h
e

C
o
m
m
i
t
t
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e

a
t

i
t
s

1
9
9
9

m
e
e
t
i
n
g

a
n
d

t
e
t
r
a
h
y
d
r
o
f
u
r
a
n
-
2
-
y
l
)

i
s
o
m
e
r
s
)

o
c
t
a
d
i
e
n
a
l

w
a
s

c
o
n
c
l
u
d
e
d

t
o

b
e

o
f

n
o

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

p
r
o
p
i
o
n
a
l
d
e
h
y
d
e

i
n
t
a
k
e
s
.

N
O
E
L

o
f

2
5
0

m
g
/
k
g

b
w

p
e
r

d
a
y

w
a
s

r
e
p
o
r
t
e
d

f
o
r

h
y
d
r
o
x
y
c
i
t
r
o
n
e
l
l
a
l

i
n

2
-
y
e
a
r

s
t
u
d
y

i
n

r
a
t
s

(
2
2
)
.

T
h
e

C
o
m
m
i
t
t
e
e

c
o
n
c
l
u
d
e
d

t
h
a
t

6
-
h
y
d
r
o
x
y
-
2
,
6
-
d
i
m
e
t
h
y
l
-
2
,
7
-
o
c
t
a
d
i
e
n
a
l

d
o
e
s

n
o
t

p
r
e
s
e
n
t

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.
H
.

2
-
P
h
e
n
y
l
p
r
o
p
a
n
o
l

d
e
r
i
v
a
t
i
v
e
s
1
4
6
5

2
-
M
e
t
h
y
l
-
3
-

9
0
%

5
%

2
-
m
e
t
h
y
l
-
3
-

T
h
e

i
n
i
t
i
a
l

s
t
e
p

i
n

t
h
e

m
e
t
a
b
o
l
i
c

p
a
t
h
w
a
y

f
o
r

t
h
e

p
r
i
m
a
r
y

m
a
t
e
r
i
a
l

i
s

(
p
-
i
s
o
p
r
o
p
y
l
p
h
e
n
y
l
)

(
p
-
i
s
o
p
r
o
p
y
l
p
h
e
n
y
l
)

o
x
i
d
a
t
i
o
n

t
o

i
t
s

c
o
r
r
e
s
p
o
n
d
i
n
g

a
c
i
d
.

A
s

t
h
e

s
e
c
o
n
d
a
r
y

m
a
t
e
r
i
a
l

p
r
o
p
i
o
n
a
l
d
e
h
y
d
e

p
r
o
p
i
o
n
i
c

a
c
i
d

i
s

t
h
e

c
o
r
r
e
s
p
o
n
d
i
n
g

a
c
i
d

o
f

t
h
e

p
r
i
m
a
r
y

m
a
t
e
r
i
a
l
,

i
t

i
s

e
x
p
e
c
t
e
d

t
o

s
h
a
r
e

t
h
e

s
a
m
e

m
e
t
a
b
o
l
i
c

f
a
t
e

o
f

c
o
n
j
u
g
a
t
i
o
n

w
i
t
h

g
l
u
c
u
r
o
n
i
c

a
c
i
d

a
n
d

e
x
c
r
e
t
i
o
n

i
n

t
h
e

u
r
i
n
e

(
2
3
)
.

T
h
e

C
o
m
m
i
t
t
e
e

c
o
n
c
l
u
d
e
d

t
h
a
t

2
-
m
e
t
h
y
l
-
3
-
(
p
-
i
s
o
p
r
o
p
y
l
p
h
e
n
y
l
)

p
r
o
p
i
o
n
i
c

a
c
i
d

d
o
e
s

n
o
t

p
r
e
s
e
n
t

s
a
f
e
t
y

c
o
n
c
e
r
n

a
t

e
s
t
i
m
a
t
e
d

c
u
r
r
e
n
t

i
n
t
a
k
e
s
.

656 ANNEX 5
L1
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ANNEX 5 657
L1
WHO FOOD
ADDITIVES
SERIES: 54
J
E
C
F
A
Safety evaluation of certain
food additives
Prepared by the
Sixty-third meeting of the Joint FAO/WHO
Expert Committee on Food Additives (JECFA)
54
S
a
f
e
t
y

e
v
a
l
u
a
t
i
o
n

o
f

c
e
r
t
a
i
n

f
o
o
d

a
d
d
i
t
i
v
e
s
International Programme on Chemical Safety
World Health Organization, Geneva
IPCS
This volume contains monographs prepared at the sixty-third meeting of the
Joint FAO/WHO Expert Committee on Food Additives (JECFA), which met in Geneva,
Switzerland, from 8 to 17 June 2004.
The toxicological monographs in this volume summarize the safety data on a
number of food additives, including benzoyl peroxide, -cyclodextrin, hexose oxidase
from Chondrus crispus expressed in Hansenula polymorpha, lutein from Tagetes
erecta L., peroxyacid antimicrobial solutions containing 1-hydroxyethylidene-1,1-
diphosphonic acid, steviol glycosides, D-tagatose, xylanases from Bacillus subtilis
expressed in B. subtilis and zeaxanthin, and a natural constituent, glycyrrhizinic acid.
Monographs on eight groups of related flavouring agents evaluated by the Procedure
for the Safety Evaluation of Flavouring Agents are also included.
This volume and others in the WHO Food Additives Series contain information
that is useful to those who produce and use food additives and veterinary drugs and
those involved with controlling contaminants in food, government and food regulatory
officers, industrial testing laboratories, toxicological laboratories, and universities.
45404 FOOD ADDITIVES 54 COV 9/2/06 12:42 pm Page 1
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59:153159.
658 ANNEX 5
L1