Carcinogenic Activity of Alkylating Agents 1,2
B. L. Van Duuren,3 B. M. Goldschmidt,3 c. Katz,3 I. Seidman,4 and J. S. Paul
SUMMARY-Administered by 1 or more routes into female
ICR/Ha Swiss mice, 17 direct-acting alkylating agents and
related compounds were tested for carcinogenic activity.
The routes of administration were skin, 2-stage tumori-
genesis on skin, subcutaneous (sc) injection, and intra-
peritoneal (ip) injection. Dimethylcarbamyl chloride, a
potent carcinogen to skin, induced a high incidence of
sarcom3S at the injection site after sc or ip injection. Four
of the other 15 compounds (ethyl bromoacetate, 2,3-
dichloro-p-dioxane, glycol sulfate, and diethyH3,'Y-epoxy-
propylphosphonate) tested through sc injection induced
significant incidences (P< 0.01) of sarcomas at the injec-
tion site. Whereas sarcomas were induced by most com-
pounds tested by sc injection, only 2 of these, dimethyl-
carbamyl chloride and 1,2,4,5,9,10-triepoxydecane, also
induced carcinomas upon application to skin.-J Natl
Cancer Inst 53: 695-700, 1974.
CHLOROMETHYL METHYL ETHER and bis-
(chloromethyl)ether are carcinogenic when applied to
mouse skin and when injected subcutaneously (sc)
into mice and rats (1, 2) ; this has led to clear evidence
linking human lung cancer and exposure to either of
these chemicals (3) (see note added in proof.). This
report describes the bioassay of a series of related
chloro-ethers and other alkylating agents administered
by several routes into mice. Some of these chemicals
are used in the chemical industry; others are fre-
quently employed in the chemical laboratory. Addi-
tional compounds were examined because of their
unique structural features which make them of interest
in the study of structure-activity relationships.
MATERIALS AND METHODS
Biologic testing methods.-Female ICRjHa Swiss mice
(A. R. Schmidt-Sprague-Dawley, Madison, Wisc.)
were vaccinated against ectromelia, and treatments
were begun when they were 6-8 weeks old. The mice
were housed, 10 to a cage, on sterile, hardwood chips
(Iso-Dry, Fisher & Son, Bound Brook, N.J.) in
stainless-steel cages 11 X 7 X 6 inches high, fed Purina
Laboratory Chow and water ad libitum, and weighed
monthly. The animal rooms were maintained at
22-24° C. There were 30 or 50 mice/group (see
"Results"). The dosages used in all experiments were
based on short-term toxicity evaluations (for 4 wk),
and the highest possible doses that gave minimal
cytotoxic effects were used. All compounds were used
in well-ventilated hoods, and the mice were housed
there for at least 3 hours after treatment. All treat-
ments were continued for the duration of the experi-
ments, except as noted in tables 4 and 5.
In the skin-application experiments, the mice were
shaved initially and whenever necessary throughout
the test. All compounds were applied in 0.1 ml
acetone in the interscapular region, 3 times a week,
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 53, NO. 3, SEPTEMBER 1974
550-859 0 - 74 - 7
5
with a micropipette (Biopette, Carworth, Inc.,
New City, N.Y.). Skin lesions were diagnosed as
papillomas when they reached 1 mm3 and persisted
for 30 days or more.
In the sc injection experiments, the mice were
given weekly injections in the left flank with a 26-
gauge, %-inch stainless-steel needle (Becton, Dickin-
son & Co., Rutherford, N.].), mounted on a I-cc
glass tip tuberculin syringe graduated to 0.01 m!.
The chemicals were injected as solutions in 0.05 ml
tricaprylin, distilled water, or Nujol [purified paraffin
oil (4)], depending on the solubility and stability
of the test compounds. The treatments were continued
throughout the tests unless otherwise stated in the
tables. The vehicle used for each compound and the
duration of the experiments are given in "Results."
Mouse-skin initiation-promotion experiments were
done as described in (4). The doses of the initiating
agents used are shown in table 3. The tumor promoter
was pure phorbol myristate acetate (PMA) prepared
from croton oil as described in (5); 2.5 J.Lg in 100 J.Ll
acetone was applied 3 times a week throughout the
tests. The interval between application of initiator
and promoter was 14 days.
In the ip series, all mice were treated once a week
by ip injection into the lower abdomen with use of
the same sizes of needles and syringes as were employed
for the sc injections. Treatments were continued for
450 days, when the surviving animals were killed.
All animals were weighed and examined regularly,
and the findings were recorded monthly. Animals in
poor health or with large tumor masses were killed.
Except for the cranial region, animals were com-
pletely autopsied at the end of the experiment or at
death. Although samples of all tissues and organs
were not taken from each animal on test, the autopsies
were done carefully and samples of all abnormal-
appearing tissues and organs were excised for histo-
pathologic diagnosis. All tissue sections were fixed in
10% formalin, processed, blocked in paraffin, and
stained with hematoxylin and eosin for histopatho-
logic examination. Included in the protocols were
groups given vehicle only and groups untreated.
1 Received December 13, 1973; revised 'May 17, 1974; ac-
cepted May 17,1974.
2 Supported by Public Health Service (PHS) contract
NOICP43221 from the National Cancer Institute, PHS grant
ES00260 from the National Institute of Environmental Health
Sciences to New York University Medical Center, PHS grant
CAl1479 from the National Institutes of Health, and grant
DRG-355-0 from the Damon Runyon Memorial Fund for
Cancer Research to the University of Texas, Southwestern
Medical School.
3 Department of Environmental Medicine, New York Uni-
versity Medical Center, New York, N.Y, 10016,
4 Department of Pathology, New York University Medical
Center.
5 Department of Pathology, University of Texas, South-
western Medical School, Dallas, Tex. 75235.
695
696 VAN DUUREN, GOLDSCHMIDT, KATZ, SEIDMAN, AND PAUL

H2 C-0
Chemicals and solvents.-The chemical structures of 12 I )=0
the test compounds are shown in text-figure 1. All o11
H2C-0
1 CI-CH 2 -C -OH
chemicals were purified before use. The sources and o
methods of purification are shown in table 1. Infrared o11 7 Y-CI
o H 2 C-0 P
and nuclear magnetic resonance (NMR) spectra were 2 CI-CH 2 -C-0-CH 2 -CH 3
Cl
13 I 's/'\
H2 C-0 0
obtained on all compounds to check purity. Where o
8 &CI
appropriate, gas chromatograms were also obtained. 11
3 Br-CH 2 -C-0-CH 2 -CH J H3 C-O,,o
s
Chemical stability of acetone solutions.-Since acetone 14
H3C-0
I\,
0
CI
solutions for the mouse-skin applications were pre- ~ /CH 2 -CH J
ri
pared freshly once a month, it was necessary to in-
Cf=o
4 CI-CH 2-C-N, HO OH
CH 2 - CH)
sure that compounds 3, 5, 7, 8, 13, and 16 did not 15
11
degrade. This was done by gas chromatography of o o
freshly prepared and I-month-old solutions of these 5 CI-N? 10.~ 9CH2CH3
compounds at the concentrations used for the bio- o CH)
16 O~~=O
assays, except for compound 16, which was assayed OCH 2 CH J

by infrared spectroscopy . No compound showed more

O~
/CH J
CI-C-N
than 1% degradation. Compounds 1, 2, 4, 6, 9~ 12, ~ 'CH J 17 N (CH 2 COOH)J

and 14 were stable in acetone solution.

RESULTS
The results of the bioassays are shown in tables
2-5. In the skin applications, some squamous car-
cinomas developed from papillomas; i.e., in tables 2
and 3, if a group of 40 animals with papillomas had
30 squamous carcinomas, the latter arose from 30
of the 40 papillomas.
The incidence of sarcomas at the site of injection
(table 4) and tumors at sites distant from the area of
administration was tabulated for all test and control
groups. Significance values (P) were calculated for
the most common distant tumors, i.e., papillary tumors
of the lung and lymphomas of liver, spleen, and nodes.
In both no-treatment control groups and test groups,
the cut-off point for significance was P= 0.05. If a
compound had a P value of 0.05, it was considered
borderline. P values >0.05 were considered not
significant. Any compound with a P value <0.05
was considered to have significant tumorigenic
TEXT-FIGURE I.-The chemical structures ofthe test compounds.
activity. All statistical data were computed based on 1
degree of freedom. We found no significant difference
between the incidence of these distant tumors in
treated animals and the incidence in no-treatment
control groups. The survival of animals was excellent
in all groups except those with high tumor incidences.
The results of this series of assays are best compared
by use of a summary table (table 6). There was a
large difference in tumor responses to skin application
and sc injections. Thus only 2 of 15 compounds tested
by skin application caused skin carcinomas; 14 of 16
compounds tested by sc injection caused local sar-
comas. Results with compounds 2, 4, and 9 were 1
of 50 mice in each group with a local sarcoma. One
of 50 mice had a local sarcoma in the group given
tricaprylin only. However, 11 of 16 compounds
tested by sc injection gave incidences of local sarcomas
greater than 6%. Only 2 of 16 compounds tested did
not induce local sarcomas. These were compounds

TABLE l.-Purification of test compounds

Compound No. and name 8ource* Method of purification t

1) Chloroacetic acid_ __ _ ___________________ ____ __ _ E Recrystallization, cyclohexane, mp 62-64°.

2) Ethyl chloroacetate _ _ ____ ___ _____________ _____ _
3) Ethyl bromoacetate ____________________________
4) 2-Chloro-N,N-diethylacetamide __________________
5) N-Chlorosuccinimide _ ______ __ ________ __ ________
6) Dimethylcarbamyl chloride _____________________
7) 2,3-Dichloro-p-dioxane __________________________
8) Epichlorohydrin _ __ __________ ________ ___ ____ __ _
9) 3-Chloro-1,2-propanedioL _______________________
10) 2,5-Dimethyl-1,2,5,6-diepoxyhex-3-yne ___________
11) 1,2,4,5,9, 10-Triepoxydecane ____________________
12) Glycolsulfitet ________________________________
13) Glycolsuliate§ _______________________________
14) Dimethyl suliate ______________________________
15) Butane sultone" _______________________________
16) Diethyl-i3, 'Y-epoxypropylphosphonate_ ___ __ __ __ __
17) Nitrilotriacetic acid_ _ ___ _____ ________ ___ ___ ___
E Distillation, bp 138-140°/760 mm.
E Distillation, bp 156-157°/760 mm.
E Distillation, bp 75-78%.6 mm.
A Recrystallization, benzene, mp 149-151°.
A Distillation, bp 84-85°/50 mm.
E Distillation, bp 106°/50 mm.
E Distillation, bp 114-115°/760 mm.
A Distillation, bp 142-144°/50 mm.
8(6) Distillation, bp 119-121°/50 mm.
A Distillation, bp 97 % .5 mm.
A Distillation, bp 88-90°/50 mm.
8(7) Recrystallization, benzE'ne, mp 93-94°.
E Distillation, bp 102°/50 mm.
A Distillation, bp 93°/45 mm.
8 (8) Distillation, bp 81-84%.2 mm.
E Recrystallization, water, mp 240° (decomposition).

*E = Eastman Organic Chemicals, Rochester, N. Y.; A =Aldrich Chemical Co., Milwaukee, Wisc.; S = synthesized in the laboratory by procedures described
in the references cited.
tlnfrared and NMR spectra of the purified samples confirmed the assigned structures and purity. mp=melting point; bp= boiling point.
fAlso named ethylene sulfite. Chemical Abstracts' nomenclature: 1,a,2-dioxathiolane-2-oxide.
§Also named ethylene sulfate. Chemical Abstract8' nomenclature: 1,a,2-dioxathiolane-2,2-dioxide.
11 Chemical Ab8tracts' nomenclature: I-butanesulfonic acid-4-hydroxy-ll-sultone.
 CARCINOGEN'ICITY OF ALKYLA TING AGENTS 697
TABLE 2.-Mouse-skin application

Dose in Median
mg/0.1 ml Number survival Duration N umber of mice with:
Compound No. and name acetone of mice/ time, of test,
3 times group days days Papillomas Carcinomas
a week

1) Chloroacetic acid ________________ 2.0 50 506 580 0 0

2) Ethyl chloroacetate ___________ - __ 2.0 50 552 580 0 0
3) Ethyl bromoacetate ______________ O. 5 50 526 580 0 0
4) 2-Chloro-N, N-diethy lacetamide ____ 2.0 50 510 580 0 0
5) N-Chlorosuccinimide _____________ 2. 0 50 519 580 0 0
6) Dimethylcarbamyl chloride _______ 2.0 50 386 492 40 30
7) 2,3-Dichloro-p-dioxane ___________ O. 5 50 478 580 2 0
8) Epichlorohydrin _________________ 2.0 50 506 580 0 0
9) 3-Chloro-1,2-propanedioL _________ 2.0 50 542 580 0 0
10) 2,5-Dimethyl-1,2,5,6-diepoxyhex-
3-yne ___________________________ O. 25* 30 520 665 0 0
11) 1,2,4,5,9,1O-Triepoxydecane ______ 0.1 30 507 624 1 1
0.1* 30 503 635 0 0
12) Glycol sulfite ___________________ 0.5 30 >456 456 0 0
13) Glycol sulfate __________________ 0.1 30 >456 456 0 0
14) Dimethyl sulfate _______________ 0.1 20 437 475 0 0
16)nate
Diethyl-i3,'Y-epoxypropylphospho-
____________________________ 5.0 30 >456 456 0 0
Acetone only ___________________ 0.1 ml 50 543 580 0 0
No treatment __________________ lOO 526 580 0 0

*Indicates experiments were carried out at the University of Texas; all others were done at New York University.

TABLE 3.- Mouse-skin 2-stage. carcinogenesis *

Dose in Days to Median Number of mice with:

Compound No. and namet mg/0.1 ml first survival
acetone papillomat time, days Papilloma Carcinoma
once only

3) Ethyl bromoacetate __________________________ 0.5 229 >385 8 1

6) Dimethylcarbamyl chloride. ___________________ 2. 0 79 >385 6 0
7) 2,3-Dichloro-p-dioxane ________________________ 0.5 48 >385 8 2
8) Epichlorohydrin --------------------------- 2.0 92 >385 9 1
12) Glycolsulfite _______________________________ 0.5 99 >385 9 2
13) Glycol sulfate ______________________________ 0.1 140 >385 4 1
14) Dimethyl sulfate ____________________________ 1.0 120 >385 2 0
16) Diethyl-i3, 'Y-epoxypropylphosphonate __________ 5.0 98 >385 5 2
7, 12-Dimethylbenz[a]anthracene§ _____________ O. 02 40 300 30 19
No treatment __________________________________ >385 0 0

*30 female ICR/Ha mice/group; except for compound 14 (20 mice) and no treatment (100 miee) groups. Experiment tenninated at 385 days.
tFollowed 2 weeks later by 3 applications/wk of PMA (2.5 p.g/O.1 ml acetone) for the duration of the expt. Numbers refer to structures in text-figure 1. There
were 3 mice with papillomas in the group given PMA only; the first papilloma appeared at 224 days. Solvent control groups and the no-treatment group yielded
no tumors. Also, no tumors developed in the groups which received a single treatment of the compounds only, Le., without application of PMA.
tFrom beginning of promoting treatment.
§Positive control.

12 and 17. However, compound 17, 2 sarcomagenic
compounds (7 and 8), and the positive control {3-
propiolactone resulted in 1 or 2 adenocarcinomas of
breast origin near the injection site.
There were few carcinomas when 8 compounds
were tested as initiating agents with PMA as the
promoting agent. Of the 7 compounds tested in 4
test systems, only compound 6 resulted in significant
numbers of local tumors in all 4 tests, though in the
initiation-promotion experiments papillomas, but not
carcinomas, were induced.
The 2 epoxides, compounds 10 and 11, were tested
by sc injection into mice in 2 different laboratories;
similar results were obtained with the same strain of
mice. However, only compound 11 caused carcinoma
in 1 of 30 mice when given by skin application. In the
test carried out at the University of Texas, no skin
tumors were observed with either compound.
DISCUSSION
We previously reported preliminary results on the
carcinogenicity of compound 6 (9) and showed that
it was carcinogenic to mice when applied to skin or
injected sc. The present report gives complete details
on those studies and includes carcinogenicity assay
data for 16 other compounds administered by 1 or
more routes in IeR/Ha mice. An unexpected finding
was the disparity between tumor induction in mouse
skin and sc tissue by a variety of chemicals. The
results suggest that one must be cautious in inter-
preting the carcinogenicity of these agents in other
tissues and species.
 CJ)
(!)
co

TABU;
T ABLI<~ 4.-Miee
4.- Mice given se injections*

Number of mice with local malignant Significance

Dose in Median tumors: value (P,
Compound No. and name mg/0.05 ml Number of survival Duration of - - - - - - - - - - - - - - - - - - for total <:
tricaprylin t mice/group time, days test, days Squamous Adenocar- local :>
>
Sarcomas cell cinomas malignant Z
carcinomas tumors) Ij
tj
c::c::
1) Chloroacetic acid ____________________________ :;u
~
0.5 .50
50 454 580 3 0 0 >0.05 t>1
t!l
2) Ethyl chloroacetate __________________________ 1.0 50 471 580 1 0 0 >0.05 _z
~z
3)
3) Ethyl bromoacetate __________________________ 0.1 .')0
50 441 580 9 0 0 <0.01 Q
Q
4) 2-Chloro-N,N-diethylacetamide ________________ 1.0 ,1)0
.50 480 580 1 0 0 >0.05 0
5)
5) N-Chlorosuccinimide _________________________ 0.3 .50
50 490 580 5 1 0 >0.05 t-<
Ij
tj
6) Dimethylcarbamyl chloride ____________________ .5.0
5.0 50 280 427 36 3 0 <0.01 Ul
r/l
0
Cl
7) 2,3-Dichloro-p-dioxane ________________________ O..1)5 50 444 .580
580 14t 1 2 <0.01 :I:
::r:
8) _ _ _ __ _ _ __ __ __ _ _ __ _ __________
8) Epichlorohydrin _____________________________ 1.0 50 486 ;")80
!180 6 0 1 <0.05 is::
~
3-Chloro-1,2-propanediol ______
9) 3-Chloro-l,2-propanedioL . _______ .. _______
_____________________ 1.0 50 487 580 1 0 0 >0.05 8
S
2,5-Dimethyl-l,2,.'i,6-diepoxyhex-3-yne_________
10) 2,5-Dimethyl-l,2,.5,6-diepoxyhex-3-yne _________ 0.5 30 461 580 2 1 0 >0.05 ~>-:l
(a) 1. O~ 30 403 6-65
665 4 2 0 <0.025
-""
~
11) __________________
1,2,4,5,9,10-Triepoxydecane _______ . __________ l. O§
1. 30 475 .580
580 4 1 0 <0.025 :>
>
>-:l
(a)1.0§
(a) 1. O§ 30 .1)12
512 635 5 1 0 <0.025 N
~N
12)
12) Glycol sulfite _______________________________ O. 3 30 468 580 0 0 0 >0.05 -""
13)
13) Glycol sulfate ______________________________
____________________ --------- .•
.55 30 295 413 21 1 0 <0.01 f{l
~
15)
15) Butane suIsultone
tone _____________________________ 1.0 30 292 580 3 1 0 >0.05 S
16) Diethyl-,B,I'-epoxypropylphosphonate
Diethyl-/3, 'Y-epoxypropylphosphonate __________ 5.0 30 441 580 4 2 0 <0.0111 i~
s::
17) Nitrilotriacetic
Ni trilotriacetic acid _________________________ 3.5
3. .5 30 498 580 0 0 1 >0. 05
>0.05 :>
>
/3-Propiolactonc ____________________________
,B-Propiolactone _Z
~Z
0.3 .50
50 241 385 28 4 2 <0. 01
<0.01
Tricaprylin
Trica prylin ________________________________ .05 ml .1)0
.50 493 580 1 0 0 :>
>
N uj oL ____________________________________
Nuj .05 ml 50 523 .1)80
.580 0 0 0 zIjZtj
Distilled water _____________________________ .05 ml 30 .504 .1)80
.580 0 0 0 .."
"tI
No treatmen _____________________________
treatrnentL______________________________ 100 fi26
626 580 0 0 0 :>
>
c::
t-<
'Female
*Female ICR/Ha Swiss mice. Chemicals injected sc once a week in the left flank for the duration of expt. except where noted. (a) Texas; all others New York University; see text.
tTricaprylin used as the vehicle except where solubility was limited, i.e., for compound 3 (dissolved in Nujol) and compound 17 (dissolved in distiUed
distil)ed water).
tOne mOUse
mouse with a metastasis to the lung, liver, and nodes.
§Treated for 26 wk.
IiOne tumors to 7.
I\One other animal with a lymphoma at the injection site brought total number of local malignant tUmors
 CARCINOGENICITY OF ALKYLATING AGENTS 699
TABLE 5.-Mice given ip injections*

Number of mice with:

Compound No. and name Dose in mg/0.05
ml vehicle once a Papillary tumors Local sarcomas
weekt of lung

3) Ethyl bromoacetatet- __ - - - - - - - - - - - - - - - - - - - - - - - - - - -- 0.1 9 o

6) Dimethylcarbamyl chloride_ - -- - - - - - - - - - - - - - - - - - - - - -- 1.0 14 8§
7) 2,3-Dichloro-p-dioxane_ - - - - - -- - - - - - - -- - - - -- - - -- ---- 0.5 12 011
8) Epichlorohydrin __________ - _- - - _- - - - - - - - - - - - - - - - - -- 1.0 11 o
12) Glycol sulfite ___ - - - ___ - - - - - - - - - - - - - - - - - - - - - - - - - -- 0.3 17 o
13) GI~'col sulfate ___________ - _____ -- - - - -- - - - - - - - - - - -- O. 05 9 511
16) Diethyl-fj, 'Y-epoxypropylphosphonate_ - - - - - - - - - - - - - - - 5.0 19 2
lJrethane~ ______________________________________ _ 20.0 20 o
Nujol __________________________________________ _ 0.05 ml 12 o
Tricaprylin _____________________________________ _ 0.05 ml 10 1
No treatment (100 mice) _________________________ _ 29 o
*30 female le R/Ha mice/group except the no-treatment group, which included 100 mice; expt terminated at 450 days. Treatments were continued through-
out the expt.
tTricaprylin except where noted.
tNujol as vehicle.
§One other animal with a local squamous carcinoma.
1I0ne animal with undifferentiated malignant tumor.
~Inlection once only.

TABLE 6.-Carcinogenicity of chloro compounds and other alkylating agents in JeR/Ha Swiss mice

Number animals with local malignant tumors

Total No. animals on test
Compound No. and name
Skin Injection
Whole Initiating sc ip
carcinogen agent

1) Chloroacetic acid _______________________________________ _ 0/50 3/50

2) Ethyl chloroacetate _____________________________________ _ 0/50 1/50
3) Ethyl bromoacetate _____________________________________ _ 0/50 1/30 9/50 0/30
4) 2-Chloro-N,N-diethylacetamide ___________________________ _ 0/50 1/50
5) N-Chlorosuccinimide ____________________________________ _ 0/50 6/50
6) Dimethylcarbamyl chloride ______________________________ _ 30/50 0/30 39/50 9/30
7) 2,3-Dichloro-p-dioxane___ - _______________________________ _ 0/50 2/30 17/50 1/30
8) Epichlorohydrin ________________________________________ _ 0/50 1/30 7/50 0/30
9) 3-Chloro-l, 2-propanedioL ________________________________ _ 0/50 1/50
10) 2,5-Dimethyl-1,2,5,6-diepoxyhex-3-yne ___________________ _ 0/30 9/60
11) 1,2,4,5,9,10-Triepoxydecane _____________________________ _ 1/60 11/60
12) Glycol sulfite _________________________________________ _ 0/30 2/30 0/30 0/30
13) Glycol sulfate ___________________________________________ _ 0/30 1/30 22/30 6/30
14) Dimethyl sulfate ______________________________________ _ 0/20 0/20 -
15) Butane sultone ________________________________________ _ - 4/30
16) Diethyl-fj, 'Y-epoxypropylphosphonate _____________________ _ 0/30 2/30 7/30 2/30
17) Nitrilotriacetic acid ____________________________________ _ 1/30

I t is nevertheless clear that compounds 3, 6, 7, 10,
11, 13, and 16 showed notable tumorigenicity in
ICR/Ha mice, based on the P values calculated for
local malignant tumor incidence. Compound 8 in-
duced sarcomas, but the P value was borderline (0.05).
Some effects of structure on carcinogenic activity are
worthy of notice. Thus, while compound 12 showed
no activity by sc injection, the related compound 13
induced local malignant tumors in 22 of 30 mice.
Possibly this happened - because the sulfate, when
reacting with nucleophiles in neutral media, under-
goes C-O-alkyl scission (7, 10), while the sulfite under
comparable conditions undergoes S-O scission. That
probably also explains why propane sultone (11-13),
a cyclic sulfonate, and compound 15 (14) are carci-
nogenic, whereas the sulfolanes, i.e., cyclic sulfones,
are not (J 1). In these instances, one requirement for
biologic activity seems to be ready cleavage of the
carbon-oxygen bond. However, neither compound 13
nor its open chain analogue, compound 14 (J 5), caused
any skin tumors.
Compound 15, which has 1 more methylene group
in its ring system than the carcinogen propane sultone
(11), has weak carcinogenicity. The lower reactivity
of the 6-membered ring system in compound 15 com-
pared to the 5-membered ring system in propane
sultone may account for this difference in biologic
activity.
Compounds 10 and 11, open-chain diepoxides and
triepoxides, respectively, were tumorigenic. Com-
700 VAN DUUREN, GOLDSCHMIDT, KATZ, SEIDMAN, AND PAUL
pound 16 may be regarded as an open-chain epoxide
with a nearby functional group and, hence, was
expected to show carcinogenic activity. Seven of 30
animals bore local malignant tumors after sc injection
with this compound. These results are consistent with
our previous findings on the structure-activity rela-
tionships of epoxides (16).
Compounds 6 and 7, like bis(chloromethyl)ether,
are rapidly hydrolyzed with half-life times of minutes
or less at physiologic pH and temperature. This is in
contrast to the carcinogenic sulfates, sultones, epoxides,
and lac tones, some of which are listed in this report,
which generally have half-life times at cellular pH and
temperature much longer than those of the halo-
genated compounds described here.
Compound 8 is widely used in industry in the manu-
facture of copolymers. It is not a mouse-skin carcinogen
but induces a significant number of local malignant
tumors through sc injection. It has a low order of
activity in initiation-promotion experiments. Further
experimentation, particularly inhalation exposure,
with this compound would be necessary to determine
whether it is an occupational hazard.
Since compound 17 has been suggested for use as a
biodegradable detergent, it was included in this test
series. However, despite the high dose used, it re-
sulted in 1 adenocarcinoma close to the injection site.
Lijinsky et al. (17) recently reported that feeding
compound 17 to rats caused no significant difference
in tumor incidences at various sites between treated
animals and untreated controls.
Note added in proof: Since this manuscript was sub-
mitted for publication, 3 additional papers have been
published that deal with lung cancer in people who
work with chloromethyl methyl ether and bis( chloro-
methyl)ether. These papers, including one from
Germany and one from Japan, are as follows:
BROWN SM, SELVIN S: Lung cancer in ch10ro-
methyl methyl ether workers. N Engl J Med
289:693-694, 1973
THIESS AM, HEY W, ZELLER H: Zur Toxil),ologie
von Dich10rdimethyHither-Verdacht auf kanzero-
gene Wirkungauch beim Menschen. Zentralbl
Arbeitsmed 23 :97-102, 1973
SAKABE H: Lung cancer due to exposure to bis
(ch10romethyl)ether. Ind Health 11: 145-148,
1973
REFERENCES
(1) VAN DUUREN BL, GOLDSCHMIDT B1\f, KATZ C, et al:
Alpha-haloethers: A new type of alkylating carcinogen.
Arch Environ Health 16:472-476, 1968
(2) VAN DUUREN BL, SIVAK A, GOLDSCHMIDT B:"l, et al:
Carcinogenicity of halo-ethers. J Natl Cancer Inst
43 :481-486, 1969
(3) FIGUEROA WG, RASZKOWSKI R, WEISS W: Lung cancer
in chloromethyl methyl ether workers. N Engl J 1\Ied
288: lO96-lO97, 1973
(4) V AN DUUREN BL, KATZ C, GOLDSCHMIDT B1\1, et al:
Carcinogenicity of halo-ethers. n. Structure-activity
relationships of analogs of bis (ehloro1l1ethyl kther. J
Natl Cancer Inst 48:1431-1439, 1972
(5) V AN DUUREN BL, SIVAK A, SEGAL A, et al: Dose response
studies with a pure tumor-promoting agent, phorbol
myristate acetate. Cancer Res 33:2166-2172,1973
(6) HERSHTEIN NA: Isomeric transformations of acetylenic
oxides and dioxides. J Gen Chem (USSR) 12:132-148,
1942
(7) KAISER ET, PANAR 11, VVESTHEIMER FH: The hydrolysis
of some cyclic esters of sulfuric acid. J Am Chem Soc
85 :602-607, 1963
(8) GRIFFIN CE, KUNDl: SK: Phosphonic acids and esters.
XX. Preparation and ring-opening reactions of (X,/3-
and /3, y-epoxyalkylphosphonates. The proton magnetic
resonance spectra of vicinally substituted ethyl- and
propylphosphonates. J Org Chem 34: 1532-1539, 1969
(9) V AN DUUREN BL, GOLDSCHMIDT B1\I, KATZ C, et al:
Dimethy1carbamyl chloride, a multipotential carcino-
gen. J Natl Cancer Inst 48:1539-1541, 1972
(10) DAVIS RE: Hydrolysis of ethylene and dimethyl sulfite
and the origin of strain in cyclic esters. J Am Chem
Soc 84 :599-604, 1962
(11) V AN DUUREN BL, 11ELCHIONNE S, BLAIR R, et al: Car-
cinogenieity of isosters of epexides and lac tones:
Aziridine ethanol, propane sultone, and related com-
pounds. J Natl Cancer Inst 46:143-149, 1971
(12) ULLAND B, FINKELSTEIN 1\1, WEISBURGER EK, et al:
Carcinogenicity of industrial chemicals propylene
imine and propane sultone. Nature (Lond) 230:460-
461, 1971
(13) DRUCKREY H, KRUSE H, PREUSSMANN R: Propanesultone,
a potent carcinogen. Naturwissenschaften 55 :449, 1968
(14) DRUCKREY H, KRUSE H, PREUSSMANN R, et al: Cancer-
ogene alkylierende Substanzen. IV. 1,3-Propansulton
und 1,4-Butansulton. Z Krebsforsch 75 :69-84, 1970
(15) DRUCKREY H, PREUSSMANN R, NASHED N, et al: Car-
cinogenic alkylating substances. I. Dimethyl sulfate
carcinogenic action in rats and probable cause of
occupational cancer. Z Krebsforsch 68: 103-111, 1966
(16) VAN DUUREN BL, LANGSETH L, GOLDSCHMIDT BM, et al:
Carcinogenicity of epoxides, lac tones, and peroxy
compounds. VI. Structure and carcinogenic activity.
J Natl Cancer Inst 39:1217-1228,1967
(17) LIJINSKY W, GREENBLATT 1\1, KOMMINENI C: Feeding
studies 'of nitrilotriacetic acid and derivatives in rats.
J Natl Cancer Inst 50:lO61-lO63, 1973