Report To Efsa - 3mcpd

90-day toxicological study of 3-MCPD and its dipalmitate
SCIENTIFIC REPORT submitted to EFSA
“Comparison between 3-MCPD and its palmitic esters
in a 90-day toxicological study”1
Prepared by
E. Barocelli, A. Corradi, A. Mutti and P.G. Petronini
University of Parma, Parma, Italy
Abstract
A 90-day toxicological study was carried out administering equimolar doses of either
3-MCPD (respectively 29.5, 7.37, and 1.84 mg/kg b.w. per day) or 3-MCPD dipalmitate
(respectively 156.75, 39.19, and 9.78 mg/kg b.w. per day) to both male and female rats
(10 animals per group). Urinary 3-MCPD and 3-MCPD mercapturate were used to monitor
exposure to both 3-MCPD and its dipalmitate, and to assess the bioavailability of the latter
(equimolar doses of dipalmitate gave rise to urinary metabolites lower by 30 % as compared
to the administration of 3-MCPD). Histopathological examination confirmed that the kidney
and, in male rats, the testes are critical organs for 3-MCPD. Changes observed after treatment
with dipalmitate were similar, but milder and proportional to the urinary excretion of
metabolites. The overall picture of nephrotoxicity was consistent with tubulotoxicity, which
in female rats was severe enough to cause acute renal failure in 20 to 50 % of animals
receiving high doses of 3-MCPD (29.5 mg/kg b.w. per day). BMD10 and BMDL10 for
mortality in female rats were 7.4 and 2.3 mg/kg b.w. per day, respectively. At such high
doses, male rats showed extensive testicular toxicity, with extensive cell depletion.
Nephrotoxicity was milder and apparently chronic in nature. Benchmark doses (BMD10) for
severe damage to renal and testicular structures in male rats were 5.6 and 8.4 mg/kg b.w. per
day, respectively. The corresponding BMDL10 were 2.5 and 6.0 mg/kg b.w. per day. Different
BMDs were obtained for 3-MCPD dipalmitate, depending on the contribution of the 3-MCPD
moiety to the molecule and probably a slower and/or lower bioavailability and excretion rate.
In male rats, BMD10 for severe renal and testicular damage were 41.1 and 64.4 mg/kg b.w. per
day, respectively. The corresponding BMDL10 were 17.4 and 44.3 mg/kg b.w. per day.
1
CFP/EFSA/CONTAM/2009/01. Accepted for publication on 22 August 2011.
The present document has been produced and adopted by the bodies identified above as author (s). In accordance with Article
36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author (s) in the context of a grant
agreement between the European Food Safety Authority and the author (s). The present document is published complying with
the transparency principle to which the European Food Safety Authority is subject. It may not be considered as an output
adopted by EFSA. EFSA reserves its rights, view and position as regards the issues addressed and the conclusions reached in
the present document, without prejudice to the rights of the authors.
1
Summary
The final report covers the whole 90-day study with either 3-MCPD (respectively 29.5, 7.37,
and 1.84 mg/kg b.w. per day) or 3-MCPD dipalmitate (respectively 156.75, 39.19, and
9.78 mg/kg b.w. per day). The results can be summarized as follows: (i) urinary excretion of
3-MCPD and 3-MCPD mercapturate can be used to monitor exposure to both 3-MCPD and
its dipalmitate, whereas only trace amounts of β-chloro-lactic acid were recovered in urine
samples from treated animals; (ii) dipalmitate exposure was associated with urinary excretion
rates of both 3-MCPD and 3-MCPD mercapturate about 30 % lower as compared to the same
urinary biomarkers observed after exposure to equimolar doses of 3-MCPD; (iii) after daily
treatment with high doses of 3-MCPD dissolved in corn oil, five female rats (50 %) died from
acute renal failure after weeks (1-5) since the beginning of exposure, whereas only one out of
ten female rats died towards the end of the 90-day treatment with the intermediate dose. In a
subsequent animal loading phase with the same dose (29.5 mg/kg b.w. per day), only 2 out of
10 animals died from acute renal failure, the difference from the first load was not statistically
significant; (iv) 100 % survival was observed in the remaining treatment groups; (v) surviving
females receiving 3-MCPD and the other treatment groups showed changes in nephrotoxicity
biomarkers: the overall picture was consistent with mild tubulotoxicity and hyperfiltration,
consistent with the observed increase in kidney weight and histopathological changes;
(vi) mild and dose-related normochromic anemia was another finding common to all
experimental groups; (vii) histopathological examination confirmed that in male rats the testes
are critical organs: indeed, extensive testicular toxicity was observed in males treated with the
high dose of 3-MCPD and 3-MCPD dipalmitate, with impressive cell depletion. Moderate
testicular damage was observed with the intermediate dose, with consistent increases in the
activity of caspase 3, without any concomitant increase in markers of oxidative stress
(TBARS and carbonyl groups) which suggests apoptotic mechanisms requiring further
investigation; (viii) changes observed after treatment with dipalmitate were similar, but milder
and proportional to the urinary excretion of metabolites, which was lower by about one third
as compared to the groups treated with equimolar doses of 3-MCPD; (ix) Benchmark doses
(BMD10) for severe damage to renal and testicular structures in male rats were 5.6 and
8.4 mg/kg b.w. per day, respectively for 3-MCPD. The corresponding BMDL10 were 2.5 and
6.0 mg/kg b.w. per day, respectively; (x) in parallel with the low dose experiment, the high
dose load was repeated for female rats: although the mortality was somehow lower (20 %), it
was not significantly different from the 50 % mortality observed in the first experiment.
BMD10 and BMDL10 for mortality in female rats were 7.4 and 2.3 mg/kg b.w. per day,
respectively. Different BMDs were obtained for 3-MCPD dipalmitate, depending on the
contribution of the 3-MCPD moiety to the molecule and probably a slower and/or lower
excretion rate. In male rats, BMD10 for severe renal and testicular damage induced by
3-MCPD dipalmitate were 41.1 and 64.4 mg/kg per day, respectively. The corresponding
BMDL10 were 17.4 and 44.3 mg/kg per day.
Key words: 3-MCPD, 3-MCPD dipalmitate, toxicity, 90-day study

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Table of contents
Abstract .................................................................................................................................................... 1
Summary .................................................................................................................................................. 2
Table of contents ...................................................................................................................................... 3
Background .............................................................................................................................................. 4
Terms of reference ................................................................................................................................... 5
Acknowledgements .................................................................................................................................. 6
1. Introduction and objectives ................................................................................................................. 7
1.1. Introduction ............................................................................................................................. 7
1.2. Objectives ............................................................................................................................... 7
2. Materials and methods ........................................................................................................................ 9
2.1. Materials ................................................................................................................................. 9
2.1.1. Synthesis of the 3-MCPD di-esters (dipalmitate) ................................................................... 9
2.1.2. Synthesis of the 3-MCPD mercapturate.................................................................................. 9
2.2. Methods .................................................................................................................................. 9
2.2.1. Animals and housing............................................................................................................... 9
2.2.2. Treatment .............................................................................................................................. 10
2.2.3. Animal observation ............................................................................................................... 11
2.2.4. Analytical methods for biological monitoring ...................................................................... 12
2.2.5. Haematology and clinical chemistry ..................................................................................... 12
2.2.6. Pathology .............................................................................................................................. 13
2.2.7. Statistical analysis ................................................................................................................. 13
3. Results ............................................................................................................................................... 13
3.1. Pathology and histopathology of female rats dying during the experiment .......................... 15
3.2. Gross changes ....................................................................................................................... 16
3.3. Histopathological evaluation................................................................................................. 16
3.4. Body weight .......................................................................................................................... 18
3.5. Water consumption ............................................................................................................... 18
3.6. Food consumption ................................................................................................................. 22
3.7. Biomonitoring results............................................................................................................ 23
3.8. Haematology and clinical chemistry ..................................................................................... 26
3.9. Pathology .............................................................................................................................. 30
3.10. Dose-response and dose-effect analysis ................................................................................ 37
4. Discussion ......................................................................................................................................... 42
Conclusions ............................................................................................................................................ 46
Recommendations .................................................................................................................................. 46
References .............................................................................................................................................. 47
Appendices ............................................................................................................................................. 49
Appendix I .............................................................................................................................................. 49
Appendix II ............................................................................................................................................ 57
Appendix III ........................................................................................................................................... 62
Appendix IV ........................................................................................................................................... 65
Appendix V ............................................................................................................................................ 66
Appendix VI ........................................................................................................................................... 88
Appendix VII.......................................................................................................................................... 98

3
Background
3-Chloropropane-1,2-diol (3-MCPD) is a food processing contaminant that belongs to a group

of compounds called chloropropanols. 3-MCPD was first detected in acid-hydrolyzed
vegetable proteins (acid-HVP) and related products, as a reaction product of phospholipids,
glycerol with hydrochloric acid (Davídek et al., 1982). Since then, studies have shown that
3-MCPD may also occur in products other than HVP such as bakery products, malt,
cooked/cured fish or meat. The Scientific Committee on Food established a tolerable daily
intake (TDI) of 2 μg/kg body weight (b.w.) in 2001 (EC, 2001), based on the lowest-
observed-adverse effect-level (LOAEL) for renal tubule hyperplasia of 1.1 mg/kg b.w. per
day from a rat chronic study (Sunahara et al., 1993). In 2002, the Joint FAO/WHO Expert
Committee on Food Additives also established a provisional maximum TDI (PMTDI) of
2 μg/kg b.w. (Schlatter et al., 2002). A European harmonised maximum level of 20 μg/kg has
been laid down for HVP and soy sauce2.
In December 2007 an official German food and feed control laboratory (Chemisches und
Veterinäruntersuchungsamt Stuttgart, CVUA) reported, for the first time, on findings of high
levels of 3-MCPD esters in edible refined plant oils and fats as well as infant formula
(CVUA, 2007). In addition, the occurrence of 3-MCPD esters in human breast milk has also
been reported (Zelinková et al., 2008). In most foodstuffs the majority of the 3-MCPD present
is ester-linked with fatty acids and only a small percentage is present as free (or unesterified)
3-MCPD (Svejkovska et al., 2004). Since 3-MCPD can be released from the esters by the
action of lipases (Hamlet and Sadd, 2004), 3-MCPD esters can be regarded as a new type of
food contaminant. The extent of the hydrolysis is not yet known, although intestinal lipase
model studies point to a faster release of 3-MCPD from monoesters and a slower release from
diesters (Seefelder et al., 2008).
In 2008, the EFSA Panel on Contaminants in the Food Chain issued a statement regarding
these findings on high levels of 3-MCPD esters in a wide range of foodstuffs (EFSA, 2008).
The statement concluded that, at that moment, there was no scientific evidence to dispute the
assumption that 100 % of 3-MCPD is released from its esters in humans, as estimated by the
German Federal Institute for Risk Assessment (BfR) (BfR, 2007). The Panel also recognised
the need for kinetic studies with 3-MCPD esters to gain further information on the time-
course and site of the release of 3-MCPD from its esters in vivo. Studies on the bioavailability
of 3-MCPD esters are currently being commissioned by the German Federal Ministry of
Consumers Protection (BMELV) (Weiβhaar, 2008).
Regarding the toxicological profile, there are a number of animal toxicological studies on free
3-MCPD, showing that this compound induces infertility in rats, suppression of the immune
function and it has been found to be genotoxic in most in vitro assays, although negative
results were obtained in vivo. In addition, clear evidence of carcinogenic activity in male rats
and some evidence of carcinogenicity activity in female rats has been reported (Cho et al.,
2
Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain
contaminants in foodstuffs. OJ L 364, 20.12.2006, p 5-24.

4
2008). However, there is a lack of toxicological data on the 3-MCPD esters. This means it is
difficult to predict the impact of these esters on human health when such compounds are
present in foodstuffs.
A risk assessment on 3-MCPD esters has not yet been conducted at an European level, but it
is expected that the European Commission may ask EFSA to assess the risks to human health
related to 3-MCPD esters in the near future. To carry out this risk assessment a hazard
identification and hazard characterization is required for the 3-MCPD esters that can be
compared to free (or unesterified) 3-MCPD.
Terms of reference
EFSA sought proposals for a 90-day rat study to evaluate the toxicological profile of 3-MCPD
esters (mono- and di-ester) and compare it to that of free (or unesterified) 3-MCPD (Figure 1).
Figure 1: Structure of 3-MCPD and its mono- and di-esters.
Studies must be preferably conducted using internationally agreed protocols (test methods
described by OECD or in European Commission Directives) and preferably carried out
according to the principles of good laboratory practices (GLP). The applicant must describe in
detail his proposed approach including the following aspects:
1. Selection and description of the test substances (3-MCPD and 3-MCPD esters). Reasoning
for the selection of particular 3-MCPD fatty acids mono- and di-esters should be given as
well as details of the preparation of the stock solutions and further dilutions of the test
substances.
2. Detailed description of the 90-day study in Wistar rats to be followed, such as:
• test animals (gender, number of animals per experimental group),
• treatment (administration route, doses and rationale for the choice - including details on
the range finding study, duration and frequency of the treatment, negative and positive
control groups).
3. Animal husbandry and maintenance.
4. Quality assurance and quality control (QA/QC) measures.
5. Type and frequency of general clinical observations (including body weight and food/water
consumption), haematology examinations, clinical biochemistry determinations and type of
pathology tests, such as gross necropsy and histopathology.

5
6. Information on the analytical method used for the determination of 3-MCPD and its esters
in biological tissues. Performance of the method to achieve the required limits of detection
in biological samples. Method validation and QC/QA measures.
Acknowledgements
This grant was awarded by EFSA to: Department of Experimental Medicine, University of
Parma
Beneficiary: Professor Pier Giorgio Petronini
Grant title: Comparison between 3-MCPD and its palmitic esters in a 90-day toxicological
study.
Grant number: CFP/EFSA/CONTAM/2009/01
The Department o Experimental Medicine, University of Parma, acknowledges the following

persons involved in the project:
Prof. Marco Mor and Dr. Stefano Vezzosi (University of Parma) have been involved in the
project with the specific task to synthesize the 3-MCPD di-esters (dipalmitate).
Dr. Martina Cirlini (post-doctoral research fellow at the University of Parma) and Dr. Roberta
Andreoli collaborated to synthesize 3-MCPD mercapturate and to set-up a suitable analytical
method for its analysis in urine as a biomarker of exposure.
Dr. Francesca Saccani and Dr. Andrea Cavazzoni were directly involved in animal husbandry,
treatment and observation.
Dr. Matteo Goldoni, PhD, was responsible for the database maintenance, statistical analysis,
and BMD modelling.
Dr. Rossella Alinovi, Dr. Silvana Pinelli, Dr. Stefania Cavazzini, and Dr. Paola Mozzoni
collaborated to measure nephrotoxicity biomarkers, and haematology and clinical chemistry
parameters.
Rats have been necropsied by staff of the Veterinary Medicine of University of Parma (Prof.
Anna Maria Cantoni, and Drs. Rosanna Di Lecce, Cristina Marchetti, Elena Muzzoni,
Benedetta Passeri, Mrs. Paola Gianelli).

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1. INTRODUCTION AND OBJECTIVES
1.1. INTRODUCTION
The present study is in response to the EFSA call for proposals for a 90-day rat study to
evaluate the toxicological profile of 3-chloropropane-1,2-diol (3-MCPD) esters (mono- and
di-ester) and compare it to that of free (or unesterified) 3-MCPD.
1.2. OBJECTIVES
The aim of this study is to compare the toxicity of 3-MCPD dipalmitate and free 3-MCPD in
a repeated dose 90-day oral toxicity study performed on male and female rats.
Considering that only a small part (<15 %) of the 3-MCPD bound in esters is in fact bound in
monoesters (Seefelder et al., 2008), the studies were performed using only di-ester form.
Palmitic acid as fatty acid was proposed due to the fact that highest levels of 3-MCPD were
found in palm oil and it is the most used di-ester to study the formation and the composition
of 3-MCPD esters in vitro (Svejkovskà et al., 2006).
The Gantt scheme depicted in Table 1 summarizes the main objectives achieved by the end
of the project. The milestones proposed in the project have been achieved and the deliverables
are available from the individual partners as outlined below. Owing to the high mortality rate
(50 %) in female rats treated with high doses of 3-MCPD (29.5 mg/kg b.w. per day), this
experiment was repeated.

7
Table 1: Activities and milestones.
Milestone\Month 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Kick off meeting x
Synthesis of di-esters x x
Dosing animals (ctrl and 29.5 mg/kg and 156.75
x x x
mg/kg for 3-MCPD and its di-ester, respectively)
Animal observation x x x
Collection of 24h urine, and then sacrifice for
x
Pathology, Haematology and Clinical Biochemistry
Analysis of 3-MCPD and 3-MCPD metabolites x
st
1 Interim report x
Interim meeting 1 x
Dosing animals (ctrl and 7.37 mg/kg and 39.19 mg/kg
x x x
for 3-MCPD and its di-ester, respectively)
x
Analysis of 3-MCPD and
x
3-MCPD metabolites
nd
2 Interim report x
Interim meeting 2 x
Dosing animals (ctrl and 1.84 mg/kg and 9.78 mg/kg
x x x
for 3-MCPD and its di-ester, respectively)
x x x
Statistical analysis x
rd
3 Interim report x
Interim meeting 3 x
Dosing animals (ctrl and 29.5 mg/kg mg/kg for 3- x x x
MCPD)
x
th
4 Interim report x
Interim meeting 4 x
Statistical analysis x
Final report x

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2. MATERIALS AND METHODS
2.1. MATERIALS
3-MCPD 98 % pure was purchased from Sigma Aldrich Inc. (Cas No. 96-24-2). Palmitic di-
ester (dipalmitate) of 3-MCPD was synthesized by Pharmaceutical Department, University of
Parma.
2.1.1. SYNTHESIS OF THE 3-MCPD DI-ESTERS (DIPALMITATE)

In order to implement the project, the 3-MCPD dipalmitate had to be synthesized. More than
100 g were required and have been produced with a satisfactory degree of purity.
Details are given in Appendix I.
2.1.2. SYNTHESIS OF THE 3-MCPD MERCAPTURATE

Likewise, it was necessary to synthesize 3-MCPD mercapturate and to set-up a suitable
analytical method for its measurement in urine as a biomarker of exposure. The development
of this analytical method was considered necessary because in addition to the oxidative
metabolism of 3-MCPD – leading to β-chlorolactic acid and oxalic acid, respectively – a
second pathway of 3-MCPD biotransformation proceeds through detoxification by
conjugation with glutathione, yielding S-(2,3-dihydroxypropylcysteine) and the
corresponding mercapturic acid [N-acetyl-S-(2,3-dihydroxypropyl)cysteine].
Notably, the mercapturate pathway is known to be very important in rats. Moreover, some
mercapturates could be bioactivated by the action of cysteine S-conjugate β-lyases in the
kidney, which makes the exploration of this pathway mandatory for better understanding a
potential mechanism of nephro-carcinogenicity (Monks et al., 1990; Newman et al., 2007;
Rankin et al., 2008).
For details on the synthesis, see Appendix II.
2.2. METHODS
2.2.1. ANIMALS AND HOUSING

Experiments (authorization n. 50/2010B from Ministero della Salute) started on March 10th,
2010 with the 5 control males.
Fifty young healthy adult Wistar rats (200-220 g) (Charles River Laboratories) of both sexes
(25 male and 25 female nulliparous and non-pregnant) were employed, after an initial period
of 7 days, during which the animals acclimated to laboratory conditions. This strain was
selected to facilitate comparisons and interpretation of results in terms of kinetics respect to
Fischer 344, which on the other hand have been used in the published 2-year carcinogenicity
study (Cho et al., 2008).

9
Rats were housed in monitored environmental conditions: temperature 22±3°C, relative

humidity 50-60 %, light/dark cycles of 12 hours each. Each rat was identified with a unique
number and housed individually. All cages were arranged in such a way that possible effects
due to cage placement were minimized.
Animals were fed with a standard diet for rats (pellets 4RF21 from Mucedola S.r.l., Settimo
Milanese, Italy) whose GLP certificate does not include 3-MCPD among its components.
Analyses for possible 3-MCPD contamination by LC-MS/MS indicated that the pellets
contain 60 μg/kg, whereas the corn oil used as a vehicle contains about 30 μg/kg. No
3-MCPD was found in drinking water.
After the conclusion of the 90-day treatment with the high doses of 3-MCPD and its
dipalmitate we started the treatment with a four times lower dosage of the compounds. This
second part of the experiment (authorization n. 50/2010B from Ministero della Salute) started
on July 6th, 2010 with 5 male rats used as control. Fifty additional young healthy adult Wistar
rats (200-220 g) (Charles River Laboratories) of both sexes (25 male and 25 female
nulliparous and non-pregnant) were employed, after an initial period of 7 days, during which
the animals acclimated to laboratory conditions.
After the 90-day treatment with the intermediate doses of 3-MCPD and its dipalmitate, the
last treatment was carried out with a four times lower dosage of the compounds, i.e. with 1/16
of high doses. This third part of the experiment started on November 9th, 2010 with 5 male
rats used as control.
Fifty additional young healthy adult Wistar rats (200-220 g) (Charles River Laboratories) of
both sexes (25 male and 25 female nulliparous and non-pregnant) were employed, after an
initial period of 7 days, during which the animals acclimated to laboratory conditions. In order
to confirm the findings obtained with 3-MCPD high dose in female rats, 10 additional female
Wistar rats were challenged with 3-MCPD high dose after a 7 day period of acclimatisation.
2.2.2. TREATMENT
Both 3-MCPD and its dipalmitate were dissolved daily in corn oil and administered daily by
oral gavage in 2 mL/kg. The choice of using corn oil instead of drinking water was based on
the reasoning that a different vehicle could result in variable exposure duration and absorption
rate, thus introducing a bias in comparisons between the effects of 3-MCPD and its
dipalmitate. Rats included in the study were randomly divided into three groups:
‐ 10 rats (5 male and 5 female) receiving daily by oral gavage 2 mL/kg of corn oil, served
as control (Male Control n. 1-5; Female Control n. 26-30).
‐ 20 rats (10 male and 10 female) receiving 3-MCPD (29.5 mg/kg b.w. per day). The dose
level was based on data available from the literature: see the 2-year study on rats
performed by Cho et al. (2008), who used 29.5 mg/kg b.w. per day (400 ppm) in drinking
water as the high dose level (Male 3-MCPD n. 16-25; Female 3-MCPD n. 41-50).
‐ 20 rats (10 male and 10 female) receiving dipalmitate [156.75 mg/kg b.w. per day: in
order to achieve equimolar doses of free 3-MCPD, taking into account their different
molecular weight (MW) and assuming 100 % hydrolysis by intestinal lipases. Male
3-MCPD dipalmitate n. 6-15; Female dipalmitate n. 31-40].

10
Rats included in the study for the experiment with intermediate doses were randomly divided
into three groups:
‐ 10 rats (5 male and 5 female) receiving daily oral 2 ml/kg of corn oil, served as control
(Male Control n. 51-55; Female Control n. 76-80).
‐ 20 rats (10 male and 10 female) receiving orally 3-MCPD (7.37 mg/kg b.w. per day). The
dose level was four times lower than the dose administered during the first
experimentation (Male 3-MCPD n. 66-75; Female 3-MCPD n. 91-100).
‐ 20 rats (10 male and 10 female) receiving orally dipalmitate [39.19 mg/kg b.w. per day:
in order to achieve equimolar doses of free 3-MCPD, taking into account their different
molecular weight (MW). Male dipalmitate n. 56-65; Female dipalmitate n. 81-90].
Rats included in the study for the experiment with the lowest doses were randomly divided
into four groups:
‐ 15 rats (5 male and 10 female) receiving daily oral 2 ml/kg of corn oil, served as control
(Male Control n. 101-105; Female Control n. 126-135).
‐ 20 rats (10 male and 10 female) receiving orally 3-MCPD (1.84 mg/kg b.w. per day). The
dose level was four times lower than the dose administered during the previous
experimentation (Male 3-MCPD n. 116-125; Female 3-MCPD n. 146-155).
‐ 20 rats (10 male and 10 female) receiving orally dipalmitate [9.78 mg/kg b.w. per day: in
order to achieve equimolar doses of free 3-MCPD, taking into account their different
molecular weight (MW). Male dipalmitate n. 106-115; Female dipalmitate n. 136-145].
‐ 10 additional female rats were included in this third treatment group, receiving the high
dose of 3-MCPD tested (29.5 mg/kg b.w. per day) in order to confirm or rule out the
survival data previous obtained (Female 3-MCPD n. 156-165).
The dosing of all experimental groups is summarized in Table 2.
Table 2: Summary of experiments indicating dosage and periods of 90-day studies.

Compound Dose Dose Conc Starting treatment day
(mmol/kg) (mg/kg) solution (male/female)
per day per day (mg/ml)
3-MCPD 0.267 29.5 14.75 11 March/18 March
3-MCPD dipalmitate 0.267 156.75 70.00 12 March/19 March
3-MCPD 0.067 7.37 3.68 8 July/15 July
3-MCPD dipalmitate 0.067 39.19 19.59 7 July/14 July
3-MCPD 0.017 1.84 0.92 11 November/18 November
3-MCPD dipalmitate 0.017 9.78 4.89 10 November/17 November
Except for treatment with the test substances, animals in the control group were handled in an
identical manner to those in the test groups.
2.2.3. ANIMAL OBSERVATION

During the period of administration, the animals were observed closely for any sign of
toxicity. General clinical conditions were assessed and recorded once a day. Moreover, twice

11
daily, usually at the beginning and at the end of each day, all rats were inspected for signs of
morbidity and mortality. Signs included changes in skin, fur, eyes, mucous membranes,
occurrence of secretions and excretions and autonomic activity. All animals were weighed
once a week, whereas food and water consumption was measured and recorded daily.
Body weight
The animals’ body weight was measured weekly and reported as mean week value for each
group.
Water consumption
The daily volume of water consumed (mL) by each animal was measured throughout the
experiment. The mean week consumption for each rat was calculated and the average value of
every group was reported.
Food consumption
The quantity of food eaten daily (g) by each rat was measured and recorded during the whole
study. The mean week consumption for each rat was calculated and the average value of
every group was reported.
2.2.4. ANALYTICAL METHODS FOR BIOLOGICAL MONITORING

A Tandem Liquid Chromatography Mass Spectrometry (LC-MS/MS) method has been
developed and validated (details are given in Annex II) for use both for biological monitoring
purposes and to address mechanistic issues. Briefly, LC-MS/MS analysis was carried out a
AB Sciex API 4000 triple-quadrupole mass spectrometer (Sciex, Concord, Canada) equipped
with a Turbo Ion SprayTM interface. Chromatography was performed on an Atlantis®dC18
column (100 × 2.0 mm i.d., 3µm; Waters, Milford, MA, USA) using variable proportion of
20 mM aqueous acetic acid and methanol/acetonitrile (MeOH/AcCN, 95/5,v/v) mixture as the
mobile phase. The analytes were ionized in negative ion mode and the detection was obtained
in selected-reaction mode (SRM). Details on method development and validation are given in
Appendix III.
Owing to the implication of the formation of glycidol as intermediate metabolite in terms of

genotoxic potential of both 3-MCPD and its esters, the mercapturate pathway is particularly
important.
2.2.5. HAEMATOLOGY AND CLINICAL CHEMISTRY

In blood samples, collected in tubes containing potassium EDTA as an anticoagulant, the
following haematological parameters were measured using a Coulter LH 500 (Beckman
Coulter): haematocrit, haemoglobin concentration, erythrocyte count, total and differential
leukocyte count, and platelet count. By an ILab 300 Plus Clinical Chemistry System
(Instrumentation Laboratory) the following clinical chemistry parameters were measured in
serum: total proteins, triglycerides, cholesterol, glucose, creatinine, direct bilirubin, total

12
bilirubin, alkaline phosphatase, urea, aspartate aminotransferase, alanine aminotransferase,

lactate dehydrogenase, iron.
2.2.6. PATHOLOGY
Ninety days after each treatment rats have been killed in saturated chamber of vapour of ethyl
ether and CO2 and necropsied by staff of the Veterinary Medicine of Parma University.
During necropsies cerebrum, cerebellum and medulla/pons, spinal cord, pituitary, thyroid,
parathyroid, thymus, oesophagus, salivary glands, stomach, small and large intestines
(including Peyer’s patches), liver, pancreas, kidneys, adrenals, spleen, heart, trachea and
lungs, aorta, gonads, uterus, accessory sex organs, female mammary gland, prostate, urinary
bladder, lymph nodes, peripheral nerve (sciatic or tibial) in close proximity to the muscle, a
section of bone marrow and a fresh bone marrow aspirate, skin and eyes have been collected
and formalin fixed 10 %. Thymus, heart and aorta, salivary glands, spleen, liver, kidneys,
adrenals, gonads and accessory sex glands were weighted (Appendix VII).
Paraffin sections, 4-5 µm thick, were stained with Hematoxylin and Eosin and PAS reaction.
Slides have been observed using a Nikon Eclipse E800 microscope (Nikon Corporation,
Japan). Images were captured at 4x, 10x, 20x magnifications using DIGITAL SIGHT DS-Fi1
Camera.
The rats that died during the testing period were necropsied by staff of the Veterinary
Medicine of Parma University (Appendix VI).
2.2.7. STATISTICAL ANALYSIS

Data were expressed as mean (SD) when the deviations from normality were not significant.
In the case of asymmetry in the distributions, data were log-transformed and geometric mean
(GM) (Geometric Standard Deviations – GSD) were reported. To compare different groups, a
two-way ANOVA using treatment and weeks of treatment, gender and treatment, or treatment
and dose as factors was performed, considering either raw or log-transformed data if
necessary to obtain a normal distribution. Linear regression parametrical models were used on
linear-linear scale, linear-log scale or log-log scale on the basis of the distribution of residues.
The comparison between the dipalmitate and 3-MCPD was always performed using the
Tukey’s or Bonferroni’s post hoc test. A p<0.05 was always considered as significant.
Statistical tests were performed by means of PASW 18 (SPSS, USA) or Graphpad Prism 5.0
(Graphpad, La Jolla, CA, USA). Finally, all the nonlinear regression models coherent with the
data were tested by means of BMDS 2.1.2 (EPA, USA) and the calculation of BMD (BMDL)
was performed on the best fit curve among the selected models (Analysis of deviance table).
3. RESULTS
Only 13 out of 20 female rats treated with 3-MCPD high dose survived until 13th week and
7 died respectively on the 7th, 8th, 9th, 9th, 38th, 48th and 83rd day of treatment (Figure 2).
However, all survived female rats treated with 3-MCPD 29.5 mg/kg b.w. per day showed
signs of morbidity during the 90 days of the study, such as impaired aspect and marked loss of

13
fur, increase of water daily intake and also an episode of severe respiratory crisis (rat #164 on
65th day of treatment).
Regarding the intermediate dose of 3-MCPD (7.37 mg/kg b.w. per day) only one out of ten
females died during the last week of treatment, whereas all animals receiving the lower dose
of 3-MCPD (1.84 mg/kg b.w. per day) survived until 13th week. The total incidence of
mortality in the female group was 10 % (1 out of 10 rats) at the intermediate dose and 35 %
(7 out of 20 animals) at the high dose. The latter mortality rate resulted from 50 % (5/10) and
20 % (2/10) observed in two separate experiments with the same loadings.
The survival curve for female rats receiving 29.5 mg/kg b.w. per day results from two
separate doses giving rise to mortality rates of 50 and 20 %, respectively (Figure 2). Owing to
the small sample size, such a difference was not statistically significant, which prompted us to
combine the results in a single survival curve (Figure 3).
Figure 2: Survival curves: 5 out of 10 Figure 3: Combined survival curves

female rats treated with the shows that 7 out of 20 died
high dose of 3-MCPD died during the 90-d observation
within the first two months of period. One female rat
treatment, whereas only 2 out treated with intermediate
of 10 died upon repetition of doses also died in proximity
the experiment. of the end of the experiment.
Male rats of all the experimental groups as well as female rats of control and dipalmitate
groups survived until the end of 90-day treatment in good general clinical conditions. Male
and female rats treated with the high dose of dipalmitate sometimes showed transitory sign of
discomfort (orripilation, eye and nasal mucosa anomalous secretion) like rats treated with
3-MCPD high dose.
During treatments periods, rats were daily monitored for signs of discomfort, such as scruffy
haircoat, changes in food intake, impaired mobility and red or brow staining around eyes or

14
nose (chromodacryorrhea: secretion of so-called "bloody tears" from the Harderian gland
which nearly circumscribes the eye within the bony orbit).
Episodes of stress and discomfort were detected in treated animals of both sexes, (especially
at high dose) and displayed an intermittent trend: rats usually recovered from this condition in
few days. Total days of discomfort during the 13 weeks treatment were counted for each rat
and represented in Figure 4.
Treatment with 3-MCPD 29.5 mg/kg b.w. per day significantly increased the total days of
discomfort in both female and male rats compared to control, whereas the corresponding
dosage of 3-MCPD dipalmitate (156.75 mg/kg b.w. per day) significantly augmented it only
in female animals.
Figure 4: Duration of discomfort episodes in male and female rats treated with the
different dose levels of 3-MCPD and its dipalmitate compared to control
group. *P<0.05, **P<0.01, ***P<0.001 ANOVA followed by Tukey’s post-hoc
test.
3.1. PATHOLOGY AND HISTOPATHOLOGY OF FEMALE RATS DYING DURING

THE EXPERIMENT
Seven female rats (rats 46, 47, 48, 49, 50, 162 and 163) treated with 3-MCPD highest dose
(29.5 mg/kg b.w. per day) died on the 7th, 8th, 9th, 9th, 38th, 48th and 83rd day of treatment.
During the administration of the intermediate dose of 3-MCPD (7.37 mg/kg b.w. per day),
only one (rat 94) out of ten females died in the last week of treatment.

15
The rats that died during the experiment were necropsied and histopathology was performed
by staff of the Veterinary Medicine of University of Parma (see Appendices V to VII). No
samples for histopathology were collected from female rats 46 and 49 because they were
submitted frozen.
3.2. GROSS CHANGES

All animals that died at different stages of the experimental protocol showed renal cardiac and
pulmonary lesions; kidney were pale, yellowish and enlarged; passive hyperemia and edema
were observed in the lungs, the heart was frequently dilated and degenerated. In female rats
died after few administrations, haemorrhagic suffusions in the neck and sternal regions were
observed. One rat (# 94) showed pancreatic haemorrhages.
3.3. HISTOPATHOLOGICAL EVALUATION

The morphological changes were consistent with acute nephrosis and were located mostly in
the proximal and distal tubules in the cortex (Figure 5).

16
Kidney: tubular necrosis and mineralisation Kidney: epithelial tubular

PAS 20x. cells necrosis PAS 20x
Kidney: hydropic-vacuolar degeneration of Heart: myocardosis

epithelial cells and mineralisation HE 20x.
HE20x.
Lung: congestion and alveolar edema Liver: centrolobular hepatocyte

HE 20x. degeneration H-E 10x.
Figure 5: Histopathological picture of kidney, heart, lung, and liver of female rats died
after administration of repeated doses of 3-MCPD (29.5 mg/kg b.w. per day).

17
The nephrosis was characterized by swelling and vacuolation of the tubular epithelium,
tubular dilation and multifocal areas of tubular epithelial cell degeneration and necrosis. Lung
parenchima shows congestion end alveolar oedema; the heart shows degeneration of
myocardial fibres and infiltrates of inflammatory cells.
3.4. BODY WEIGHT

Overall, the body weight of control, 3-MCPD-treated and dipalmitate-treated groups
increased gradually and progressively with age, in accordance with the standard growth curve
provided by Charles Rivers Lab. for the Wistar rat strain. Male rats gained approximately
150 % body weight, whereas female rats showed an increase of 50 % body weight. No
significant differences in body weight gain were detected among treatment groups of the same
sex (Figure 6). A minimal, though significant reduction of body weight gain was observed in
the last weeks of treatment of male rats with 3-MCPD 1.84 mg/kg b.w. per day.
3.5. WATER CONSUMPTION

Treatment with 3-MCPD (29.5 mg/kg b.w. per day) induced a significant increase in daily
water consumption at the 13th week of exposure in male rats, whereas administration of its
dipalmitate (156.75 mg/kg b.w. per day) significantly increased it on week 12 (Table 3 and
Figure 7).
In female rats, 3-MCPD administration was associated with significant increased water
consumption at 6th, 7th, 8th, 9th and 13th weeks of treatment, while 3-MCPD dipalmitate
administration significantly increased water assumption on weeks 1, 2, 3, 5, 6, 11 and 12.
This finding is confirmed by data obtained with comparable treatment performed in
10 additional Wistar female rats in the last phase of the study [Comparison among groups was
made using analysis of variance, two-way ANOVA, followed by Bonferroni’s post-test.
*p<0.05, **p<0.01].
Thirteen weeks treatment with 3-MCPD (7.37 mg/kg b.w. per day or 1.84 mg/kg b.w. per
day) or its dipalmitate (39.19 mg/kg b.w. per day or 9.78 mg/kg b.w. per day) did not induce
any notable changes in daily water consumption of both male and female rats compared to
controls at variance with the results obtained during high doses treatment.

18
Figure 6: Effects on body weight gain in male and female rats as a function of duration of
exposure to either 3-MCPD or its dipalmitate. Top panels represent high
dosage, middle panels the intermediate doses and the bottom panels the low
doses and from an additional group of 10 female rats receiving 3-MCPD
29.5 mg/kg b.w. per day. *p<0.05, two-way Anova followed by Bonferroni’s
post-hoc test.

19
Table 3: Daily water consumption (ml) of male and female rats treated with 3-MCPD
(29.5 mg/kg b.w. per day) and its dipalmitate (156.75 mg/kg b.w. per day)
(values are expressed as mean±SEM). *p<0.05; **p<0.01; two-way Anova
followed by Bonferroni’s post-hoc test.
Week Control 3-MCPD 3-MCPD Control 3-MCPD 3-MCPD

No. (males) (males) dipalmitate (females) (females) dipalmitate
(males) (females)
1 42.4±2.7 45.1±1.0 46.1±1.2 31.1±0.6 37.2±2.3 43.6±1.9*
2 38.1±1.6 50.3±4.8 47.6±2.9 34.5±0.6 44.9±3.8 50.1±2.4**
3 38.9±1.2 47.8±6.0 52.4±3.3 41.7±0.7 50.4±4.3 54.4±1.7*
4 42.7±1.9 56.6±3.5 54.1±2.8 37.2±1.2 49.4±4.2 47.5±2.1
5 37.8±1.8 54.9±5.5 50.7±2.9 35.0±1.6 47.0±2.9 49.2±2.9**
6 35.1±2.5 51.8±4.2 49.8±2.9 35.7±0.7 50.6±1.5* 49.3±2.6*
7 37.3±2.3 57.2±5.2 55.0±3.6 38.7±2.3 53.9±4.7* 49.8±2.1
8 37.9±2.8 57.5±5.6 56.6±3.6 36.7±1.0 51.0±2.8* 46.5±1.8
9 37.4±3.0 57.0±7.4 54.2±4.0 37.7±1.8 51.6±4.6* 48.1±2.6
10 37.6±2.3 54.1±6.0 54.8±3.7 39.3±2.3 52.1±3.6 52.5±3.1
11 39.5±2.5 57.4±6.4 58.6±4.4 38.8±1.9 50.6±3.5 54.4±2.2*
12 36.8±1.5 56.7±7.6 60.5±4.8* 38.6±2.2 48.7±4.4 49.8±2.4**
13 39.5±2.2 61.3±6.7* 57.9±5.1 39.2±2.5 56.7±3.8** 47.8±2.4

20
Figure 7: Upper graphs show the time course of daily water consumption (ml) in male
and female rats, as a function of duration of exposure 3-MCPD (29.5 mg/kg per
day) and its dipalmitate (156.75 mg/kg b.w. per day): *p<0.05; **p<0.01; two-
way Anova followed by Bonferroni’s post-test. Graphs in the middle show data
recorded in rats receiving either 3-MCPD (7.37 mg/kg b.w. per day) or its
dipalmitate (39.19 mg/kg b.w. per day), whereas graphs on the bottom refer to
3-MCPD (1.84 mg/kg b.w. per day) or its dipalmitate (9.78 mg/kg b.w. per day)
treatments and from an additional group of 10 female rats receiving 3-MCPD
29.5 mg/kg b.w. per day.

21
3.6. FOOD CONSUMPTION

No significant difference in food consumption was detected between the groups except a
scattered but significant reduction of food consumption of male rats treated with 3-MCPD
1.84 mg/kg b.w. per day compared to control on weeks 5, 8 and 9 (Figure 8). Upper panels
refer to the high dose experiments implying administration of either 3-MCPD (29.5 mg/kg
b.w. per day) or its dipalmitate (156.75 mg/kg b.w. per day), middle panels show data of the
intermediate dose experiments, i.e. exposure to either 3-MCPD (7.37 mg/kg b.w. per day) and
its dipalmitate (39.19 mg/kg b.w. per day) and lower panels display data from rats treated
with either 3-MCPD (1.84 mg/kg b.w. per day) or dipalmitate (9.78 mg/kg b.w. per day) and
from an additional group of 10 females receiving 3-MCPD 29.5 mg/kg b.w. per day.
Figure 8: Changes in daily food consumption (grams) by both male and female
rats as a function of either duration of exposure or dose of either
3-MCPD or its dipalmitate. *p<0.05; **p<0.01; two-way Anova
followed by Bonferroni’s post-hoc test.

22
3.7. BIOMONITORING RESULTS

In urine samples, the presence of glucuronated metabolites was excluded, and β-chloro-lactic
acid (ppb or μg/L) was detectable in trace amounts only in the first aliquot collected for 5 h
after dosing. Of the other metabolites, only mercapturates were determined and found to occur
in quantitatively relevant rates (Tables 4a to 4c).
Gender had a significant effect, values recorded in females being one third lower compared to
males. A gender by treatment interaction was noted for urinary mercapturates, but not by
urinary 3-MCPD. The most striking result was that mercapturate levels recorded in surviving
females was lower by 50 % compared to the corresponding values measured in males.
Table 4a: Urinary excretion of 3-MCPD and its mercapturate in male and female rats
treated with 29.5 mg/kg b.w. per day of 3-MCPD or 156.75 mg/kg b.w. per day
of its dipalmitate; values are expressed as geometric mean (GSD).
Control Dipalmitate 3-MCPD Statistical significance (p)

Total Gender Treatment Gender* Dipalmitate
Treatment vs. 3-MCPD
3-MCPD M 0.003 (2.0)* 0.27 (1.6)* 0.38 (1.4)* ns <0.001 ns 0.001
(mg/24h) F 0.004 (2.2)* 0.15 (1.3)* 0.45 (2.2)*
Mercapturate M 0.027 (2.0)* 2.13 (1.9)* 3.87 (1.4)* <0.001 <0.001 0.004 ns
(mg/24h) F 0.021 (1.6)* 1.38 (1.2)* 1.21 (1.4)*
β-Chlorolactic M <0.01 <0.01 0.15 Statistics not feasible
acid (mg/24h) F <0.01 <0.01 0.15
3-MCPD (% of M - 1.9 (1.6)* 2.6 (1.3)* 0.021 <0.001 0.014 (only 2
dose) F - 1.8 (1.3)* 5.8 (2.1)* groups)
Mercapturate M - 6.9 (2.0)* 12.5 (1.4)* ns 0.041 0.021 (only 2
(% of dose) F - 7.6 (1.2)* 7.3 (1.4)* groups)
Table 4b: Urinary excretion of 3-MCPD and its mercapturate in male and female rats
treated with 7.37 mg/kg b.w. per day of 3-MCPD or 39.19 mg/kg b.w. per day
of its dipalmitate; values are expressed as geometric mean (GSD).

Treatment vs. 3-MCPD
3-MCPD M 0.003 (2.0)* 0.09 (1.4)* 0.13 (1.3)* 0.04 <0.001 0.03 ns
(mg/24h) F 0.004 (2.2)* 0.06 (1.4)* 0.05 (5.7)*
Mercapturate M 0.027 (2.0)* 0.71 (1.3)* 0.96 (1.3)* <0.001 <0.001 0.002 ns
(mg/24h) F 0.021 (1.6)* 0.31 (1.1)* 0.25 (2.9)*
β-Chlorolactic M <0.01 0.02 (1.5) 0.03 (1.6) Statistics not feasible
acid (mg/24h) F 0.03 (1.8) 0.03 (2.0) 0.10 (9.8)
3-MCPD (% of M - 2.3 (1.3)* 3.4 (1.3)* ns ns ns (only 2
dose) F - 3.1 (1.4)* 2.3 (5.1)* groups)
Mercapturate M - 8.7 (1.2)* 11.5 (1.2)* 0.007 ns ns (only 2
(% of dose) F - 7.1 (1.1)* 5.4 (2.8)* groups)

23
Table 4c: Urinary excretion of 3-MCPD and its mercapturate in male and female rats
treated with 1.84 mg/kg b.w. per day of 3-MCPD or 9.78 mg/kg b.w. per day of
its dipalmitate; values are expressed as geometric mean (GSD).

Treatment vs 3-MCPD
3-MCPD M 0.003 (2.0)* 0.06 (1.7)* 0.05 (2.4)* ns <0.001 ns ns
(mg/24h) F 0.004 (2.2)* 0.05 (1.5)* 0.05 (1.7)*
Mercapturate M 0.027 (2.0)* 0.41 (1.3)* 0.51 (1.6)* <0.001 <0.001 <0.001 ns
(mg/24h) F 0.021 (1.6)* 0.12 (1.2)* 0.11 (1.3)*
β-Chlorolactic M <0.01 <0.01 <0.01 Statistics not feasible
acid (mg/24h) F <0.01 <0.01 <0.01
3-MCPD (% of M - 6.4 (1.6)* 6.3 (2.4)* 0.035 ns ns (only 2
dose) F - 9.2 (1.5)* 9.9 (1.6)* groups)
Mercapturate M - 20.4 (1.2)* 27.5 (1.6)* <0.001 ns ns (only 2
(% of dose) F - 11.0 (1.2)* 10.5 (1.3)* groups)
β-Chlorolactic acid was found to represent a minor pathway. Indeed, measured values were
below the LOD (<0.3 micrograms/l) in controls and in male rats treated with 3-MCPD
dipalmitate. Due to too many values below LOD, statistical analysis was not feasible. When
measurable, urinary β-chlorolactic acid accounted for less than 1 % of the administered dose.
As a whole, these data suggest that urinary 3-MCPD can be used to monitor exposure to both
3-MCPD and 3-MCPD dipalmitate. The corresponding mercapturate has the advantage of
being quantitatively higher, but the disadvantage of being less specific, as this metabolite may
be derived from either 3-MCPD or glycidol.
For practical reasons, urine collection was fractionated after treatment: a first urine sample
was approximately representative of the first five hours following administration, while a
second urine sample covered the subsequent 19 hours. Of course, spontaneous voiding makes
any kinetic study impossible. In other words, both 5-h and subsequent 19-h urine samples can
only represent incomplete collections, as we cannot exclude that one voiding shifted from the
period under observation to the subsequent one, the opposite being impossible. Despite these
limitations, we verified that the amount of individual metabolites excreted over the first
5 hours is approximately the same excreted during the subsequent 19 hours, which from a
kinetic point of view seems to make sense. As a whole, biomarkers measured in treated
animals were two orders of magnitude higher than background levels (Tables 3a to 3c).
In male rats, about 30 % of 3-MCPD was excreted either unchanged (3 %) or as 3-MCPD

mercapturate (27 %). Urinary metabolites recovered after treatment with 3-MCPD dipalmitate
were about two thirds of those measured after administration of 3-MCPD. We have no means
to assess whether such a lower proportion is due to a reduced absorption of equimolar
amounts or to metabolic factors, e.g. partial hydrolysis by intestinal lipases or retarded
excretion due to this intermediate metabolic step.
Lower proportions were observed in female rats, with a significant sex by treatment
interaction (Figure 9). After administration of low doses of 3-MCPD both as free 3-MCPD
and 3-MCPD dipalmitate, the proportion of 3-MCPD excreted as mercapturate in urine from

24
male rats was much higher (20-27 %) than that observed in females (10-11 %). At high and
intermediate doses, the proportions of 3-MCPD excreted as mercapturate were overlapping,
ranging from 7 to 10 %. At all dose levels, the concentrations of free 3-MCPD were higher in
female than in male rats, whereas the opposite trend was observed for 3-MCPD mercapturate.
Figure 9: 24-h urinary excretion of 3-MCPD and its mercapturate expressed as a

percentage of three administered doses of equimolar amounts of either
3-MCPD (29.5-7.37-1.84 mg/kg b.w. per day) or its dipalmitate (156.75-39.19-
9.78 mg/kg b.w. per day).
A close relationship was noted between urinary excretions of 3-MCPD and its mercapturate,
after administration of both 3-MCPD and 3-MCPD dipalmitate. No differences were seen
between the two treatments, although mercapturate levels in urine were slightly higher after
administration of 3-MCPD as compared to its dipalmitate (Figure 10).
2
3-MCPD Dipalmitate: Merc=-113+8.5*3-MCPD, R =0.86
2
3-MCPD: Merc=-193+9.5*3-MCPD, R =0.83
6000
5000
Urinary Merc (μg/24h)
4000
3000
2000
1000
0
0 100 200 300 400 500 600 700
Urinary 3-MCPD (μg/24h)
Figure 10: Correlation between 3-MCPD and its mercapturate after administration of
either 3-MCPD (red dots) or its dipalmitate (black squares) with both
intermediate and high dose taken together.

25
3.8. HAEMATOLOGY AND CLINICAL CHEMISTRY

Values obtained in control rats for haematology parameters and clinical chemistry are within
the range reported in “Clinical Laboratory Parameters for Ctrl. WI (Han) Rats (Charles River
Laboratories, 2008). Statistical analysis revealed a number of significant differences, depicted
in Table 5.
Haematology parameters (Table 5a and Table 5d for males and females, respectively) showed
a mild normocytic and normochromic anaemia, with a consistent reduction of red blood cell
number, haemoglobin concentration and haematocrit. There seems to be a discrepancy
between haemoglobin concentration and haematocrit, the latter being relatively unchanged.
This finding, together with increased protein concentrations in serum (Table 5b) suggests de-
hydration, possibly masking the actual level of anaemia.
De-hydration would also explain the increase in water consumption following some weeks of
treatment (Table 3 and Figure 7). Such a de-hydration would be accounted for by a relative
polyuria noted when 24-h urine collection was made in metabolic cages both at the end of the
90-d exposure period and in studies on 3-MCPD metabolism using deuterated compounds
described in Appendix III. Both 3-MCPD and 3-MCPD dipalmitate caused significant
changes, without any difference between treatments.
Both polymorphonuclear leukocytes and platelets were increased in male rats treated with
either 3-MCPD or its dipalmitate, whereas only platelets were increased in the corresponding
female groups.
Clinical chemistry revealed a number of changes (Table 5b and Table 5e for males and
females, respectively). Increased (indirect) bilirubinemia could indicate that anaemia is
mainly haemolytic in nature.
Renal damage (in surviving animals) seems to be mild and mainly consisting of glomerular
hyperfiltration, a change usually associated with incipient nephropathy (e.g. diabetic and both
Cd- and Pb-induced nephropathy), possibly due to moderate tubular damage. Whereas minor
increases in both low and high molecular weight proteinuria (Table 5c and Table 5f for males
and females, respectively) can be explained by hyperfiltration, without significant lesions to
the first segment of the proximal tubule (S1), increased urinary volume and other
minor/inconsistent changes (uric acid, NAG, etc.) are still difficult to interpret at the moment.
Abnormalities in surviving animals were mild, though 5/10 and 2/10 female rats treated with
high doses of 3-MCPD in two separate experiments died from acute renal failure.

26
Table 5a: Haematology parameters in male rats. *GM (GSD)

dipalmitate vs
Dose Treatment Dose*Treatment
Males Control Dose Dipalmitate 3-MCPD 3-MCPD
(p) (p) (p)
(Tukey)
Low 8.34 (3.29) 8.93 (1.70)
WBC
9.55 (2.50) Int 8.94 (2.61) 9.16 (1.92) <0.001 ns ns ns
(103/mm3)
High 12.30 (2.67) 11.24 (2.47)
Low 8.16 (0.27) 8.02 (0.36)
RBC
8.21 (0.35) Int 7.86 (0.38) 7.84 (0.32) <0.001 ns ns ns
(106/mm3)
High 7.49 (0.43) 7.57 (0.34)
Low 16.2 (0.6) 15.9 (0.6)
Hb
15.4 (0.4) Int 15.2 (0.5) 15.3 (0.7) <0.001 ns ns ns
(g/dL)
High 14.2 (0.9) 14.1 (0.5)
Low 47.0 (4.5) 44.9 (2.3)
Hct 44.3 (1.6) Int 43.2 (2.0) 42.8 (2.1) <0.001 ns ns ns
High 39.9 (2.7) 39.6 (2.0)
Low 871 (87) 894 (170)
Plt
908 (210) Int 945 (100) 934 (76) <0.001 ns ns ns
(103/mm3)
High 1162 (131) 1211 (112)
Table 5b: Clinical chemistry in male rats. *GM (GSD)

dipalmitate vs
(p) (p) (p)
(Tukey)
Low 1.50 (0.81) 1.45 (0.44)
Uric Acid
1.65 (0.60) Int 1.85 (0.85) 1.98 (1.00) ns 0.049 0.047 ns
(mg/dL)
High 1.30 (0.20) 2.30 (0.70)
Low 180 (36) 202 (32)
Glucose
197 (33) Int 180 (23) 213 (29) ns 0.001 ns 0.002
(mg/dL)
High 162 (22) 188 (25)
Low 38.3 (4.8) 44.6 (4.1)
Urea-N
42.3 (10.3) Int 40.8 (2.9) 38.8 (6.6) ns 0.035 ns ns
(mg/dL)
High 37.6 (3.7) 44.1 (6.5)
Low 46.5 (12.6) 52.3 (12.6)
Cholesterol
51.7 (13.4) Int 40.3 (8.2) 50.0 (13.3) <0.001 0.02 ns ns
(mg/dL)
High 62.1 (10.2) 68.9 (11.3)
Low 113 (80) 101 (49)
Triglycerides
138 (70) Int 124 (52) 132 (50) ns ns ns ns
(mg/dL)
High 90 (40) 89 (29)
Low 6.49 (0.87) 6.10 (0.59)
Total Proteins
6.28 (0.46) Int 6.58 (0.39) 6.75 (0.42) 0.036 ns ns ns
(g/dL)
High 6.16 (0.25) 6.41 (0.41)
Low 0.06 (0.02) 0.14 (0.02)
Bilirubin (Tot.)
0.11 (0.04) Int 0.12 (0.03) 0.19 (0.02) <0.001 <0.001 <0.001 <0.001
(mg/dL)
High 0.17 (0.04) 0.15 (0.05)
Low 0.026 (0.013) 0.060 (0.015)
Bilirubin (Dir.) 0.035
Int 0.045 (0.011) 0.064 (0.020) <0.001 <0.001 ns <0.001
(mg/dL) (0.018)
High 0.022 (0.012) 0.041 (0.022)
Low 165 (28) 131 (29)
Alk. Phosph.
136 (16) Int 152 (27) 120 (25) ns <0.001 ns <0.001
(UI)
High 142 (19) 139 (14)
Low 0.61 (0.02) 0.56 (0.07)
Creatinine
0.67 (0.07) Int 0.65 (0.06) 0.71 (0.12) <0.001 0.017 <0.001 0.048
(mg/dL)
High 0.53 (0.06) 0.66 (0.05)
Low 81.5 (15.7) 71.4 (8.9)
AST GOT
82.9 (16.3) Int 67.1 (11.3) 47.2 (11.4) <0.001 0.009 ns 0.010
(UI)
High 124.1 (26.5) 118.4 (21.7)
Low 36.5 (6.3) 30.7 (7.2)
ALT GPT
35.7 (7.1) Int 30.5 (5.8) 23.7 (6.0) <0.001 0.032 ns 0.048
(UI)
High 44.5 (9.2) 45.0 (7.3)

27
Table 5b: Continued

dipalmitate vs
(p) (p) (p)
(Tukey)
Low 778 (523) 693 (348)
LDH
823 (501) Int 307 (166) 492 (326) <0.001 ns ns ns
UI
High 1737 (442) 1646 (217)
Low 1.49 (0.17) 1.57 (0.17)
Serum Cys-C
1.82 (0.42) Int 2.05 (0.59) 1.93 (0.44) 0.004 ns ns ns
mg/L
High 1.86 (0.64) 1.57 (0.30)
Serum B2M 3.75 (0.42) Low 3.83 (0.46) 3.77 (0.62)
mg/L Int 3.42 (0.64) 3.66 (0.46) ns ns ns ns
High 3.59 (0.70) 3.52 (0.55)
Table 5c: Urinary parameters in male rats. *GM (GSD)

Males Control Dose Dipalmitate 3-MCPD Dose Treatment Dose*Treatment dipalmitate vs
(p) (p) (p) 3-MCPD
(Tukey)
7.5 (1.0) Low 8.0 (0.6) 7.9 (0.5)
Urinary pH Int 8.0 (0.6) 7.8 (0.8) <0.001 ns ns ns
High 7.0 (1.2) 6.4 (0.9)
Density 1013 (5) Low 1011 (7) 1014 (7)
g/mL Int 1008 (3) 1011 (5) 0.003 0.02 ns ns
High 1014 (7) 1018 (8)
Volume* 21.2 (2.3) Low 28.1 (2.4) 28.8 (2.1)
µL/min Int 22.4 (2.0) 18.3 (1.7) ns ns ns ns
High 36.1 (1.6) 19.0 (2.2)
U-NAG* 24.4 (1.9) Low 23.3 (2.4) 15.6 (1.7)
(uU/min) Int 16.3 (1.6) 11.5 (1.3) <0.001 0.006 ns 0.011
High 63.4 (1.9) 36.5 (1.7)
U-KIM-1* 11.1 (2.1) Low 13.3 (1.9) 15.6 (1.9)
pg/min Int 9.7 (2.1) 9.1 (1.7) <0.001 ns ns ns
High 24.7 (1.4) 21.3 (1.9)
U-UB2M* 16.7 (2.1) Low 31.1 (1.8) 17.1 (1.3)
ng/min Int 11.7 (1.4) 9.6 (1.2) <0.001 0.017 ns 0.039
High 19.8 (1.6) 16.1 (1.7)
Total Proteins* 138 (2.0) Low 162 (1.7) 101 (1.4)
µg/min Int 136 (1.2) 192 (1.6) <0.001 ns ns ns
High 354 (1.9) 344 (1.9)
U-Alb* 26.9 (3.2) Low 53.7 (3.3) 19.5 (2.1)
µg/min Int 26.9 (2.8) 64.6 (4.0) 0.03 ns 0.028 ns
High 79.4 (3.0) 83.2 (2.1)
U-αGST* 272 (1.7) Low 175 (1.5) 162 (1.6)
pg/min Int 479 (2.1) 347 (1.6) <0.001 ns ns ns
High 389 (1.4) 357 (1.5)
U-Cys-C* 7.0 (1.9) Low 5.2 (1.3) 5.1 (1.7)
ng/min Int 7.2 (1.4) 6.4 (1.3) <0.001 ns ns ns
High 16.9 (1.5) 14.5 (1.7)

28
Table 5d: Haematology parameters in female rats. *GM (GSD)

Females Control Dipalmitate 3-MCPD Dose Treatment Dose*Treatment dipalmitate vs
(p) (p) (p) 3-MCPD
(Tukey)
WBC 4.69 (0.82) Low 4.16 (1.41) 4.34 (0.93)
(103/mm3) Int 5.54 (1.70) 5.50 (1.68) 0.006 ns ns ns
High 4.63 (1.31) 4.28 (1.25)
RBC 7.57 (0.44) Low 7.69 (0.65) 7.62 (0.36)
(106/mm3) Int 7.47 (0.69) 7.01 (0.74) 0.005 0.032 ns ns
High 7.29 (0.58) 6.87 (0.55)
Hb 15.4 (0.5) Low 15.3 (0.7) 15.2 (0.6)
(g/dL) Int 15.0 (1.1) 14.4 (1.3) <0.001 0.042 ns ns
High 14.4 (0.7) 13.8 (0.9)
Hct 43.5 (1.6) Low 43.3 (2.7) 43.0 (2.0)
Int 42.1 (3.3) 40.2 (4.4) <0.001 ns ns ns
High 39.8 (2.0) 38.4 (2.7)
Plt 960 (121) Low 976 (127) 1064 (167)
(103/mm3) Int 1056 (176) 1080 (206) <0.001 ns ns ns
High 1277 (120) 1270 (197)
Table 5e: Clinical chemistry in female rats. *GM (GSD)

Females Control Dipalmitate 3-MCPD Dose (p) Treatment Dose*Treatment dipalmitate vs
(p) (p) 3-MCPD
(Tukey)
Uric Acid 1.65 (0.26) Low 1.81 (0.45) 2.08 (0.66)
(mg/dL) Int 1.35 (0.32) 1.34 (0.14) <0.001 ns ns ns
High 2.13 (0.35) 2.05 (0.83)
Glucose 202 (42) Low 212 (27) 243 (47)
(mg/dL) Int 198 (18) 210 (40) <0.001 0.014 ns ns
High 158 (23) 187 (43)
Urea-N 39.4 (5.7) Low 39.5 (6.5) 38.5 (7.4)
(mg/dL) Int 40.0 (6.6) 46.0 (9.1) ns ns ns ns
High 37.3 (4.5) 38.7 (8.8)
Cholesterol 52.7 (8.3) Low 51.8 (9.3) 62.0 (24.7)
(mg/dL) Int 44.5 (10.6) 55.0 (12.6) ns 0.014 ns 0.027
High 56.2 (17.8) 62.4 (12.4)
Triglycerides 86.8 (49.5) Low 61.4 (26.7) 127.5 (98.1)
(mg/dL) Int 54.1 (27.7) 90.8 (63.1) ns 0.005 ns 0.013
High 87.3 (52.2) 101.9 (24.8)
Total Protein 6.08 (0.58) Low 6.18 (0.39) 7.05 (0.91)
(g/dL) Int 5.81 (0.59) 5.89 (0.43) <0.001 0.004 ns 0.009
High 5.90 (0.62) 6.26 (0.31)
Bilirubin (Tot.) 0.12 (0.04) Low 0.14 (0.03) 0.12 (0.03)
High 0.13 (0.04) 0.15 (0.06)
Bilirubin (Dir.) 0.035 (0.02) Low 0.037 (0.013) 0.031 (0.015)
High 0.036 (0.034) 0.035 (0.018)
Alk. Phosph. 98.8 (25.4) Low 89.8 (26.9) 100.2 (29.2)
(UI) Int 73.4 (13.1) 66.3 (20.7) 0.005 ns ns ns
High 78.0 (16.1) 80.5 (24.3)
Creatinine 0.65 (0.06) Low 0.72 (0.05) 0.69 (0.06)
(mg/dL) Int 0.74 (0.06) 0.69 (0.10) <0.001 <0.001 ns <0.001
High 0.61 (0.04) 0.50 (0.04)
AST GOT 117 (44) Low 163 (65) 122 (80)
(UI) Int 112 (49) 114 (40) 0.046 0.043 ns ns
High 125 (19) 88 (23)
ALT GPT 42.0 (17.0) Low 64.8 (23.0) 49.6 (22.8)
(UI) Int 41.0 (20.8) 46.7 (16.3) <0.001 ns ns ns
High 38.1 (5.9) 30.1 (4.0)

29
Table 5e: Continued

(p) (p) 3-MCPD
(Tukey)
Serum Cys-C 1.56 (0.30) Low 1.46 (0.16) 1.54 (0.26)
mg/L Int 2.36 (0.92) 1.89 (0.49) <0.001 ns ns ns
High 1.23 (0.20) 1.13 (0.15)
Serum B2M 3.38 (0.47) Low 3.91 (0.53) 3.92 (0.79)
mg/L Int 3.76 (0.81) 3.73 (0.70) <0.001 ns ns ns
High 2.95 (0.63) 2.76 (0.68)
Table 5f: Urinary parameters in male rats. *GM (GSD)

(p) (p) 3-MCPD
(Tukey)
Urinary pH 8.02 (0.71) Low 7.95 (0.60) 7.95 (0.69)
Int 7.65 (0.34) 8.06 (0.39) <0.001 <0.001 <0.001 <0.001
High 5.35 (0.67) 7.69 (0.88)
Density 1014 (6) Low 1013 (8) 1020 (6)
g/mL Int 1013 (4) 1012 (4) 0.006 ns 0.002 ns
High 1021 (3) 1015 (6)
Volume* 16.1 (1.7) Low 28.2 (1.9) 15.0 (1.6)
µL/min Int 17.3 (2.0) 23.8 (1.8) 0.031 ns <0.001 ns
High 9.1 (1.4) 20.6 (1.7)
U-NAG* 12.8 (1.6) Low 12.0 (1.6) 12.3 (1.6)
(uU/min) Int 12.2 (1.5) 11.0 (1.5) <0.001 ns ns ns
High 18.3 (1.4) 19.2 (1.4)
U-KIM-1* 7.6 (1.6) Low 11.9 (1.9) 6.0 (1.6)
pg/min Int 8.3 (2.4) 9.4 (1.8) ns ns 0.001 ns
High 6.5 (1.3) 14.1 (1.9)
U-UB2M* 2.7 (1.8) Low 3.7 (1.4) 6.1 (3.2)
ng/min Int 2.9 (2.3) 8.7 (4.2) <0.001 0.001 ns 0.008
High 1.2 (2.6) 2.5 (2.4)
Total Proteins* 85.1 (1.5) Low 87.0 (1.3) 89.7 (1.8)
µg/min Int 118.5 (2.0) 190.5 (4.3) 0.028 ns ns ns
High 108.6 (1.3) 157.8 (1.5)
U-Alb* 14.6 (2.9) Low 15.6 (2.3) 23.2 (4.1)
µg/min Int 32.8 (4.2) 86.1 (7.7) 0.049 ns ns ns
High 29.6 (2.4) 29.7 (3.3)
U-αGST* 219 (1.5) Low 162 (1.9) 207 (1.4)
pg/min Int 485 (2.1) 542 (2.2) <0.001 ns ns ns
High 229 (1.4) 233 (1.6)
U-Cys-C* 3.5 (1.5) Low 3.3 (1.6) 2.9 (1.3)
ng/min Int 4.6 (1.7) 6.7 (3.4) 0.002 ns ns ns
High 2.9 (1.8) 3.1 (1.6)
3.9. PATHOLOGY
At the end of the experimental protocols (high, intermediate and low administration of
3-MCPD and 3-MCPD dipalmitate), all rats were euthanized in saturated chamber of vapour
of ethyl-ether and CO2, and then necropsied.
Thymus, heart and aorta, salivary glands, spleen, liver, kidneys, adrenals, gonads and
accessory sex glands were removed and weighted respectively in male (Table 6) and female
rats (Table 7).
A consistent and dose-dependent increase in kidney weight was observed both in male and
female rats. The weight of liver was also increased both in males and females, but such a
change was observed only at high doses of either 3-MCPD or 3-MCPD dipalmitate.

30
Table 6: Total body weight and weight of most important organs in male rats.
Dose * dipalmitate vs
Dose Treatment
Males Control Dose Dipalmitate 3-MCPD Treatment 3-MCPD
(p) (p) (Tukey)
(p)
Low 509 (51) 476 (31)
Total Body
514 (38) Int 518 (53) 547 (31) 0.003 ns ns ns
(g)
High 489 (58) 487 (38)
Low 19.0 (2.5) 16.7 (2.7)
Liver
18.6 (2.8) Int 19.0 (1.3) 19.2 (2.4) 0.001 ns ns ns
(g)
High 20.5 (3.2) 21.7 (2.0)
Low 3.9 (0.3) 3.4 (0.5)
Kidney+Adrenal Int 5.3 (1.1) 4.3 (0.7)
4.3 (0.9) <.001 ns ns ns
(g)
High 7.0 (2.3) 7.5 (1.9)
Low 8.8 (1.1) 7.9 (1.5)
Gonads
8.2 (1.5) Int 7.3 (0.9) 7.4 (1.2) ns 0.039 ns ns
(g)
High 9.1 (2.2) 7.5 (1.7)
Low 4.5 (1.0) 4.5 (1.2)
Accessory Sex
4.2 (1.0) Int 5.3 (0.9) 4.3 (0.9) ns ns ns ns
Glands (g)
High 5.2 (0.9) 5.4 (1.1)
Table 7: Total body weight and weight of most important organs in female rats.
Dose * dipalmitate vs
Dose Treatment
Females Control Dose Dipalmitate 3-MCPD Treatment 3-MCPD
(p) (p) (Tukey)
(p)
Low 267 (20) 269 (25)
Total Body
284 (29) Int 275 (13) 262 (26) 0.007 ns ns ns
(g)
High 291 (25) 292 (37)
Low 8.7 (0.9) 9.7 (1.8)
Liver
9.5 (2.0) Int 8.7 (0.9) 8.9 (0.9) <.001 ns ns ns
(g)
High 12.5 (1.3) 13.4 (2.3)
Low 2.3 (0.3) 2.4 (0.4)
Kidney+Adrenal Int 2.4 (0.4) 2.6 (0.6)
2.6 (0.7) <.001 ns ns 0.031
(g)
High 3.4 (0.2) 3.9 (0.5)
Low 2.0 (0.7) 1.6 (0.4)
Gonads
2.3 (1.1) Int 1.4 (0.3) 1.5 (0.2) 0.049 ns ns ns
(g)
High 1.9 (0.7) 2.3 (1.0)

31
Histopathology and scoring of kidneys lesions
Microscopic evaluation of the kidneys of the experimental groups (male and female rats)
treated with the highest doses of 3-MCPD and its dipalmitate, highlighted degenerative
lesions involving different tubular segments of one or more parts of the kidney parenchyma.
The main findings were: multifocal hypertrophy and swelling of epithelial cells of proximal
tubules, hydropic-vacuolar degenerations and pyknosis of tubular epithelial cells, hyaline and
basophilic casts scattered among tubules in the overlying cortex and subjacent medulla,
tubular regenerative hyperplasia, kariomegaly. Glomeruli, more resistant to treatments
showed mild morphological changes.
The renal findings of male rats treated with the intermediate doses of 3-MCPD and its
dipalmitate (39.19 mg/kg b.w. per day 3-MCPD dipalmitate and 7.37 mg/kg b.w. per day
3-MCPD) showed moderate multifocal hypertrophy and swelling of epithelial cells of
proximal tubules, mild hydropic-vacuolar degenerations and pyknosis of tubular epithelial
cells, hyaline and basophilic casts scattered among tubules in the overlying cortex, mild
tubular regenerative hyperplasia, mild atrophy or hypertrophy of glomeruli. The female rats
treated with 39.19 mg/kg b.w. per day 3-MCPD dipalmitate and 7.37 mg/kg b.w. per day
3-MCPD showed degenerative and inflammatory changes, as multifocal interstitial nephritis
and pyelitis (3/10).
Microscopic evaluation of the kidneys of the male and female rats treated with the lowest
doses of 3-MCPD and its dipalmitate highlighted mild or moderate degeneration hypertrophy
and swelling of epithelial cells of proximal and distal tubules,; mild or moderate hyaline and
basophilic casts in tubules cortex; mild tubular regenerative hyperplasia; rare or mild
interstitial cellular infiltrated.
In the second group of female Wistar rats treated with 3-MCPD (29.5 mg/kg b.w. per day)
(rats 156 and 165), moderate to severe degeneration, hypertrophy and swelling of epithelial
cells of proximal and distal tubules were observed (rats 156, 158, 159 and 165), associated
with hydropic-vacuolar degenerations of tubular epithelial cells and multiple foci of
interstitial nephritis.
Histopathological changes of the kidneys were graded according to standard scores (See
Appendix VII). Summary scores are reported as mean (SD) and range in Table 8. For the total
pathological score, statistically significant changes were observed only at high doses of either
3-MCPD (29.5 mg/kg b.w. per day) or equimolar amounts of 3-MCPD dipalmitate. Similar
changes were observed involving both the glomerulus and tubular structures, but tubular
epithelial degeneration showed a gradual dose-dependent trend and was more severe after
administration of 3-MCPD than after equimolar amounts of its dipalmitate, thus suggesting
that the proximal tubule is the target of 3-MCPD or its metabolite(s) and that the effect of its
dipalmitate is subsequent and proportional to the concentration of hydrolysed 3-MCPD.

32
Table 8: Score for histopathology of the kidney in male rats. Values are reported as
mean (SD) and range.
Dose * dipalmitate
Treatment
Males Control Dose Dipalmitate 3-MCPD Dose (p) Treatment vs 3-MCPD
(p) (Tukey)
(p)
Low 0 0
0.2 (0.4) 0.4 (0.5)
Glomerular 0.4 (0.5) Int
0-1 0-1 nc nc nc nc
lesions 0-1
1.2 (0.4) 1.2 (0.8)
High
1-2 0-3
0.4 (0.3) 0.3 (0.3)
Tubular Low
0-1 0-0.5
epithelial
0.2 (0.4) 0.4 (0.7)
hyperplasia Int 0 nc nc nc nc
0-1 0-2
or
3.0 (0.9) 3.0 (1.0)
proliferation High
2-4 2-4
1.4 (0.5) 1.4 (0.6)
Low
1-2.5 0.5-2.5
Tubular
1.6 (1.0) 2.6 (0.5) 2.1 (0.7)
epithelial Int <0.001 ns 0.001 ns
0-4 2-3 1-3
degeneration
1.8 (1.3) 3.6 (1.2)
High
1-5 2-6
2.1 (1.4) 1.5 (1.0)
Low
Cylinders 0-4 0-3
basophils or 0.8 (0.8) 1.2 (0.4) 1.2 (1.1)
Int <0.001 ns ns ns
hyaline 0-2 1-2 0-3
material 3.9 (1.6) 4.0 (1.3)
High
1-6 2-6
0.3 (0.4) 0.4 (0.6)
Low
0-1 0-1
Cellular
0.3 (0.6) 0.3 (0.5)
infiltration Int 0 nc nc nc nc
0-2 0-1
interstitial
0.8 (0.8) 0.4 (0.5)
High
0-2 0-1
Low 0 0
0.4 (0.5)
0.2 (0.4) Int 0
Cystis ectasia 0-1 nc nc nc nc
0-1
0.3 (0.5) 0.2 (0.7)
High
0-1 0-2
Low 0 0
Karyomegaly
Int 0 0
tubular 0 nc nc nc nc
1.0 (0.8) 1.4 (0.7)
epithelial cells High
0-2 0-2
Low 0 0
Fibrosis 0 Int 0 0 nc nc nc nc
High 0 0
4.1 (1.9) 3.7 (0.9)
Low
1-7 2.5-5
Total Renal 3.5 (1.7) 4.7 (0.9) 4.1 (0.9)
Int <0.001 ns ns ns
Score 1-6 3-6 3-5
12.0 (3.1) 13.9 (3.6)
High
8-16 9-21
Renal pathology was also examined in female rats, though scoring in animals treated with
high doses of 3-MCPD (29.5 mg/kg b.w. per day) was only possible in those surviving acute
tubular necrosis and subsequent renal failure. Data reported in Table 9 could therefore
underestimate actual nephrotoxicity.

33
Table 9: Score for histopathology of the kidney in female rats. Values are reported as
mean (SD) and range.
Dose* dipalmitate
Dose Treatment
Females Control Dose Dipalmitate 3-MCPD Treatment vs 3-MCPD
(p) (p) (Tukey)
(p)
0.6 (0.4) 0.7 (0.2)
Low
0-1 0.5-1
Glomerular 0.3 (0.5) 0.1 (0.3)
Int 0 nc nc nc nc
lesions 0-1 0-1
1.6 (0.5) 1.0 (0.8)
High
1-2 0-2
0.1 (0.2) 0.3 (0.6)
Tubular Low
0-0.5 0-2
epithelial
0.1 (0.2) 0.8 (0.8) 1.8 (2.3)
hyperplasia Int <.001 0.043 ns ns
0-1 0-2 0-6
or
1.1 (1.0) 1.7 (1.5)
proliferation High
0-3 0-4
3.0 (1.3) 3.4 (1.2)
Low
1-5 2-6
Tubular
1.6 (1.6) 3.3 (1.4) 1.9 (0.8)
epithelial Int 0.038 ns 0.027 ns
0-6 1-5 0-3
degeneration
1.9 (0.6) 2.4 (0.9)
High
1-3 1-4
0.4 (0.8) 2.2 (2.5)
Low
Cylinders 0-2 0.5-9
basophils or 0.6 (1.0) 0.7 (0.8) 1.4 (2.0)
Int 0.006 0.002 ns 0.002
hyaline 0-4 0-2 0-6
material 1.9 (1.2) 3.2 (1.8)
High
0-4 1-6
0.6 (1.0)
Low 0
0-3
Cellular
0.4 (0.7) 0.4 (0.8) 2.1 (2.3)
infiltration Int nc nc nc nc
0-2 0-2 0-6
interstitial
0.6 (0.7) 1.2 (1.2)
High
0-2 0-3.5
0.4 (0.7)
Low 0
0-2
0.4 (0.7) 1.1 (2.1)
Cystis ectasia 0 Int nc nc nc nc
0-2 0-6
0.9 (1.0) 0.6 (0.7)
High
0-3 0-2
Low 0 0
Karyomegaly
0.1 (0.3) Int 0 0
tubular nc nc nc nc
0-1 0.3 (0.5) 0.6 (0.8)
epithelial cells High
0-1 0-2

34
Table 9: Continued
Dose* dipalmitate
Dose Treatment
Females Control Dose Dipalmitate 3-MCPD Treatment vs 3-MCPD
(p) (p) (Tukey)
(p)
Low 0 0
0.4 (0.5)
Int 0
Fibrosis 0 0-1 nc nc nc nc
0.2 (0.3)
High 0
0-1
4.0 (2.2) 7.5 (4.8)
Low
1.5-8.0 3.5-20.0
Total Renal 3.1 (2.2) 5.7 (1.8) 8.8 (7.9)
Int 0.009 0.004 ns 0.006
Score 0-8 3-8 2-23
8.3 (3.7) 10.8 (4.0)
High
3-16 6-17
Testis lesions
Histopathological changes of the testes were also graded according to standard scores (see
Appendix VII). In control groups the testes are normal, or with mild degenerative phenomena
in seminiferous tubules. Nine out of ten rats treated with high doses of 3-MCPD (29.5 mg/kg
b.w. per day) show total degeneration of seminiferous tubules and limphomononuclear
infiltrates in the epididymus. Rat 6 showed abscess of the epididymis and rat 24 displayed
focal hyperplasia of Leydig cells.
In 6 out of 10 rats receiving 7.37 mg/kg b.w. per day of 3-MCPD, mild decrease of
spermatids, atrophy of spermatogenic and supporting cells in the seminiferous tubules were
observed. Low doses of 3-MCPD (1.84 mg/kg b.w. per day) caused minimal degenerative
phenomena, with decreased spermatids density (9/10), slight atrophy of supporting cells
(4/10) and slight atrophy of spermatogenic cells (3/10).
In group treated with high dose of 3-MCPD dipalmitate (156.75 mg/kg b.w. per day), 4 rats
showed moderate degenerative features in the seminiferous tubules, total degeneration of
seminiferous tubules in 2 out of 10 rats; the other rats showed atrophy (3/10) and necrosis of
germinative cells (3/10) and Sertoli cells (3/10).
Summary pathology scores for the testes are reported as mean (SD) and range in Table 10.
For the total pathological score, statistically significant changes were observed only at high
doses but were three times more severe after administration of 3-MCPD (29.5 mg/kg b.w. per
day) than after administration of equimolar amounts of 3-MCPD dipalmitate.
In rats treated with intermediate and low dose of 3-MCPD dipalmitate (39.19 mg/kg b.w. per
day and 9.78 mg/kg b.w. per day), mild degenerative change with decrease of spermatids,
slight atrophy of supporting cells and slight atrophy of spermatogenic cells were observed.
After 90-day of treatment with high doses (29.5 mg/kg b.w. per day), male rats showed a
much higher total score than the controls, with no substantial differences between

35
intermediate and low doses. On the contrary, female rats showed a dose-related trend: scores
observed at low doses (1.84 mg/kg b.w. per day) are twice as control values.
Table 10: Score for histopathology of testes in male rats. Values are reported as
mean(SD) and range.
Dose* dipalmitate
Dose Treatment
Males Control Dose Dipalmitate 3-MCPD Treatment vs 3-MCPD
(p) (p) (Tukey)
(p)
0.3 (0.3) 0.5 (0.2)
Low
0-1 0-1
Decrease of 0.2 (0.4) 0.3 (0.5) 0.3 (0.5)
Int <.001 <0.001 <0.001 0.001
spermatids 0-1 0-1 0-1
0.9 (1.2) 2.7 (0.7)
High
0-3 1-3
0.2 (0.3) 0.3 (0.3)
Low
0-1 0-1
Atrophy of
0.1 (0.3) 0.3 (0.5)
supporting Int 0 nc nc nc nc
0-1 0-1
cells
0.6 (1.1) 1.9 (1.2)
High
0-3 0-3
0.2 (0.3) 0.2 (0.3)
Low
0-1 0-1
Spermatogenic 0.1 (0.4) 0.6 (0.5)
Int 0 nc nc nc nc
cell atrophy 0-1 0-1
0.7 (1.2) 2.6 (1.0)
High
0-3 0-3
Low 0 0
Int 0 0
Mineralization 0 nc nc nc nc
1.0 (0.5)
High 0
0-2
Low 0 0
Hyperplasia Int 0 0
0 nc nc nc nc
Leydig cells 0.1 (0.3) 0.8 (0.7)
High
0-1 0-2
Low 0 0
0.1 (0.3)
Inflammatory Int 0
0 0-1 nc nc nc nc
infiltrates
0.6 (0.7) 0.9 (0.9)
High
0-2 0-3
0.6 (1.0) 1.0 (0.9)
Low
Total Testis 0-3 0-3
Score 0.4 (0.8) 0.3 (0.5) 1.3 (1.4)
Int <.001 <0.001 <0.001 <0.001
Decrease of 0-2 0-1 0-4
spermatids 2.9 (3.3) 9.78 (2.7)
High
0-9 4-14

36
3.10. DOSE-RESPONSE AND DOSE-EFFECT ANALYSIS
Correlation between biomarkers of exposure and effect in urine
Urinary biomarkers of internal dose were correlated with markers of renal damage. Whereas
in male rats almost all markers renal damage and dysfunction were correlated with both free
3-MCPD and its mercapturate, in female animals such relationships were not always apparent
(Table 11), probably because of the greater sensitivity of the latter to acute kidney injury and
the greater subsequent variability associated with tissue repairing at the time of sampling.
Table 11: Correlations between urinary free 3-MCPD and Mercapturate.
3-MCPD Mercapturate 3-MCPD Mercapturate

NAG 0.62 0.48 NAG 0.62 0.51
Kim1 0.46 0.37 Kim1 0.43 Ns
UB2M Ns Ns UB2M Ns -0.39
Prot tot 0.75 0.61 Prot tot 0.41 0.32
Albumin 0.49 0.29 Albumin Ns Ns
GST 0.42 0.40 GST Ns Ns
Cys C 0.67 0.69 Cys C Ns Ns
Males (Pearson on log-log scale) Females (Pearson on log-log scale)
Mortality in female rats
The dose-response relationship for mortality rates observed in females exposed to free
3-MCPD is shown in Figure 11. A high mortality rate (5 out of 10) was observed in the first
group of female rats treated with 29.5 mg/kg b.w. per day. Therefore, this experiment was
repeated at the end of the study and a mortality of 2 out of 10 animals was observed. Due to
the small sample size, the difference between the two experiments was not statistically
significant and hence the two groups were merged in a single one, thereby increasing the
statistical power of the dose-response relationship depicted in Figure 11. Applying a gamma
function on a log scale, the BMD10 and BMDL10 were 7.4 and 2.3 mg/kg b.w. per day,
respectively. The model used was the best fitting one (see statistical analysis section).
No mortality was observed in control rats and in animals treated with 3-MCPD dipalmitate.
As markers of adverse effects, a semi-quantitative assessment of pathological changes in the
two target organs (i.e. the kidneys and the testes) was used, relying in both cases a threshold
score denoting severe toxicity.
Details on scoring criteria are given in the Appendix VII.

37
90-Day toxicological study of 3-MCPD and its dipalmitate
40
35
30
25
Mortality (%) 20
15
10
0
Control 1.84 mg/Kg 7.37 mg/Kg 29.5 mg/Kg
Daily dose of 3‐MCPD
Figure 11: Dose-response relationship for mortality observed in female rats receiving
daily doses of 3-MCPD by gavage for 90 days.
Extrapolation of Benchmark doses from dose-response curves in female rats
Since the excretion of urinary 3-MCPD was similar at low and intermediate doses of both
3-MCPD and 3-MCPD dipalmitate, BMDs were calculated starting from nominal doses of
exposure, separating the two exposures.
3-MCPD dipalmitate: BMD and the corresponding BMDL were extrapolated using dose-
response relationships for pathology scores describing nephrotoxicity (Figure 12).
80
70
% of Rats with renal score >6
60
50
40
30
20
10
0
1 10 100
3-MCPD Dipalmitate (mg/Kg)
Figure 12: Dose-response relationship for renal toxicity observed in female rats receiving
daily doses of 3-MCPD dipalmitate by gavage for 90 days.
38
BMD10 and BMDL10 were 7.9 and 3.6 mg/kg, respectively. The corresponding 3-MCPD
doses based on molecular weight were 1.5 and 0.7 mg/kg, well below administered doses.
3-MCPD: we can only conclude that BMD and BMDL were <1.84 mg/kg b.w. per day.
Owing to the relatively high prevalence of pathological scores denoting renal damage even at
the lowest nominal dose, it was impossible to identify a threshold. Nor was it possible to
identify a statistically significant fitting curve (Figure 13). In order to calculate a BMD,
further experiments in the low dose range are necessary.
100
% of Rats with renal score >6
80
60
40
20
0
0.1 1 10
3-MCPD (mg/Kg)
Figure 13: Dose-response relationship for renal toxicity observed in female rats receiving
daily doses of 3-MCPD by gavage for 90 days.
Relationship between nominal and urinary dose of 3-MCPD for male rats
On the basis of data presented in Table 3a-c, showing that different urinary concentrations of
3-MCPD and 3-MCPD mercapturate were observed at equimolar doses of either 3-MCPD or
its dipalmitate, after normalization for m.w. and considering that estimated background intake
of 3-MCPD in controls was about 1/20 of the lowest dose, i.e. 0.05 mg/kg b.w. per day of
3-MCPD or 0.26 mg/kg b.w. per day for 3-MCPD dipalmitate, we found equations describing
the relationship between the nominal dose of exposure (ND) and urinary 3-MCPD (UD) in
males.
Dipalmitate: log(ND)=1.64*log(UD)-2.49 => log(UD)=1.52+0.61*log(ND) (1)
3-MCPD: log(ND)=1.52*log(UD)-2.39 => log(UD)=1.57+0.66*log(ND) (2)
The validity of these equations is for ND exceeding 3 mg/ kg b.w. per day. Below such a
dose, there is no difference between free and esterified 3-MCPD. Using these equations,
urinary biomarkers were converted into administered doses, thereby obtaining the dose-
response relationships used for BMD calculations.
39
Extrapolation of Benchmark doses from dose-response curves in male rats
BMD and the corresponding BMDL were extrapolated using dose-response relationships for
pathology scores describing nephrotoxicity and testicular damage, respectively (Figures 14a
and 14b).
A
B
Figure 14: Dose-response relationships for renal (A) and testicular toxicity (B) observed
in male rats.
Whereas both dose-effect and dose-response relationships were observed for renal damage,
only dose-response relationship was observed for testicular damage. Moreover, the use of
urinary markers of dose gave rise to a better fitting for the prevalence of nephrotoxicity,
whereas no such relationship was found to occur for testicular damage. In the latter case,
BMD calculations were based directly on the administered dose.
(1) Quartiles of urinary 3-MCPD (0-43-85-161-396 µg/24 h of urinary 3-MCPD) and

prevalence of rats with total renal score >6 (on the basis of the scores assigned at the
previous paragraph, considering all the domains together and considering that in the
control group none of the rats had a score >6);
(2) ND of 3-MCPD and prevalence of rats with total testis score ≥3 (on the basis of +
assigned at the previous paragraph, considering all the domains together and considering
that in the control group none of the rats had a score ≥3).
For renal changes, using an extra risk of 10 % and the gamma function, we obtained a BMD10
of 100.5 μg/24h and a BMDL10 of 67.6 µg/24 h, which correspond to ND of 5.6 and
2.5 mg/kg b.w. per day, respectively.
On the basis of equations 1 and 2 (above), we supposed the same effective dose after exposure
to free 3-MCPD (1.84 mg/kg b.w. per day) and its ester (9.78 mg/kg b.w. per day). On the
other hand, a nominal dose of 39.19 mg/kg per day of dipalmitate corresponds to 5.35 mg/kg
b.w. per day of 3-MCPD (c: 7.37 mg/kg per day) and 156.75 mg/kg b.w. per day to
17.54 mg/kg b.w. per day (expected: 29.5 mg/kg b.w. per day). Therefore, nominal doses used
to build up dose-response curves were based on the following groups: Controls, 1.84, 5.35,
7.37, 17.54, 29.5 mg/kg b.w. per day (Figures 14 and 15).
Using an extra risk of 10 % for testicular toxicity and the logistic function, we obtained a
BMD10 of 8.4 mg/kg b.w. per day and a BMDL10 of 6.0 mg/kg b.w. per day.
40
Figure 15: Dose-effect relationship between 3-MCPD and red blood cell count.
Administered dose was back-calculated from the urinary excretion of free
3-MCPD, according to equations (1) and (2).
Extrapolation of Benchmark doses from dose-effect relationships in male rats
In male rats, dose-effect relationships were established for several parameters of clinical
biochemistry and nephrotoxicity biomarkers. As an example, the relationship between
nominal doses of either 3-MCPD or 3-MCPD dipalmitate and red blood cell count is shown in
Figure 15. Similar calculations were made for other end-points, resulting in several possible
BMDs and BMDLs, summarized in tables 12 and 13 for male and female rats, respectively.
Table 12: BMDs and BMDLs calculated according to different criteria in male rats.
3-MCPD (mg/kg b.w. per day) dipalmitate*(mg/kg b.w. per day)

BMDL10 BMD10 BMDL10 BMD10
a
Renal pathology 2.5 5.6 17.4 41.1b
Testicular pathology 6.0 8.4 44.3 64.4
Total Proteinuria 2.7 6.4 18.7 47.2
Urinary KIM-1 6.5 10.6 48.3 84.1
RBC (5 % loss) 3.5 7.2 24.8 53.5
RBC (10 % loss) >High Dose >High Dose >High Dose >High Dose
a
equivalent to 3.27 mg/kg b.w. per day of 3-MCPD.
b
41
Table 13: BMDs and BMDLs calculated according to different criteria in female rats.
3-MCPD (mg/kg b.w. per day) dipalmitate*(mg/kg b.w. per day)

BMDL10 BMD10 BMDL10 BMD10
a
Renal pathology Between 0.1 and Between 0.1 and 3.6 7.9b
1.84 1.84
Urinary NAG NC 7.37-29.5 NC 39.2-156.75
RBC (5 % loss) 2.6 4.5 90.4 187.0
RBC (10 % loss) 5.8 10.8 >High Dose >High Dose
a
b
4. DISCUSSION
No studies on the potential toxicological properties of esters of 3-MCPD and glycidol have
been reported to date. The primary toxicological concern is the possible hydrolysis of
3-MCPD and glycidol esters by lipases in the gastrointestinal tract during the digestion.
The most striking result of the present study is the high mortality rate observed in female rats
treated with 29.5 mg/kg b.w. per day. Such a high mortality rate could be partly ascribed to
the administration mode (bolus by gavage) likely to result in peak blood (and tissue)
concentrations not reached during exposure through drinking water.
Relatively mild changes observed in surviving animals might derive from repeated acute
injuries, rather than from chronic effects. It should to be noted that recovery from
tubulonecrosis makes animals less susceptible to additional acute damage.
Both 3-MCPD and glycidol have been found to induce tumors in long-term studies in rodents,
though results with 3-MCPD showed some inconsistencies. Moreover, 3-MCPD is known as
a food process contaminant, and in 2001 it was classified by the European Scientific
Committee on Food as a nongenotoxic, threshold carcinogen with a tolerable daily intake
(TDI) of 2 μg/kg b.w. per day (European Commission, 2001). In 2002, Joint FAO/WHO
Expert Committee on Food Additives (JECFA) set a provisional maximum tolerable daily
intake (PMTDI) of 2 μg/kg b.w. (Schlatter et al., 2002). On the contrary, glycidol is a
genotoxic carcinogen classified by IARC as group 2A, “probably carcinogenic to human”
(IARC, 2000).
The initial risk assessments of the esters conducted by the German Federal Institute for Risk
Assessment (BfR) revealed a possible safety concern particularly for infants fed commercial
infant formulas (BfR, 2009). Since there were no reliable in vivo data on the metabolism of
3-MCPD esters and glycidol esters, the risk assessment was based on the “worst case”
assumption of 100 % hydrolysis of the esters and the bioavailability of released 3-MCPD and
glycidol assumed to be the same as that of the unbound compounds ingested orally.
42
Figure 16: Metabolic fate of 3-MCPD: in addition to the parent compound, its
mercapturate is a major metabolite in rat. β-chlorolactic acid was found only
in trace amounts (Lynch et al., 1998).
Figures 16-17 depict the metabolic pathways leading to the urinary excretion of metabolites.
Only a small fraction is excreted unchanged in urine.
Data obtained in the present study confirm that at low doses (1.84 mg/kg b.w. per day) there
are no differences between rats treated with either 3-MCPD or its dipalmitate as far as the
urinary excretion of 3-MCPD and 3-MCPD mercapturate is concerned. At higher doses, the
recovery of 3-MCPD and 3-MCPD mercapturate from animals treated with 3-MCPD
dipalmitate was lower for males basing on equations 1 and 2, and gender-dependent. In fact,
values recorded in females being one third lower compared to males, and the most striking
result was that mercapturate levels recorded in surviving females treated with 3-MCPD was
lower by 50 % compared to the corresponding values measured in males. Finally, even at low
dose levels, female rats excreted less mercapturate mainly when treated with free 3-MCPD
(and appeared to be more susceptible to renal injury than male animals).
We speculate that metabolic differences can explain the greater sensitivity of female rats to
acute injury and that part of 3-MCPD or cysteine radical could be either retained or fixed to
the renal tissue, thereby causing acute tubulonecrosis, which was observed in 50 % of animals
belonging to this group.
An in vivo study on the metabolism and bioavailability of 3-MCPD esters in rats is currently
in progress at the BfR (Berger-Preiss et al., 2010). The study will compare the release of free
3-MCPD from 3-MCPD-diesters and its toxicokinetic behavior with the toxicokinetic fate of
43
free 3-MCPD. This study is being conducted in Wistar rats, which will enable us to better
interpret metabolic data obtained in the same strain.
Regarding nephrotoxicity, the most striking result obtained in the present study is the
markedly higher sensitivity of female rats compared to male animals. Also, in contrast with
previously published studies, suggesting a lower prevalence and reduced severity of renal
damage in females than in males, we observed mortality from acute renal failure due to
tubulonecrosis in females only. Moreover, histopathological changes were observed in
females even at the lowest dose (1.84 mg/kg b.w. per day), at which no appreciable changes
were observed in males.
Long-term studies published in the literature were carried out either on Fischer 344 (Sunahara
et al., 1993) or Sprague-Dawley rats (Cho et al., 2008), whereas the present study was carried
out in Wistar rats, the same strain used for the toxicokinetic study at BfR. Apart from the
different strain, another important difference introduced in our study was the administration
methods: owing to the failure of alternative ways to administer the diester per os, 3-MCPD
dipalmitate was administered daily by gavage. For consistency, the same method of
administration was used for the administration of 3-MCPD. Such an approach ensures a
complete intake of the administered dose, but the bolus effect, causing peak levels in serum,
certainly not attained when intake occurred with drinking water throughout the day, should be
noted.
Brush border peptidases are known to degrade GSH conjugated. The resulting cysteine
conjugate undergoes either cleavage by intracellular β-lyases, thereby forming intermediate
metabolites which are either glucuronated to form sulfo-glucuronides or reactive species (thus
covalently binding nucleophilic cell macromolecules, e.g. DNA).
This pathway is known to be more important in rodents than in other mammals. Indeed,
mercapturate concentration is higher by one order of magnitude as compared to free 3-MCPD
(Table 3). In urine from treated animals, both free 3-MCPD and its mercapturate were
increased by two orders of magnitude over background levels, with sex- and treatment-related
differences. Administration of 3-MCPD dipalmitate resulted in urinary excretions reduced by
50 % as compared to those associated with equimolar doses of free 3-MCPD (Table 3).
44
3-MCPD GLU-CIS-GLY (GSH)
Glutathione-S-Transferase
2,3-dihydroxypropyl-GLU-CYS-GLY
γ-Glutamyltransferase
H2O
GLU
2,3-dihydroxypropyl-CYS-GLY
Cysteinylglycinase
H2O (Aminopeptidase M)
GLY
2,3-dihydroxypropyl-CYS
N-Acetyl-Transferase
Acetyl-Co A
2,3-dihydroxypropyl-N-Acetyl-CYS
(MERCAPTURATE)
Figure 17: Pathway leading to the formation of 3-MCPD mercapturate, which is a major
metabolite in rat. Brush border peptidase lead to subsequent de-amination to
form a cysteine conjugate which is then acetylated prior to urinary excretion.
45
CONCLUSIONS
The present study confirms that the kidneys and the testes are the main target organs for
3-MCPD and suggests that 3-MCPD dipalmitate may cause similar effects, despite a lower
recovery of urinary metabolites at intermediate and high doses, reduced by about one third
after administration of the diester as compared to equimolar doses of free 3-MCPD.
In contrast to previous experiments (Cho et al., 2008), i.e. chronic studies on 3-MCPD
carcinogenicity documenting nephrotoxicity and cancerous renal lesions in male rats
following exposure to 29.5 mg/kg b.w. per day in drinking water, in the current study the
administration of the same doses by gavage (thus, as a bolus) caused death from acute renal
failure in seven out of twenty females. In addition, one female out of ten receiving the
intermediate dose (7.37 mg/kg b.w. per day) died. Surviving females and males showed mild
tubular toxicity associated with hyperfiltration. Similar changes were seen after administration
of equimolar doses of 3-MCPD dipalmitate, which however showed a lower acute
nephrotoxicity, probably because of kinetic factors. No mortality was observed in male rats
during the 90-day experiments.
In males, the most striking effects were seen on testes, which at the end of the 90-day study
were almost devoid of cellularity both in rats receiving 3-MCPD and in those treated with
3-MCPD dipalmitate at the high doses.
In a subchronic toxicity study of 3-MCPD administered by drinking water to B6C3F1 mice

(Cho et al., 2008), it was concluded that the target organs were kidney, testis and ovary. The
no-observed-adverse-effect level (NOAEL) was found to be 100 ppm (18.05 mg/kg b.w. per
day for males and 15.02 mg/kg b.w. per day for females). In a study using much lower doses
of 3-MCPD (5 mg/kg) by gavage daily for 4 weeks in Sprague-Dawley male rats, Kwack et
al. (2004) observed significantly reduced sperm motility, copulation, fertility indices, and the
number of live fetuses showed steep dose-response curves. Spermatogenesis was not affected
nor were hormonal changes induced in the blood and testes of male rats.
In the present study, severe effects on testes were caused by daily gavage administration of
29.5 mg/kg b.w. per day of 3-MCPD or equimolar doses of 3-MCPD dipalmitate for 90 days.
RECOMMENDATIONS
• Further studies are necessary in female Wistar rats to identify a threshold for
nephrotoxicity, which after administration of 3-MCPD occurs at doses well below the
lowest dose used in the present study (1.84 mg/kg b.w. per day).
• Kinetic studies should clarify gender differences in distribution volume, bioavailability
and biotransformation rate, and any metabolic differences accounting for the observed
differences in the sensitivity to nephrotoxic injury in male and female rats.
• A comparison of bioavailability and toxicity of 3-MCPD after oral administration in
drinking water should clarify whether observed acute nephrotoxic injury observed in the
present study, particularly in female rats, can be ascribed to daily dose or to peak levels of
3-MCPD attained only by gavage.
46
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3-MCPD fatty acid esters in human breast milk. Food Additives and Contaminants, Part A,
25, 669-676.

48
APPENDICES
APPENDIX I
Synthesis of
(±)-1,2-di-O-palmitoyl-3-chloropropane
49
Chemistry
(±)-1,2-di-O-palmitoyl-3-chloropropane was synthesized by acylation of (±)-3-chloropropane-

1,2-diol, using commercially available palmitoyl chloride as acylating reagent.
OH O C15H31
i
O
OH O
Cl Cl
O C15H31
1 2
Figure 1: Reagents and conditions: (i) Dry pyridine, palmitoyl chloride, dry DCM , R.T.
16h, 80-90 %.
Synthetic methods reported in literature for this compound present some potential problems,
due to the use of dangerous solvents or to low conversion yields.3
In our synthesis, dichloromethane (CH2Cl2) was preferred, as dry reaction solvent, to

chloroform (CHCl3),3 carbon tetrachloride (CCl4)4 or ethyl ether (Et2O)5 because it is less
toxic or less flammable. However, Et2O was also considered as a second choice, and it was
employed to optimize and scale-up the synthesis.
Pyridine2a, 3 was preferred, compared to other reported general bases, because can be more
easily purified and dried. Moreover, pyridine hydrochloride has low solubility in
dichloromethane and can be precipitated during reaction, favouring reaction yield.
Characterization of obtained (±)-1,2-di-O-palmitoyl-3-chloropropane was carried out by m.p.

determination, thin-layer chromatography (TLC, on SiO2), 1H-NMR and elementary analysis.
Initially, in order to select the best method for the synthesis of (±)-1,2-di-O-palmitoyl-
3-chloropropane, in terms of yield and process scalability, some reaction trials were carried
out using either freshly-distilled dry dichloromethane or dry Et2O as reaction solvent (as
reported in “Experimental section”). Even if all these attempts gave the desired final
compound in high yield (from 80 to 95 % of yield), the use of dry dichloromethane was
preferred because it produced softer reaction mixture, causing less stirring problems.
Considering that this may be an issue for reaction scalability, freshly distilled dry
3
J. Am. Chem. Soc. 1941, 63, 3244-3248.
4
a) J. Chem. Soc. 1950, pp. 2663-2667; b) J. Am. Chem. Soc. 1950, 72, 942-949.
5
J.Prakt. Chemie. 1979, 756-768.
50
dichloromethane was adopted as the standard dry reaction solvent for the production of
(±)-1,2-di-O-palmitoyl-3-chloropropane.
The best method for compound purification resulted flash chromatography. Distillation at
reduced pressure or crystallization gave product decomposition or unchanged purity profile,
respectively. On the other hand, chromatographic purification of crude reaction product was
very time consuming and required huge amounts of n-hexane (about 2.10 L of n-hexane for
10g of crude). The yield of purification was low, with only 30-40 % of loaded compound
recovered as pure product to be delivered to animals.
Crude batches contained some impurities, mainly palmitic acid or contaminants of reaction
solvents. Reaction and purification procedures were setup to reduce their presence.
Purity of product batches was assessed by m.p. determination, 1H-NMR, elementary analysis
and TLC with spot visualization by potassium permanganate. Reference samples were
prepared adding small amounts of palmitic acid and 3-chloro-1,2-propandiol (1 % or
2 % w/w) to a purified sample of (±)-1,2-di-O-palmitoyl-3-chloropropane. We verified that
analytical data and spectra for each and all delivered batches were significantly better
(impurities in non-detectable or significantly lower amounts) than those of reference samples.
This means that all delivered batches had purity > 98 % w/w. Different batches were noot
pooled.
To achieve good yields with the required purity, the reaction had to be performed on a few-
grams scale.
Experimental section
Unless otherwise noted, all reagents and solvents were purchased from SigmaAldrich and
used without further purification.
Reactions were monitored by TLC SiO2 on Kiesegel 60 F 254 (DC-Alufolien, Merk).

Compounds were purified by flash chromatography, using SiO2 (SiO2 60, 40-63µm, Merck).
Melting points of final compounds were determined by a Tottoli (Buchi) melting point
apparatus. The purity of each released product batch was evaluated by TLC SiO2 on Kiesegel
60 F 254 (DC-Alufolien, Merk).
The NMR spectra were recorded on Bruker 300 (300 MHz) and 400 (400 MHz) Avance
spectrometers; chemical shifts (δ scale) were reported in parts per million (ppm) relative to
tetrameylsilane as internal standard. Elementary analyses were performed by ThermoQuest
FlashEA 1112 Elemental Analyser.
Synthesis
A solution of palmitoyl chloride (24.567 g; 89.386 mmol) in freshly distilled dry

dichloromethane (38 ml) was dropwise added under a N2 atmosphere to a stirring 0°C cooled
solution of 3-Chloro-1,2-propandiol 1 (4.704 g; 42.56 mmol) and dry pyrdine (7.406 g;
51
93.632 mmol) in freshly distilled dry dichloromethane (47 ml). During the addition the
reaction temperature was maintained between 0°C and 10°C. The reaction mixture was
allowed to reach the room temperature and stirred for 16 h.
The reaction solvent was distilled away under reduced pressure and the resulting pale yellow
residue was dissolved into Et2O (200 mL). The organic solution was washed with 1 % HCl
aqueous solution (2 X 100 mL), Na2CO3 saturated aqueous solution (2 X 50 mL) and water
(2 X 100 mL). After drying over anhydrous Na2SO4, the organic solution was filtered and the
solvent was evaporated under reduced pressure.
MeOH (200 mL) was added to the obtained white solid residue and the mixture was heated to
52°C under stirring condition for 1h. The obtained well dispersed emulsion was gently cooled
back to R.T. and the precipitated white solid was filtered. The crude was triturated in MeOH
(200 ml), filtered, dried under reduced pressure and purified by flash chromatography (SiO2,
SiO2/Loading ratio: 3/1, n-hexane/dichloromethane from 99/1 to 84/16). The recovered pure
product was characterized by m.p. determination, direct phase TLC, 1H-NMR and elementary
analysis.
m.p. = 50-51 6
Not scheduled costs
The low purification efficiency of flash chromatography used to purify the reaction crudes
required large amounts of n-hexane. At this date, about 56 L of n-hexane (7.76 E/L +20 %
VAT) were used to produce and deliver 71.88 g of (±)-1,2-di-O-palmitoyl-3-chloropropane.
Recycling: 30 %.
6
46-47°C (J.Prakt. Chemie. 1979, pp. 756-768), 51-52,5°C (J. Am. Chem. Soc. 1971, 49, 281-282), 48-50°C
(Chem. Ber. 1905, 38, p. 2286).
52
Typical TLC comparative analysis (product versus reference samples, KMnO4 aqueous
solution was used as TLC stain):
n-hexane/ethylacetate 95/5 n-hexane/ethylacetate 80/20
Product Product
Reference Reference
Rf Product = 0.30 7 Rf Product = 0.61
1
H-NMR (CDCl3, 300 MHz): δ 0.90 (t, J= 6.4 Hz, 6H, -CH3), 1.27 (m, 48H, -CH2-), 1.63 (m,
4H, -CH2-), 2.34 (t, J=7.8 Hz, 2H, -O(CO)CH2-), 2.35 (t, J = 7.2 Hz, 2H, -O(CO)CH2-), 3.64
(dd, J=11.7 , 5.4 Hz, 1H –CH2-Cl), 3.71 (dd, J=11.7 , 5.4 Hz, 1H –CH2-Cl), 4.23 (dd, J =
11.9, 5.6 Hz, 1H -CH2O-), 4.36 (dd, J = 11.9, 4.3 Hz, 1H, -CH2O-), 5.23 (tt, J = 5.5, 5.4 Hz,
1H, -CH-).
7
Product Rf value was comparable with Rf reported for commercially available (±)-1,2-di-O-palmitoyl-3-
chloropropane supplied by Toronto Research Chemicals (Certificate of Analysis).
53
Delivered product batches
Batches Code Laboratory Workbook Reference Code Quantity (g)

UPR1281.00/1001 Q20-263-21 16.5
UPR1281.00/1002 Q20-271-05 7.1
UPR1281.00/1003 Q20-271-10 12.685
UPR1281.00/1004 Q20-277-03 14.109
UPR1281.00/1005 Q20-277-04 6.605
UPR1281.00/1006 Q20-285-10 6.025
UPR1281.00/1007 Q20-285-19 1.479
UPR1281.00/1008 Q20-285-21 4.071
UPR1281.00/1009 Q20-285-24 3.306
Attachments
1- Typical 1H-NMR product spectra

2- Certificate of Analysis of (±)-1,2-di-O-palmitoyl-3-chloropropane from Toronto
Research Chemicals supplier
54
55
56
APPENDIX II
Synthesis of 3-MCPD Mercapturic Acid (N-acetyl-S-(2,3-

dihydroxypropyl)cysteine) and validation of HPLC-MS/MS method
In order to develop an analytical method for the quantification of 3-MCPD and its metabolites
it is necessary to have the standards.
The standards of 3-MCPD, 3-MCPD-d5 and β-chlorolactic acid were purchased from Sigma-
Aldrich (98 %), while for the mercapturic acid the standard was not commercially available
and chemical synthesis was necessary.
Synthesis of N-acetyl-S-(2,3-dihydroxypropyl)cysteine
The synthesis of N-acetyl-S-(2,3-dihydroxypropyl)cysteine (S-DHPMA) was performed on

the basis of another study8, using L-cysteine hydrochloride and 3-chloropropan-1,2-diol
(Sigma-Aldrich) as reagents and triethylamine and distilled water as solvents and two steps
were necessary.
Step 1
First, the reagents were mixed in a reaction test-tube and then, the homogeneous solution was
kept at room temperature for at least 3 days. The solution was reduced under vacuum at
40-45°C obtaining a white residue, purified in 10-20 mL of methanol (boiled for 3 hours).
Then, the solution was centrifuged at 4000 r.p.m. for 5 minutes and filtered.
The S-(2,3-dihydroxypropyl)cysteine, product of the first step of the process, was obtained by
dissolving the residue in 1 mL of distilled water and precipitated with 60-80 mL of acetone.
The solvent was eliminated drying the product under vacuum (40-45°C).
The first reaction product was controlled by TLC (Thin Layer Chromatography) technique.
For TLC control few mg of reaction product were dissolved in 200 µL of distilled water. For
this analysis L-cysteine was used as reference. Two spots were pointed on the TLC plate (one
of reference and one of hypothesized S-(2,3-dihydroxypropyl)cysteine) and developed with a
8
A.R. Jones, The metabolism of 3-chloro-, 3-bromo- and 3-Hydropropan-1,2-diol in rats and mice, (1975)
Xenobiotica, 5 (3), 155-165.
57
mixture of buthanol/water/acetic acid (3.5/1/0.5) as eluent. The TLC plate was then treated
with a solution of sulphuric acid in ethanol (10 %) and heated at 150°C in order to evidence
the spots of the analytes. The RF (Retention Factor) of L-cysteine results 0.18, while the S-
(2,3-dihydroxypropyl)cysteine remains at the base of the deposition on TLC plate. After the
TLC the result was confirmed by tandem mass spectrometry technique (AB Sciex API4000).
Step 2
S‐DHPMA
The second step of the reaction brigs to the formation of N-acetyl-S-(2,3-

dihydroxypropyl)cysteine as mercapturic acid (S-DHPMA): S-(2,3-dihydroxypropyl)cysteine,
obtained from the first step of reaction, was dissolved with 1 mL of distilled water, put in a
test-tube and added with 0.5 mL of acetic anhydride. The solution was shaken for 2 minutes
and then kept in a laboratory oven at 30°C for 16 hours. The solution was dried under vacuum
at 40-45°C obtaining a yellow-brown residue: the crude mercapturic acid.
The reaction product was characterized both by tandem mass spectrometry (AB Sciex API
4000) and 1H-NMR spectroscopy (Varian INOVA-600 mHz), confirming that the pure N-
acetyl-S-(2,3-dihydroxypropyl)cysteine was effectively formed (see figure below).
This product was used as standard for the development of the analytical method and for the
analysis of real urinary samples.
Same procedure was applied on the 3-MCPD-d5 standard to obtained S-DHPMA-d5.
58
HOOC
S OH
HN OH
COCH3
5000
4.50 4.00 3.50 3.00 2.50

ppm (f1)
1H-NMR spectrum of N-acetyl-S-(2,3-dihydroxypropyl)cysteine interpretation:
Proton groups (n°) Number of proton atoms Corresponding signals (ppm)
1 3 2.04
2 1 4.60
3 2 2.70
4 2 3.10
5 1 3.83
6 2 3.60
59
LC-MS/MS method
Liquid chromatography was carried out on an Agilent HP 1100 Series HPLC apparatus,
consisting of a binary pump, a thermostated autosampler and a vacuum degasser. The LC
system was coupled with a AB Sciex API 4000 triple-quadrupole mass spectrometer (Sciex,
Concord, Canada) equipped with a Turbo Ion SprayTM interface. Chromatography was
performed on an Atlantis®dC18 column (100 x 2.0 mm i.d., 3µm; Waters, Milford, MA,
USA) using variable proportion of 20 mM aqueous acetic acid and methanol/acetonitrile
(MeOH/AcCN, 95/5,v/v) mixture as the mobile phase. Elution program: 0 % MeOH/AcCN,
hold for 3.5 min; from 0% to 90% MeOH/AcCN in 5 min (linear gradient); 90 %
MeOH/AcCN, hold for 1 min; the back to the starting condition in 0.30 min. The flow-rate
was 0.2 mL/min. The injection volume was 5 µL and each analysis required 22 min, including
the re-equilibration time. The first (0-2 min) and the last (8-22 min) parts of the run were
diverted to waste using a 10-port valve (Valco Systems, Houston, Texas, USA).
The analytes were ionized in negative ion mode and the detection was obtained in selected-
reaction mode (SRM) after optimization of TISP-MS/MS parameters by infusing a 1 mg/L
solution of 3-MCPD in 0.1 % aqueous acetic acid and a 1 mg/L solution of S-DHPMA and β-
Cl-lactic Acid in 50/50 (v/v) 0.1% aqueous acetic acid/MeOH. For 3-MCPD and β-Cl-lactic
Acid, [M+CH3COOH-H]- was selected by first mass filter. After collision activation, the
higher specific ions were selected by the last mass filter for each analytes. SRM transitions,
the limit of quantifications (LODs, S/N=3, μg/L) and the calibration curve ranges (μg/L) are
summarized in Table I for all analytes and ISs.
Table I: SRM transitions, LODs and calibration curve concentrations for all analytes and
ISs.
SRM transition LOD Range SRM transition

Compound Q1 Q3 (μg/L) (μg/L) IS Q1 Q3 (μg/L)
169 95
3-MCPD a 1.73 10-200 3-MCPD-d5a 174 95 50
171 97
107
S-DHPMA b 236 0.71 20-400 S-DHPMA-d5b 241 112 100
128
β-Cl-lactic-acid a 183 123 9.0 40-800
Legend: a standard commercially available;

b
standard obtained by synthesis.
Calibration standard curves were obtained by spiking a 100 fold diluted urine sample with
standard mixtures at five concentration levels and with two labelled ISs mixture. Calibration
curves were constructed by linear regression analysis of the area ratios analyte/IS versus the
concentration of analytes injected, but not for β-Cl-lactic Acid. In all samples we are able to
quantify 3-MCPD and S-DHPMA. Before the injection, urines of untreated rats and rats
treated with low doses of both 3-MCPD and 3-MCPD diesters were diluted 1:100, urines of
rats treated with middle and high doses were diluted 1:1000.
60
A chromatogram of a 100 fold diluted urine sample of a rat treated with the lowest dose of
3-MCPD is reported in Figure 1. In particular, the transitions related to 3-MCPD and its IS are
on the left of the figure and the ones related to the S-DHPMA and its IS are on the right.
Figure 1: Chromatogram of urine sample diluted 1:100 of rat treated with the lowest
dose of 3-MCPD. Chromatograms on the left are related to 3-MCPD, whereas
those on the right refers to S-DHPMA.
61
APPENDIX III
IDENTIFICATION AND QUANTIFICATION OF 3-MCPD METABOLITES IN RATS

TREATED WITH DEUTERATED 3-MCPD AND ITS DIPALMITIC ESTER
INTRODUCTION
The aim of this experiment is to study the metabolism of free 3-MCPD and 3-MCPD palmitic
diester, using deuterated compounds and to evaluate hypothetic interferences.
METHODS
Substances:
3-MCPD-d5 98 % pure was purchased from Sigma Aldrich Inc. Deuterated palmitic di-ester
of 3-MCPD was synthesized (starting from 3-MCPD-d5) by Pharmaceutical Department,
University of Parma.
Animals:
Four young healthy adults rats of both sexes (2 males and 2 females nulliparous and non-
pregnant) were employed, for a period of 4 days. Rats were housed in monitored
environmental conditions: temperature 22±3°C, relative humidity 50-60 %, light/dark cycles
of 12 hours each.
Each rat was housed individually for consecutive four days. All cages were arranged in such a
way that possible effects due to cage placement were minimized.
Both 3-MCPD-d5 and its deuterated palmitic diester were dissolved in corn oil and
administered by oral gavage in a single dose, the second day. Firstly, 2 male rats and 2 female
rats were treated with free 3-MCPD-d5 (29.5 mg/kg for males and 20 mg/kg for females), a
week later, the same rats were treated with the 3-MCPD-d5 palmitic diester (156.7 mg/kg).
One male and one female were treated only with corn oil and used as controls.
Sampling:
Urine samples were collected for four days (each sample corresponded to 24hrs urine), one
day before the treatment and for two days after it.
LC-MS/MS METHOD
Liquid chromatography was carried out with an Agilent HP 1100 Series HPLC apparatus,
consisting of a binary pump, a thermostated autosampler and a vacuum degasser. The LC
system was coupled with a AB Sciex API 4000 triple-quadrupole mass spectrometer (Sciex,
Concord, Canada) equipped with a Turbo Ion SprayTM interface. Chromatography was
performed on an Atlantis®dC18 column (100 × 2.0 mm i.d., 3µm; Waters, Milford, MA,
USA) using variable proportion of 20 mM aqueous acetic acid and methanol/acetonitrile
(MeOH/AcCN, 95/5,v/v) mixture as the mobile phase. Elution program: 0 % MeOH/AcCN,
hold for 3.5 min; from 0 % to 90 % MeOH/AcCN in 5 min (linear gradient);
90 % MeOH/AcCN, hold for 1 min; the back to the starting condition in 0.30 min. The flow-
rate was 0.2 mL/min. The injection volume was 5 µL and each analysis required 22 min,
62
including the re-equilibration time. The first (0-2 min) and the last (8-22 min) parts of the
chromatographic run were diverted to waste using a 10-port valve (Valco Systems, Houston,
Texas, USA).
The analytes were ionized in negative ion mode and the detection was obtained in selected-
reaction mode (SRM) after optimization of TISP-MS/MS.
In Table I we reported the mass spectrometric transitions, the limit of quantifications (LODs,
S/N=3, μg/L), the calibration curve ranges (μg/L). The glucuro- and sulfo-conjugated
metabolites are not commercially available, so for semi-quantitative analysis we used the
commercially available ones, namely Phenyl-glucuronide (PhG), p-nitro-Phenyl-glucuronide
(NO2-PhG), Naphtyl-glucuronide (NG), p-nitro-Phenyl-sulfate (NO2-PhS) and Naphtyl-
sulfate (NS) as references.
Table I: Transitions used to quantify relevant analytes and corresponding LODs and
dynamic ranges.
SRM transition LOD Range

Compound Q1 Q3 (μg/L) (μg/L)
3-MCPD-d5a 174 95 1.73 10-200
b 112
S-DHPMA-d5 241 0.71 20-400
128
c
β-Cl-lactic-acid d3 286 126 - -
a
β-Cl-lactic-acid 283 123 9.0 40-800
113
3-Cl-2-propanolG-d5 c 285
175
- -
3-Cl-2-propanolS-d5 c 190 110 - -
a 113
PhG 269 0.28 50-1000
175
113
NO2-PhGa 314
175
0.25 50-1000
319 113
NGa 175
0.30 50-1000
NO2-PhSa 218 138 0.05 50-1000
NSa 223 143 0.06 50-1000
Legend: a standard commercially available;

b
standard obtained by synthesis;
c
hypothized metabolite;
G: glucuronide; S: sulfate; Ph: Phenyl; N: Naphtyl.
Calibration standard curves were obtained by spiking urine samples after a 100 fold dilution
with standard mixtures at five concentration levels. For all the standards the % CV calculated
on all the calibration levels were in the 2-6 % range.
63
RESULTS
The main objective of this experiment was the identification of metabolites shared by
3-MCPD-d5 and of its deuterated palmitic diester. For this purpose, we looked for the
following possible metabolites: free 3-MCPD-d5, the N-acetyl-S-(2,3-
dihydroxypropyl)cysteine as mercapturic acid (S-DHPMA-d5), β-chloro-lactic acid-d3, 3-
chloro,2-propanolglucuronide-d5 and 3-chloro,2-propanolsulfate-d5.
LODs were calculated in matrix. For 3-MCPD-d5 and S-DHPMA-d5 we used standards
obtained one by Sigma and the other by our own synthesis, respectively. LOD for β-chloro-
lactic acid-d3 was assumed to be the same of the unlabelled β-chloro-lactic acid. The standard
of the glucuro- and sulfo-conjugates of 3-MCPD-d5 are not commercially available. For a
semi-quantitative analysis, we assumed that their LODs were the same of commercially
available products, namely Phenyl-glucuronide, p-nitro-Phenyl-glucuronide, Naphtyl-
glucuronide, p-nitro-Phenyl-sulfate and Naphtyl-sulfate.
In Table II, we reported the amounts (mg/24 h) and of the % of the dose of the founded
metabolites in the four 24 h urine samples. β-chloro-lactic acid-d3, and both glucuro- and
sulfo-conjugates-d5 were below the LODs in all the urinary samples.
Table II
Treatment
controls 3-MCPD-d5 palmitic diester 3-MCPD-d5
Metabolite day M (n=1) F (n=1) M (n=2) F (n=2) M (n=2) F (n=2)
1 <LOD <LOD <LOD <LOD <LOD <LOD
3-MCPD-d5 2 <LOD <LOD 0.696 0.605 1.835 0.251
(mg/24hrs) 3 <LOD <LOD 0.039 0.008 0.029 0.008
4 <LOD <LOD 0.007 0.004 0.006 0.001
1 <LOD <LOD <LOD <LOD <LOD <LOD
S-DHPMA-d5 2 <LOD <LOD 0.216 0.090 0.429 0.061
(mg/24hrs) 3 <LOD <LOD 0.038 0.008 0.067 0.010
4 <LOD <LOD 0.010 0.005 0.014 0.005
1 N.A. N.A. N.A. N.A. N.A. N.A.
3-MCPD-d5 2 N.A. N.A. 6.69 9.36 22.31 3.81
(% dose) 3 N.A. N.A. 0.38 0.12 0.35 0.11
4 N.A. N.A. 0.07 0.07 0.07 0.02
1 N.A. N.A. N.A. N.A. N.A. N.A.
S-DHPMA-d5 2 N.A. N.A. 2.12 1.35 5.20 0.93
(% dose) 3 N.A. N.A. 0.37 0.12 0.81 0.15
4 N.A. N.A. 0.10 0.08 0.17 0.07
In the case of one acute dose and even if the sample number is low (only two rats for each
treatment), the obtained data confirm the previous results.
64
APPENDIX IV
65
APPENDIX V
PATHOLOGY
Ninety days after each treatment rats have been killed in saturated chamber of vapour of ethyl
ether and CO2 and necropsied by staff of the Veterinary Medicine of Parma University.
During necropsies cerebrum, cerebellum and medulla/pons, spinal cord, pituitary, thyroid,
parathyroid, thymus, oesophagus, salivary glands, stomach, small and large intestines
(including Peyer’s patches), liver, pancreas, kidneys, adrenals, spleen, heart, trachea and
lungs, aorta, gonads, uterus, accessory sex organs, female mammary gland, prostate, urinary
bladder, lymph nodes, peripheral nerve (sciatic or tibial) in close proximity to the muscle, a
section of bone marrow and a fresh bone marrow aspirate, skin and eyes have been collected
and formalin fixed 10 % v/v.
Thymus, heart and aorta, salivary glands, spleen, liver, kidneys, adrenals, gonads and
accessory sex glands were weighted (Appendix VII).
Paraffin sections, 4-5 µm thick, were stained with Hematoxylin and Eosin and PAS reaction.
Slides have been observed using a Nikon Eclipse E800 microscope (Nikon Corporation,
Japan). Images were captured at 4x, 10x, 20x magnifications using DIGITAL SIGHT DS-Fi1
Camera.
The rats that died during the testing period were necropsied by staff of the Veterinary
Medicine of Parma University (Appendix VI).
FIRST TREATMENT- 3-MCPD DIPALMITATE 156.75 mg/kg b.w. per day

3-MCPD 29.5 mg/kg b.w. per day
Necropsy
During experimentation rats n 46, 47, 48, 49 and 50 (5/10) died and were subject to necropsy
(Appendix VI).
Sacrifices were scheduled on:

‐ June 8: 5 control male rats (1-5)
‐ June 9: 10 treated with 3-MCPD male rats (16-25)
‐ June 10: 9 treated with 3-MCPD dipalmitate male rats (6-15 except n14)
‐ June 15: 5 control female rats (26-30)
‐ June 16: 10 treated with 3-MCPD dipalmitate female rats (31-40)
‐ June 17: 5 treated with 3-MCPD female rats (41-45) and 1 male rat treated with
3-MCPD dipalmitate (rat 14)
All rats have shown congestive splenomegaly and lung congestion. Lung and spleen lesions
are related to agonic state of rats during euthanasia.
Control male group (rats 1-5): did not show gross lesions.
66
3-MCPD dipalmitate (156.75 mg/kg b.w. per day) male group (rats 6-15): the presence of
white gelatinous material was noted in urinary bladder of rats 7, 9 and 14 (3/10).
3-MCPD (29.5 mg/kg b.w. per day) male group (rats 16-25): showed several changes:
heart: slight hypertrophy, rat 20 (1/10)
kidneys: small cyst (right kidney) and renal pelvis dilation (left kidney), rat 21 (1/10);
discolouration renal cortex, rat 17 (1/10);
gonads: fluid collection in tonaca albuginea (10/10); white material in epididymis, rat 16
(1/10);
urinary bladder: presence of white gelatinous material, rats 18, 19, 21, 22, 23 (5/10).
Female control group (rats 26-30): presence of numerous ovarian formation resembling
follicles/cysts was noted.
3-MCPD dipalmitate (156.75 mg/kg b.w. per day) female group (rats 31-40):
heart: hemorrhage observed at interventricular sulcus, rat 37 (1/10);
liver: hepatomegaly, rats 31, 32, 33, 34 (4/10); slightly yellow parenchyma colour, rat 35
(1/10); pale parenchyma, rat 37 (1/10);
gonads: presence of numerous ovarian formation resembling follicles/cysts (10/10).
3-MCPD (29.5 mg/kg b.w. per day) female group (rats 41-45):
gonads: presence of numerous ovarian formation resembling follicles/cysts (5/5):
skin: right flank self-induced alopecia (broken hair). Scotch test: negative for fungus and
mites, rats 41 and 44 (2/5).
Histopathological evaluation of kidneys lesions (scores in appendix VII)
Kidney general description:

Histopathological evaluation of the kidneys of the experimental groups highlighted
progressive degenerative lesions involving different tubular segments of one or more parts of
the kidney parenchyma. The main findings can be summarized as follows: multifocal
hypertrophy and swelling of epithelial cells of proximal tubules, hydropic-vacuolar
degenerations and picnosis of tubular epithelial cells, hyaline and basophilic casts scattered
among tubules in the overlying cortex and subjacent medulla, tubular regenerative
hyperplasia, kariomegaly. Glomeruli, more resistant to treatments, showed mild
morphological changes.
Control male and female groups (rats 1-5 and 26-30): mild or rare hydropic-vacuolar
degeneration, swelling of epithelial cells of proximal tubules were observed in all animals
(10/10), mild picnosis in male rats (4/5) and mild presence of granular basophilic casts in
proximal tubules was observed in all rats (10/10). No other lesions were found (Figures 1a
and 1b).
3-MCPD dipalmitate (156.75 mg/kg b.w. per day) male group (rats 6-15): mild hydropic-
vacuolar degeneration was present in all animals (10/10); tubular regenerative hyperplasia
was mild or moderate in all rats (10/10); mild tubular ectasia was observed in 2/10 rats; mild
glomerular hypertrophy or atrophy was present in 8/10 rats; moderate or mild karyomegaly
67
of epithelial cells was present in 7/10 rats and mild-moderate presence of granular basophilic
cast were observed in all animals (10/10) (Figure 2).
3-MCPD (29.5 mg/kg b.w. per day) male group (rats 16-25): severe hypertrophy or
swelling of the epithelial cells of proximal tubules was present in rats (10/10); moderate to
severe hydropic-vacuolar degeneration was present (10/10); moderate to severe karyomegaly
of epithelial cells (10/10) and moderate or severe presence of granular basophilic cast were
observed (10/10); focal regenerative epithelial hyperplasia was recorded (10/10) (Figure 3).
3-MCPD dipalmitate (156.75 mg/kg b.w. per day) female group (rats 31-40): mild
epithelial cell swelling, hydropic-vacuolar degeneration and presence of granular basophilic
casts within tubules were observed in all rats (10/10). Six rats showed mild tubular
regeneration (6/10). Nuclear pyknosis and karyomegaly, glomerular nephrosis, and tubular
ectasia were rare or absent in all rats (Figure 4).
3-MCPD (29.5 mg/kg b.w. per day) female group (rats 41-50): mild and moderate swelling
of the epithelial cells of tubules were observed in all rats (10/10); hydropic-vacuolar
degeneration was mild in three rats (3/10); pyknosis of nuclei of tubular cells was absent in all
animals (10/10); severe and moderate accumulation of granular cast in tubular lumen was
present in all rats (10/10); glomerular hypertrophy/atrophy and tubular regeneration were mild
or moderate in all rats (10/10) (Figure 5).
a b
Figure 1: Kidney control group,

a: male rat 1- PAS 10x; b: female rat 28- PAS 10x.
68
Figure 2: 3-MCPD dipalmitate male group.

kidney: mild/moderate presence of granular basophilic casts (arrows)
a: rat 12 PAS 10x; b: rat 13 PAS 20x.
a b
Figure 3: 3-MCPD male group, kidney:

a) hyperplasia and kariomegaly tubular epithelium and tubular epithelial hypertrophy rat 20
PAS 20x and b: basophilic casts in tubular lumina rat 24 PAS 10x.
69
a b
Figure 4: 3-MCPD dipalmitate female group, kidney:

a) epithelial tubular regeneration and b) tubular ectasia (arrowhead)
H-E 20x.
Figure 5: 3-MCPD female group, kidney:

tubular epithelial hypertrophy and basophilic casts (arrows)
in rat 44 H-E 10x (a) and 20x (b).
Histopathological evaluation of testis lesions (scores in Appendix VII)
Control group (rats 1-5): in control rats the testes are normal, there is no degenerative or
inflammatory phenomena in seminiferous tubules, Sertoli cells or ductal structures of the
epididymis (Figure 6).
3-MCPD dipalmitate (156.75 mg/kg b.w. per day) group (rats 6-15): rats 9 to 12 slight to
moderate degenerative features in the seminiferous tubules (4/10); rats 7 and 8 show total
degeneration of seminiferous tubules (2/10) (Figure 7a); the other rats show atrophy (3/10),
necrosis of germinative cells (3/10) as well as of Sertoli cells (3/10) (Figure 8). Presence of
multinucleated cells (Figure 7b).
70
3-MCPD (29.5 mg/kg b.w. per day) group (rat 16-25): rats 17, 18, 19, 20, 21, 22, 23, 24
and 25 (9/10) show total degeneration of seminiferous tubules and limphomononuclear
infiltrates in the epididymus. Rat n 16 show abscess of the epididymis (1/10) and rat n 24
display focal hyperplasia of Leydig cells (1/10) (Figures 9 and 10).
a b
Figure 6: Control group, testis rat 1: H-E 10x (a) and H-E 20x (b).
Figure 7: 3-MCPD dipalmitate group, testis rats 7 and 8 show severe degenerative change of
seminiferous tubules with Sertoli and germinal cells atrophy.
Presence of multinucleated giant cells.
H-E-10x (a) and H-E-20x (b)
71
a b
Figure 8: 3-MCPD dipalmitate, testis: absence of spermatozoa in epididymis duct (a) and
degeneration and atrophy of seminiferous tubulus (b) H-E 20x.
Figure 9: 3-MCPD group, testis: a) mineralization, Leydig cells hyperplasia and b) tubules
seminiferous degeneration with atrophy and depletion of germinal and Sertoli cells.
72
Figure 10: 3-MCPD group, testis: a) absence of spermatozoa in the lumina of epididymal
ducts and b) atrophy of seminiferous tubules.
SECOND TREATMENT- 3-MCPD DIPALMITATE 39.19 mg/kg b.w. per day

Necropsy
During experimentation female rat 94 died in October, 8 2010, and was subjected to necropsy
(Appendix VI).

‐ October 4: 5 control male rats (51-55)
‐ October 5: 10 treated with 3-MCPD dipalmitate 39.19 mg/kg, male rats (56-65)
‐ October 6: 10 treated with 3-MCPD 7.37 mg/kg, male rats (66-75)
‐ October 11: 5 control female rats (76-80), and six rats treated with deuterio
‐ October 12: 10 treated with 3-MCPD dipalmitate 39.19 mg/kg, female rats (81-90)
‐ October 13: 9 treated with 3-MCPD 7.37mg/kg, female rats (91-100), except 94 died 08-
10-2010
All rats showed congestive splenomegaly and lung congestion. Lung and spleen lesions are
related to euthanasia.
Control male group (rats 51-55): did not show gross lesions. In the urinary bladder white
gelatinous material was observed (rats 53, 54 and 55) (3/10).
3-MCPD dipalmitate (39.19 mg/kg b.w. per day) male group (rats 56-65): no gross
lesions were observed unless a moderate hyperplasia of Payer's patches.
3-MCPD (7.37 mg/kg b.w. per day) male group (rats 66-75): showed several changes:
heart: dilated cardiomyopathy in rat 69 (1/10);
kidney: small cyst in rat 66 and rat 68 (2/10);
gonads: enlarged in rat 72 and 74 (2/10); smaller in rat 75 (1/10);
urinary bladder: presence of white gelatinous material, rat 73 (1/10).
73
Control female group (rats 76-80): no gross lesion was observed
3-MCPD dipalmitate (39.19 b.w. mg/kg per day) female group (rats 81-90):
kidney: rat 86 dilation of pelvis of the right kidney; left kidney is cystic and transformed in a
thin-walled sac, filled with yellow fluid (1/10) (Figure 11);
gonads: right ovary cyst, rat 82 (1/10); atrophic uterine horn, rat 86 (1/10).
3-MCPD (7.37 mg/kg b.w. per day) female group (rats 91-100):
Kidney: yellow kidney and cortical scarring with capsular depression were observed in rat
number 96 (1/10) (Figures 12-13) and wrinkled kidney in rat 100 (1/10);
gonads: ovary were smaller in all rats (10/10).
Figure 11: Female rat 86 treated with 3-MCPD dipalmitate: left kidney filled with yellow
fluid.
12 13
Figures 12-13: Female rat 96 treated with 3-MCPD yellow kidney and scar.
74
Kidneys general description:
The renal findings of male rats treated with 39.19 mg/kg per day 3MCPD dipalmitate and
7.37 mg/kg per day 3-MCPD can be summarized as follows: moderate multifocal hypertrophy
and swelling of epithelial cells of proximal tubules, mild hydropic-vacuolar degenerations and
pyknosis of tubular epithelial cells, hyaline and basophilic casts scattered among tubules in
the overlying cortex, mild tubular regenerative hyperplasia, mild atrophy or hypertrophy of
glomeruli.
The female rats treated with 39.19 mg/kg per day 3MCPD dipalmitate and 7.37 mg/kg per
day 3-MCPD showed degenerative and inflammatory changes, as multifocal interstitial
nephritis and pyelitis.
Control male and female groups (rats 51-55 and 76-80): in male control rats mild features
of glomerular atrophy/hypertrophy (2/5) and hypertrophy and cellular swelling (4/5) were
observed; in 4/5 female rats mild hydropic-vacuolar degeneration and swelling of epithelial
cells of proximal tubules was observed; features of multifocal interstitial nephritis was present
in 2/5 female rats.
3-MCPD dipalmitate (39.19 mg/kg b.w. per day) male group (rats 56-65): mild to
moderate hydropic-vacuolar degeneration, hypertrophy and cellular swelling was present in
9/10 animals. Mild tubular ectasia was observed in 4/10 rats. Mild-moderate presence of
granular basophilic casts were also observed in all animals.
3-MCPD (7.37mg/kg b.w. per day) male group (rats 66-75): moderate or severe
hypertrophy and swelling of the epithelial cells of proximal tubules was present in all rats.
Mild or moderate presence of granular basophilic cast were observed. Focal regenerative
epithelial hyperplasia was recorded in 3/10 rats.
3-MCPD dipalmitate (39.19mg/kg b.w. per day) female group (rats 81-90): mild and
moderate picnosis (5/10), hydropic-vacuolar degeneration ((9/10) hypertrophy and swelling of
the epithelial cells of proximal tubules (8/10) were observed. Mild accumulation of hyaline
granular casts in tubular lumen was present in 4/10 rats. Multifocal interstitial cellular
infiltrates and tubular cystic ectasia were observed in 2/10 and 3/10 female rats.
3-MCPD (7.37mg/kg b.w. per day) female group (rats 91-100): hydropic-vacuolar
degeneration (7/10) hypertrophy and swelling of the epithelial cells of proximal tubules (5/10)
were observed. Mild accumulation of hyaline granular casts in tubular lumen was present in
4/10 rats. Moderate regenerative hyperplasia was present in 4/10 rats. Features of multifocal
interstitial chronic nephritis (3/10) (Figure 15) pyelitis (1/10) (Figure 14) and tubular cystic
ectasia (4/10) were observed in female rats number 93, 95, 96 and 100 (Figure 16).
75
Figure 14: Kidney: pyelitis in female rat 97 treated with 3-MCPD a) H-E 4x; b) H-E 10x.
Figure 15: Kidney: interstitial nephritis in female rat 95 treated with 3-MCPD H-E 10x.
76
a b
Figure16: Kidney: cystic tubules in rat 96 treated with 3-MCPD

a) H-E 4x; b) H-E 10x.
Histopathological evaluation of testis lesions (scores in Appendix VII)
Control male groups (rats 51-55): in rat number 51, 52 and 54 (3/10) the testes slight mild
decrease of spermatids, atrophy of spermatogenic and supporting cells in the seminiferous
tubules.
3-MCPD dipalmitate (39.19 mg/kg b.w. per day) male group (rats 56-65): rats n 60, 63
and 65 (3/10) presents mild degenerative change with decrease of spermatids.
3-MCPD (7.37mg/kg b.w. per day) male group (rats 66-75): rats 66, 67, 70, 71, 72 and 74
(6/10) show mild decrease of spermatids, atrophy of spermatogenic and supporting cells in the
seminiferous tubules (Figure 17, 18, 19, and 20). Rat 72 present also mild inflammatory
infiltrates in epididymis.
77
Figure 17: Testis rat 70 treated with 3-MCPD H-E 10x.
Figure 18: Testis rat 71 treated with 3-MCPD with giant cells in tubules H-E 10x.
78
19
20
Figure 19 and 20: testis: decrease of spermatids cells in rat74 treated with 3-MCPD
H-E 10x.
79
THIRD TREATMENT- 3-MCPD DIPALMITATE 9.78 mg/kg b.w. per day

Necropsy
During experimentation female rat 162 died in January 6, 2011 (47th day of treatment) and
female rat 163 died in February 13, 2011 (82nd day of treatment) (2/10). Both were subject to
necropsy (Appendix VI).
- February 7: 5 control male rats

- February 8: 10 male rats (106-115) treated with 3-MCPD dipalmitate
- February 9: 10 male rats (116-125) treated with 3-MCPD
- February 14: 10 control female rats (126-135)
- February 15: 10 female rats (136-145) treated with 3-MCPD dipalmitate
- February 16: 10 female rats (146-155) treated with 3-MCPD
- February 21: 10 female rats (156-165) treated with 3-MCPD
All rats showed an acute congestive splenomegaly and lung congestion.

Lung and spleen lesions are related to euthanasia with vapor of ethyl ether and CO2.
Male control group (rats 101-105): no gross lesions was observed.
3-MCPD dipalmitate (9.78 mg/kg b.w. per day) male group (rats 106-115): rat 108 shows
white spot into liver (1/10) and on rat 111 petechial bleeding in thymus was observed (1/10)
3-MCPD (1.84 mg/kg b.w. per day) male group (rats 116-125): rat 117 shows heart
dilation (1/10)
Female control group (rats 126-135): rats 127, 130 and 132 show clear fluid in the uterus
and enlargement of uterus (oestrus) (3/10).
3-MCPD dipalmitate (9.78 mg/kg b.w. per day) female group (rats 136-145): rats 137,
138, 141 and 143 show clear fluid in the uterus and enlargement of uterus (oestrus) (4/10)
(Figure 21); the rat 144 has atrophy of right uterine horn (isolated malformation) (1/10).
3-MCPD (1.84 mg/kg b.w. per day) female group (rats 146-155): rats 151 and 155 show
clear fluid in the uterus and enlargement of uterus (oestrus) (2/10).
3-MCPD (29.5 mg/kg b.w. per day) female group (rats 156-165): the management history
of rat 156 report oligodipsia and anorexia for one week and weight loss, the clinical history
80
of rats 156, 157, 161 and 164 report clinical signs of discomfort at various moment of
experiment like oligodipsia, anorexia, weight loss, respiratory failure and orripilation (4/10);
the rats 156, 157, 158, 160, 161 and 165 show kidneys pale and large (6/10) (Figure 22); the
rats n. 159, 161 and 164 display edema of uterus (oestrus) (3/10); rat 165 show cardiac
dilation (1/10) (Figure 23); rat n. 156 has one lobe of liver in thoracic cavity and the trachea is
dilated (1/10); rat n. 162 died January 6, 2011 and rat 163 died February 13, 2011.
81
Figure 21: Female Wistar rats

3-MCPD dipalmitate (9.78
mg/kg b.w. per day) group, rat
138: enlargement of uterus.

3-MCPD (29.5 mg/kg b.w. per
day) group, rat 156: kidneys pale
and large.

3-MCPD (29.5 mg/kg b.w. per
day) group, rat 165: cardiac
dilation.
82
Male control group (rats 101-105): regenerative hyperplasia of proximal tube is rare (1/5);
hypertrophy and swelling of epithelial cells of proximal and distal tubules are mild or
moderate in all rats (5/5); hyaline and basophilic casts in proximal tubules are mild or
moderate (4/5) and rare in distal tube (1/5); cystic tubules are rare or mild (2/5) and rare are
also the interstitial infiltrated (1/5).
3-MCPD dipalmitate (9.78 mg/kg b.w. per day) male group (rats 106-115): hypertrophy
and swelling of epithelial cells of proximal and distal tubules are mild or moderate in all rats
(10/10); hydropic-vacuolar degenerations of tubular epithelial cells are rare (3/10); hyaline
and basophilic casts in tubules cortex are mild or moderate (9/10); tubular regenerative
hyperplasia are rare or mild (4/10); cystic tubules are rare or mild (5/10); glomerular
hypertrophy/atrophy is present in four rats (4/10); interstitial cellular infiltrated are rare or
mild (3/10).
3-MCPD (1.84 mg/kg b.w. per day) male group (rats 116-125): hypertrophy and swelling
of epithelial cells of proximal and distal tubules are mild or moderate in all rats (10/10);
hydropic-vacuolar degenerations of tubular epithelial cells are rare (3/10); hyaline and
basophilic casts in tubules cortex are mild or moderate (9/10); tubular regenerative
hyperplasia is mild (5/10); cystic tubules are rare or mild (2/10); tubular ectasia is rare or mild
(3/10), interstitial cellular infiltrates are rare or mild (4/10).
Female control group (rats 126-135): hypertrophy and swelling of epithelial cells of
proximal and distal tubules are mild or moderate (8/10); glomerular hypertrophy/atrophy is
mild (4/10); interstitial cellular infiltrates are rare or mild in 2/10 rats, tubular ectasia is rare
(2/10) and cystic tubules are also rare (2/10).
3-MCPD dipalmitate (9.78 mg/kg b.w. per day) female group (rats 136-145): hypertrophy
and swelling of epithelial cells of proximal and distal tubules are mild or moderate in all rats
(10/10); hydropic-vacuolar degenerations of tubular epithelial cells are mild (6/10); hyaline
and basophilic casts in tubules cortex are mild or moderate (9/10); tubular regenerative
hyperplasia is rare (2/10); cystic tubules are rare or mild (2/10); glomerular
hypertrophy/atrophy is mild in all rats (10/10), hyaline or basophils granular casts are mild
(9/10).
3-MCPD (1.84 mg/kg b.w. per day) female group (rats 146-155): hypertrophy and
swelling of epithelial cells of proximal and distal tubules is mild or moderate in all rats
(10/10); hydropic-vacuolar degenerations of tubular epithelial cells is mild (7/10) (Figure 24);
hyaline and basophilic casts in tubules cortex are moderate or severe in all rats (10/10);
tubular regenerative hyperplasia is rare (3/10) (Figure 24); cystic tubules are moderate (2/10)
(Figure 24); glomerular hypertrophy/atrophy is mild in all rats (10/10); interstitial cellular
infiltrates are mild or moderate (3/10) (Figure 25), picnosis rare or moderate (4/10).
3-MCPD (29.5 mg/kg b.w. per day) female group (rats 156-165): hypertrophy and
swelling of epithelial cells of proximal and distal tubules are mild in all rats (10/10);
hydropic-vacuolar degenerations of tubular epithelial cells is severe to mild (9/10); epithelial
tubular necrosis is severe (2/10); interstitial cellular infiltrates are severe or moderate (9/10)
83
(Figure 26), atrophy or hypertrophy is rare (6/10), hyalinosis is rare (2/10), regenerative
hyperplasia is mild (5/10), picnosis is rare or severe (2/10), hyaline or basophils granular
casts are rare to moderate in all rats (10/10), tubular ectasia is moderate (8/10), cystic tubules
are moderate to severe (6/10), nuclear changes are rare (1/10) and rare is also the interstitial
fibrosis (3/10) .
Figure 24: Female Wistar rats 3-MCPD (1.84 mg/kg b.w. per day) group, kidney rat 148:
hydropic degeneration; cystic ectasia; tubular regenerative hyperplasia PAS 20x.
84
Figure 25: Female Wistar rats 3-MCPD (1.84 mg/kg b.w. per day) group, kidney rat n. 151:
pyelitis a: HE 4x; b: HE 10x.
Figure 26: Female Wistar rats 3-MCPD (29.5 mg/kg b.w. per day) group, kidney rat 158:
interstitial nephritis H-E 4x.
85
Histopathological evaluation of testis lesions (scores in appendix VII)
Male control group (rats 101-105): no lesions (Figure 27); only rare edema (3/5).
3-MCPD dipalmitate (9.78 mg/kg b.w. per day) male group (rats 106-115): slight
decrease of spermatids (4/10) (Figure 28); slight atrophy of supporting cells (2/10) (Figure
28); slight atrophy of spermatogenic cells (2/10) (Figure 28) and slight edema (4/10).
3-MCPD (1.84 mg/kg b.w. per day) male group (rats 116-125): slight decrease of
spermatids or sperm (9/10) (Figure 29); slight atrophy or supporting cells (4/10) (Figure 29);
slight atrophy of spermatogenic cells (3/10) (Figure 29); mild edema (7/10).
Figure 27: Male Wistar rats control group, testis rat 103
PAS 10x.
86
Figure 28: Male Wistar rats 3-MCPD dipalmitate (9.78 mg/kg b.w. per day) group
testis rat 115: rare or mild decrease of spermatids, atrophy and detachment of supporting cells
and spermatogenic cell atrophy - PAS 10x.
Figure 29: Male Wistar rats 3-MCPD

(1.84 mg/kg b.w. per day) group testis rat
121: a and b mild tubular degeneration,
in b a giant cell into the degenerated
tubule PAS 10x.
87
APPENDIX VI
NECROPSIES RATS DIED DURING THE EXPERIMENTATION
NECROPSIES FEMALE WISTAR RATS NUMBER 46, 47, 48, 49 and 50
Wistar rat adult 46
Rat of group treated with 3-MCPD, female nulliparous, 240g body weight, died on March 28,
2010 (8th day of treatment) and was necropsied. Delivered frozen.
Gross changes
The animal, after thawing, shows skin cyanosis of the extremity. Perioral area is stained by
brown material. Skin and subcutaneous connective tissue shown dehydration. Presence of
serum-haematic liquid in abdominal cavity expression of physical phenomena by thawing.
Abdominal organs “in situ”. Presence of oily material in oral cavity compatible with gastro-
esophageal reflux. The stomach is full of feed while the intestine shows putrefactive post-
mortal alterations. The liver show congestive hepatomegaly. Presence of hemorrhage in the
thoracic cavity as well as in the mediastinum; lung congestion and pulmonary oedema.
Hematic congestion of ventricular and atrial myocardium. Diffuse congestion of muscles.
Muscular hemorrhage observed in the ventral neck region and presternal region.
Wistar rat adult 47
Rat of group treated with 3-MCPD, female nulliparous, 246 g body weight, died March 29,
2010 (9th day of treatment) was necropsied.
Gross changes
Perioral area is stained by brown material. The mucous membranes are anaemic. The skin
and subcutaneous connective tissue show dehydration. Normal topography of organs in
presence of serous fluid in the abdominal cavity. The stomach appears full of feed. Presence
of dark brown material in jejunum and intestinal lumen. The liver appear congested. The
parenchyma of kidneys appear pale and the urinary bladder is empty. The thymus is
moderately hyperemic. Lung emphysema. Presence of voluminous blood red clots in atria and
ventricula. Cerebral vessels slightly congested.
Histopathology
Kidney: proximal and distal tubular epithelial necrosis. Presence of proteinaceous material in
the tubular lumen.
Liver: congestion. Hepatocellular megakaryosis and increase number of hepatocytes showing

karyokinesis activity. Karyolisis.
88
Lung: slight pulmonary congestion. Presence of subpleuric emphysema.

Heart: myocardial infiltrates of polymorphic and lymphomononuclear cells. Vacuolar
degeneration of myocardial fibres.
Wistar rat adult 48
Rat of the group treated with 3-MCPD, female nulliparous, 247g body weight, died March 28,
2010 (8th day of treatment) and was necropsied.
Hypostatic spots are present in the ventral region of body. Cyanosis of extremity as well as of
oral mucous membranes. Subcutis dehydration. Presence of serous fluid in the abdominal
cavity. No alteration of abdominal organs topography. The stomach is enlarged with oedema
of gastric and enteric walls. Liver shows congestion and hepatomegaly. Slight discoloration
of the kidneys parenchyma. Thymus hemorrhages. Lung congestion and oedema. Presence of
voluminous blood red clots in atria and ventricula. Meningeal and cerebral vascular
congestion.
Histopathology
Kidneys: proximal and distal tubular epithelial necrosis. Presence of proteinaceous material
in the tubular lumen. Vacuolar-hydropic degeneration of the epithelium of the pars recta and
collecting tubules.
Liver: congestion and hepatocellular megakaryosis.
Lung: slight pulmonary congestion. Presence of sub-pleuric emphysema.
Thymus: diffuse cytolysis.
Heart: myocardial infiltrates of polymorphic and lympho-mononuclear cells. Myocardial
congestion, oedema and haemorrhages.
Wistar rat adult 49
Rat of the group treated with 3-MCPD, female nulliparous, 348 body weight, died March 27,
2010 (7th day of treatment) and was necropsied. Delivered frozen
Gross changes
The animal, after thawing, show cyanosis of skin of the extremity. Perioral area is stained by
brown material. Subcutis dehydration. Presence of serum-haematic liquid in abdominal cavity
expression of physical phenomena by thawing. Abdominal organs “in situ”. Presence of oily
material in oral cavity compatible with gastroesophageal reflux. The stomach is full of feed
while the intestine shows putrefactive post-mortal alterations. Congestive hepatomegaly.
Muscle haemorrhage observed in the ventral neck region and presternal region. Presence of
haemorrhage in the intercostal muscles on the right side of the thoracic cavity. Lung
congestion and pulmonary oedema. Presence of haemorrhagic suffusions at carina level.
Haematic congestion of ventricular and atrial myocardium. Diffuse congestion of muscles.
89
Wistar rat adult 50
Rat of the group treated with 3-MCPD, female nulliparous, 230g body weight, died April 25,
2010 (38th day of treatment) and was necropsied.
Gross changes
Presence of rigor mortis on the initial phase. Anemia of mucosal membrane of oral cavity.
Subcutis dehydration. No evidence of alterations of abdominal organs topography.
Colon enlargement. The stomach is full of feed while the intestine shows putrefactive post-
mortal alterations. The liver is congested with hypostatic spots as the spleen. Enlargement of
kidneys associated to a gray yellow color of the medulla and cortex. Congestion of the genital
organs. Intratracheal foam and slight serous fluid collection in the thorax cavity are observed.
Pulmonary oedema. Discoloration of ventricular myocardium. Presence of haematic clots in
the atrium as well as in the vena cava. Slight muscles hypotrophy.
Histopathology
Kidneys: proximal and distal tubular epithelial necrosis. Presence of proteinaceous material
in the tubular lumen. Vacuolar hydropic degeneration of the epithelium of the pars recta and
collecting tubules. Steatonecrosis of adipose capsule associated to polimorphous and
mononuclear cell infiltrates.
Liver: congestion and centrolobular hepatocyte degeneration. Hepatocellular mytosis and
hepatocellular megacariosis.
Lungs: congestion and alveolar oedema.
Heart: myocardial congestion and oedema.
COMMENT
No samples for histopathology were collected from rats number 46 and 49 because they were
submitted frozen. Causa mortis of female rats number 47, 48 and 50 may be related to diol
nephrotoxicity.
90
Gross changes in rat 47: presence of dark brown material in jejunum and intestinal lumen;
liver congested; kidneys pale.
External examination of rat 46: perioral area is stained by brown material.
91
Kidney: tubular necrosis and mineralisation Kidney: epithelial tubular

PAS 20x. cells necrosis PAS 20x.
Kidney: hydropic-vacuolar degeneration of Heart: myocardosis

epithelial cells and mineralisation HE 20x.
HE20x.
Lung: congestion and alveolar edema Liver: centrolobular hepatocyte

HE 20x. degeneration H-E 10x.
92
NECROPSY FEMALE WISTAR RAT NUMBER 94
Wistar rat adult 94 group treated with 7.37 mg/kg b.w. per day 3-MCPD, female nulliparous,
died October 8, 2010 (85th day of treatment) was necropsied.
Gross changes
Oily brown material was observed in the perioral area.
Abdominal cavity: normal topography of organs; gastrointestinal tract appears congested;
Liver: congestion. Pancreas: haemorragic; Kidneys: the parenchyma appear pale.
Thoracic cavity: the thymus is moderately hyperaemic. Trachea and lungs: lung congestion.
Nervous system: cerebral vessels slightly congested.
Female rat 94 treated with 3MCPD (died October 8, 2010).
Histopathology
Pancreas: interstizial hemorrhage and small focal areas of necrosis. Kidney: acute tubular
epithelial necrosis. Liver: congestion, hepatocellular megacariosis and increase number of
hepatocytes showing caryocinetic activity, single cell necrosis, cystic vascular spaces. Small
intestine; iperemia, atrophy of villi, erosion, increased number of intraepithelial lymphocytes.
Lung: slight pulmonary congestion. Heart: degeneration of myocardiocites.
93
Kidney: tubular necrosis rat 94 treated with 3-MCPD (died October 8, 2010) H-E 10x.
NECROPSIES FEMALE WISTAR RATS NUMBER 162 AND 163
Necropsy Wistar rat 162
died January 6, 2011 (48th day of treatment) was necropsied.
Gross changes
The rat shows cyanosis of the skin of the extremities, perioral area stained with blood,
kidneys pale and large, lung congestion and pulmonary oedema. The other organs were
autolytic.
Causa mortis may be related to 3-MCPD nephrotoxicity and cardiopulmonary lesions.
Kidneys histopathology
Mild hypertrophy and swelling of epithelial cells of proximal and distal tubules; severe
hydropic-vacuolar degenerations of tubular epithelial cells; severe epithelial tubular necrosis;
various hyaline or basophilic granular casts; interstitial cellular infiltrated are slight, mild
tubular ectasia and various cystic tubuls.
94
Female Wistar rats 3-MCPD (29.5 mg/kg b.w. per day) group, kidney rat 162:
epithelial tubular necrosis and calcifications
H-E 20x.
Necropsy Wistar rat 163
died February 13, 2011 (83rd day of treatment) was necropsied.
Gross changes
The rat shows cyanosis of the skin of the extremities, perioral area smeared of blood, kidney
pale and large, lung congestion and pulmonary edema. The other organs were autolityc.
Causa mortis may be related to 3-MCPD nephrotoxicity and cardiopulmonary lesions.
95
a) Necropsy of Wistar rat 163 (29.5 mg/kg b.w. per day 3-MCPD) female;
b) kidneys pale and large, lung congestion and pulmonary edema.
Kidneys histopathology
light hypertrophy and swelling of epithelial cells of proximal and distal tubules; mild
hydropic-vacuolar degenerations of tubular epithelial cells; severe epithelial tubular necrosis;
various hyaline or basophilic granular casts; interstitial cellular infiltrated are severe, mild
tubular ectasia and various cystic tubuls.
96
Female Wistar rats 3-MCPD (29.5 mg/kg b.w. per day) group, kidney rat 163: epithelial
tubular necrosis (arrows) PAS 20x.
COMMENT
Causa mortis of female rats 162 and 163 may be related to 3-MCPD nephrotoxicity.
97
APPENDIX VII
TABLES OF BODY AND ORGAN WEIGHTS
First treatment: 3-MCPD dipalmitate 156.75 mg/kg b.w. per day

Control group M
Weight (g) R1 R2 R3 R4 R5 average DS
Body 450 501 581 468 470 Body 494 51,97
Liver 15,78 19,93 23,76 14,54 15,9 Liver 17,98 3,81
Kidneys + adrenals 4,15 4,35 6,65 3,52 6,05 Kidneys+ Adrenals 4,94 1,34
Gonads 6,37 8,97 9,63 8,97 9,29 Gonads 8,65 1,3
Accessory sex glands 1,62 3 5,1 2,96 4,9 Accessory sex glands 3,52 1,46
3-MCPD dipalmitate
M
Weight (g) R6 R7 R8 R9 R 10 R 11 R 12 R 13 R 14 R 15
Body 372 542 425 515 512 505 479 453 570 515
Liver 16,21 24,78 15,74 19,41 21,31 23,08 19,75 17,83 22,55 24,31
Kidneys + Adrenals 4,6 8,95 4,51 7,63 9,18 5,72 8,78 4,75 5,1 10,78
Gonads 6,31 8,25 5,99 9 7,76 10,3 9,87 11,96 8,88 12,73
Accessory sex glands 4,08 7,08 5,94 4,98 5,42 4,52 4,73 4,61 4,9 5,48
average DS
Body 479,78 54,04
Liver 20,27 3,35
Kidneys + Adrenals 7,21 2,36
Gonads 9,13 2,33
Accessory sex glands 5,2 0,9
3-MCPD, M
Weight (g) R 16 R 17 R 18 R 19 R 20 R21 R 22 R 23 R 24 R 25
Body 520 446 520 523 409 490 510 450 477 486
Liver 23,56 18,5 24,89 21,66 18,7 23,41 19,55 20,95 20,41 22,57
Gonads 10,6 8,73 6,3 8,81 6,58 7,39 8,2 8,89 5,77 5,29
average DS
Body 483,1 38,14
Liver 21,42 2,18
Gonads 7,66 1,67
98
Control group, F
Weight (g) R 26 R 27 R 28 R 29 R 30 average DS
Body 298 333 308 329 322 Body 318 14,68
Spleen 1,13 1,02 0,95 0,87 0,69 Spleen 0,93 0,16
Liver 12,33 12,78 12,04 11,22 12,68 Liver 12,21 0,63
Kidneys + Adrenals 2,86 3,73 3,17 3,3 3,15 Kidneys + Adrenals 3,24 0,32
Gonads 3,78 3,8 3,2 3,1 5,56 Gonads 3,89 0,99
3-MCPD dipalmitate,
F
Weight (g) R 31 R 32 R 33 R 34 R 35 R 36 R 37 R 38 R 39 R 40
Body 286 325 313 263 302 317 273 292 247 292
Spleen 0,58 0,61 0,77 0,66 0,81 0,7 0,55 0,68 0,6 0,64
Liver 11,32 14,03 13,69 13,25 12,01 13 11,7 14,37 10,91 11,08
Gonads 1,5 1,36 1,8 1,75 2,66 1,06 2,47 1,27 3,06 1,78
average DS
Body 291 24,74
Spleen 0,66 0,08
Liver 12,54 1,28
Gonads 1,87 0,65
3-MCPD, F
Body 283 268 258 300 332 240 246 247 348 230
Spleen 0,78 0,65 0,64 0,68 0,78 nd nd nd nd nd
Liver 12,51 12,92 12,43 14,87 15,57 nd nd nd nd nd
Kidneys + Adrenals 4,06 3,91 3,73 4,36 4,89 nd nd nd nd nd
Gonads 1,76 2,13 2,01 4,12 3,7 nd nd nd nd nd
average DS
Body 288,2 29,17
Spleen 0,71 0,07
Liver 13,66 1,46
Gonads 3,19 1,26
99
Second treatment: 3-MCPD dipalmitate 39.19 mg/kg b.w. per day (56-65/66-75)
3-MCPD 7.37 mg/kg b.w. per day (81-90/91-100)
Control group M
Weight (g) R 51 R 52 R 53 R 54 R 55 average DS
Body 540 525 529 508 507 Body 522 14,17
Thymus 0,57 0,43 0,55 0,33 0,58 Thymus 0,49 0,11
Heart + aorta 1,5 1,31 1,57 1,55 1,7 Heart + aorta 1,53 0,14
Salivary glands 2,3 2,24 3,06 1,61 2,11 Salivary glands 2,26 0,52
Spleen 1,08 1,2 0,99 0,94 0,89 Spleen 1,02 0,12
Liver 20,37 21,69 19,75 16,98 14,69 Liver 18,7 2,52
Kidneys 4,26 3,57 3,62 2,98 3,64 Kidneys 3,61 0,45
Adrenals 0,17 0,67 0,54 0,54 0,55 Adrenals 0,49 0,19
Gonads 5,53 6,78 6,37 7,12 7,03 Gonads 6,57 0,65
Accessory sex glands 3,94 5,03 4,78 4,54 4,03 Accessory sex glands 4,46 0,47
3-MCPD dipalmitate M
Weight (g) R 56 R 57 R 58 R 59 R 60 R61 R62 R63 R64 R65
Body 575 541 623 530 487 447 516 519 465 480
Thymus 0,63 1,07 0,58 0,61 0,64 0,37 0,5 0,47 0,57 0,57
Heart + aorta 1,86 1,44 1,56 3,41 2,16 3,47 1,32 1,34 1,51 3,44
Salivary glands 1,98 1,48 3,84 1,53 1,85 1,58 1,61 1,41 1,85 1,99
Spleen 1,31 1,11 1,45 2,71 1,2 2,94 0,99 1,04 0,79 2,88
Liver 20,37 18,06 20,03 19,63 17,07 17,55 19,79 20,7 18,08 19,21
Kidneys 4,5 4,41 4,66 6,58 4,19 5,47 3,96 3,52 5,94 5,88
Adrenals 0,32 0,5 0,52 0,54 0,55 0,44 0,13 0,19 0,54 0,47
Gonads 7,75 5,85 8,24 6,95 7,07 8,77 7 6,02 8,1 7,72
average DS
Body 518,3 52,92
Thymus 0,6 0,18
Heart + aorta 2,15 0,92
Salivary glands 1,91 0,71
Spleen 1,64 0,85
Liver 19,05 1,27
Kidneys 4,91 0,1
Adrenals 0,42 0,15
Gonads 7,35 0,95
100
3-MCPD group M
Body 534 582 486 552 513 583 542 544 578 555
Thymus 0,64 0,41 0,65 0,65 0,59 0,69 0,67 0,47 0,46 0,47
Heart + aorta 1,86 1,38 1,63 1,84 1,61 2,02 1,97 1,71 1,5 1,62
Salivary glands 1,67 1,71 1,75 2,41 1,9 1,78 1,95 2,26 1,84 1,92
Spleen 1,06 0,9 0,65 1,47 1,13 1,32 1,27 1,48 1,02 1,15
Liver 18,79 24,4 17,67 19,76 15,56 20 19,96 20,33 16,34 19,35
Kidneys 3,9 3,96 3,66 3,81 3,28 5,11 5,13 4,91 3,41 4,73
Adrenals 0,14 0,2 0,11 0,2 0,09 0,16 0,15 0,13 0,17 0,14
Gonads 8,05 5,08 6,72 7,21 6,22 7,58 9,55 7,32 8,5 7,6
average DS
Body 546,9 31
Thymus 0,57 0,1
Spleen 1,14 0,26
Liver 19,22 2,44
Kidneys 4,19 0,71
Adrenals 0,15 0,03
Gonads 7,38 1,23
Control group F
Weight (g) R 76 R 77 R 78 R 79 R80 average DS
Body 281 258 310 262 316 Body 285,4 26,73
Thymus 0,29 0,34 0,52 0,4 0,45 Thymus 0,4 0,09
Heart + aorta 0,93 0,99 1,05 1,25 1,19 Heart + aorta 1,08 0,13
Spleen 0,87 2,68 2,86 0,75 0,88 Spleen 1,61 1,06
Liver 8,97 8,13 9,45 8,94 11,49 Liver 9,4 1,26
Kidneys 1,97 4,25 2,17 2,32 2,46 Kidneys 2,63 0,92
Gonads 1,93 1,72 1,67 2,03 1,95 Gonads 1,86 0,16
101
3-MCPD dipalmitate
group F
Body 247 278 281 275 267 280 275 294 266 287
Thymus 0,11 0,26 0,23 0,17 0,32 0,22 0,16 0,2 0,24 0,03
Heart + aorta 0,68 0,95 0,71 1,09 1,17 0,87 1,09 1 0,86 0,97
Salivary glands 0,69 1,06 0,92 0,77 0,96 0,92 1,04 1,04 0,92 1,02
Spleen 0,37 0,36 0,7 0,58 0,64 0,61 0,29 0,6 0,71 0,41
Liver 7,31 10,21 9,16 8,54 7,28 8,76 8,17 9,41 9,14 8,62
Kidneys 1,75 2,19 2,58 2,25 2,03 2,11 1,95 2,94 2,26 2,27
Adrenals 0,24 0,1 0,26 0,2 0,18 0,26 0,11 0,25 0,26 0,2
Gonads 0,95 1,93 1,21 1,55 1,23 1,6 1,43 1,44 1,5 1,38
average DS
Body 275 12,93
Thymus 0,19 0,08
Spleen 0,53 0,15
Liver 8,66 0,91
Kidneys 2,23 0,33
Adrenals 0,21 0,06
Gonads 1,42 0,26
3-MCPD group F
Body 305 236 253 303 290 221 258 266 273 261
Thymus 0,49 0,17 0,19 0,42 0,29 0,07 0,17 0,22 0,17 0,27
Heart + aorta 1,16 1,05 0,72 1,39 0,7 1,12 0,97 0,99 0,9 1,16
Salivary glands 0,88 0,84 0,81 1,05 1,01 0,59 0,85 0,79 1,01 0,95
Spleen 0,49 0,49 0,46 0,55 0,3 0,73 0,44 0,48 0,7 0,73
Liver 9,72 8,51 8,29 14,4 8,74 10,22 7,71 8,28 8,88 10,11
Kidneys 2,82 2,27 2,32 3,16 2,66 4,07 2,01 2,31 2,03 2,45
Adrenals 0,18 0,07 0,11 0,35 0,11 0,11 0,12 0,04 0,06 0,12
Gonads 2,04 1,39 1,44 2,89 1,31 1,34 1,43 1,45 1,59 1,61
102
average DS
Body 262,55 24,09
Thymus 0,23 0,12
Spleen 0,53 0,85
Liver 8,94 0,88
Kidneys 2,55 0,63
Adrenals 0,1 0,04
Gonads 1,51 0,22
Third treatment: 3-MCPD dipalmitate 9.78 mg/kg b.w. per day (106-115/136-145)
3-MCPD 29.5 mg/kg b.w. per day (156-165)
control Male average DS

Weight (g) R 101 R 102 R 103 R 104 R 105 Body 525,4 38,26
Body 507 536 470 570 544 Thymus 0,76 0,12
Thymus 0,61 0,81 0,69 0,91 0,8 Heart + aorta
Spleen 0,76 0,86 0,65 0,78 0,71 Spleen 0,75 0,08
Liver 18,22 21,3 16,16 20,34 19,68 Liver 19,14 2,01
Kidneys 4,12 4,09 3,39 3,11 4,01 Kidneys 3,74 0,46
Gonads 9,67 10,36 8,76 8,58 9,19 Gonads 9,31 0,72
Accessory sex glands 4,51 4,9 3,95 5,06 5,12 Accessory sex glands 4,71 0,48
3-MCPD dipalmitate,
Male

Body 485 483 554 532 594 504 477 561 418 484
Thymus 0,73 1,05 0,8 0,78 0,45 0,66 0,59 0,67 0,49 0,54
Heart + aorta 1,71 1,85 1,49 1,56 2,15 1,66 1,54 1,76 1,54 1,69
Salivary glands 1,97 1,71 2,21 1,19 2,07 1,65 1,84 1,91 1,56 1,67
Spleen 1,17 1,07 0,87 0,57 1,1 1,12 0,9 0,79 0,71 0,91
Liver 18,22 19,25 22,75 17 23,03 16,01 18,09 20,07 15,8 20,32
Kidneys 3,46 3,83 3,8 4,18 4,25 3,46 3,42 3,79 3,62 3,98
Adrenals 0,15 0,16 0,13 0,11 0,17 0,17 0,1 0,11 0,11 0,11
Gonads 9,01 8,55 9,85 7,8 10,91 8,83 8,46 8,88 6,99 9,26
103
3-MCPD
dipalmitate, Male

average DS
Body 509,2 51,3
Thymus 0,68 0,18
Spleen 0,92 0,19
Liver 19,05 2,53
Kidneys 3,78 0,29
Adrenals 0,13 0,03
Gonads 8,8 1,07
3-MCPD, Male

Weight (g) R116 R 117 R 118 R 119 R 120 R 121 R 122 R 123 R 124 R 125
Body 456 450 465 450 485 465 468 467 552 504
Thymus 0,57 0,56 0,6 0,49 0,82 0,7 0,58 0,72 0,82 0,44
Heart + aorta 1,38 1,43 1,43 1,35 1,63 1,78 1,61 1,28 2,24 1,45
Salivary glands 1,56 1,47 1,9 1,53 1,62 1,53 1,43 1,58 1,65 1,6
Spleen 0,78 0,49 0,7 0,75 0,77 1,04 0,63 0,79 1,11 0,75
Liver 13,57 14,29 16,39 15,62 16,34 17,67 14,8 16,98 23,35 17,57
Kidneys 2,8 2,73 3,01 2,93 3,97 3,5 3,09 3,21 3,96 3,9
Adrenals 0,09 0,08 0,11 0,09 0,14 0,11 0,04 0,06 0,11 0,2
Gonads 7,5 4,75 9,45 6,8 10,15 8,74 7,78 8,04 8,81 7,19
Accessory sex glands 4 4,18 4,21 3,66 4,06 7,94 5,04 4,44 3,75 4,11
3-MCPD, Male

average DS
Body 476,2 31,23
Thymus 0,63 0,13
Spleen 0,78 0,18
Liver 16,66 2,72
Kidneys 3,31 0,49
Adrenals 0,1 0,04
Gonads 7,92 1,52
104
control, F
Body 287 270 267 230 280 255 294 261 241 283
Thymus 0,72 0,41 0,46 0,35 0,33 0,47 0,49 0,25 0,34 0,42
Heart + aorta 1,35 1,23 1,1 0,79 0,97 1,27 1,3 0,75 1,42 1,36
Salivary glands 1,2 1,28 0,93 0,71 0,92 1,19 0,93 0,7 1,06 1,45
Spleen 0,82 0,86 0,47 0,45 0,68 0,83 0,53 0,29 0,77 0,93
Liver 8,59 8,1 10,1 6,46 8,23 7,33 8,64 7,9 6,84 9,13
Kidneys 2,14 2,19 1,84 1,49 2,15 2,21 1,82 1,96 2,05 2,3
Adrenals 0,15 0,18 0,19 0,14 0,24 0,11 0,17 0,19 0,13 0,19
Gonads 1,69 2,1 1,39 1,35 1,84 1,6 2,7 1,29 1,61 1,67
control, F
average DS
Body 266,8 20,5
Thimus 0,42 0,13
Salivary
glands 1,03 0,24
Spleen 0,66 0,21
Liver 8,13 1,08
Kidney 2,01 0,24
Adrenal 0,17 0,04
Gonads 1,72 0,42
3-MCPD dipalmitate, Female

Body 230 236 284 275 269 288 267 289 258 275
Thymus 0,36 0,23 0,5 0,5 0,46 0,35 0,35 0,46 0,54 0,38
Heart + aorta 1,12 0,83 1,04 0,77 0,79 0,9 0,79 1,39 0,93 1,17
Salivary glands 0,99 0,7 0,98 0,92 0,95 1,01 0,99 1,15 1,14 0,97
Spleen 0,86 0,66 0,54 0,51 0,49 0,37 0,73 0,65 0,38 0,48
Liver 7,83 7,96 9,14 10,61 7,72 8,49 8,79 9,3 8,46 9,16
Kidneys 2,05 1,73 2,5 2,56 1,86 2,31 2,64 2,13 2 2,13
Adrenals 0,16 0,19 0,19 0,22 0,14 0,16 0,1 0,18 0,07 0,09
Gonads 1,61 2,31 3,99 1,69 1,71 1,88 1,81 1,7 2,04 1,6
105
3-MCPD dipalmitate, Female
average DS
Body 267,1 20,43
Thymus 0,41 0,09
Spleen 0,57 0,16
Liver 8,74 0,87
Kidneys 2,19 0,3
Adrenals 0,15 0,05
Gonads 2,03 0,72
3-MCPD, Female

Body 269 239 288 277 316 258 277 238 285 245
Thymus 0,3 0,4 0,32 0,3 0,35 0,18 0,53 0,45 0,37 0,39
Heart + aorta 1,16 1,23 1,04 0,81 1,21 1,16 0,84 0,87 1,16 0,99
Salivary glands 1,01 1,11 1,11 1,22 1,13 0,87 1,05 0,98 1,38 1,03
Spleen 0,58 0,33 0,91 2,55 0,59 0,55 0,63 0,42 0,66 0,56
Liver 8,94 9,33 14,05 8,13 11,11 9,53 8,84 8,52 10,29 8,12
Kidneys 2,27 1,62 2,81 2,15 2,43 2,17 2,37 1,93 2,65 1,87
Adrenals 0,11 0,13 0,14 0,14 0,17 0,08 0,1 0,09 0,19 0,09
Gonads 2,28 1,08 1,73 1,66 1,6 1,2 1,53 2,43 1,19 1,47

average DS
Body 269,2 24,75
Thymus 0,36 0,09
Spleen 0,78 0,64
Liver 9,69 1,8
Kidneys 2,23 0,36
Adrenals 0,12 0,04
Gonads 1,61 0,44
106
3-MCPD, Female
29.5 mg/kg b.w. per day
Body 268 243 260 298 304 302 n.d. n.d. 285 296
Thymus 0,21 0,33 0,2 0,24 0,32 0,29 n.d. n.d. 0,2 0,38
Heart + aorta 1,19 0,88 1,07 1,37 1,17 1,13 n.d. n.d. 1,52 1,58
Salivary glands 0,8 1 1,09 1,41 1,32 0,99 n.d. n.d. 0,99 1
Spleen 0,53 0,48 0,4 0,5 0,42 0,68 n.d. n.d. 0,48 0,7
Liver 18,41 8,89 13,13 14,29 13,92 14,1 n.d. n.d. 11,05 12,31
Kidneys 3,42 2,44 3,44 3,3 3,76 3,89 3,62 5,7 3,69 4,07
Adrenals 0,09 0,1 0,16 0,2 0,18 0,11 n.d. n.d. 0,25 0,14
Gonads 1,56 1,5 1,54 2,03 1,26 2,97 n.d. n.d. 2,84 1,84
3-MCPD,
Female

average DS
Body 282 22,05
Thymus 0,27 0,07
Salivary
glands 1,07 0,2
Spleen 0,52 0,11
Liver 13,26 2,77
Kidneys 3,5 0,5
Adrenals 0,15 0,05
Gonads 1,94 0,64
107
TABLES OF KIDNEYS LESIONS SCORES
SCORE OF EVALUATION KIDNEY LESIONS
natural morphology 0
glomerular <25 % of glomerular tuft showing thickening of basement membrane and/or hyalinization 1
sclerosis <50 % 2
>50 % 3
normal morphology 0
interstitial <25 % of field occupied by hyaline connective tissue and/or structures surrounded by thickened basement membranes 1
fibrosis <50 % 2
>50 % 3
no clusters 0
interstitial <5 clusters 1
cellular
infiltrates <!0 2
>10 3
no cystic tubules 0
cystic <5 cystic tubules 1
tubules <10 2
>10 3
normal 0
<5 tubule section filled with hyaline material 1
hyalinosis
<10 2
>10 3
108
First treatment: 3-MCPD dipalmitate 156.75 mg/kg b.w. per day (6-15/31-40)
control group M 1 2 3 4 5
glomerulopathy 0 0 0 0 0
sclerosis 0 0 0 0 0
glomerular
lesions
atrophy/hypertr 0 1 1 1 1
hyalinosis 0 0 0 0 0
prox tube 1 0 0 1 1
regenerative
dist tube 0 0 0 0 0
hyperplasia
collecting tube 0 0 0 0 0
picnosis 1 0 1 1 1
tubular
hydropic/vacuolar
degenerative 1 0 1 1 1
degeneration
change
hypertrophy or cells
0 0 0 0 0
swelling
prox tube 1 1 1 1 0
hyaline or dist tube 0 0 1 1 0
basophils
granular casts collecting tube 0 0 0 0 0
glomerular urinary
0 0 0 0 0
space
interstitial cortical 1 1 0 0 1
cellular
infiltrated medullary 0 1 0 0 0
cortical 0 0 0 0 0
tubular ectasia
medullary 0 0 0 0 0
nuclear changes: cortical 0 0 0 0 0
karyomegaly of
tubular
epithelial cells medullary 0 0 0 0 0
cortical 0 0 0 0 0
interstitial
fibrosis
medullary 0 0 0 0 0
109
3‐MCPD dipalmitate group M 6 7 8 9 10 11 12 13 14 15
glomerulopathy 1 0 1 1 1 0 0 0 0 0
sclerosis 0 0 0 0 0 0 0 0 0 0
glomerular
lesions
atrophy/hypertr 0 1 1 0 1 1 1 1 1 1
hyalinosis 0 0 0 0 0 0 0 0 0 0
prox tube 1 2 1 2 2 2 2 1 1 2
regenerative
dist tube 1 1 1 2 1 2 2 1 1 2
hyperplasia
collecting tube 0 0 0 0 0 0 0 0 0 0
picnosis 0 0 1 0 1 1 2 1 0 0
tubular
hydropic/vacuolar
degenerative 1 1 0 1 0 1 1 1 0 1
degeneration
change
0 0 0 0 0 0 2 1 1 1
swelling
prox tube 1 1 1 2 1 3 1 2 3 2
hyaline or dist tube 1 1 0 2 1 2 1 2 3 2
basophils
granular casts collecting tube 0 0 0 0 0 0 0 0 0 0
glomerular urinary
1 1 0 1 1 1 1 0 0 1
space
interstitial cortical 1 1 1 1 0 1 0 0 0 1
cellular
infiltrated medullary 0 0 1 0 0 1 0 0 0 0
cortical 0 1 0 0 0 0 0 0 0 1
tubular ectasia
medullary 0 0 0 0 0 0 0 0 1 0
nuclear changes: cortical 0 0 0 2 1 1 2 1 1 2
karyomegaly of
tubular
epithelial cells medullary 0 0 0 0 0 0 0 0 0 0
cortical 0 0 0 0 0 0 0 0 0 0
interstitial
fibrosis
medullary 0 0 0 0 0 0 0 0 0 0
110
3‐MCPD group M 16 17 18 19 20 21 22 23 24 25
sclerosis 0 0 0 0 0 0 0 0 0 0
glomerular
lesions
hyalinosis 2 0 0 0 0 0 0 0 0 0
prox tube 2 2 2 2 2 1 2 1 1 1
regenerative
dist tube 1 1 2 2 2 1 2 1 1 1
hyperplasia
picnosis 1 1 1 2 1 0 0 1 0 1
tubular
hydropic/vacuolar
degenerative 1 1 1 1 1 0 1 1 1 1
degeneration
change
0 2 2 3 2 2 2 2 2 2
swelling
prox tube 2 1 2 3 2 1 3 3 2 1
basophils
glomerular urinary
0 1 0 0 0 0 1 0 0 0
space
cellular
cortical 0 0 0 1 0 0 0 0 0 0
tubular ectasia
medullary 0 0 0 1 0 0 0 0 0 0
karyomegaly of
tubular
cortical 0 0 0 0 0 0 0 0 0 0
interstitial
fibrosis
medullary 0 0 0 0 0 0 0 0 0 0
111
control group F 26 27 28 29 30
sclerosis 0 0 0 0 0
glomerular
lesions
prox tube 0 0 1 0 0
regenerative
dist tube 0 0 0 0 0
hyperplasia
picnosis 0 0 0 0 0
tubular
hydropic/vacuolar
degeneration
change
0 0 0 0 0
swelling
prox tube 1 1 1 2 0
basophils
glomerular urinary
0 0 0 0 0
space
cortical 0 1 0 0 1
interstitial
cellular infiltrated
medullary 0 0 0 0 0
cortical 0 0 0 0 0
tubular ectasia
medullary 0 0 0 0 0
karyomegaly of
tubular
cortical 0 0 0 0 0
interstitial
fibrosis
medullary 0 0 0 0 0
112
3‐MCPD dipalmitate group F 31 32 33 34 35 36 37 38 39 40
sclerosis 0 0 0 0 0 0 0 0 0 0
glomerular
lesions
hyalinosis 0 0 0 0 0 0 0 0 0 0
prox tube 0 1 0 0 1 1 1 1 0 1
regenerative
dist tube 0 1 0 0 0 1 1 1 0 1
hyperplasia
picnosis 0 0 1 0 1 1 1 0 0 1
tubular
hydropic/vacuolar
degenerative 1 1 1 1 1 1 1 1 1 1
degeneration
change
0 0 0 0 0 0 0 0 0 0
swelling
prox tube 1 1 0 0 1 1 1 2 1 1
basophils
glomerular urinary
0 0 0 0 0 0 0 0 0 0
space
cortical 0 0 0 0 0 1 0 0 0 1
interstitial
medullary 0 1 0 1 0 0 0 2 0 0
cortical 0 1 0 1 1 0 0 2 0 0
tubular ectasia
medullary 0 1 0 0 0 1 0 1 0 1
karyomegaly of
tubular
cortical 0 0 0 0 0 0 0 0 0 0
interstitial
fibrosis
medullary 0 0 0 0 0 0 0 0 0 0
113
3‐MCPD group F 41 42 43 44 45 46 47 48 49 50
glomerulophaty 0 0 0 0 0 na 0 0 na 0
sclerosis 0 0 0 0 0 na 0 0 na 0

glomerular
lesion
atrophy/hipertr 1 2 2 2 2 na 2 1 na 1
hyalinosis 0 0 0 0 0 na 0 0 na 0
prox tube 2 2 1 2 2 na 0 0 na 0
regenerative
dist tube 1 2 0 2 1 na 0 0 na 0
hyperplasia
collecting tube 0 0 0 0 0 na 0 0 na 0
picnosis 0 0 0 0 0 na 0 0 na 0
hydropic
tubular 1 1 0 1 0 na 2 2 na 3
degeneration
degenerative
change necrosis 0 0 0 0 0 na 3 3 na 3
hypertrophy or
1 2 1 2 2 na 1 1 na 2
cells swelling
prox tube 2 3 2 3 2 na 3 3 na 3
hyaline or dist tube 2 3 2 3 2 na 3 3 na 3

basophils
granular
casts collecting tube 0 0 0 0 0 na 0 0 na 0
glomerular
0 0 1 0 1 na 2 1 na 1
urinary space
interstitial cortical 0 1 1 1 0 na 1 1 na 3

cellular
infiltrated medullary 0 0 0 0 0 na 0 0 na 0
cortical 0 0 0 0 0 na 0 0 na 0

tubular
ectasia
medullary 0 0 0 0 1 na 0 0 na 0
nuclear
changes: cortical 2 1 2 1 2 na 1 1 na 2
karyomegaly
of tubular medullary 0 0 0 0 0 na 0 0 na 0
epithelial cells
cortical 0 0 0 0 0 na 0 0 na 0

interstitial
fibrosis
medullary 0 0 0 0 0 na 0 0 na 0
114
Second treatment: 3-MCPD dipalmitate 39.19 mg/kg b.w. per day (56-65/66-75)
control group 51 52 53 54 55
sclerosis 0 0 0 0 0
glomerular
lesions
prox tube 0 0 0 0 0
regenerative
dist tube 0 0 0 0 0
hyperplasia
picnosis 0 0 1 2 0
tubular
hydropic/vacuolar
degeneration
change
1 1 2 2 0
swelling
prox tube 0 1 0 0 0
basophils
glomerular urinary
0 0 0 0 0
space
interstitial cortical 0 0 0 0 0
cellular
cortical 0 0 0 1 1
tubular ectasia
medullary 0 1 0 0 0
karyomegaly of
tubular
cortical 0 0 0 0 0
interstitial
fibrosis
medullary 0 0 0 0 0
115
sclerosis 0 0 0 0 0 0 0 0 0 0
glomerular
lesions
hyalinosis 0 0 0 0 0 0 0 0 0 0
prox tube 0 0 0 0 0 0 0 0 0 0
regenerative
dist tube 0 0 0 0 0 0 0 0 0 0
hyperplasia
picnosis 0 0 0 0 0 0 0 0 0 0
tubular
hydropic/vacuolar
degenerative 1 1 1 1 0 1 1 1 1 1
degeneration
change
1 2 2 2 2 1 1 2 2 2
swelling
prox tube 1 1 1 1 2 1 2 1 1 1
basophils
glomerular urinary
0 0 0 0 0 0 0 0 0 0
space
cellular
cortical 0 0 0 0 0 1 0 0 0 0
tubular ectasia
medullary 0 0 0 0 1 0 1 0 1 0
karyomegaly of
tubular
cortical 0 0 0 0 0 0 0 0 0 0
interstitial
fibrosis
medullary 0 0 0 0 0 0 0 0 0 0
116
3‐MCPD group M 66 67 68 69 70 71 72 73 74 75
sclerosis 0 0 0 0 0 0 0 0 0 0
glomerular
lesions
hyalinosis 0 0 0 0 0 0 0 0 0 0
prox tube 0 0 0 2 1 1 0 0 0 0
regenerative
dist tube 0 0 0 0 0 0 0 0 0 0
hyperplasia
picnosis 0 0 0 0 0 0 0 0 0 0
tubular
hydropic/vacuolar
degenerative 0 0 0 0 0 0 0 0 0 0
degeneration
change
3 2 1 2 2 1 3 2 3 2
swelling
prox tube 0 1 1 0 0 1 1 2 0 1
basophils
glomerular urinary
0 0 0 0 0 0 0 0 0 0
space
cellular
cortical 0 0 0 0 0 0 0 0 0 0
tubular ectasia
medullary 0 0 0 0 0 0 0 0 0 0
karyomegaly of
tubular
cortical 0 0 0 0 0 0 0 0 0 0
interstitial
fibrosis
medullary 0 0 0 0 0 0 0 0 0 0
117
3‐MCPD group F 76 77 78 79 80
sclerosis 0 0 0 0 0
glomerular
lesions
prox tube 0 0 0 0 0
regenerative
dist tube 0 0 0 0 0
hyperplasia
picnosis 0 0 0 2 0
tubular
hydropic/vacuolar
degeneration
change
2 2 2 2 0
swelling
prox tube 1 0 0 0 0
basophils
glomerular urinary
0 0 0 0 0
space
cortical 0 0 0 0 2
interstitial
medullary 2 0 0 0 0
cortical 0 0 0 0 0
tubular ectasia
medullary 0 0 0 0 0
karyomegaly of
tubular
cortical 0 0 0 0 0
interstitial
fibrosis
medullary 0 0 0 0 0
118
sclerosis 0 0 0 0 0 0 0 0 0 0
glomerular
lesions
hyalinosis 0 0 0 0 0 0 0 0 0 0
prox tube 0 0 0 0 0 0 0 0 1 0
regenerative
dist tube 0 0 0 0 0 0 0 0 1 0
hyperplasia
picnosis 1 2 0 1 1 0 2 0 0 0
tubular
hydropic/vacuolar
degenerative 2 2 2 2 2 1 2 1 2 0
degeneration
change
0 2 2 2 2 1 3 0 2 2
swelling
prox tube 0 0 0 1 0 0 0 0 0 0
basophils
glomerular urinary
0 0 0 1 0 0 0 0 0 0
space
cortical 0 0 0 0 0 0 0 0 0 0
interstitial
medullary 0 0 0 0 0 0 0 2 0 0
cortical 0 0 0 0 0 2 0 0 0 0
tubular ectasia
medullary 0 0 0 0 0 2 0 1 0 1
karyomegaly of
tubular
cortical 0 0 0 0 0 0 0 0 0 0
interstitial
fibrosis
medullary 0 0 0 0 0 0 0 0 0 0
medullary 0 0 0 0 0 0 0 0 0 0
interstitial fibrosis
cortical 0 0 0 0 0 0 0 0 0 0
119
3‐MCPD group F 91 92 93 94 95 96 97 98 99 100
sclerosis 0 0 0 0 0 0 0 0 0 0
glomerular
lesions
atrophy/ hypertrophy 0 0 0 0 0 0 0 0 0 0
hyalinosis 0 0 0 0 0 0 0 0 0 0
prox tube 0 0 1 0 2 3 0 0 0 2
regenerative
dist tube 0 0 1 0 2 3 0 0 0 2
hyperplasia
picnosis 0 0 0 0 0 0 0 0 0 0
hydropic/vacuolar
tubular 1 2 1 3 0 2 0 2 2 0
degeneration
degenerative
change hypertrophy or cells
1 0 1 2 0 0 2 0 0 3
swelling
necrosis 0 0 0 3 0 0 0 0 0 0
prox tube 1 0 0 0 0 1 0 0 0 3
basophils
glomerular
0 0 0 0 0 0 0 0 0 0
urinary space
cellular
cortical 1 0 0 0 0 3 0 0 0 3
tubular ectasia
medullary 0 0 0 0 0 3 0 0 0 0
cistic tubules 0 0 1 0 1 3 0 0 0 3
karyomegaly of
tubular
cortical 0 0 1 0 1 1 0 0 0 1
medullary 0 0 0 0 0 0 0 0 0 0
120
Third treatment: 3-MCPD dipalmitate 9.78 mg/kg b.w. per day (106-115/136-145)
control group M 101 102 103 104 105
sclerosis 0 0 0 0 0
glomerular
lesions atrophy/
0 0 0 0 0
hypertrophy
prox tube 0 0 0,5 0 0
regenerative
dist tube 0 0 0 0 0
hyperplasia
picnosis 0 0 0 0 0
hydropic/vacuolar
tubular 0 0 0 0 0
degeneration
degenerative
hypertrophy or
change 1 1 2 1 1
cells swelling
necrosis 0 0 0 0 0
prox tube 1 0 1 1 0,5
basophils
glomerular
0 0 0 0 0
urinary space
interstitial cortical 0 0 0,5 0 0

cellular
cortical 0,5 0 1 0 0
tubular ectasia
medullary 0 0 0 0 0
cistic tubules 0,5 0 1 0 0
nuclear changes:
cortical 0 0 0 0 0
karyomegaly of
tubular
medullary 0 0 0 0 0
epithelial cells
cortical 0 0 0 0 0
interstitial
fibrosis
medullary 0 0 0 0 0
121
sclerosis 0 0 0 0 0 0 0 0 0 0
glomerular
lesions atrophy/
0 0 0 0 0 0 0 0 0 0
hypertrophy
hyalinosis 0 0 0 0 0 0 0 0 0 0
prox tube 0 0 0,5 0 0 0,5 0 0,5 0,5 1
regenerative
dist tube 0 0 0 0 0 0 0 0 0 0
hyperplasia
picnosis 0 0 0 0 0 0 0 0 0 0
hydropic/vacuolar
tubular 0 0 0 0,5 0 0,5 0,5 0 0 0
degeneration
degenerative
change hypertrophy or
1 1 1 1 2 1 1 1 1 1
cells swelling
necrosis 0 0 0 0 0 0 0 0 0 0
prox tube 0 1 1 1 1 1 2 1 2 2
basophils
glomerular
0 0 0 0 0 0 0 0 0 0
urinary space
interstitial cortical 0 0 0 0 1 0 0 0 0,5 1

cellular
cortical 0 0 0 0 0 0 0 0 0 0
tubular ectasia
medullary 0 0 0 0 0 0 0 0 0 0
cistic tubules 0,5 0,5 1 0,5 1 0 0,5 0 0 0
karyomegaly of
tubular
cortical 0 0 0 0 0 0 0 0 0 0
interstitial
fibrosis
medullary 0 0 0 0 0 0 0 0 0 0
122
3‐MCPD group M 116 117 118 119 120 121 122 123 124 125
sclerosis 0 0 0 0 0 0 0 0 0 0
glomerular
lesions atrophy/
0 0 0 0 0 0 0 0 0 0
hypertrophy
hyalinosis 0 0 0 0 0 0 0 0 0 0
prox tube 0 0,5 0,5 0,5 0,5 0 0 0 0 0,5
regenerative
dist tube 0 0 0 0 0 0 0 0 0 0
hyperplasia
picnosis 0 0 0 0 0 0 0 0 0 0
hydropic/vacuolar
tubular 0 0 0 0 0,5 0,5 0,5 0 0 0
degeneration
degenerative
change hypertrophy or
1 1 1 2 2 1 1 1 2 0,5
cells swelling
necrosis 0 0 0 0 0 0 0 0 0 0
prox tube 1 0 1 1 1 1 1 2 2 1
basophils
glomerular
0 0 0 0 0 0 0 0 0 0
urinary space
interstitial cortical 0,5 0,5 0,5 0 0 2 0 0 0 0

cellular
cortical 0 0,5 0 0 0 0 0 0,5 0 1

tubular ectasia
medullary 0 0 0 0 0 0 0 0 0 0
cistic tubules 0 0,5 0 0 0 0 0 0,5 0 0
karyomegaly of
tubular
cortical 0 0 0 0 0 0 0 0 0 0
interstitial
fibrosis
medullary 0 0 0 0 0 0 0 0 0 0
123
control group F 126 127 128 129 130 131 132 133 134 135
sclerosis 0 0 0 0 0 0 0 0 0 0
glomerular
lesions
atrophy/ hypertrophy 0 0 0 1 1 0 0 0,5 1 0
hyalinosis 0 0 0 0 0 0 0 0 0 0
prox tube 0 0 0 0 0 0 0 0 0 0
regenerative
dist tube 0 0 0 0 0 0 0 0 0 0
hyperplasia
picnosis 0 0 0 0 0 0 0 0 0 0
hydropic/vacuolar
tubular 0 0 0 0 0 0 0 0 0 0
degeneration
degenerative
1 1 1 1 2 1 0 1 1 0
swelling
necrosis 0 0 0 0 0 0 0 0 0 0
prox tube 0 0 0 0 0 0 0 0 0 0
basophils
glomerular
0 0 0 0 0 0 0 0 0 0
urinary space
interstitial cortical 1 0 0 0 0,5 0 0 0 0 0

cellular
cortical 0 0 0,5 0 0,5 0 0 0 0 0

tubular ectasia
medullary 0 0 0 0 0 0 0 0 0 0
cistic tubules 0 0 0,5 0 0,5 0 0 0 0 0
karyomegaly of
tubular
cortical 0 0 0 0 0 0 0 0 0 0
medullary 0 0 0 0 0 0 0 0 0 0
124
sclerosis 0 0 0 0 0 0 0 0 0 0
glomerular
lesions
atrophy/ hypertrophy 0,5 0,5 0,5 0,5 0,5 0,5 1 1 0,5 1
hyalinosis 0 0 0 0 0 0 0 0 0 0
prox tube 0 0 0,5 0 0 0 0 0 0 0
regenerative
dist tube 0 0 0,5 0 0 0 0 0 0 0
hyperplasia
picnosis 0 0 0 0 0 0 0 0 0 0
hydropic/vacuolar
tubular 0 1 1 1 0,5 0 0 1 1 0
degeneration
degenerative
1 1 2 2 1 0,5 2 2 2 2
swelling
necrosis 0 0 0 0 0 0 0 0 0 0
prox tube 0 0,5 0 1 0 1 1 2 1 2
hyaline or dist tube 0 0,5 2 1 0,5 0 2 2 2 2

basophils
glomerular
0 0 0 0 0 0 0 0 0 0
urinary space
cellular
cortical 0 0 0 0 0 0 0 0 0 0
tubular ectasia
medullary 0 0 0 0 0 0 0 0 0 0
cistic tubules 1 0 0 1 0 0 0 0 0 0
karyomegaly of
tubular
cortical 0 0 0 0 0 0 0 0 0 0
medullary 0 0 0 0 0 0 0 0 0 0
125
3‐MCPD F 146 147 148 149 150 151 152 153 154 155

sclerosis 0 0 0 0 0 0 0,5 0 0 0
glomerular
lesions
atrophy/ hypertrophy 0,5 1 1 1 0,5 0,5 0,5 0,5 0,5 0,5
hyalinosis 0 0 0 0 0 0 0 0 0 0
prox tube 0 0 1 0 0 0,5 0 0 0 0
regenerative
dist tube 0 0 1 0 0 0 0 0 0 0
hyperplasia
picnosis 1 0 1 1 0,5 0 0 0 0 0
hydropic/vacuolar
tubular 0 1 2 1 1 1 1 0 0 1
degeneration
degenerative
2 2 3 3 2 2 2 2 3 2
swelling
necrosis 0 0 0 0 0 0 0 0 0 0
prox tube 0,5 0,5 3 1 1 1 0,5 0,5 1 0,5
hyaline or dist tube 0,5 0,5 3 1 1 0,5 0 0,5 0 0,5

basophils
granular casts collecting tube 0 0 3 0 0 0,5 0 0 0 0
glomerular
0 0 0 0 0 0 0 0 0 0
urinary space
cellular
infiltrated medullary 0,5 2 0 0 0 2 0 0 0 0
cortical 0 1 1 0 0 0 0,5 0 0,5 0

tubular ectasia
medullary 0 0 1 0 0 0 0 0 0,5 0
cistic tubules 0 1 2 0 0 0 0 0 0 0
karyomegaly of
tubular
cortical 0 0 0 0 0 0 0 0 0 0
medullary 0 0 0 0 0 0 0 0 0 0
126
3‐MCPD F 156 157 158 159 160 161 162 163 164 165

sclerosis 0 0 0 0 0 0 0 0 0 0
glomerular
lesions
atrophy/ hypertrophy 0,5 1 1 0,5 0,5 0,5 0 0 0 0
hyalinosis 0 0 0 0 0 0 0,5 0,5 0 0
prox tube 0 0 1 0 0 1 0 2 0,5 1
regenerative
dist tube 0 0 1 0 0 1 0 2 0 1
hyperplasia
picnosis 0 0 0 0 0 0 3 1 0 0
hydropic/vacuolar
tubular 1 1 2 0 1 1 3 2 0,5 2
degeneration
degenerative
2 2 1 1 1 2 2 2 1 2
swelling
necrosis 0 0 0 0 0 0 3 3 0 0
prox tube 1 0,5 0,5 0,5 1 1 3 3 0,5 0,5
hyaline or dist tube 1 0,5 0,5 0,5 1 1 3 3 0,5 0,5

basophils
granular casts collecting tube 0 0 0,5 0,5 1 1 3 3 0 1
glomerular
0 0 0 0 0 0 0,5 0,5 0 0
urinary space
interstitial cortical 2 0 3 0,5 0,5 0,5 1 2 2 1

cellular
infiltrated medullary 0 0 0,5 3 0 0 1 2 0 0
cortical 1 1 1 0 0 0,5 2 2 0 0
tubular ectasia
medullary 0 0 1 0 0,5 1 2 1 0 1
cistic tubules 0 0 1 0 1 1 2 3 0 1
karyomegaly of
tubular
cortical 0,5 0 0,5 0 0 0 0 0 1 0

medullary 0 0 0 0 0 0 0 0 0 0
127
TABLES OF TESTIS LESIONS SCORES
TESTIS
0 no lesions
1 mild
2 moderate
3 severe (injuries over 90 % of seminiferous tubules)
First treatment: 3-MCPD dipalmitate 156.75 mg/kg b.w. per day (6-15)
atrophy of spermato‐ inflamma‐

decrease of mineraliza‐ hyperplasia
supporting genic cell tory
spermatids tions Leydig cells
RAT cells atrophy infiltrates
1 0 0 0 0 0 0
2 0 0 0 0 0 0
3 0 0 0 0 0 0
4 0 0 0 0 0 0
5 0 0 0 0 0 0

6 0 0 0 0 0 2
7 3 2 3 0 1 0
8 3 3 3 0 0 0
9 1 0 0 0 0 0
10 1 0 0 0 0 0
11 1 0 0 0 0 1
12 0 1 1 0 0 1
13 0 0 0 0 0 0
14 0 0 0 0 0 1
15 0 0 0 0 0 1
128

16 1 0 0 0 0 3
17 2 2 2 2 0 2
18 3 0 3 1 1 1
19 2 2 2 1 1 1
20 3 2 3 1 0 0
21 3 2 3 1 1 0
22 3 3 3 1 1 0
23 3 3 3 1 0 1
24 3 3 3 2 2 1
25 3 2 3 1 1 1
Second treatment: 3-MCPD dipalmitate 39.19 mg/kg b.w. per day (56-65)
decrease of atrophy of spermatoge‐ inflamma‐

calcifica‐ hyperplasia
spermatids/ supporting nic cell tory edema
tions Leydig cells
RAT sperm cells atrophy infiltrates
51 1 0 1 0 0 0 1
52 1 0 1 0 0 0 1
53 0 0 0 0 0 0 1
54 1 1 0 0 0 0 1
55 0 0 0 0 0 0 0

tions Leydig cells
56 0 0 0 0 0 0 1
57 0 0 0 0 0 0 1
58 0 0 0 0 0 0 1
59 0 0 0 0 0 0 1
60 1 0 0 0 0 0 1
61 0 0 0 0 0 0 0
62 0 0 0 0 0 0 0
63 1 0 0 0 0 0 0
64 0 0 0 0 0 0 0
65 1 0 0 0 0 0 0
129

tions Leydig cells
66 1 0 1 0 0 0 1
67 0 0 1 0 0 0 1
68 0 0 0 0 0 0 1
69 0 0 0 0 0 0 1
70 0 1 1 0 0 0 1
71 0 0 1 0 0 0 2
72 1 1 1 0 0 1 1
73 0 0 0 0 0 0 1
74 1 1 1 0 0 0 1
75 0 0 0 0 0 0 1
Third treatment: 3-MCPD dipalmitate 9.78 mg/kg b.w. per day (106-115)
tions Leydig cells
101 0 0 0 0 0 0 0,5
102 0 0 0 0 0 0 0,5
103 0 0 0 0 0 0 0
104 0 0 0 0 0 0 0
105 0 0 0 0 0 0 0,5

tions Leydig cells
106 0 0 0 0 0 0 0
107 0 0 0 0 0 0 0
108 0 0 0 0 0 0 0
109 0 0 0 0 0 0 0,5
110 0,5 0 0 0 0 0 0,5
111 1 1 1 0 0 0 0,5
112 0 0 0 0 0 0 0
113 0 0 0 0 0 0 0
114 0,5 0 0 0 0 0 0
115 0,5 0,5 0,5 0 0 0 0,5
130

tions Leydig cells
116 0,5 0 0 0 0 0 1
117 0,5 0 0 0 0 0 2
118 0 0 0 0 0 0 0,5
119 0,5 0 0 0 0 0 0,5
120 0,5 0 0 0 0 0 0,5
121 1 1 1 0 0 0 0,5
122 0,5 0,5 0,5 0 0 0 0
123 0,5 0,5 0,5 0 0 0 0
124 0,5 0,5 0 0 0 0 0
125 0,5 0 0 0 0 0 0,5
131

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90-day toxicological study of 3-MCPD and its dipalmitate

SCIENTIFIC REPORT submitted to EFSA

“Comparison between 3-MCPD and its palmitic esters

in a 90-day toxicological study”1

E. Barocelli, A. Corradi, A. Mutti and P.G. Petronini

University of Parma, Parma, Italy

Key words: 3-MCPD, 3-MCPD dipalmitate, toxicity, 90-day study

3-Chloropropane-1,2-diol (3-MCPD) is a food processing contaminant that belongs to a group

Figure 1: Structure of 3-MCPD and its mono- and di-esters.

Beneficiary: Professor Pier Giorgio Petronini

Grant number: CFP/EFSA/CONTAM/2009/01

The Department o Experimental Medicine, University of Parma, acknowledges the following

1. INTRODUCTION AND OBJECTIVES

Table 1: Activities and milestones.

2. MATERIALS AND METHODS

2.1.1. SYNTHESIS OF THE 3-MCPD DI-ESTERS (DIPALMITATE)

Details are given in Appendix I.

2.1.2. SYNTHESIS OF THE 3-MCPD MERCAPTURATE

For details on the synthesis, see Appendix II.

2.2.1. ANIMALS AND HOUSING

Rats were housed in monitored environmental conditions: temperature 22±3°C, relative

The dosing of all experimental groups is summarized in Table 2.

Table 2: Summary of experiments indicating dosage and periods of 90-day studies.

2.2.3. ANIMAL OBSERVATION

2.2.4. ANALYTICAL METHODS FOR BIOLOGICAL MONITORING

Owing to the implication of the formation of glycidol as intermediate metabolite in terms of

2.2.5. HAEMATOLOGY AND CLINICAL CHEMISTRY

bilirubin, alkaline phosphatase, urea, aspartate aminotransferase, alanine aminotransferase,

2.2.7. STATISTICAL ANALYSIS

Figure 2: Survival curves: 5 out of 10 Figure 3: Combined survival curves

3.1. PATHOLOGY AND HISTOPATHOLOGY OF FEMALE RATS DYING DURING

3.2. GROSS CHANGES

3.3. HISTOPATHOLOGICAL EVALUATION

Kidney: tubular necrosis and mineralisation Kidney: epithelial tubular

Kidney: hydropic-vacuolar degeneration of Heart: myocardosis

Lung: congestion and alveolar edema Liver: centrolobular hepatocyte

3.4. BODY WEIGHT

3.5. WATER CONSUMPTION

Week Control 3-MCPD 3-MCPD Control 3-MCPD 3-MCPD

1 42.4±2.7 45.1±1.0 46.1±1.2 31.1±0.6 37.2±2.3 43.6±1.9*

2 38.1±1.6 50.3±4.8 47.6±2.9 34.5±0.6 44.9±3.8 50.1±2.4**

3 38.9±1.2 47.8±6.0 52.4±3.3 41.7±0.7 50.4±4.3 54.4±1.7*

4 42.7±1.9 56.6±3.5 54.1±2.8 37.2±1.2 49.4±4.2 47.5±2.1

5 37.8±1.8 54.9±5.5 50.7±2.9 35.0±1.6 47.0±2.9 49.2±2.9**

6 35.1±2.5 51.8±4.2 49.8±2.9 35.7±0.7 50.6±1.5* 49.3±2.6*

7 37.3±2.3 57.2±5.2 55.0±3.6 38.7±2.3 53.9±4.7* 49.8±2.1

8 37.9±2.8 57.5±5.6 56.6±3.6 36.7±1.0 51.0±2.8* 46.5±1.8

9 37.4±3.0 57.0±7.4 54.2±4.0 37.7±1.8 51.6±4.6* 48.1±2.6

10 37.6±2.3 54.1±6.0 54.8±3.7 39.3±2.3 52.1±3.6 52.5±3.1

11 39.5±2.5 57.4±6.4 58.6±4.4 38.8±1.9 50.6±3.5 54.4±2.2*

12 36.8±1.5 56.7±7.6 60.5±4.8* 38.6±2.2 48.7±4.4 49.8±2.4**

13 39.5±2.2 61.3±6.7* 57.9±5.1 39.2±2.5 56.7±3.8** 47.8±2.4

3.6. FOOD CONSUMPTION

3.7. BIOMONITORING RESULTS

Control Dipalmitate 3-MCPD Statistical significance (p)

Control Dipalmitate 3-MCPD Statistical significance (p)

Control Dipalmitate 3-MCPD Statistical significance (p)

In male rats, about 30 % of 3-MCPD was excreted either unchanged (3 %) or as 3-MCPD

Figure 9: 24-h urinary excretion of 3-MCPD and its mercapturate expressed as a

3.8. HAEMATOLOGY AND CLINICAL CHEMISTRY

Table 5a: Haematology parameters in male rats. *GM (GSD)

Table 5b: Clinical chemistry in male rats. *GM (GSD)

Table 5b: Continued

Dipalmitate: log(ND)=1.64log(UD)-2.49 => log(UD)=1.52+0.61log(ND) (1)

3-MCPD: log(ND)=1.52log(UD)-2.39 => log(UD)=1.57+0.66log(ND) (2)