Carbamate, Thiocarftamate and Substituted Urea Compounds
CARCINOGEN1CITY AND STRUCTURE-ACTIVITY
-RELATIONSHIPS. OTHER BIOLOGICAL PROPERTIES.
ACTIVATING METABOLISM. ENVIRONMENTAL SIGNIFICANCE
Yin-tak Woo, Ph. D.,
Joseph C. Arcos, D. Sc., and
Mary F. Argus, Ph. D.
Prepared for the Chemical Hazard
Identification Branch "Current
Awareness" Program
-------
Table of Contents;
5.2.1.6 Carbamates, Thiocarbamates and Substituted Urea Compounds
5.2.1.6.1 Historical Background
5.2.1.6.2 Physical and Chemical Properties and Biological Effects
5.2.1.6.2.1 Physical and Chemical Properties
5.2.1.6.2.2 Biological (Other Than Carcinogenic) Effects
5.2.1.6.3 Carcinogenicity and Structure-Activity Relationships
5.2.1.6.3.1 Overview
5.2.1.6.3.2 Urethan and Related Compounds
5.2.1.6.3.3 Acetylenic Carbamates
5.2.1.6.3.4 N-Carbamoyl Aziridines
5.2.1.6.3.5 Carbamate Pesticides
5.2.1.6.3.6 Thiocarbamate Pesticides
5.2.1.6.3.7 Substituted Urea Compounds
5.2.1.6.3.8 Transplacental and "Lactational" Carcinogenesis
5.2.1.6.3.9 Modification of Carcinogenesis by Carbamate and
Related Compounds
5.2.1.6.4 Metabolism and Mechanism of Action
5.2.1.6.5 Environmental Significance
References
*
-------
574
5 2 1 6 Carbamate, Thiocarbamate and Substituted Urea Compounds
52161 Historical Background Caibamates are esters or salts of a
i
simple organic chemical, carbarruc a'cid, NH COOH Substitution of one or
both oxygen atoms of carbamate with sulfur gives rise to thiocarbamate or di-
thiocarbamate, respectively. Replacement of the alkoxy group of carbamate
by[aminogroup yields urea type compounds. Carbamate and related compounds
A
have long been known to possess a variety of toxicological and pharmacologic-
i
al properties A naturally occurring carbamate alkaloid present in the seeds
of Calabar bean was reportedly used several hundreds of years ago by the na-
tives (the Efiks) of Old Calabar along the coastline of West Africa in their
witchcraft trials The accused was reportedly forced to swallow a milky po-
tion containing macerated Calabar bean seeds and ordered to walk around un-
i
til justice was seived — he either regurgitated the potion, recovered and was
declared innocent or he died quickly of the toxic effects thus establishing his
guilt (1) "Pfar tl im ni'Mi j \>y Fnin^M^im i-rfThp native use of the bean see'ds in-
^ , " "- "
itiated a flurry of researchjEurope which eventually led to the identification
i
and synthesis of the active carbamate alkaloid, Rim 1< u.^.. n* nn-rf physotigmine.
Extensive investigations of physotigmine, through its neurotoxic effects, led
to the elucidation of the mechanism of transmission of nerve impulse and laid
the foundation of toxicology and pharmacology of carbamate compounds Studies
of physotigmine analogs stimulated an intensive interest in the search and
development of carbamate and related compounds as pesticides." The hastor c-
a] development of carbamate, thiocarbamate and urea type pesticides has been
-------
575
described (2-4). To date, hundreds of these compounds have been developed,
many of which are used commercially in very large amounts
Urethan (ethyl carbamate), one of the simplest carbamates, was first syn-
thesized from urea a.nd ethanol by Wohler in 1840. It has since found uses tn
human and veterinary medicineJmdustrial chemical intermediate, solubilizer
and co-solvent. Like many carbamates, urethan has narcotic action It was
formerly used as a hypnotic in humans. In fact, it was recommended as "a
very safe hypnotic, . excellent for children . " in a pharmacology textbook
published in 1940 (5). At high doses (in the order of 1 g/kg), urethan was used
I
as an anesthetic for laboratory animals and it was at these high doses that the
carcinogenic effect of urethan was discovered in 1943 In a study designed to
i
investigate the effect of X-ray irradiation on the initiation of skin tumors in
C3H mice, Nettleship, Henshaw and Meyer (6) noted an unexpectedly high fn-
cidence of multiple lung tumors in both experimental and control animals. On
further examination, it occurred to them that during irradiation the mice had
been under urethan anesthesia, and it was urethan treatment that was respon-
sible for the lung tumorigenesis. For some time, urethan was considered as
a specific lung carcinogen However, in 1953, it was independently demon-
strated by Graffi et. al (7) and Salaman and Roe (8) that urethan, when applied
to mouse skin,
-------
576
administration of urethan (9). These findings he lped)ff irmly substantiate the
(DrpcessofJ
concept of initiation and promotion in theytwo-stage skin carcmogenesis4
onr> Intrigued by reports of skin carcinogenicity of urethan, Tannenbaum and
I
associates (10-14) undertook a series of long-term studies in several strains
of mice and demonstrated that urethan has a much broader spectrum of car-
cinogenic action In addition to lung adenomas, it induced or potentiated mam-
mary carcinomas, malignant mesenchymal tumors m the interscapular fat pad,
and cystadenomas of the harderian gland These results have been confirmed
by various investigators (see Section 52163) using different species and
strams Furthermore, several other tissues of some strains have also been
found susceptible to the carcinogenic action of urethan thus clearly establish-
ing ure_fhan as a multipotential agent . Urethan is now one of the'mo-st ex-
I
tensively used experimental carcinogens. =•
The study of the structure-activity relationships of urethan and related
compounds was initiated by the pioneering studies of Larsen (15, 16) a-nd Beren-
blum et al (17) Small changes in the molecular structure may have profound
effect on the carcinogenic potential of the carbamates To date, close to a
hundred carbamates and related compounds have been tested for carcinogen-
icity. Of great importance is the necessity to assess the carcinogenic risk of
carbamates and related compounds used as pesticides. The ever-increasing
demand for pest control, the dwindling supply of naturally QecurringJipesticid'es
and the recent governmental banning'of certain organochlonne" pesticides have
placed an increasingly prominent role on carbamates and related compounds
-------
577
l
in crop and animal protection. A large-scale preliminary carcinogemcity study
«
on 130 commercial pesticides was undertaken by the National Cancer Institute
i
at Bionetics Laboratories in 1963 and completed in 1968 Close to 30 carba-
mate, thiocarbamate and substituted urea compounds were included in the study
l
Of these compounds, three (Diallate, Ethyl selenac, and Potassium bis-2-hy-
droxyethyl-dithiocarbamate) were considered carcinogenic Six (Sodium dieth-
yldithiocarbamate, Monuron, Ethyl tellurac, Ledate, Disulfiram, Zectran) had
Section
* • —^±s^ ~~
marginal activity and required further evaluation (18, 19) (seep 2. 1 6 3). Fur-
ther studies of some of these and related compounds have been completed or
are in progress in the current carcinogenesis testing program of the National
Toxicology Program This chapter reviews these and other carcinogemcity
studies wiih special emphasis on the structure-activity relationships
52162 Physical and Chemical Properties and Biological Effects ~
5.2 1 6 2.1 Physical and Chemical Properties The physical and chemical
properties of carbamate, thiocarbamate and substituted urea compounds have
been described in detail in several reviews (20-23) and monographs (2, 3, 24).
The phys ical constants of some of the more well known compounds are sum- / & £> J. &.
marized in Table CXIII. In general, simple alkyl carbamates are water sol-
uble and slightly volatile, both the solubility and volatility decrease with the
increase of the size of the alkyl group and/or replacement of the ammo hydro-
gens by alkyl or aryl groups
The chemical properties of simple alkyl carbamates (20) and carbamate pes-
ticides (3, 24) have been reviewed The basic backbone of all these compounds
-------
Table CXIII
p. 1 of 3 pp
Physical Constants of
Carbamates, Thiocarbamates and Substituted Urea Compounds'
Compound
m. p.
b. p.
Density
Refractive
index
Vapor
pressure
Solubility
Urethan [Ethyl carbamate
Methyl carbamate
n-Propyl carbamate
Zectran [Mexacarbate,
4-(dimethylami-
no)~3, 5-dimethylphenyl
N-methylcarbamate]
Propoxur [Baygon,
2-isopropoxyphenyl
N-methylcarbamate J
Carbaryl [Sevin, 1-naph-
thyl N-methylcarbamate]
48-50*
54°
60°
85°
84-87°
142'
182-184
177
196'
= 1.4125
volatile
1 mm Hg
(52.4°)
0. 1 mm Hg
(139°)
0. 01 mm Hg
(120°)
4. IxlO"5
mm Hg (25°)
Soluble in water (1 g in 0. 5 ml at
25°), ethanol, chloroform, ether
Soluble in water, ethanol, ether
Soluble in water, ethanol, ether
Practically insoluble in water, (100
ppm at 25°), soluble in aeet-oie
ethanol, benzene, acetonitrile,
methylene chloride
Slightly soluble in water (0. 2%
at 20 ), soluble in most
organic solvents
Practically insoluble in water
(40 ppm at 25°), moderately
soluble in dimethylformamide,
acetone, isophorone
-------
Table CXIH, continued
p. 2 of 3 pp
Caibofuran [Furadan, 153-154°
2, 3-dihydro-2, 2-di-
methyl-7-benzofuranyl
N-methylcarbamate ]
Propham [IPC, Isopropyl 87-87.6'
N-phenylcarbamate ]
Chloropropham [CIPC, 40. 7-4l'
Isopropyl N-(3-chloro-
phenyl)-carbamatel
Primicarb [2-(dimeth- 90.5°
ylammo)-5, 6-dimeth-
y Ipy rimidi n- 4 -y 1
dimethylcarbamatel
Aldicarb [Temik, 2-meth- 98-100°
yl-2-(methylthio)-pro-
pnonaldehyde O-(meth-
ylcarbamoyl)-oximel
Diallate [Avadex, S-(2, 3-di- —
chloroallyl) N, N-dnso-
propylthiocarbamatel
Sulfallate [CDEC, I
2-chloroallyl N, N-di-
ethyldithibcarbamate}
45-1.180
=1.4989
= 1.5395
= 1 195
150
(9 mm Hg)
128-130°
(9 mm Hg)
- 1.188
= 1.088
s 1.5822
2 x 10
-5
mm Hg (33 )
io-6-io-5
mm Hg (25°)
'3 x 10
-5
mm Hg {30 )
1 x 10
-4
mm Hg (25°)
1. 5xlO"4
mm Hg (25°)
2.2x10
-3
mm Hg (20°)
Sparingly soluble in water (0. 07%)
at 25 ); soluble in dimethylform-
amide, dimethyl sulfoxide,
N-methy 1-2-pyr roll done
Practically insoluble in water
(32-250 ppm at 20-25°), soluble
in most organic solvents
Practically insoluble in water
(88 ppm at 25°), miscible
with most organic solvents
Slightly soluble in water (0. 27% at
25°), soluble in most
organic solvents
Slightly soluble in water
(0. 9% at 30°), soluble in
most organic solvents
Practically insoluble in water
(14 ppm at 25°), miscible with
most organic solvents
Practically insoluble in water
(100 ppm at 25°), miscible with
most organic solvents
-------
Table CXIII, continued
p. 3 of 3 pp
Thiram [TMTD, tetra- 155-156*
methylthiuram disulfide]
Disulfiram [Tetraethylthi- 70'
uram disulfide, Antabuse]
Monaron [N'-(4-chlorophen- 174-175'
yl)-N, N-dimethylurea]
Diuron [N1-(3, 4-dichloro- 158-159*
phenyl)-N, N-di-
methylurea]
d = l. 30
5 x 10"?
mm Hg (25°)
3. 1x10
-6
mm Hg (50°)
Practically insoluble in water
(30 ppm at room temperature),
soluble in acetone, chloroform
Sparingly soluble in water (0. 02%),
soluble in ethanol, ether,
acetone, benzene,
chloroform, carbon disulfide
Sparingly soluble in water
(0. 023% at 25°), slightly soluble
in polar organic solvents
such as acetone (5. 2% at 27°)
Practically insoluble in water (42
ppm at 25°), slightly soluble
in polar organic solvents such
as acetone (5. 3% at 27°)
Data summarized from H. Martin, "Pesticide Manual", British Crop Protection Council, 1973, 3rd edn, IARC, IARC Monog. Vol. 7,
International Agency for Research on Cancer, Lyon, France, 1974, IARC, IARC Monog. Vol. 12, International Agency for Research on
Cancer, Lyon, France, 1976, R. J. Kuhn and H. W. Dorough, "Carbamate Insecticides Chemistry, Biochemistry and Toxicology", CRC
Press, Cleveland, Ohio, 1976.
The structural formulas are depicted in Tables CXXI, CXXIII and CXXVIII.
-------
578
is carbamic acid which is_,extremely unstable and does not exist in free form, it
spontaneously decomposes to carbon dioxide and ammonia In contrast to the
free acid, the esters and salts of carbamic acid are quite stable. The thermal
stability of carbamates increases withlthe extent of N-substitution Thus,
N, N-disubstituted carbamates are quite resistant to thermal decomposition,
N-monosubstituted carbamates decompose at elevated temperatures primarily
i
to alkyl isocyanate, whereas unsubs touted carbamates break down to deriva-
tives of cyanic acid without heating. In aqueous solution, carbamates are very
susceptible to alkaline hydrolysis. The half-lives of Carbaryl, Zectran and
i
Propoxur are 0 5, 2 3 and 3 1 hrs., respectively, at pH 9 3 and 25 C (3)
Also, resistance to alkaline hydrolysis is substantially enhanced by alkyl N-sub-
stitution—N, N-Disubstituted carbamates are even more resistant, the hydro-
' i
3-7
lytic rate of some aromatic N, N-dimethylcarbamates is as much as 10 to 1 0
times slower than their N-methyl counterparts. Among the phenyl N-subsii-
tuted carbamates studied, resistance to alkyl hydrolysis follows the order
i
N-phenyl
-------
579
urethan has been reviewed by Mirvish (21). In many respects, N-hydroxy ure-
than, N-hydroxyurea and hydroxylamine have sirnilar chemical properties that
i
are different from those of urethan. They all directly react with DNA under
in vitro .conditions probably via the involvement of free radical reaction.
The chemical properties of dithiocarbamate and related compounds have
been reviewed (2, 22-24) Dithiocarbamates are quite reactive, they chelate
metals, interact with sulfhydryl groups and undergo many reactions involving
the oxidation and loss of sulfur Metallic dialkyldithiocarbamates (je £ , Ziram)
may be oxidized under mild conditions to yield thiuram disulfide (Thiram)
i
in a manner analogous to the formation of disulfide from mercaptans Acid
hydrolysis of dialkyldithiocarbamates yields dialkylamine and carbon disulfide
The chemical properties of a number of chlorinated phenyldimethylurea com-
pounds have been described by Melnikov (24) They are stable and may with-
stand heating up to the melting point, without decomposition. Upon heating in
the presence of alkali or mineral acids, the compounds break down to dimeth-
i
ylamine and chloroanihne or chlorophenyl isocyanate
5 2.1.6.2 2 Biological (other than carcinogenic) Effects Carbamate, thiocar-
bamate and substituted urea compounds have a wide variety of biological activ-
ities. Only the toxic, mutagenic and teratogemc effects of those compounds
that have been tested or suspected for carcinogenicity are discussed in the fol-
lowing paragraphs.
Toxic Effects Most of the toxicity studies in this area have been drevoted
to the study of carbamate pesticides. fhe acute toxicity data of a variety of
-------
carbamates and related compounds are summarized in Table CXIV The
values range from 0. 6 mg/kg for Aldicarb to 12. 0 g/kg for disulfiram The
route of administration, the sex, species and the diet all have significant ef-
fects on-the toxicity of these compounds. The symptoms of carbamate poison-
ing in mammals are essentially identical to those of organophosphorus com-
pounds (see Section 5 2. 1. 4 1). The principal toxic actions are due to the in-
hibition of cholinesterase, thus causing excessive stimulation of the nervous
system The duration of carbamate poisoning is, however, much shorter than
that of organophosphorus compounds,b'ecause of the reversibility of carbamate-
-cholinesterase inhibition Several case histories of human overexposure to
carbamate pesticides have been reported (26, 27), the exposed individuals re-
covered from the symptoms of carbamate poisoning in a relatively short peri-
od of time The toxicology of carbamate insecticides (3) and dithiocarbamate
fungicides (22), and the peripheral neuropathic effects caused by dithiocarba-
mates (28) have been extensively reviewed
Mutagenic Efects. The mutagenicity of carbamates and related compounds
has been extensively tested in a variety of test organisms, such as bacteria
and yeast (29), molds (30, 31), higher plants (32), Drosophila (33, rev 34),
cultured mammalian cells (35-38), and experimental animals (4, 39-44). The
mutagenic actions of urethan and related compounds (34) and dithiocarbamate
i
pesticides (23) have y p p o r^ly. been reviewed. The current governmental ban-
ning of certain organochlorine pesticides brought about an increase in the use
of carbamate pesticides and stimulated a new surge of interest in assessing
-------
Table CXV p. 1 of 3 pp.
Mutagemcity of Carbamate, Thiocarbamate and Substituted Urea Compounds in Salmonella Strains and Correlation to Carcinogenicity
Compound
Urethan (ethyl carbamate)
Methyl carbamate
1, 1 -Diphenyl-2-propynyl-N-cy-
Strain TA
no activation
-(41, 50, 51)
-(50)
+ (49)
a
Mutagemcity
100 or 1535 Strain TA 98, 1537 or 1538 Carcinogemcityb
with activation no activation with activation
-(41,49-51) -(41,50,51) -(41,49-52) +
+ (52)
-(49, 50) -(50) -(49, 50)
n. t. n. t. n. t. +
clohexy lea rba mate
1, 1 -Diphenyl-2-butynyl-N-cy-
clohexy lea rba mate
1 -Phenyl-1 -(3, 4-xylyl)-2-propynyl
N-cy clohexy lea rbamate
Barban
Chlo r bupham
Piopoxur (Baygon)
Carbaryl (Sevin)
+ (49)
+ (49)
-(30, 47, 100)
-(47)
-(47, 101, 102)
- (47, 101-103)
n. t.
n. t.
-(47)
-(47)
-(47)
-(47, 49, 103)
n. t.
n. t.
-(30, 47, 100)
-(47)
- (47, 101, 102)
- (47, 101-103)
n. t.
n. t.
-(47)
-(47)
-(47)
-(47, 49, 103)
n t.
n. t.
-------
L'cible CXV, continued
p 2 of 3 pp
Propham (IPC)
Chloropropham (CIPC)
c
Benomyl (Benlate)
Diallate (Avadex)
Sulfallate (CDEC)
Triallate (Vegadex)
Sodium dimethyldithiocarbamate
Sodium diethyldithiocarbamate
Ziram (zinc dimeth-
yldithiocarbamate)
Ethylzimate (zinc diethyl
dithiocarbamate)
Arsenic dimethyldithiocarbamate
Ferbam (ferric dimeth-
yldithiocarbamate)
-(47, 100)
-(47, 100)
-(55, 56)
+ (54)
-(47, 100,
104-106)
-(47, 104)
+ (30, 106)
-(47, 104)
+(56, 106)
+ (46)
-(46)
-(47)
+ (45, 46)
-(46)
+ (46)
-(47)
+ (45, 46)
-(47)
-(47)
-(55, 56)
+ (47, 104, 105)
+ (47, 104)
+ (47, 56, 104)
n. t.
n. t.
-(47)
n. t.
n t.
-(47)
-(47, 100)
- (47, 100)
-(54, 56)
-(47, 100
104, 106)
-(30, 47,
104, 106)
-(30, 56,
104, 106)
-(46)
-(46)
-(46, 47)
-(46)
-(46)
-(46,47)
-(47)
-(47)
-(56)
- (47, 104)
-(47, 104)
-(47, 56, 104)
n. t.
n t.
-(47)
n. t.
n. t.
-(47)
n. t.
n. t.
n. t.
-------
Table CXV, continued
p 3 of 3 pp
cine diethyldithiocarbamate -(46)
hiram +(45, 46, 48)
isulfiram -(46)
abam - (47)
[aneb - (47)
ineb -(47)
ydioxyurea -(41)
;onuron - (57, 100)
luron - (100)
n. t.
-(48)
n. t.
-(47)
-(47)
-(47)
-(41)
-(57)
+ (53)
+ (53)
-(46)
-(46,48)
-(46)
-(47)
-(47)
i
-(47)
-(41)
- (57, 100)
-(100)
n t. n. t.
+ (48)
n t.
- (47)
-(47) -, +
- (47) - , +
-(41)
-(57) -, +
n t
Mutagenicity in strain TA 100 or 1535 (missense mutants) or strain TA 98, 1537 or 1538 (frameshift mutants) either in the presence or
absence of hepatic microsomal activation systems. The numbers in parentheses indicate the reference numbers. '+' =positive; '-' = negative,
'n t ' = not tested.
Carcmogenicity. '+'=positive, '-'= negative, '+_' = equivocal, 'n. t '= not tested. See Section 5. 2. 1. 6. 3 for details
Q
Benomyl was reported to be positive without activation in strains his G46 and TA 1530 [J. P. Seller, Experientia 29. 622 (1973)3, however,
the results could not be confirmed in a more recent study using the same strains [G. Fiscor, S. Bordas and S. J. Stewart, Mutation Res. 51,
151 (1978)].
-------
Table CXIV . p 1 of 3 pp
Acute Toxicity of Carbamates, Thiocarbamates aad Substituted Urea Compounds
Compound
Urethan [Ethyl
carbamate]
Methyl carbamate
N-Propyl carbamate
Zectran [Mexacarbate]
Propoxur [Baygon]
Carbaryl [Sevin]
Species and route
Mouse, s. c.
Hamster, i p.
Mouse, oral
Mouse, s c.
Rat, oral
topical
Mouse, oral
Rat, oral
1. V.
i. p
topical
Guinea pig, oral
Mouse, oral
Rat, oral
oral
1. V.
topical
Rabbit, oral
Cat, oral
LD5Q (mg/kg)
2,230, 1, 750
2,460
6,200
1,300
37 (M), 25 (F)
1, 500-2, 500
24
83-86
10.6
44 (M), 33 (F)
800-1,000
40
438, 540
500-850
575 (NP), 89 (LP)
41 9
4,000
710
150
References
(61, 76)
(77)
(78)
(76)
(7Q)
(79)
(SO)
(79)
(81)
(82)
(83)
(83)
(80, 84)
(79, 85, 86)
(87)
(8!)
(79)
(86)
(88)
-------
Table CXIV, continued
p. 2 of 3 pp.
Carbofuran [Furadan] Mouse, oral
Rat, oral
topical
inhalation
Guinea pig,
inhalation
Dog, oral
inhalation
(80)
Propham
Chloropropham
Benomyl [Benlate]
Primicarb
Aldicarb [Termk]
Diallate [Avadex]
Mouse, oral
Rat, oral
Rat, oral
Rat, oral
Mouse, oral
Rat, oral
Mouse, oral
Rat, oral
topical
Rat, oral
Rabbit, topical
Dog, oral
8.2-14. 1
10,200
LC =85-133 mg/m3
LC5Q =43-74 mg/m3
19
LC Q =52 mg/m
(exposure time
not specified)
52
1,000-9,000
10, 390 (NP),
2, 590 (LP)
>10, 000
107
147
0.3-0.5
0. 8 (M), 0.6 (F)
3 (M), 2.5(F)
395
2,000-2,500
510
(26)
(26)
(26)
(26)
(26)
(26)
(89)
(90)
(87)
(40)
(83)
(83)
(80)
(79)
(79)
191)
(90)
(83)
Sulfallate
Rat, oral
850
(91)
-------
Table CXIV, continued
p 3 of 3 pp
Sodium diethyldi-
thiocarbamate
Ziram [Zinc dimeth-
yldithioca rbamate 3
Ferbam [Ferric dimeth-
yldithioca rbamate]
Thiram [TMTD]
Disulfiram
Nabam [Sodium
ethylenebisdi-
thioca rbamate ]
Ma neb [Manganese
ethylenebisdi-
thioca rbamate ]
Dulcin [4-Ethoxy-
phenyl urea]
Monuroi
Diuron
Mouse, i. p
Rat, i. p.
Rat, oral
Rat, oral
Mouse, oral
Rat, oral
topical
Rabbit, oral
Mouse, oral
Rat, oral
Rat, oral
Mouse, oral
Rat, oral
Rat, oral
Rat, oral
Rat, oral
1,000
1,500
1,400
4, 000
2,050-2,500 "
620-640
2,000
350
12,000
8,600
395
4, 100
4, 500
3, 200 (adult)
2, 800 (NP),
950 (LP)
2, 390 (NP) »
437 (LP)
(92)
(93)
(94)
(94)
(75)
(79)
(79)
(75)
(95)
(96)
(97)
(98)
(98)
(99)
(S7;
(87)
M = male, F =female, NP = normal protein diet (26% casein), LP = protem-defici-
ent diet (3 5% casein)
-------
581
their mutageruc potential. ^ Various studies of the mutagenicity of carbamates
and related compounds have been reported in the past few years. The follow-
ing discussion focuses only on studies involving the use of the Ames Salmonel-
la test, .which is widely regarded as a very useful predictive tool for carcin-
ogenicity (see Suppletory Note 1 for Section 5211)
The majority of the mutagenicity studies of carbamates and related com-
pounds used the Salmonella strains TA 100 and 1535 (which detect base-substitu-
tion mutagens) and TA 98, 1537 and 1538 (which detect frame-shift mutagens). /VX I/
The major findings of most of these studies are summarized in Table CXV. With
the exception of Thiram, none of the compounds displays any significant frame-
-shift mutagenic activity either in the presence or absence of a liver microsomal
activatiqn_system Only the TA 100 and 1535 strains indicated SEE mutagenicity
of some of these compounds. With a few notable exceptions (e_ g , urethan),
there is a reasonably good correlation between bacterial mutagenicity and_an-
imal carcinogenicity.
Three of the acetylenic N-cyclohexylcarbamates, which have been shown
to be potent carcinogens (see Section 5.2 1 6. 3. 3), also possess potent muta-
genic activity Consistently with the direct-acting, alkylating activity of these
compounds (25), they do not require metabolic activation for mutagenicity It
is interesting to note, however, that Barban and Chlorbupham, which are close-
ly related to the acetylenic jpfiyy.ylftfl UTW HflH tfF^f^do not appear to be mutagenic.
The two compounds have been extensively used as pesticides, their careinogen-
ic potential has yet to be assessed
-------
582
S-Chloroallyl thiocarbamates (£ £. , Diallate, Sulfallate, Triallate) rep-
resent another class of carbamate compounds that show definite mutagenicity.
Although there is some disagreement regarding the mutagenicity of these com-
pounds in the absence of activation, there is little doubt that they are all mu-
tagenic following metabolic activation. The structural moiety common to these
compounds is the 2-chloroallyl group, a metabolic intermediate of which is
probably responsible for the mutagenic activity Both Diallate and Sulfallate
have been unequivocally shown to be carcinogenic In the light of this, it would
seem compelling to consider also Triallate to be carcinogenic
The mutagenic ity of a series of dithiocarbamate derivatives and related
compounds has been tested by Shirasu ^t al. (45) and Moriya _et al (46). An
interesting structural requirement for mutagenicity has been noted Thiram,
i
Ferbam, Ziram, sodium dimethyldithiocarbamate, arsenic dimethyldithio-
carbamate each of which possesses two methyl groups on the ammo nitro-
gens have been found mutagenic for strain TA 100 without activatio~n. In
contrast, the N, N-diethyl derivatives (Disulfiram, ferric diethyldithiocarba-
mate, ethyl zimate, sodium diethyldithiocarbamate) and a N-monomethyl de-
rivative (zinc monomethyldithiocarbamate) are all inactive It has been sug-
gested (46) that N, N-disubstitution of the ammo nitrogen(s) with methyl groups
is essential for the mutagenicity of these compounds It should be pomted out
that the mutagenicity of Ferbam and Ziram has not been confirmed by the study
of De Lorenzo e^ al (47) The mutagenicity of Thiram in base-subs titurtion
mutants has been confirmed by Zdzienicka e^ a_l_ (48) Interestingly, these
-------
583
authors (48) also found that the liver metabolic activation system abolishes the
base-substitution mutagenic activity, but activates the frame-shift mutagenic
activity of the compound.
A number of carbamates and related compounds showed variable results
in mutagenicity assays In contrast to its proven carcinogenic activity, ure-
than kas been found by various groups of investigators (41, 49-51) to be inac-
tive in all 5 strains, both in the presence and the absence of a liver activation
system, Probably the only positive result was reported by Anderson and Styles
(52). However, even in this study only the TA 100 strain was sensitive, where-
as the TA 1535 strain was not Inconsistent findings have also been reported
in the mutagenicity tests of urethan by detecting point mutation (37, 44), micro-
nucleus formation (41-43), and sister chromatid exchange (35, 38) in mammali-
an cells. The reason for the variability is not known Benomyl is another
compound of some controversy. It was reported to be positive in Salmonejla
strains his G46 and 1530 (53) and 1535 (54). However, these results could not
be confirmed by two subs^equejit studies (55, 56) Even in the presence of
various types of activation systems (including liquid culture assay, host-me-
diated assay), Benomyl proved to be inactive (55) The disagreement in 1.1m
attributed to the possible contamination of the chemical used in the
earlier studies (55). Disagreement also arose in the case of Monuron. It was
found to be inactive by Simmon et al. (57). In a more recent study, it displayed
weak but significant mutagenic activity (4) A number of related compounds have
also been tested in this study and found to be mutagenic (4)
-------
584
Teratogenic Effects _The teratogemcity of urethan in the mouse has been
extensively investigated by Nomura and ( coworkers (58-63). Urethan ap-
pears to be capable of freely penetrating the placental barrier (62). High in-
cidences of malformations have been observed after the administration to preg-
nant mice of single, high doses (1. 5 or 1 0 g/kg) of urethan during day 8 to day
12 of the gestation period (58, 6l) As with other teratogenic agents (see Sup-
pletory Note 2 in Section 5 2. 1 1), the induction of malformations in any given
organ seems to be totally dependent on the time of differentiation of the organ
concerned (58, 61) and, therefore, is determined by the time (during gestation
period) of the treatment Malformations of the external appearance are ob-
served with the urethan treatment from day 9 to 12, whereas anomalies of the
internal organs occur if urethan is given on day 8 or 9 External malforma-
tions frequently observed include tail anomalies (kinky, short, and/or tuber-
cular tail), cleft palate, syndactyly and polydactyly of the limbs. The most
affected internal organs are the liver and the lung with interruption of'lobula-
tion and sometimes with mtiathoracic livers, diaphragmatic hernias and asplenia
The dose-response relationship of internal organ anomalies shows a striking
non-linearity, the incidence of anomalies drop from 44 to 93% at the dose of
1. 5 g/kg to near zero at 1 0 g/kg (58, 59) The teratogenic effects of urethan
may be significantly reduced by the administration, within 24 hrs , of caffeine (60)
There is some evidence of similarities between the mechanisms of urethan-m-
j
duced teratogenesis and carcinogenesis (60) The teratogenic effects 1>f ure-
than on the mouse limbs may also be demonstrated by _m vitro organ culture
systems (64)
-------
585
The teratogemcity of.urethan has also been shown in Syrian golden ham-
sters (65). Treatment of pregnant hamsters with urethan on day 8 of gestation
induces a variety of malformations (including exencephaly, encephalocele, mi-
crocephaly, anophthalmia, microphthalmia, omphalocele, anomalies of ex-
tremities) and growth retardation. Nine other related compounds havealso been tested
by the same authors (65). Of the compounds structurally modified at the car-
boethoxy end of urethan, ji-propyl carbamate is as potent as urethan, p-hydroxy-
ethyl carbamate has marginal activity, whereas allyl- and ji-butylcarbamate
are inactive Of the compounds modified at the carbamyl end, N-methyl ethyl
carbamate is more potent than urethan, diethyl carbonate is as potent, whereas
N, N-dimethyl ethyl carbamate is inactive. N-Hydroxyurethan , proved to be the
most potent teratogen of the group, producing malformations of extremities
and anophthalmia in 1 7 to 44% of the fetuses With a few exceptions, the tera-
togenicity of the carbamates in the hamster correlates well with their car-cin-
ogenicity in the mouse (65).
In addition to the above compounds, the teratogemcity of different carba-
mate pesticides has been investigated Carbaryl, the most extensively studied
carbamate pesticide, has been tested in a variety of animal species, such-as the
mouse (66, 67), rat (39), hamster (68), guinea pig (68, 69), rabbit (67, 68), dog
(70) and monkey (71) Inconsistent findings, due largely to differences in the
species, strains and dosages, have been reported The teratogemc action of
carbaryl has been demonstrated in the mouse (66), guinea pig (68), rabbit (67)
and dog (70). In most cases, high doses, often maternally toxic, are required
to exert the teratogemc effect. The positive findings in the guinea pig (68) and
rabbit (67) are not in agreement with those of Weil £t al (69) and Robens (68),
-------
586
respectively. Negative findings have been reported with CF-1 mice (67), rat
(39), hamster (68), and monkey (71). Another carbamate pesticide, Benomyl,
fed at a dietary level of up to 0 5%, was found to be inactive as a teratogen
(40). Rropham (IPC) has been reported to have a positive but somewhat incon-
sistent teratogenic activity in the mouse (66)
Among the thiocarbamates and related compounds, Maneb and Zineb have
been found teratogenic in the rat, when given a_t maternally toxic doses How-
ever, at doses of 0 5 g/kg (for Maneb) and 1 0 g/kg (for Zineb), they produce
no teratogenic effects (72). The teratogenicity of Maneb in the rat has been
confirmed by Larsson^t aj^ (73), who further demonstrated that the teratogen-
ic effects of Maneb may be reduced by simultaneous treatment with zinc acetate
In the NMRI mouse, Maneb has no teratogenic activity (73). Thiram has been
found teratogenic in the Syrian golden hamster (68), NMRI (74, 75) and SW mice
(74) In the hamster, the malformations include exencephaly, spma bifida,
fused ribs, shortened maxila and mandible, and tail and limb anomalies (68).
In the mice, cleft palate, wavy ribs, distorted bones of extremities and micro-
gnathy are the most frequently observed effects (74, 75) The NMRI strain is
more sensitive to the teratogenic action of Thiram than the SW strain (74) In
contrast to Thuam, Disulfiram has no significant teratogenic effects (68),
apparently, replacement of the methyl groups with ethyl groups abolishes the
teratogenic activity
5 2. 1 63 Carcinogenicity and Structure-Activity Relationships -
5.2.1 631 Overview Since the discovery of carcinogenicity of urethan
in 1943, close to 100 carbamates and related compounds have been tested for
-------
587
carcinogerucity. On the basis of the chemical structure and the properties
and/or biological functions and uses, these compounds may be classified into
the following groups (a) urethan and related compounds, (b) acetylenic car-
bamates, (c) N-carbamoyl azindines, (d) carbamate pesticides, (e) thiocar-
bamate pesticides, and (f) substituted urea compounds. These categories are
v y
discussed in the subsequent Sections 52163 3-6.2.1 6 3. 7
AX
Early research efforts were devoted to the elucidation of structure-
-activity relationships of urethan and related compounds The pioneering studies
of Larsen (15, 16) showed that small changes in the chemical structure can
have a profound effect on the carcinogenicity of the compound The structural
feature which is particularly sensitive to change is the alkyl group, substitu-
tion of the ethyl group of urethan by a methyl group completely abolishes car-
cinogenicity With the exception of N-hydroxylation, N-substitution of urethan
also diminishes the carcinogenicity, although to a lesser extent The demon-
stration by Berenblum et al (17) that N-hydroxy-urethan is almost as potent
as urethan,raised the interesting possibility that N-hydroxylation may be a
metabolic activating pathway This possibility has been extensively tested by
various investigators, in particular by Mirvish and his group, who concluded
that N-hydroxylation is not a likely activating pathway (rev 21). One inter-
esting recent finding has been the demonstration of substantially greater car-
cinogenicity of vinyl carbamate than urethan (107) It underscores the impor-
tance of the vinyl group in chemical carcinogenesis and suggests in VIVQ de-
hydrogenation as a possible activating pathway
-------
588
Both acetylenic carbamates and N-carbamoyl azindmes have been dis-
cussed separately because of the presence of different highly reactive func-
tional groups in these two classes of compounds From studies with diaryl
acetylenic carbamates it appears that the phenyl groups have a crucial role in
determining the carcinogenic potency of the compound, whereas changes in the
ammo group may alter the tissue target specificity. Several N-carbamoyl aziri-
dmes are 10-20 times more potent (on a molar basis) than urethan, as a carcin-
ogen The presence of a carbamoyl group renders the aziridine (ethyleneimine)
group more reactive by forming, through resonance, an ethyleneimmonium ion
which may readily react with nucleophilic sites in cellular macromolecules to
initiate carcmogenesis
Ten carbamate pesticides representing various classes such as aryl N-meth-
i
ylcarbamates, alkyl N-aryl carbamates, N, N-dimethylcarbamates, and oxims
of carbamates, have been tested for carcinogenicity Among these compounds,
p-Sevm (2-naphthyl N-methylcarbamate) is the only compound that has been
unequivocally shown to be carcinogenic. This is in sharp contrast to the gen-
eral lack of carcinogenicity of Carbaryl (1-naphthyl N-methylcarbamate). Ap-
parently, in close analogy to naphthylammes, the introduction of a functional
group into the ^-position (but not the opposition) of naphthalene confers car-
cinogenicity to the molecule In addition to [2-Sevin, there is some (although
not convincing) evidence that Zectran may be carcinogenic
The twenty thiocarbamate pesticides that have been tested for carcino-
genicity may be subc lassified into 4 groups S-chloroallyl thiocarbamates,
-------
589
dialkyldithiocarbamates, thiocarbamyl disulfide, and ethylenebisdithuocarba-
mate. Among these, Diallate and Sulfallate (both S-chloroallyl thiocarbamates)
have been unequivocally shown to be carcinogenic On the basis of structural
and metabolic considerations, the carcinogenicity of these compounds is due
to the S-chloroallyl group. It is expected that this class of compounds may be
carcinogenic There is no firm evidence to indicate the carcinogenicity of di-
alkyldithiocarbamates. Five of the 13 dialkyldithiocarbamates tested in the
preliminary NCI bioassay were either shown or suspected to be carcinogenic,
three of these five have subsequently been shown to be inactive in && more
thorough MVGat NCI bioassays Only potassium bis(2-hydroxyethyl)dithiocar-
bamate appears to be carcinogenic in more than one study. No simple structure-
-activity relationships may be established to associate carcinogenic potential
to specific chemical structure It is possible that the metal ion plays a role in
the biological activity of some of these compounds The two thiocarbamyl di-
sulfides, Thiram and Disulfiram, do not seem to be carcinogenic Among the
ethylenebisdithiocarbamates there is some suggestive evidence for the carcin-
ogenicity of Maneb and Zineb, however, it is believed that the carcinogenicity
of these may be due to ethylenethiourea present as a metabolite or as impurity
Among five substituted ureas tested, two aliphatic compounds (hydroxyurea
and Carbromal) are inactive and two with an aromatic ring (Dulcin and Monuron)
show evidence of carcmogenicity, whereas Diuron does not appear to have been
adequately tested. Thus, from the limited information available, substituted
urea compounds with an aromatic ring are suspect and should be more exten-
sively studied
-------
590
Urethan has been shown to easily penetrate the placental barrier, the trans-
placental carcinogenicity of urethan has been reviewed in Section 5. 2. 1 6. 3. 8.
Urethan is one of the most extensively used experimental carcinogens. A num-
ber of carbamates (_£._g , Disulfiram) may alter the carcinogenicity of other
agents. Many examples of synergism or enhancement of urethan carcmogen-
esis have been observed.
5.2.1 6. 3 2 Urethan and Related Compounds The field of investigations on
the carcinogenicity of urethan has been the subject of several comprehensive
cxvi
reviews (11, 21, 108) The major findings of the representative studies in var-
^__— a
ious species and strains of animals are summarized in Tables CXVI and CXVII.
Urethan is a multipotential carcinogen in mice, rats, and hamsters Consid-
erable species-, strain- and age-differences have been observed The rcuate
of administration does not seem to affect the organotropism of tumorigene""-
s is to any great extent, although the doses and schedule of treatment may play
some role.
The carcinogenicity of urethan has been tested in over 30 different strains
and substrains of mice In most of these strains, the lung is the most affected
organ. The induction of lung tumors may occur irrespective of whether urethan
is administered by oral, i p., i v , s c , topical or inhalational route In ad-
dition to the lung, the hematopoietic system and the liver are often affected, es-
pecially in younger mice. Other carcinogenicity targets in some specific
strains include the mammary gland, Harderian gland, forestomach, fat pad,
intestines, skin and salivary gland
-------
Table CXVI
p. 1 of 4 pp.
Carcmogenicity of Urethan in Adult Animals
Species
Mouse,
Mouse,
Mouse,
Mouse,
Mouse,
Mouse,
Mouse,
Mouse,
Mouse,
Mouse,
Mouse,
and strain
A
A/ Jax
AK
AKR
Balb/c
Bagg
BLH
(Bagg X DBA)FJ
C
C3H
C57
B oute
oral, i. p
or i. v
i. p.
t. p.
i.p.
i. p.
i. p.
inhalation
i. p.
8. C.
oral
i. p.
topical
i. p.
i. p.
Principal organs affected
Lung
Lung
Lung
Hematopoietic system
Lung
Lung
Lung
Lung
Lung
Lung, hematopoietic system, fat pad
Mammary gland, lung t
Mammary gland, lung, fat pad
Lung
Liver, intestines
References
(263)
(116)
(110)
(264)
(199, 265)
(109, 110)
(266)
(109)
(267)
(13)
(10)
(10)
(110)
(268)
-------
Table CXVI, continued
p 2 of 4 pp
Mouse, C57BL
Mouse, C58
Mouse, (C57 X A/J)F
1
Mouse, (C57 X C3H)F
1
Mouse, CBA
Mouse, CTM
Mouse, Db
Mouse, DBA
Mouse, DBA/2eBDE
Mouse, dd
Mouse, FA
Mouse, FB
inhalation
i. p.
oral
i. p. or
topical
i. p.
i. p.
oral
i. p.
oral, i p. or
topical
i. p. or
topical
oral
i. p.
i. p.
Lung
Hematopoietic system, liver, intestines
Lung
Lung, mammary gland,
fat pad, Hardenan gland
Lung, Harderian gland, liver
Lung
Lung, hematopoietic system, mammary
gland, liver, Harderian gland
Lung, hematopoietic system, liver
Lung, mammaiy gland, fat pad
i
Liver, hematopoietic system,
lung, fat pad, Harderian gland
Lung
None
t
Lung
(266)
(264)
(188)
(10, 12)
(189)
(109)
(270, 271)
(265)
(10, 13)
(269)
(272)
(109)
(109)
-------
Table CXVI, continued
p 3 of 4 pp.
Mouse, Hall
Mouse, NH
Mouse, NMRI
Mouse, NZO/B1
Mouse, Stock albino 'S1
Mouse, Stiong A
Mouse, Swiss
Mouse, "White-footed"
Mouse, Zb
Rat, MRC
Rat, Sprague
8. C.
1. P.
inhalation
i. p.
topical
i. p.
oral
oral
i. p
s, c.
i. p.
i. p.
i. p.
oral
Lung, liver, hematopoietic system, skin
Lung
Lung
/
Skin
None
Lung
Lung, hematopoietic system
Forestomach
Lung
Lung
None
Lung, hematopoietic system,
liver, mammary gland
Nervous system, thyroid gland, liver
Liver, adrenal cortex, hematopoietic
system, mammary glancl
(273)
(109)
(266)
(111)
(8)
(109)
(186, 274)
(9)
(109)
(267)
(110)
(265)
(126)
(125)
-------
Table CXVI, continued
Rat, Sprague-Dawley
Hamster, Syrian golden
Hamster, European
oral or i p.
oral
oral or
topical
s. c.
i. p.
Guinea pig oral
Chicken (Brown Leghorn) oral or i p.
Mammary gland, ear duct (Zymbal's
gland), hematopoietic system, kidney
Skin, forestomach, intestines,
lung, mammary gland, liver
Skin, mammary gland, ovary
Skin, forestomach, intestines
Subcutaneous and peritoneal tissues, (with
lower incidence liver, lung, adrenal
gland, nasal cavity, kidney, forestomach)
None
None
p. 4 of 4 pp
(14)
(275, 276,
277)
(278-281)
(134, 282)
(77)
(136)
(136)
-------
Table CXVII
p 1 oi 2 pp
Carcmogenicity of Urethan in Newborn or Pre-weanling Animals
Species and strain
Route Principal organs affected
Reference
Mouse, AKR
Mouse, Balb/c
Mouse, C3Hf/Lw
Mouse, C57B1
Mouse, (C57xA/J)F
1
Mouse, (C57xC3H)F
1
Mouse,
Charles River CD-I
Mouse, DBA/f
Mouse, dd
Mouse, dd/I
Mouse, Swiss
Mouse, XVII/G
Rat, August hooded
s.c. Hematopoietic system (192)
s. c. Lung (112)
s.c. Liver, lung (112)
s.c. Hematopoietic system (283)
i. p. Lung, hematopoietic system (284)
i. p. Lung, liver, hematopoietic (188, 196)
system
f
i. p. Liver, lung, hematopoietic (113, 189,
system, Harderian 193, 198)
gland, ovary
i. p. Hematopoietic system, (285)
liver, lung
s.c. Liver, lung (112)
s c. Hematopoietic system (201, 202)
s. c. Hematopoietic system, lung, (287)
Hardenan gland, liver
s.c. Hematopoietic system (192, 286)
s. c. Liver (190)
s. c. Lung (186)
s.c. Lung, hematopoietic system, (171)
salivaiy gland, spleen
s c. Eye (130, 131)
-------
Table CXVII, continued
p 2 oi 2 pp
Rat, MRC
Hamster,
Syrian golden
Hamster,
Syrian golden
Hamster,
Syrian white
Guinea pig,
Hartley albino
i. p. Nervous system, liver
up. Liver, pituitary gland,
uterus, nervous system,
mammary gland, and
other various sites *
s. c. None (single low dose)
s. c. Adrenal cortex, liver,
forestomach, pancreas
s.c. Forestomach, skin,
intestine, thyroid gland
i. p. Skin, stomach,
liver, kidney
s. c. Lung, ovary
(126)
(128, 129)
(132)
(133)
(134)
(135)
(137)
-------
591
The considerable strain differences in the susceptibility of mice to the car-
cinogenic action of urethan may be best illustrated by the studies of Shapiro and
Kirschbaum (109) and Gross jit al (110) In the former study (109), mice of
8 different strains were given weekly i. p. injections of urethan (1 g/kg) for 6
weeks, starting at the age of 10 weeks, and were killed 6-9 months later The
(average
lung tumor incidence and multiplicityVnumber of nodules/mouse) were, in de-
creasing order of susceptibility, as follows Strong A, 100%, 15 nodules, Bagg,
100%, 10, (Bagg X DBA)F , 100%, 8, NH, 100%, 7, CBA, 96%, 4, DBA, 16%,
2, FB, 12%, 1, and FA, 0%, 0. In the study of Gross^tal_ (110), the lung tu-
mor incideice in Efe? six different strains was as follows Swiss albino, 100%
(36/36), Bagg albino, 100% (16/16), C3H, 95% (1 89/1 99), AK, 71% (1 7/24), C57
BL, 70% (33/47), "white-footed" field mice, 0%(0/79) In addition to FA.strain
and "white-footed" field mice, stock albino 'S' mice were reported to be re-
sistant to the carcinogenic action of urethan applied topically (8) On the other
hand, Bielschowsky et al (111) noted that an unusual strain (NZO/B1) of mice
developed skin tumors after receiving i p administration of urethan alone, in
all other strains, the application of a promoter, such as croton oil, is needed
for the expression of skin carcinogenicity of urethan Strain differences in the
susceptibility to liver carcinogenes is by urethan have also been noted, Trainin
_e_t a_l_ (112) reported that after administration of 2 mg urethan subcutaneously
shortly after birth, 100% of C3Hf/Lw, 86% of male DBAf but none of BALB/c
mice developed hepatomas. However, the lung tumor incidence was highest in
BALB/c mice (76%) followed by DBAf (34%) and C3Hf (17%).
-------
592
Newborn and infant mice are more susceptible to the carcinogenic action
of urethan than are adults Thus, whereas tumors of the hematopoietic sys-
tem and liver rarely develop in adult mice (Table CXVI), such tumors are read-
ily observed in mice treated with urethan at newborn age (Table CXVII). Sus-
ceptibility generally decreases as the animal ages (this topic will be discussed
in more detail in Section 5. 2. 1. 6 3 9)
Several investigators have noted that the dosage and schedule of treatment
may affect the carcinogenicity of urethan For example, Vesselinovitch and
Mihailovich (113) observed that continuous treatment with urethan, starting at
the newborn stage, is significantly more efficient in inducing leukemia than if
such treatment is interrupted for various periods of time. About 32% of
(C57 X C3H)F mice developed leukemia after receiving 6 doses of urethan at
1 i
3-day intervals starting on the 1st day of their life. The incidence droppe'd to
13% and 4% if the interval between the 3rd and 4th injections was extended to
9 or 21 days, respectively. It is possible that this is due to the effecfof age
rather than to specific dosage effect In an experiment by Gubareff [as re-
ported by Shimkm et al. (114) ]dis tribution of a given dose of urethan into sev-
eral smaller doses caused either an increase or a decrease in the tumor yield,
the direction of change depended on the number of the doses and the time interval
of spacing However, this finding was only partially confirmed by Shimkm e^
al (114) and White £t al (115), who noted only a decrease in tumor yield upon
fractionation and spacing of a g iven dose of urethan Age was not considered
to be a factor, since the decreasing effect of fractionation occurred in mice of
different ages (4J, b~z and 87 weeks) (116)
-------
593
As mentioned earlier, the route of administration appears to have very
little effect on the organotropism of tumongemcity of urethan This also holds
for the initiation of skin tumors by urethan. Whether administered topically
(7, 8, 117), orally, mtraperitoneally or subcutaneously (9, 118-122), urethan
induced skin tumors, after promotion by promoters, such as croton oil. The
promotion with croton oil may be delayed for 8 weeks with no significant change
in tumor yield (9), a delay of 24-30 weeks may result in. a decrease of about
50% in tumor incidence (120, 123) An additional application of croton oil short-
ly before urethan initiation may substantially enhance the skin carcinogenicity
of urethan (120, 122) Goerttler and Lohrke (124) have recently reported an
interesting finding that urethan-induced skin tumor initiation may occur trans-
placentally and may be promoted postnatally by 1 2-O-tetradecanoylphorbol-l 3-
-acetate, the active ingredient of croton oil (see Section 521638)
The multipotential carcinogenicity of urethan in the rat has been well es-
tablished and considerable strain differences have also been observed" In
young adult Sprague-Dawley rats the principal tissues affected are the mam-
mary gland, the ear duct (Zymbal's gland), the hematopoietic system and the
kidney (14), the overall tumor incidence was as high as 85% In female Sprague
rats (G if-sur-Yvette strain), treated with urethan from the age of 4-5 months,
82% developed tumors predominantly in the liver, adrenal cortex, hematopoietic
system and mammary gland (125) In the study of Kommmem etal (126) using
young adult MRC rats, tumors were mainly found in the neural tissues; (neurilem-
momas), thyroid gland and liver In a study designed to investigate the synergistic
-------
594
effect between urethan and X-ray, three different strains of rats were used,
the average number of mammary tumors (induced by urethan alone) per female
rat was 2. 3 in Sprague-Dawley rats (control 0. 4), 1.8 in Long-Evans rats \no
control)-, and 0 1 in Collip rat5 (control 0 02) suggesting different susceptibility
of the tissue to the carcinogenic action of urethan among these strains (127).
The carcinogenic effects of urethan in newborn rats may be quite differ-
ent from those in adults. In the study of Kommineni ^_t al (126) mentioned
above, much higher incidences of neurilemmomas and liver tumors were ob-
served in rats treated at newborn age. Notably, the thyroid gland was not af-
fected in these rats. It was suggested that the "functional status" of the tis-
sue may play a significant role in determining the susceptibility of the tissue
to carcinogenesis In the study of Vessehnovitch and Mihailovich (128, 12.9),
newborn MRC rats responded to urethan treatment with the development of" a
variety of tumors in the liver, pituitary gland, uterus, nervous system, mam-
mary gland and various other sites In newborn August hooded rats.s c injec-
tions of urethan led to the induction of an unusual type of tumors (melanoma of
the eye). The iris, ciliary body and/or choroid were affected No tumors at-
tributable to the administration of urethan occurred at other sites (130, 131)
It is not known whether this peculiar target tissue is specific to the newborn
of this strain
The carcinogenicity of urethan has also been investigated in different strains
of hamsters In adult Syrian golden hamsters, the induction of melandlic tu-
mors of the skin appears to be the most predominant carcinogenic effect of
-------
595
urethan Skin tumors developed irrespective of whether urethan was given or-
ally, i. p. or s. c (see Table CXVI) The induction of papillomas and carcinomas
of the forestomach was also frequently observed after oral or s. c administra-
tion Other susceptible tissues are the mammary gland, ovary, intestines,
lung and liver Thus, urethan is also a multipotential carcinogen in the ham-
ster. In the wild European hamster, up to 80% of the animals developed tumors
after receiving i p injections of urethan (77) Most of these tumors were sub-
cutaneous, int raperitoneal, and subperitoneal fibrosarcomas About 10% of the
treated animals developed adrenal pheochromocytomas, and in a few cases tu-
mors of the respiratory system, liver and forestomach also occurred
Newborn hamsters exhibited a somewhat different carcinogenic response to
urethan. A single s. c dose of 150 jjg was ineffective in inducing tumors, _prob-
!
i
ably because the dose was too low (132) Six weekly s. c. injections of 1 g/kg
starting at the age of 7 days led to the induction of adrenal cortical tumors in
25-30% of the animals, a few tumors also occurred in the liver, forestomach and
pancreas (133) Toth (134) compared the carcinogenic response of newborn and
adult Syrian golden hamsters to urethan More intestinal tumors developed in
newborns, whereas the reverse was true for papillomas of the forestomach.
The incidence of other types of tumors was not significantly affected by age.
Vesselinovitch et^ al (135) administered ani p doseofO 5g/kg urethan to new-
born Syrian white hamsters and continued injections at 3-day intervals until a total
dose of 2. 5 g/kg was reached About 46% of the males and 27% of the females
developed tumors, mostly malignant melanomas of the skin
-------
596
In addition to mice, jrats and hamsters, the carcinogenicity of urethan was
tested in adult guinea pigs and chickens (136) These latter two species proved
to be resistant to the carcinogenic effect of urethan The refractoriness of the
guinea pig may be, however, reduced if urethan is given at neonatal age. Toth
(137) reported that 33-35% of Hartley albino guinea pigs developed tumors,
mostly in the lung and ovary, after receiving 5 s. c doses of 1 g/kg urethan
starting within 24 hours after birth.
The elucidation of the relationships between the chemical structure and
carcinogenicity of urethan and related compounds has been of great interest
for several decades Larsen (15, 16), Berenblum _e_t aj_ (17), Shimkin et al
(138), and Pound and Lawson (139, 140) have made significant contributions in
synthesizing and testing a variety of urethan analogs. Most of these studies
were carried out in the mouse using lung tumor induction and skin tumor initi-
ation as the indicators of carcinogenicity The results of these studies are ^-
-_^-
summarized in Table CXVIII. Compounds are assigned arbitrary ratings for
the purpose of comparison It is salient from the table that minor modification
of the chemical structure can have profound effect on the carcinogenicity of the
compound. In general, modification of the ester group appears to bring about
a more dramatic effect on the carcinogenicity, whereas substitution at the ammo
group produces a more gradual change
The effect of modification of the ester group may be illustrated by com-
pounds listed in group (A) Table CXVIII Substitution of the ethyl group-by
any other alkyl group either greatly diminishes or completely abolishes the
-------
Table CXVIII
p. 1 of 5 pp
Relative Carcinogenic Potency of Urethan and Its Structural Analogs in the Mouse
Compound
Structure
Pulmonary
- '
tumongenicity
Skin tumor-initiating
activity
Other
Urethan (ethyl carbamate)
(A) Modification of the
Ester Group
Methyl carbamate
j^-Piopyl carbamate
iso-Propyl carbamate
£>-Butyl carbamate
sec-Butyl carbamate
R, 0
K 1!
N-C-O-R.
/
R
R = R = H-, R =C H -
1
R =CH_CH_CH_-
R3 = (CH3)2CH-
R =CH,(CH,),CH,-
R =CH,CH,CH-
J J & I
CH.
+++ (15-17,107,
138, 139, 226,
273, 288)
(15, 138, 273)
(15, 17)
(138)
(273)
(15, 138, 273)
(15, 138,
226, 273)
(138)
+++ (17, 139, 107,
273, 288)
(273)
(17)
(273)
(273)
(273)
Liver 444 (273)
Mammary +4+ (141)
Lymphosarcoma 444
(284)
Liver- - (273)
Liver - (273)
Liver 4 (273)
Liver- 4 (273)
Mammary 444(141)
n. t.
-------
lable CXVIII, continued
p 2 of 5 pp
iso-Amyl carbamate
ji-Hexyl carbamate
Vinyl carbamate
Allyl carbamate
Methylallyl carbamate
Phenyl carbamate
Benzyl carbamate
(?>-Hydroxy ethyl carbamate
pj-Hydroxypropyl carbamate
b-A minoethyl carbamate
A-Chloroethyl carbamate
i\»fl>»ft- Trichloroethyl carbamate
(B) Modification of the
Ammo Group
N-Methyl ethyl carbamate
N, N-Dimethyl ethyl carbamate
—CH-
R3=HOCH2CH2-
R, =CH,-CH(OH)-CH
J J
(15, 226)
(138)
++++ (107)
(17)
++ (138)
(138)
(138)
(138)
(17, 138)
(138)
(17)
(15, 138)
(15)
++ (16, 17, 273)
+ (16)
(17)
n. t.
n. t.
++++ (107)
+ (17)
n. t.
n. t.
n. t.
± (17)
n. t.
(17)
n. t.
n. t.
++ (17, 273)
(17)
Liver ++ (273)
-------
Table CXVIII, continued
p. 3 of 5 pp
N-Ethyl ethyl carbamate
N, N-Diethyl ethyl carbamate
N-ji-Propyl ethyl carbamate
N, N-Di-]i-propyl ethyl
carbamate
N-ji-Butyl ethyl carbamate
N, N-Di-ji-butyl ethyl
carbamate
N, N-Diphenyl ethyl carbamate
N-Hydroxy ethyl carbamate
N-Hydroxy-N-methyl ethyl
carbamate >
N-Acetyl ethyl carbamate
N-Cyanoacetyl ethyl carbamate
Uiethan phosphate
Carboethoxyglycine
R =C H -; R =H-
JL LJ ^ £4
R1=R2 =
~D — <^tJ f'T-T
R1-CH3CH2
R1=R2 =
Rj = CH3CH2CH2CH2- ,
R2=H-
R1=R2=CH3CH2CH2CH2.
=HO-,
=HO-,
R,
j
R
R
R
= CH co-, RZ = H-
= N=CCH CO-, R =H-
Lt fL
= PO(OH) -, R =H-
LJ Ct
= HOOCCH -, R =H-
L* L*
•f
H-f
(16)
(273)
(16)
(16)
(273)
(16)
(16)
(16)
(16)
++ (17, 288)
+++ (284)
n t
(138)
(138)
(17)
(17)
++ (273)
n. t.
+ (273)
n. t.
n. t.
n. t.
n. t.
++ (17, 284)
+++ (139)
(139)
n. t.
n. t.
± (17)
(17)
Liver ++ (273)
Liver ++ (273)
Lymphosarcoma
(284)
-------
Table CXVIII, continued
p. 4 of 5 pp
(C) Other Structurally
Related Compounds
Thiourethan
O
(17)
(17)
Caibamyl phosphate
O OH
II /
NH -C-O-P=O
2 \
OH
(17)
(17)
Xanthogenamide
(17)
(17)
Oxazolidone
H O
\ II
N-C-O-CH_CH
I 2|
(17)
(17)
Diethyl carbonate
O
H
C,H -0-C-0-C,H
L b £.
(17)
(17)
2-Methyl-2-ji-propyl
1, 3-propanediol dicarbamate
NHCOOCH- CH0
2 2s / 3
C
NH COOCH, C H
& £+ J I
(17)
(17)
Methylene diurethan
NHCOOC,.H
2
H7C
2 \
NHCOOC H
+++ (16)
n. t.
-------
Table CXVIII, continued
Ethyiidene diurethan
NHCOOC H
/
-CH
"'"'"'"
p. 5 of 5 pp
n< fc>
Ethylene diurethan
N- Ethyl 1, 3-dichloro-
isopropyl carbamate
N, N-Diethyl 1, 3-dichloro-
isopropyl carbamate
CH0NHCOOC0H_
l 2 25
CH
H
U J
c H NH-COO-CH
CH2C1
(CH ) N-COO-CH
CH2C1
++++(142)
++++(142)
n. t.
n. t.
Relative potency ++++ = considered stronger than urethan, +++ = of comparable activity to urethan, ++ = less potent than urethan, + = slight
carcinogenic activity, jf = questionable carcinogenic activity, -= inactive.
-------
597
carcinogenicity. This trend is observed in the induction of lung tumors and
initiation of skin tumors, as well as production of liver tumors. Only ji-propyl
and isopropyl carbamates exhibit some, although often not convincing, evidence
of carci-nogenicity. The induction of mammary gland tumors may, however, be
an exception to the general observation of lower carcinogenicity of alkyl carba-
mates other than urethan ri-Butyl carbamate was found to be as potent as
urethan in inducing mammary tumors in C3H mice (141)
Substitution of the ethyl group with a vinyl yields a highly carcinogenic
compound Dahl £t al_ (107) have recently shown that vinyl carbamate is about
10-50 times more potent than urethan in inducing lung tumors and initiating
skin tumors. The potent carcinogenicity of vinyl carbamate is not due to the
miej . .
presence of [double bond alone. Allyl, methylallyl, phenyl and benzyl carba-
mates have all been shown to be either inactive or much less active than ure-
than Apparently, a two-carbon moiety may be the optimal size for carcino-
genic activity in carbamates. Vinyl carbamate has been suggested to be a pos-
sible active metabolite of urethan through in vivo
-------
598
The effect of structural modification of the ammo group is illustrated by
compounds in group (B) of Table CXVIII. N-Monosubstitution with alkyl groups
generally reduces the carcinogenicity of urethan The lowering effect on lung
tumor igenicity and skin tumor initiating activity appears to be dependent on the
size of the alkyl group, the decrease being greater with larger alkyl groups.
The ability of N-alkyl ethyl carbamates to induce liver tumors, however, does
not seem to vary among the derivatives tested. N, N-Disubstitution with alkyl
groups further diminishes or abolishes the carcinogenicity of urethan.
N-Hydroxy urethan is the only N-subs tituted compound that displays com-
parable or slightly less carcinogenicity than urethan itself This observation,
coupled with the knowledge that N-hydroxy urethan is chemically more reactive
w
than urethan, led some investigators to propose N-hydroxylation as a poss-ible
I
metabolic activating pathway of urethan (see Section 5 2 1 6. 4) In contract
/"N ^
to N-hydroxy urethan, N-hydroxy-N-methyl-ethyl carbamate is completely de-
void of skin tumor initiating activity, suggesting that di-substitution abolishes
carcinogenicity. N-Substitution with acetyl or cyanoacetyl group decreases
a
the carcinogenicity, whereas substitution with phosphate or carboxyethyl group
completely abolishes activity.
The compounds listed in group (C) of Table CXVIII substantiate the view
that the ester group of urethan probably plays a significant role in the carcino-
genicity of the compound Compounds with modified ester groups (such as thio-
urethan, carbamyl phosphate, xanthogenamide, oxazolidone and 2-me thy 1-2-
-ji-propyl 1, 3-propanediol-dicarbamate) are all inactive as lung caicmogens,
-------
599
and marginally active as_skm-tumor initiators It is interesting to point out
that replacement by sulfur of either the carbonyl or ethereal oxygen of urethan
yields completely inactive compounds. In contrast to the above, compounds
with a slightly modified ammo group (e_ £., methylene diurethan, ethyhdene di-
urethan, ethylene diurethan) display carcmogenicity comparable to or slightly
less than urethan. Nonetheless, the ammo group is required as can be shown
by the lack of carcmogenicity of diethylcarbonate Two chlorinated derivatives
of urethan (N-ethyl 1, 3-dichloroisopropyl carbamate and N, N-diethyl 1,3-di-
chloroisopropyl carbamate) were claimed to be more potent carcinogens than
urethan (142), however, the details of the study are not available On the basis
of the known structure-activity relationships of urethan derivatives, the sup-
posedly higher activity of the two compounds remains questionable.
5 2 1.6 3.3 Acetylenic Carbamates Diaryl acetylenic carbamates were orig-
inally developed as a new class of potential antineoplastic agents (143) A 90-day
subacute toxicity study of 1, 1-diphenyl-2-propynyl N-cyclohexyl-carbamate
[compound (i) in Table CXIX J revealed the remarkable carcmogenicity of the
compound (144) Harlan rats fed diets containing 0.1, 0 25 or 0 5% of the com-
pound developed lymphoblastoma affecting mainly the spleen, liver, adrenals,
and lung In a subsequent chronic study (475 days duration), using dietaiy
levels of 0 025 to 0. 1%, carcinomas of the mammary gland, duodenum, Zym-
bal's gland (m ear duct) and liver were detected Daily s c injections of 12 5
to 50 mg/kg of the compound for 11 weeks induced m this decreasing order of inci-
dence local sarcomas (38/60 rats), mammary tumors (24/60), lymphoblastoma
-------
600
(5/60) and carcinomas of the Zymbal's gland (3/60) Swiss albino and AK mice
and Mongolian gerbils also developed lymphoblastoma after mgestion of the
compound (144).
Intrigued by the above finding, Harris and associates (145-147), extended
their study to nine other compounds of the same class The results are sum-
«£r—
marized in Table CXiX. Interesting structure-activity relationships may be
derived from these data Both the carcinogenic potency and organotropism of
the compound are dependent on the nature of the substituent groups The R
and R groups seem to play an important role in determining the carcinogenic
potency of the compound The replacement of one of the phenyl groups of com-
pound (i) with a methyl group [giving rise to compound (vi) ] completely abol-
ished the carcinogenicHy in male rats and diminished the potency in female
t
rats (146) Introduction of electron-donating groups (e_ g_ , methyl) into the
phenyl ring tends to decrease the carcinogenicity, whereas ring substitu-
tion with electronegative groups (e_ g_ , chlorine, fluorine) has the opposite ef-
fect Thus, compound (vu) was found to be a weaker carcinogen than compound
(i) (146), whereas compounds (in), (vm) and (ix) appeared to be more potent
Compounds (vm) and (ix) were so potent that dietary levels as low as 0 05-0 25%
and 0. 01-0 05% were sufficient to induce tumors in as early as 64 and 54 days,
respectively (145) The ammo group seems to determine the carcinogenicity
target of the compound, N, N-disubstitution shifts the organotropism as well as
enhances the potency Compound (v) induces a high incidence of hepatacarcm-
cinomas in rats receiving diets containing only 0 005% of the compound (146)
-------
Table CXIX
p. 1 of 2 pp
Carcmogenicity of Acetylenic Carbamates in the Rat aftei Oral Administration
Structure
R. O R_
k II I3
N-C-O.-C-CHC-R,.
X I 5
Compound
R
R.
R
R Principal carcinogenicity targets
Reference
(i) 1, 1 -Diphenyl-2-propy-
' nyl-N-cyclohexylcarbamate
(n) 1, 1 -Diphenyl-2-propy-
nylcarbamate
(>JL) 1-(4-Chlorophenyl)-l-phen-
yl-2-propynyl carbamate
(iv) 1, l-Diphenyl-2-propy-
nyl-N-ethylcarbamate
C6Hll
H-
C6H5-
C6H5- H-
H-
H- H- Cl-C.H •
6 4
H-
C,H - H-
65
H-
Hematopoietic system, mammary (144, 146)
gland, intestine, ear duct,
(Zymbal's gland), liver
Mammary gland (females), (146)
intestine (males)
Mammary gland (females), (147)
intestine (males), palate, brain
Hematopoietic system, mammary (146)
gland, intestine, ear
duct, liver
(v) 1, 1 -Diphenyl-2-propy-
nyl-N, N-dime thy lea rbamate
(vi) 1 -Phenyl-1 -methyl-2-propy-
nyl N-cyclohexylcarbamate
CH_- CH_- C,H_
5 3 b 5
C6H - H-
H-
H-
Liver
Mammary gland (females),
none (males)
(146)
(146)
-------
Table CXIX, continued
p. 2 of 2 pp
(vii) l-Phenyl-l-(3, 4-xylyl)-2-pro-
pynyl N-cyclohexylcarbamate
(vm) 1, 1-Bis-(4-fluorophen-
yl)-2-propynyl N-cyclo-
hep ty lea rba mate
(ix) 1, 1 -Bis-(4-fluorophen-
yl)-2-propynyl N-cyclo-
octylcarbamate
C6H11-
(x) 1, l-Diphenyl-2-baty-
nyl-N-cyclohexylcarbamate
H-
F-C,H
64
F-C,H
64
C6H5-
F-C,H - H-
64
F-C,H - H-
64
Intestines (moderate), liver (weak) (146)
Hematopoietic system, (145)
mammary gland
Hematopoetic system, intestines (145)
(Harlan rats)
Ear duct, intestines, hematopoietic (148)
system (F344 males)
Ear duct, mammary gland, (148)
intestines, hematopoietic system
(Sprague-Dawley rats)
Mammary gland, intestines, (145)
liver (females), liver,in-
testines (in a few males)
-------
601
Acetylenic carbamates wi_th free ammo groups [compounds (11) and (iu)] in-
duced mammary tumors in female and intestinal tumors in male rats (146, 147)
Most N-monosubs tituted compounds have similar organotropism, affecting
mainly-the hematopoietic system, the mammary gland and the intestines.
An interesting strain-difference in the carcinogenicity of compound (ix)
has been reported by Weisburger jet a_l^ (148) Male F344 rats given compound
(ix) developed squamous cell carcinomas of the ear duct, carcinomas of small
intestine and lymphomas with about the same incidences However, male Sprague-
-Dawley rats developed a greater number of ear duct tumors than at any other
sites. There was an unusually high incidence of mammary adenocarcmomas,
22/75 males developed such tumors (148) These results are quite different
from those in the study of Harris and associates using Harlan rats which,-upon
receiving compound (ix), developed lymphomas with a 100% incidence. __ Both
the ear duct and the mammary glands of the Harlan rats were not significantly
affected by the compound (145)
Two acetylenic cArbamate pesticides have recently been gaining increas-
ingly important roles in crop protection These two compounds, Barban
(R =m-Cl-C. H -, R =R =R = H-, R = C1CH ,-) and Chlorobupham
1 64234 52
(R =m-Cl-C,H - , R =R =H-, R =CH -, R =H-) are similar in structure
1 6423 435
to the carcinogenic compounds discussed above (Table CXIX) and are, there-
fore, suspect There is no information on the carcinogenicity of Barban and
Chlorobupham, Salmonella tests have, however, been consistently negative (se e
Table CXV) It is possible that replacement of the two phenyl groups gieatly
-------
602
reduces or abolishes the .potential carcmogetucity of the compounds. Never-
theless, in view of the wide use of these compounds, carcinogenicity tests are
urgently called for to ensure that they would not pose any health hazard to
humans.-
5.2 1.6.3.4 N-Carbamoyl Aziridines Like the highly reactive acetylenic
carbamates, N-carbamoyl aziridines constitute a special class of carbamoyl
compounds with a highly reactive functional group azindine (ethyleneimme)
(see also Section 52114). N-Carbamoyl aziridines are probably more re-
active than unsubstituted aziridines toward biological nucleophiles, possibly
because of the resonance structures
O CH_
C-N
\
L
<-
0 CH.
-> R-NH-C=N
\
L
The carcinogenicity of eight N-carbamoyl aziridines has been tested by Shim-
kin £t aL_ (138) using strain A/He mice The relative potencies in inducing lung
tumors are summarized in Table CXX. Several of the compounds are, on a A )(
molar basis, much more active than urethan, for example, 3, 4-dichlorophe-
nyl-N-carbamoyl azindine was estimated to be 23 times more potent than ure-
than. The nature of the substituent group (R) in the carbamoyl moiety plays a
crucial role in determining the carcinogenicity of the compound Saturation of
the phenyl ring greatly reduces carcinogenicity, the relative potency of cyclo-
hexyl-N-carbamoyl aziridine was more than 7 times less than that of phenyl-
-N-carbamoyl azindine (138) However, there is no simple relationship
-------
Table CXX
Relative Carcinogenic Potency of N-Carbamoyl Azindines in the Induction
a
. of Pulmonary Tumors in Strain A/He Mice.
O CH.,
R-NH-C-N
Compound
R group
Relative
b
potency
3, 4-Dichlorophenyl-N-car-
bamoyl aziridine
3-Chlorophenvl-N-car-
barnoyl aziridine
Phenyl-N-carbamoyl
aziridine
Cyclohexyl-N-car-
bamoyl aziridine
4-Methoxyphenyl-N-car-
bamoyl aziridine
p_-Tolyl-N-carbamoyl
aziridine
*
2-Ethoxyphenyl-N-car-
bamoyl aziridine
4-Fluorophenyl-N-car-
bamoyl aziridine
3,4-diCl-C,H,-
b j
3-Cl-C,H -
6 4
Cyclohexyl~
.4-CH30-C6H4-
4-CH -C,H -
3 64
117
56
51
4-F-C,H -
6 4
Summarized from the data of M. B. Shimkin, R. Wieder, M. McDonough,
L. Fishbem, and D. Swern, Cancer Ees 29, 2184 (1969).
b
For comparison, urethan was assigned a relative potency of 5. 0 in the
same study. The relative potencies are designated + = marginal and — = in-
acti\e The compounds were adrrunis tered mtraperiteneall^
-------
603
between ring substitution-and carcinogenicity Ring substitution with electron-
-donating groups (methyl, methoxy, ethoxy) yields compounds that are either
equivocal or inactive, whereas substitution with the electron-attracting chlor-
ine atoms greatly enhances the carcinogenicity. However, ring substitution
with the more electronegative fluorine completely abolishes activity.
It is possible that ring substitution with fluorine gives rise to compounds that
are too reactive to reach target macromolecules in the cell.
5.2.1.6 3.5 Carbamate Pesticides Carbamates have increasingly been used
as pesticides in recent years, the annual consumption of some of these com-
pounds is in excess of a million pounds (see Section 5 2. 1 6 5). The increas-
ingly prevalent use of carbamates is likely to continue as more and more or-
ganochlorine pesticides are banned. Despite extensive acute toxicity studres
of carbamate pesticides, only ten such compounds have thus far been te-sted for
carcinogenicity The structural formulas of these compounds are depicted in Ta-
ble CXXI and the carcinogenicity testing results are summa-rized in Table CXXII
x
Among the ten compounds tested, five belong in the group of aryl N-meth- ~7~g7>?f>
ylcarbamates. Zectran (Mexacarbate) is a phenyl N-methylcarbamate with two C«/vX/
4
methyl groups and one dimethylamino group attached to the ring. It was first
suspected to be carcinogenic in a preliminary NCI bioassay (18, 19) Oral ad-
ministration of the compound (4 6 mg/kg) for 77 weeks to B6C3F mice,
starting at the age of 7 days, led to a slight but significant increase in the in-
cidence of lung tumors in both sexes and in the incidence of hepatomas In male
mice In contrast to B6C3F mice, no significant carcinogenic effects in
-------
Table CXXI
Structural Formulas of Carbamate Pesticides
Tested for Carcmogenicity
8
CH,
Zectron(Mexocorbote)
£H PfOpoxur(Boyqon)
0
II
CH3-NH-C-0
Corbofuron(Furodon)
'"^X
Primicorb
CH3-NH-C-0
Corboryl (q-Sevm)
ii /C
NH-C-O-CH
X Prophom(X=H)
Chloroprophom(X=CI)
0 CH3
II I 3
CH3-NH-C-0-N=CH-C-S-CH,
a I 3
Aldicarb (Temik)
CH3
-------
Table CXXII
p 1 of 3 pp
Carcinogemcity of Carbamate Pesticides
Compound
Species and strain'
Carcinogemcity (route)
Reference
Zectran
[Mexacarbate, 4-(dimethyl-
amino)-3, 5-dunethylphen-
yl-N-methylca i hamate]
Mouse, B6C3F
1
Piopoxur
[Baygon, 2-isopi opoxy-
phenyl N-methylcarbaxnate]
Carbaiyl
[Sevin, 1 -naphthyl
N-methylcarba mate]
Mouse, B6*KF '
Rat, Osborne-Mendel
Rat, unspecified
Mouse, B6C3F
1
orB6AKF
Mouse, A/Jax or C3H
Mouse, ICR/Ha or A/J
Mouse, A/He
Rat, CF-N
Rat, random bred
Lung, liver (oral)
None (s. c.)
Liver, skin (oral)
(marginal)
None (oral or s. c.)
None (oral)
None (oral)
None (oral or s. c.)
None (s. c )
None (oral)
t
No significant
effect (i p.)
None (oral)
Connective tissue (oral)
No significant
rtfeet (c. c )
(18, 19)
(18)
(149)
(18, 19)
(149)
(reported in ref. 83)
(18, 19)
(86)
(150)
(138)
(86)
(151)
(151)
-------
Table CXXII, continued
p. 2 of 3 pp.
/J-Sevin
[2-Naphthyl
N-methy lea rba mate]
Mouse, CC57W
Rat, random bred
Caibofuran Rat, unspecified
[Furadan, 2, 3-chhy-
dro-2, 2-dimethyl-7-benzO- Dog, unspecified
furanyl N-methyIcarbamate]
Piopham
[isopropyl N-phenylcarb-
amate, IPC, Isopropyl
carbanilate ~]
Mouse, strain C
Mouse, B6C3F
or B6AKF
1
1
Mouse, A/He
Rat, Osborne-Mendel
Rat, unspecified
Liver, lung (oral)
Lung (s. c )
Mammary gland (oral)
Local sarcoma (s. c,)
None (oral)
None (oral)
None (oral, s. c., i. m.,
or intrapleural)
None (oral or s. c.)
None (i. p.)
None (oral)
Uterus (i. m. )
(questionable)
None (oral)
Hamster, Syrian golden None (oral)
(152)
(1 52)
(152)
(152)
(26)
(26)
(153)
(18, 19)
(138)
(153)
(153)
(154)
(155)
-------
Table CXXII, continued
p. 3 of 3 pp.
Chlo r op r opham
[Isopropyl N-(3-chlorophen-
yl)-carbarnate, CIPC, iso-
propyl m-chlorocarbanilate]
Benomyl
[Benlate, methyl l~(bu-
tylcarbamoyl)-2-benz-
imidazolecarbamate]
Primicarb
[2-Dimethylammo-5, 6-di-
jmethyl-pynmidine-4-yl
dime thy lea rba mate ]
Aldicarb
[Temik, 2-methyl-2-(meth-
ylthio)-propion-
aldehyde O-(methyl-
carbamoyl)-oxime ~\
Mouse, B6C3Fj or
B6AKF or Swiss
Rat, albino
Hamster, Syrian
golden
Rat, unspecified
Dog, unspecified
Rat, unspecified
Dog, unspecified
Mouse, B6C3F
1
Rat, unspecified
Rat, Greenacre Lab
Rat, F344
Dog, unspecified
None (oral or s. c.)
None (oral)
None (oral)
None (oral)
None (oral)
None (oral)
None (oral)
None (oral)
None (oral)
None (oral)
None (oral)
None (oral)
(18, 19, 155)
(156)
(155)
(reported in ref 157)
(reported in ref 157)
(reported in ref 83)
(reported in ref 83)
(159)
(Weil and Carpenter
cited in ref. 158)
(Weil and Carpenter
cited in ref. 158)
(159)
(Weil and Carpenter
cited in ref. 158)
= (C57BL x
j , B6AKF = (C57BLx AKRjFj
-------
604
similarly treated B6AKF _.mice were observed. A single s.c. injection of 10
mg/kg of the compound was without any effect in both strains (18, 19) The
carcinogenicity of Zectran has recently been re-evaluated in the NCI bioassay
program-(149) Osborne-Mendel rats and B6C3F mice were fed diets containing
two dose levels of the compounds (time-weighted average 209 or 418 ppm for
male rats, 339 or 678 ppm for female rats, 327 or 654 ppm for male mice, 68
or 135 ppm foi female mice), no significant increase in tumor incidence occurred
in dosed rats and female mice. Among male mice surviving beyond 56 weeks,
significant association between Zectran treatment and the induction of hepato-
cellular carcinoma, s.c fibrosarcomas and skin fibromas was established by
one statistical test (Cochran-Armitage) but not by another (Fisher) Thus, al-
though there is some indication of potential carcinogenicity, there is no con-
vincing evidence
Propoxur (Baygon) is a phenyl N-methylcarbamate with an isopropoxy
group in the ortho position. In a two^-year feeding study, a dietary level of 250
ppm was reported to have no ill effects in male and female rats. At 750 ppm,
the liver weight of female rats was increased although there were no other ob-
vious adverse effects (reported in ref. 83), no details of the study are available
Carbaryl (Sevin, 1-Naphthyl N-methylcarbamate) is probably the most
extensively studied carbamate pesticide. In a variety of mouse strains (includ-
ing B6C3F , B6AKF , A/Jax, A/J, A/He, C3H, and ICR/Ha), no significant
carcinogenicity could be demonstrated by various routes of administratron (oral,
i p., s c.)(l8, 19, 86, 138, 150) Similarly, Carbaryl failed to induce tumors in
-------
o05
CF-N rats after feeding of^diets containing up to 0 4% of the compound (86).
The only evidence of potential carcinogenicity was provided by Andrianova and
Alekseev (151) who showed that 4/10 rats receiving 30 mg/kg of the compound
for 22 months developed tumors (3 fibrosarcoma/ 1 osteosarcoma) Only 1/46
control rats developed a fibrosarcoma. When tested by s c. administration,
the carcinogenicity of Carbaryl was not significant (151).
In contrast to the general lack of carcinogenicity of Carbaryl, p-Sevin
(2-naphfhyl N-methylcarbamate) was shown to be unequivocally carcinogenic
in rodents (152) Daily oral administration of 10 mg $-Sevm to CC57W mice
or 25 mg to random-bred white rats for 2-2|- years led to significant increases
in the incidence of liver and lung tumors in mice and mammary tumors in rats
(152) It is uiteiestLng to note that the introduction of a functional group (^.-&.,
amine, mustard) into the /3-position of naphthalene almost always confers, car-
cinogenicity to the molecule Thus, /3-naphthylamine (Section 5 } 2.2.1) j_s
a well-known human bladder carcinogen. H-Naphthylamme nitrogen mustard
(Section 52111) induces lung tumors and local sarcomas in animals and is
suspected to be a human carcinogen. It is therefore not surprising that p-Sevin
is also carcinogenic.
Carbofuran (Furadan) is a relatively new carbamate pesticide containing
a substituted benzofuranyl ring. In an industry-sponsored two-year feeding
study, dietary levels of 25 or 20 ppm Carbofuran were reported to have no ad-
verse effects in rats and dogs, respectively (26) The details of the study are
not available The doses administered are probably well below the maximum
tolerated levels.
-------
606
Two alkyl N-pheny].carbamates, Propharn and Chloropropham, that have
been extensively used as herbicides, have been tested for carcinogenicity
Both compounds have been consistently found to have no significant carcino-
genic effect in various strains of mice, rats, and in Syrian golden hamsters
(18, 19, 86, 153-156). The only possible evidence of carcinogenicity of Propham
was provided by Hueper (153) who showed that 4/10 rats that survived 6 monthly
intramuscular injections of 400 mg/kg Propham developed tumors (2 uterine
adenomyomas, 1 adenofibroma of the groin, 1 mammary adenocarcinoma).
One mammary adenoma was detected among 10 control rats The evidence
may be of questionable significance because of the small number of animals used
Benomyl is a fungicide with a substituted benzimidazole ring linked to
the ammo group of methylcarbamate. It was reported to have very low toxicity
in chronic studies In two-year feeding studies, dietary levels of 2500, pp_m
and 500 ppm were reported to have no adverse effect in rats and dogs, re-spec-
tively (reported in ref. 157). The details of the study are not available.
Primicarb, a selective aphicide, is a N, N-dimethylcarbamate with a sub-
stituted heterocyclic aromatic ring. In two-year feeding studies, the "no ef-
fect" levels were reported to be 1 8 mg/kg for dogs and 250 ppm in the diet
for rats (equivalent to 12 5 mg/kg) (reported in ref 83), no details are available
(firsjj
Aldicarb (Temik) is theYoxime carbamate pesticide registered It has
been used as a substitute pesticide for some cancelled and suspended uses of
DDT. The carcinogenicity of Aldicarb was first tested by Weil and Carpenter.
Their unpublished data (reviewed by USEPA, ref 158) mducated that Aldicarb,
-------
607
at dietary levels equivalent to daily intake of 0. 1 mg/kg, did not cause any
significant increase in the tumor incidences in an unspecified strain of rats
ter two years This study was repeated by Weil (reviewed by USEPA, ref. 158)
using Greenacre Laboratory Controlled Flora rats with essentially the same
results. The sulfoxidized metabolites (sulfoxi.de and sulfone) of Aldicarb were
also not carcinogenic. The "no adverse effect level" was estimated to be 0. 3
mg/kg/day for the rat. The carcinogenicity of Aldicarb was also studied in
beagle dogs Four groups of 6 dogs were fed diets containing 0, 0. 83, 1. 67
and 3. 33 ppm Aldicarb (equivalent to daily intake of 0, 0. 025, 0 05 and 0 1
mg/kg, respectively), no statistically measurable deleterious effects were ob-
served in any of these groups (158) In a recent NCI bioassay (159), Aldicarb
was fed to B6C3F mice and F344 rats at two dose levels (Z or 6 ppm) for-103
weeks, no tumors occurred in either the rat or the mouse at incidences that
could be related to the administration of the pesticide It was pointed out.*.
iaowever, that the doses used may not represent the maximal tolerated doses
(159).
5.2.1. 6. 3. 6 Thiocarbamate Pesticides. Thiocarbamate pesticides may be
classified, on the basis of difference in chemical properties, into four groups
S-chloroallyl thiocarbamate, dialkyldithiocarbamate, thiocarbamyl disulfide
(or tetraalkylthiuram disulfide), and ethylenebisdithiocarbamate. Twenty such
compounds have been tested for carcinogenicity The structural formulas of
these compounds are depicted in Tables CXXIII and CXXV and the carcinogen- .
icity data are summarized in Tables CXXIV through CXXVII.
-------
Table CXXIII
Structural Formulas of Thiocarbamate Pesticides Tested
for Carcmogemcity
Cl Cl
ii ii'
N-C-S-S-C-N
R' SR
Thirom(R=CH^)
Disulf|ram]RfC2H5)
S HN-CH2-CH2-NH
CH2-NH-C-SX l/s\..xs\i
CH2-NH-C-S7 ^Sx
* II
S
Maneb(M=Mn)
Zmeb(M=Zn)
-------
Table CXXIV
Carcmogenicity of S-Chloroally 1 Thiocarbamates
Compound
Species and strain
Principal carcinogenic effect (route)
Reference
Diallate
[Avadex, S-(2, 3-di-
chloroallyl) N, N-duso-
propyl thiocarbamate ]
Mouse, B6C3F
1
Mouse, B6AKF
1
Liver, lung (oral)
Hematopoietic system (s c.)
Liver (oral)
None (s c.)
(18, 19)
(18)
(18, 19)
(18)
Rat, Charles River CD Mammary gland, various sites (oral) (160, 161)
Sulfallate Mouse, B6C3F Lung, mammary gland (oral)
[2-Chloroallyl N, N-di-
ethyl dithiocarbamate] Rat, Osborne-Mendel Forestomach, mammary gland (oial)
(162)
(162)
=(C57BLXC3H)F1> B6AKF = (C57BLX
-------
608
S-Chloroallyl thiocarbamates have recently attracted much attention be-
cause of their carcmogem.cj.ty (Table CXXIV) and/or mutagerucity (Table CXV).
S-Chloroallyl thiocarbamates have been used as selective pre-emergence
herbicid-es. In a preliminary NCI bioassay, daily oral administration of Z15
mg/kg body weight of Diallate (Avadex) was found to cause significant increase
in the incidences of liver and lung tumors in B6C3F mice and liver tumors in
B6AKF mice (18, 19). A single s. c dose (1 g/kg) induced reticulum-cell sar-
coma in B6C3F mice (18). Ulland et al. (160) reported in a 1973 abstract that
1
Diallate was carcinogenic in Charles River CD rats after feeding for two years
The details of the study were provided to the Carcinogen Assessment Group of
the U.S. Environmental Protection Agency (161), which concluded that the com-
pound was significantly carcinogenic in male rats at high dose and m female rats
at low dose Re-interpretation by this government agency (161) of-ca_r-
cinogenicity data supplied by an industrial laboratory also indicated that Dral-
late was carcinogenic, inducing a statistically significant excess of mammary
gland carcinoma in female rats These data, along with the results of muta-
gemcity and neurotoxicity studies, led the Environmental Protection Agency
(161) to propose the banning of the use of the pesticide.
Sulfallate is closely related to Diallate. Sulfallate has been tested for
carcmogenicity in a recent NCI bioassay study (162). Osborne-Mendel rats
and B6C3F mice were fed diets containing two dose levels of the compound
(the time-weighed averages are 250 and 404-410 ppm for rats and 908-949 and
1815-1897 ppm for mice) for 78 weeks The results of the study indicated that
-------
609
Sulfallate was carcinogenic in both rodent species. It induces mammary gland
tumors in the females of both species, tumors of the forestomach in male rats,
and tumors of the lung in male mice (162) Triallate is another member of
the S-chloroallyl thiocarbamate class, it is an analog of Diallate, with an ad-
ditional chlorine atom on the terminal carbon of the allyl group. The carcin-
ogenicity of the compound has not been tested. However, mutagemcity studies
of the compound revealed that it behaved in a manner closely similar to Dial-
late and Sulfallate (see Section 5. 2. 1 6 2 2). Based on structural analogy and
mutagemcity data, the evidence is compelling to suggest that Triallate would
slso be carcinogenic.
Dialkyldithiocarbamates have been used in industry as accelerators of
rubber processing and in agriculture as herbicide. Thirteen dialkyldithiocarba-
mates were included in the preliminary NCI carcinogenesis bioassay (18, 19)
Some of these compounds have also been investigated by other investigater-s
and re-evaluated in the recent NCI bioassay The results of these studj.es
are summarized in Table CXXV. In the preliminary NCI bioassay (18, 19) none
of the 13 compounds tested by a single s c injection was carcinogenic. By oral
administration, however, 5 of the 13 compounds were either weakly or margin-
ally active as carcinogens. These five included (a) sodium diethyldithiocarba-
(uy
mate which had marginal activity in inducing hepatomas ][B6C3F mice and
lung tumors in B6AKF mice, (b) potassium bis(2-hydroxyethyl) dithiocarba-
mate which induced hepatomas in both strains, (c) Ledate which had a margin-
(m,
al effect in inducing reticulum-cell sarcomas j B6C3F mice, (d) ethyl selenac
(in, l
which induced hepatomas \ B6C3F mice and (e) ethyl tellurac which induced
-------
610
lung tumors i hepatomas_in B6AKF mice. There is no simple structure-ac-
tivity relationship associating chemical structure with carcinogenicity It is
possible, however, that the metal ion may play some role in the carcinogenicity
The carcinogenicity of sodium diethyldithiocarbamate, Ledate and ethyl
tellurac has been re-evaluated in NCI bioassays (163-165) in F344 rats and
B6C3F mice. At maximally tolerated doses, none of these compounds were
unequivocally carcinogenic. Only ethyl tellurac induced a dose-related increase
in mesothelioma in male rats but the increase was not significantly different
from that of the controls In male mice, the incidence of ethyl tellurac-m-
duced adenoma of the H^rderian gland (lacnmal gland of the eye) was signifi-
cantly higher, however, a dose-related trend could not be established (165).
Potassium bis(2-hydroxyethyl)dithiocarbamate is the only compound-
shown to be carcinogenic (in Charles River CD rats) by oral administration
(160), the details of the study were not given
The lack of carcinogenicity of Ziram was shown by Chernov and Khitsen-
ko (166) in C57 and strain A mice Rochester strain rats receiving Ziram did
not have a higher incidence of pituitary and thyroid gland tumors than untreated
animals of the same strain (167) In random-bred rats Ziram was considered
carcinogenic (l5l). Given orally twice weekly for 22 months, Ziram (70 mg/kg)
induced tumors of the liver and connective tissue in 4/10 surviving rats. By
s. c. administration, 3/10 rats developed tumors in the liver, colon and sub-
cutaneous tissue Only 1/46 control rats developed a tumor. The survival
rate of the dosed animals was very low
-------
611
Two thiocarbamyldisulfides (Thiram and Disulfiram) have been tested
- <— cxxv/
for carcinogenicity (Table CXXVI). Thiram was found to be noncarcmogenic
in the preliminary NCI bioassay (18, 19). Disulfiram was, however, margin-
ally active, by oral administration to B6C3F mice it induced hepatomas and
lung tumors, and by s c. administration to B6AKF mice it induced reticulum-
-cell sarcomas in the females (18). These marginal activities of Disulfiram
could not be confirmed in the more thorough recent NCI bioassays (168)." oral
administration of maximally tolerated doses of Disulfiram to F344 rats (300 or
600 ppm) and B6C3F mice (500 or 2000 ppm for males, 100 or 500 ppm for
females) did not bring about any significant increase in tumor incidence The
lack of carcinogenicity of Disulfiram in Sprague-Dawley rats was also reported
by Schmahl et aL_ (169)
Ethylenebisdithiocarbamates have been widely used as fungicides. The
carcinogenicity of three such compounds (Nabam, Maneb and Zineb) has been
tested (Table CXXVII). In a preliminary NCI bioassay, none of these,three
compounds was found to be carcinogenic by oral administration to B6C3F and
B6AKF mice (18, 19). Only Zineb, by s. c. administration, caused a slight
increase in the incidence of reticulum-cell sarcomas (T male B6C3F mice (18).
Balm (170) administered orally 6 weekly doses of 500 mg/kg Maneb to C57BL
and strain A mice Increases in the incidences of lung tumors in both strains
were observed, however, the increase was statistically significant only in strain
A mice (170). Andrianova and Alekseev (151) administered Maneb orafly (335
mg/kg twice weekly for 22 months) and subcutaneously (12 5 mg) to random
-------
Table CXXVI
Carcmogemcity of Thiocarbamyl Di.sulfj.des
Compound
Species and strain Principal carcmogenictty
target (route)
Reference
Thiram
[Tetramethylthiuram disulf-
ide, TMTD, bis(dimethylami-
nothiocarbamyl) disulfide]
Mouse, B6C3F
or B6AKF
1
1
None (oral or s. c )
(18, 19)
Disulfiram
[Tetraethylthiuram disulfide,
bis(diethylaminothio-
caibamyl) disulfide]
Mouse, B6C3F
Mouse, B6AKF
Rat,
Sprague-Dawley
Rat, F344
Liver, lung (oral) (marginal)
None (s c )
None (oral)
None (oial)
Hematopoietic system
(s c.) (marginal)
None (oral)
None (oial)
(18, 19)
(18)
J168)
(18, 19)
(18)
(169)
(168)
B6C3F = (C57BLX C3H)F , B6AKF = (C57BLX AKR)F
-------
Table CXXV
Carcmogemcity of Metallic Dithiocarbamates
p. ] of 3 pp
Compound
Structure
R
\
N-C-S
R
n f-D
M
n
M n R
Species and strain
Principal organs affected (route)
Reference
Sodium diethyldi-
thiocarbamate
Potassium bis-
-(2-Hydroxyethyl)
dithiocarbamate
Ziram
[Zinc dimethyl-
dithiocarbamate]
Na 1 C H -
K 1 HOCH.CH
L* C
Zn 2 CH3-
Mouse, B6C3F
1
Mouse, B6AKF
1
Rat, F344
Mouse, B6C3F or B6AKF
Rat, Charles River CD
Mouse, B6C3F or
Mouse, C57 or A
Rat, Rochester
Rat, random bred
1
Liver (oral) (marginal)
None (s. c.)
None (oral)
Lung (oral) (marginal)
None (s. c.)
None (oral)
Liver (oral)
None (s. c.)
Various sites (oral)
None (oral or s. c.)
No significant effect (oral)
Pituitary, thyroid (oral) (not
considered significant)
Liver, connective tissue (oral)
Various sites (s. c. )
(18, 19)
(18)
(165)
(18, 19)
(18)
(165)
(18, 19)
(18)
(160)
(18, 19)
(166)
(167)
(151)
(151)
-------
Table CXXV, continued
Ethyl zimate Zn 2
[Zinc diethyldi-
thioca rbarnate ]
Butyl zimate Zn 2
[Zinc dibutyl di-
thiocarbamate ]
Cumate Cu 2
[Cupric dimethyl-
dithiocarbamate J
Ethyl cadmate Cd 2
[Cadmium diethyl-
dithiocarbamate^
JLedate Pb 2
[Lead dimethyl-
dithiocarbamate}.
C4H9'
C2H5'
CH3-
Mouse, B6C3F or B6AKF None (oral or s. c )
Mouse, B6C3F or B6AKF None (oral or s. c )
Mouse, B6C3F or B6AKF None (oral or s. c )
Mouse, B6C3F or B6AKF
Mouse, B6C3F
1
Mouse, B6AKF
Rat, F344
1
None (oral or s. c )
Hematopoietic system
(oral) (marginal)
None (oral)
None (s c )
None (oral or s. c.)
None (oral)
p 2 of 3 pp
(18, 19)
»
(18, 19)
(18, 19)
(18, 19)
(18, 19)
(163)
(18)
(18, 19)
(163)
-------
Table CXXV, continued
Bismate Bi
[Bismuth dimeth-
yldithiocarbamatel
Pei bam Fe
[Ferric dimethyl-
dithiocarbamate 3
Methyl selenac Se
[Selenium dimeth-
yldithiocarbamate3
CH3-
CH3-
CH3-
Mouse, B6C3F or B6AKF
Mouse, B6C3F or B6AKF
Rat, Rochester
Mouse, B6C3F or B6AKF
None (oral or s. c )
None (oral or s. c.)
None (oral)
None (oral or s. c.)
p. 3 of 3 pp
(18, 19)
(18, 19)
(94, 167)
(18, 19)
Ethyl selenac Se 4 C H -
[Selenium diethyl-
dithiocarbamate 3
Ethyl tellurac Te 4 C2Hi;~
[ Tellurium dieth-
yldithiocarbamate3
Mouse,
Mouse,
Mouse,
Mouse,
B6C3F
J.
B6AKF
B6C3F
B6AKF
Rat, F344
Liver (oral)
None (s. c.)
•
None (oral or s. c.)
None (oral or s. c.)
Harderian gland (oral)
Lung, liver (oral)
None (s. c.)
None (oral)
- (18, 19)
(18)
(18, 19)
(18, 19)
(164)
(18, 19)
(18)
(164)
B6C3F = (C57BL X C3H)F , B6AKF = (C57BL X AKR)F
-------
Table CXXVII
Carcinogenicity of Metallic Ethylenebisdithiocarbamates
Compound
Species and strain
Principal carcinogenicity
target (route)
Reference
Nabam
[Disodium ethylene-
bisdithiocarbamatel
Ma neb
[Manganese ethylene-
bisdithiocarbamate]
Zineb
[Zinc ethylenebis-
dithiocarbamate]
Mouse, B6C3F
or B6AKF
1
Mouse, B6C3F
or B6AKF
1
1
1
Mouse, C57B1
Mouse, strain A
Rat, random bred
Mouse, B6C3F
or B6AKF
1
Mouse, B6C3F
1
1
Mouse, C57B1
Mouse, strain A
Rat, unspecified
Rat, random bred
Rat, landom bred
None (oral or s. c.)
None (oral or s. c )
No significant effect (oral)
Lung (oral)
Various sites (oral or s. c.)
(significance questionable)
None (oral)
Hematopoietic system (s. c.)
Lung (oral)
No significant effect (oral)
No significant effect (oral)
No significant effect (oral)
Various sites (s. c.)
(significance questionable)
B6C3F =(C57BLXC3H)F
, B6AKF =(C57ELXAKR)F
(18, 19)
(18, 19)
(170)
(170)
(151)
(18, 19)
(18)
(166)
(166)
(97)
(151)
(151)
-------
612
bred rats, the induction of tumors in various sites (mammary gland, s c tis-
sue, thyroid gland, connective tissue) was noted. The significance of this
study may be questionable because of the low survival rate of dosed animals.
The carcmogenicity of Zineb was investigated by Chernov and Khitsenko (166).
Strain A and C57BL mice were given 6 weekly doses of Zineb (3. 5 g/kg) and
killed 3 months later, 6 of the 79 C57 mice developed lung tumors compared
to 0/87 control In strain A mice, the incidence of lung tumors in the Zineb-
-treated group (35%) was not significantly different from that of the control
group (31%) Blackwell-Smith ^_t a_l_ (97) fed groups of 10 rats of each sex diets
containing 0, 500, 1,000, 2,500, 5, 000 or 10, 000 ppm of Zineb There was no
significant carcinogenic effect associated with the administration of the chem-
ical. In the most affected group (1,000 ppm) only 4/20 rats developed tumors,
for comparison, 2/20 control rats also developed tumors The study by An-
drianova and Alekseev (151) indicated that Zineb (285 mg/kg, twice weekly for
22 months) was not significantly carcinogenic in random-bred rats after oral
administration. By s c. administration (20 mg/kg), however, 4 of the 6 sur-
viving rats developed tumors (1 hepatoma, 1 fibrosarcoma, 1 spindle cell sar-
coma, 1 subcutaneous rhabdomyosarcoma) The significance of the latter study
is questionable, however, because of the low survival rate and the small num-
ber of animals involved.
5. 2. 1. 6. 3. 7 Substituted Urea Compounds Substituted urea compounds are
closely related in structure to carbamates Like carbamates and thiocarba-
mates, substituted urea compounds have been used as pesticides Similaily
-------
613
to urethan, some substituted urea compounds (_e ^ , Carbromal) have sedative
and hypnotic activity Only a few substituted ureas have been tested for car-
cinogenicity. The structural formulas aie shown in Table CXXVIII and the car-
cinogeni-city data summarized in Table CXXIX. ^-—• _ *>*-&£ C A A Vf/(
t cxxix
Hydroxyurea was found noncarcinogenic in two strains of mice Muran-
yi-Kovacs and Rudali (171) administered intrapentoneally to 50 XVII/G mice
doses of 1, 3, 5 mg hydroxyurea at the age of 2, 8, 15 days and then 10 mg/week
from day 30 to 1 year No significant carcinogenic effects were observed, in
fact, the lung tumor incidence in the hydroxyurea-treated group (45. 7%) was
lower than that of untreated animals (60%) which had a high spontaneous tumor
incidence. In the experiments of Bhide and Sirsat (172) Swis-s mice were
treated subcutaneously with hyd^oxyurea, only one male developed lung tumors
and only one female developed a mammary fibrosarcoma (an incidence not "dif-
ferent from that of the controls).
Cabromal is a mild central nervous system depressant, similar in bio-
logical action t-3 urethan It was selected by the NCI (1?3) for carcinogenicity
testing because of its similarity to urethan. Fisher 344 rats and B6C3F mice
were given dietary levels of 1, 250 or 2, 500 ppm Carbromal for 103 weeks and
78 weeks, respectively, and were observed up to 105 weeks No significant posi-
tive association between administration of the compound and the level of tumor
incidence was noted There was some indication of dose-related incidence of
adrenal pheochromocytomasj male rats, but the effect was not statistically
significant.
-------
Table CXXVTII
Structural Formulas of Substituted Urea Compounds
Tested for Carcmogemcity
0
II
NH2-C-NHOH
Hydroxyurea
0 0 C2H5
II II r °
NHo-C-NH-C-C-Br
Carbromal
0
II
NH2-C-NH
Dulcin
OC2H5
H3C
Monuron(X=CI,Y=H)
Dmron(X=Y=CI)
-------
Table CXXIX
Carcinogenicity of Substituted Urea Compounds
Compound
Species and strain
Principal carcinogenidty target (route) Reference
Hydioxyurea
Carbromal
[N- (Ammocarbo-
nyl)-2 -bromo-2-ethyl
butanamide,
(2-bromo-2-eth-
yl-butyryl) urea]
Monuron
[N'-(4-Chlorophenyl)
N, N-dimethyl urea]
Diuron
[N'-(3,4-Di-
chlorophenyl)
N, N-dimethyl ureal
Mouse, XVII/G
Mouse, Swiss
Mouse, B6C3F
Rat, F344
1
Mouse, B6C3F.
Mouse, B6AKF
Mouse, random
bred or C57B1
Rat, random bred
Mouse, B6C3F
or B6AKF
1
1
None (i p )
No significant effect (s c )
None (oral)
None (oral)
None (o Lal or s c )
Lung (oral)
None (s. c.)
Liver, lung (oral)
Liver, lung (oral)
None (oral or s c.)
(171)
(172)
(173)
(173)
(18, 19)
(18, 19)
(18)
(174)
(174)
(18, 19)
B6C3F =(C57BLXC3H)F , B6AKF = (C57BLX AKR)F
-------
614
Dulcin (4-ethoxyphenyl urea), originally produced as an artificial sweet-
ener, may also be regarded as 4-ethoxy-N-carbamylanilme. Classified as a
substituted monocyclic aromatic amine, Dulcin has been discussed in Section
5 1.2 1- in Vol IIB. Its use as an artificial sweetener has now been banned in
most countries.
Phenylalkylureas are rapidly gaining importance as herbicides. Two such
compounds (Monuron and Diuron) have been tested for carcinogenicity In the
preliminary NCI bioassay, Monuron was found to be inactive in B6C3F mice
but induced lung tumors after oral administration to B6AKF mice (18, 19). A
more convincing demonstration of carcinogenicity of Monuron was subsequent-
ly provided by Rubenchik et al_ (174) One hundred random-bred rats were fed
diets containing 450 mg/kg of Monuron for 18 months. Fifty random-bred.mice
and 45 C57B1 mice received 6 mg Monuron in milk once a week for 15 weeks.
Significant increases in tumor incidence were observed in all treated rodents,
the liver and the lung were the most affected organs The tumor incidence was
46. 5% in treated rats (compared to 0% in controls), 56 5% in random-bred mice
(control figure not available), and 26 9% in C57B1 mice (control 3. 9%) Diuron,
the only other phenylalkylurea studied, was inactive in the preliminary NCI bio-
assay (18, 19) However, in view of the preliminary nature of the latter study
and the previous demonstration of the carcinogenicity of Monuron, further
studies of the compound should be undertaken.
5.2.1 6 3 8 Transplacental and Lactationally Mediated Carcinogenesis The
transplacental carcinogenicity of urethan in the mouse has been extensively
-------
615
studied. The demonstration by Nomura _et al_ (62) that radioactively labeled
urethan may readily cross the placental barrier throughout the gestation peri-
od, irrespectively of the presence of unlabeled compound, indicates the easy
accessibility of the carcinogen to the fetus. Larsen (175) was the first to demon-
state the transplacental carcinogenicity of urethan Pregnant strain A mice,
given a single i. p. or i. v. injection of 25 mg urethan 1 -5 days before term,
gave birth to offspring with increased incidence of lung tumors When sacri-
ficed 6 months after birth, the incidence was 100% and the multiplicity was
8.9-10 tumors/mouse among offspring whose mothers received urethan one
day before parturition. For offspring whose mothers received urethan 2-5 days
before term, the incidence was 60-80% and the multiplicity was substantially
lower (with an average of 1-2 tumors/mouse). Similar findings have been re-
i
por ted by Smith and Rous (1 76) using strain C mice, by Klein (1 77) using AxC
mice, and by DiPaolo (178) and Kolesnichenko (179) using strain A mice Ves-
selinovitch et al (180) injected urethan (0 5 g/kg) subcutaneously for 5 conse-
cutive days to Swiss or C3H mice starting on day 7 or day 11 of gestation, res-
pectively. The treatment resulted in increased incidence of hepatomas in the
C3H offspring and of ovarian tumors in the offspring of both strains. Bojan
(181) treated pregnant CFLP mice with a single dose (1 g/kg) of urethan on day
10, 11, 12 or 19 of gestation, the tumo r incidences observed among offspring
sacrificed 35 days after birth were 0%, 0%, 3-4%,and 31-42%, respectively An-
derson (182) has stressed that for some strains, the offspring must be allowed
to live for a sufficient!) long duration in order to detect transplacental carcino-
genicity In BALB/c mice, for example, a high incidence of lung tumors was
observed only if they were sacrificed 8-12 months after birth
-------
616
The chronological relationship between organogenesis and transplacental
carcinogenesis has been investigated by Nomura and associates (61, 183). Preg-
nant ICR-JCL mice were given a single s c. dose (1 g/kg) of urethan on day 5,
7, 9, 11, 13, 15, 17 or 19 of gestation. The incidence of lung tumors in the
offspring was not significantly different between control and the groups treated
before day 11. The incidence then rose to 30. 8%, 64. 3% and 71. 4% for the
groups treated on day 13, 15 and 17, respectively. Embryological studies re-
vealed that this sensitive period of carcinogenesis coincides with the period
when the lung buds appear (on day 12) and grow actively. Of the group treated
on day 19, 64. 9% of the offspring developed lung tumors Within this group,
however, there was a large difference in the incidence and multiplicity between
the offspring that were born within 24 hr of urethan treatment to mothers {IQQ%,
average 4 56) and those born over 24 hours after treatment (31 6%, average
0 47) It was suggested (61) that, due to the long retention of urethan, a signi-
ficant proportion of urethan could be transferred into the newborns, thus en-
hancing the carcinogenic effect of urethan. In addition to lung tumors, hepa-
tomas were found in male but not in female offspring. The incidences were
i
6.9, 1 5 and 7 1% if the mothers were treated on day 11, 13 or 15 of gestation,
respectively Microscopic examination of the embryos indicated that liver
"buds" first appeared on day 10.
The modifying effect of a number of exogenous and endogenous factors
on the transplacental carcinogenicity of urethan has been explored. Dipaolo
(178) showed that exposure of pregnant mice during urethan treatment (given
-------
617
within 24 hours before parturition) to an atmosphere containing either 100%
oxygen (hyperoxia) or 10% oxygen (hypoxia) led to the induction of greater num-
ber of lung tumors than in those mice kept in normal room air. Hyperoxia or
hypoxia alone did not significantly affect the lung tumor yield Apparently, a
change in oxygen concentration may alter the susceptibility of lung cells to ure-
than The role of thymus has been studied using genetically athymic mice
(BALB/c nu/nu, nu being gene for nude, hairlessness and athymia). Female
nu/+ mice, mated with nu/ + males, were given urethan on day 17 or 19 of ges-
tation and the incidences of primary lung tumors in the nude (nu/nu) and pheno-
typically normal (nu/+ or +/+) mice were compared (184). There was no sig-
nificant difference. Histologically, however, the tumors in nude mice appeared
to be more invasive and atypical, suggesting that the absence of thymus may
i
increase the metastatic tendency of primary tumors An interesting diaplacen-
al initiation-promotion experiment has been reported by Goerttler and Lohrke
(124). NMRI mice were treated prenatally with urethan (3 daily doses of 60
mg/kg between gestation day 14-21) and postnatally with an active promotor,
l2-O-tetradecanoylphorbol-13-acetate (TPA) A broad spectrum of tumors at
the site painted with TPA and in other tissues have been observed. In general,
the tumor incidences with the combined treatment exceeded those due to spon-
taneous incidence and those produced by urethan alone The skin carcinomas
developed only after combined treatment of urethan and TPA. The results in-
dicate the occurrence of transplacental tumor-initiation Furthermore^ the rela-
tively low doses required to initiate the tumor emphasize the high susceptibility
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618
of the fetus. The transplacental carcinogenicity of urethan may also be enhanced
by further exposing the suckling offspring to urethan via mother's milk (se e
also discussion below), and these carcinogenic effects appear to be additive
(61, 183).
The transplacental carcinogenicity of urethan has also been tested in
MRC rats. A low incidence (4 5%) of hepatomas and tumors of the heart were
noted in offspring whose mothers received a single dose of urethan four days
before term.
In contrast to the extensive study of urethan, the transplacental carcin-
ogenicity of other carbarnates and related compounds has been virtually unex-
plored. Zineb is probably the only dithiocarbamate that has been tested and
reported Kvitnitskaya and Kolesnichenko (185) injected a single dose of 81mg
Zineb to 1 8 strain A mice during the second half of pregnancy. Eleven of these
18 produced 38 offspring, of which only 20 survived. After 4 months, six of
these offspring were found to have lung adenomas, no such tumors were detected
in control mice of similar age.
The maternal transfer of urethan is not limited to the transplacental route
Several investigators have reported increased tumor incidence in suckling mice
whose motheis were treated with urethan during lactation, suggesting the trans-
fer of urethan via mother's milk De Benedictis et al. (186) noted first that
25/32 (78%) Swiss mice developed lung tumors 210 days after receiving milk
from mothers treated with urethan (30 mg, p. o., 1st, 3rd and 5th day atter
parturition) The possible contamination of the suckling mice from sources
-------
619
other than maternal milk may be excluded, because suckling mice caged in the
presence of untreated lactating female and urethan-treated adult male did not
develop tumois to any significant extent Similar results were obtained by
I
Nomura-(l83) with ICR/JCL mice that were suckled by urethan-treated lactat-
ing mothers. The effect was greatest if the mothers were repeatedly treated
shortly after delivery. All 10 suckling mice developed lung tumors (average
1 3. 6/mouse) 32 weeks after receiving milk from mothers treated with 4 sub-
cutaneous doses (1 g/kg) of urethan on the 2nd, 4th, 6th and 8th day after par-
turition Similar treatments on days 2, 7, 12, 17 or 14, 16, 18, 20 postpartum
led to tumor incidences of 58% and 56%, respectively For controls, the in-
cidence was only 2 4% Essentially the same finding was reported by Bojan
(181) using CFLP mice Lactating mothers were treated with 4 doses (0.5
i
g/kg) of urethan on 4 consecutive days and allowed to nurse two groups of suck-
ling mice aged 1-4 days or 12-16 days When autopsied 5 weeks after treat-
ment, the former had a tumor incidence of 12-16% whereas the latter~had a
tumor incidence of only 4-5%
5 2.1 6.3 9 Modification of Carcinogenesis Induced by Carbamates and Re-
lated Compounds. As is the case with many other chemical carcinogens, the
carcinogenicity of carbamates and related compounds may be modified by a
variety of endogenous and exogenous factors Urethan (ethylcarbamate), in
particular, has been extensively used as a model multipotential carcinogen in
the study of various modifying factors, age, sex, host immune response, diet,
physical trauma, exogenous chemicals, viral interaction, radiation, and other
physical factors In addition to urethan, a few carbamate pesticides have been
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620
investigated because of their potential risk to humans. While the major find-
ings of these studies are briefly outlined in this sub-section, detailed discus-
sion will be presented in a future volume of this series
Age. As previously mentioned in Section 5.2. 1. 6. 3. 2, newborn and pre-
weanling animals have a greater susceptibility to the carcinogenic action of ure-
than. The greater susceptibility is particularly evident in the induction of pul-
monary and hepatic tumors and leukemia. A variety of investigators have ex-
tensively studied the age effect. Some of the representative studies are des-
cribed below
Rogers (187) was among the first to demonstrate the age effect in ure-
than-induced lung tumongenesis. He administered a single i. p dose of ure-
than (1 g/kg) to Swiss mice at various ages and recorded the development of
lung tumors 7 weeks later. Significantly higher susceptibility of the younger
animals particularly during the first 6 weeks, was noted. The tumor incidences
and multiplicity were, respectively, 100% and 6 1 tumors/mouse for 2-week-
-old, 92% and 5. 4 for 4-week-old, 88% and 3. 6 for 6-week-old, 84% and 2. 6 for
8-week-old and 76% and 3 6 for 10-week-old mice The age effect was even
more dramatic at lower are than doses. At the dose of 0.25 g/kg, 16/19 (84%)
mice developed tumors after treatment at the age of 3 weeks compared to 4/17
(23%) after treatment at the age of 8 weeks. Similar results have been ob-
served with Swiss (186), A/Jax (116) and CFLP (1^1) mice. However, the age-de-
pendence of'pulmonary susceptibility to urethan is probably strain-spe'cific,
no significant age-difference has been observed in the development of urethan-
-induced lung tumors in some other strains of mice (188, 189)
-------
621
Urethan rarely induces hepatic tumors in adult mice, however, when giv-
en to neonatal or infant animals, high incidences of hepatomas have been noted
The role of age in urethan-induced hepatocarcinogenesis in Swiss mice has
been investigated by Chieco-Bianchi ^t a_l._ (190). Subcutaneous injection of ure-
than (1 g/kg) to newborn, 5-, 20- and 40-day-old mice led to the induction of
liver tumors in 87, 70, 8 and 0% male and 9, 18, 0 and 0% female mice, res-
pectively. In B6AF /J hybrid mice, the most sensitive period was reported
to be around the 7th day of age (188). Oral administration of 1 g/kg urethan
to newborn, 7-, 14-, 21- and 28-day-old mice elicited hepatomas in 45, 91,
80, 57 and 17% male and 35, 77, 43, 5 and 5% female mice, respectively.
There was no significant difference in the incidence of lung tumors (ranging
from 84-100%) in this strain (188). Vesselmovitch et al. (189) administered
i. p injections of'urethan to (C57 x C3H)F mice starting at day 1, 4 or 1 75 of
age. The incidences of hepatomas were 86%, 100%, 0-6 8% in males and 0%,
27%, 0% in females, respectively. In contrast, the lung and Harderian gland
adenomas were observed with similar incidences in the groups receiving the
same amount of urethan starting at day 4 or 1 75 of age.
The hematopoietic system also exhibits significant age-dependence in the
susceptibility to the carcinogenic action of urethan Several investigators re-
ported the lack of leukemogenic action of urethan in adult mice (e_. £ , ref. 191).
Fiore-Donati et al. (192) studied the age effect with Swiss mice Mice given a
single dose of urethan at the age of 1, 5 or 40 days developed leukemia with in-
cidences of 21 6, 17.9 and 3.2%, respectively. Vesselmovitch and Mihailovich
-------
622
(193) found that the most susceptible period is the very early neonatal stage.
They administered to (C57 x C3H)F mice total doses of 2 1, 3. 0 or 4. 2 g/kg
starting at the age of 1 day or 7 days. The incidences of leukemia for the three
doses m mice started on the first day of life were 7, 32 and 74%, whereas those
of mice started on day 7 were 0, 7 and 38%, respectively.
In addition to the mouse, the age-dependence of susceptibility to urethane
has been observed in the rat and hamster. Tannenbaum et_ a.L_ (14) showed that
urethan potentiates the formation of mammary tumors in Sprague-Dawley rats
by increasing the multiplicity and reducing the latent period. Although their
study was not designed to demonstrate age effect, it is evident from their data
that the potentiating effect was greatest if the animals were treated starting at
the first week of their life. The effect declined as the starting age of treatment
increased. With Wistar-denved MRC rats, Kommmeni e± a_l_ (126) showed that
the incidences of both neunlemmomas and hepatomas were significantly higher
in rats treated neonatally than those treated from 4^ or 6^ weeks of ag'e. In *
contrast, tumors of the thyroid gland were observed only in adult but not in
neonatally treated rats. In the Syrian golden hamster, Toth (134) noted a sub-
stantially higher number of tumors of the intestines in animals treated neonatally
than those treated starting at 8 weeks of age. On the other hand, an opposite
effect was observed in the induction of tumors of the forestomach, whereas no
age differences were found in the induction of skin tumors, thyroid, lung and
other tumors. ~
Thus, age has a significant effect on the animal susceptibility to urethan.
The effect is species-, strain- and target organ-specific In general, greater
-------
623
susceptibility is associated with younger animals, this could be attributed to
the slower rate of catabolism of urethan (194), higher proliferative activity of
the immature cells and/or lower immune competence.
Sex. The sex of the animal plays an important role in the induction of
tumors by urethan. Sex difference appears to be specific for certain organs
or tissues, and is often species- or strain-dependent Probably the most con-
sistent findings of sex difference are the greater susceptibility of male mice to
hepatocarcinogenesis and female mice to leukemogenesis by urethan. In ex-
periments with newborn or infant Swiss (190), C3Hf (195), B6AF /J (188, 196)
and C57 X C3H hybrid (197, 198) mice, the incidences of urethan-mduced he-
patomas were invariably higher in males than in females. The greater suscep-
tibility of the males has also been observed in young adult BALB/c (199) and
i
"Hall" strain (200) mice receiving urethan after partial hepatectomy. In con-
trast to hepatocarcinogenesis, higher incidences or earlier development of
neoplasms of the hematopoietic system have been found in female C3Hf (195)
and dd (201, 202) mice after neonatal exposure to urethan.
The role of sex hormones in the modification of organ susceptibility to
urethan has been studied by several investigators. Liebelt £t al_ (1 95) admin-
istered a single dose of urethan to newborn C3Hf mice and allowed them to live
out their lifespan During autopsy of the tumor-bearing animals, the type of
sex hormone stimulation in the animals was assessed on the basis of gross and
microscopic examination of the gonads, reproductive tract, submaxillary glands
(a good indicator of andiogenic stimulation) and kidneys There was a high
-------
624
correlation between the oq.cur ence of hepatomas and evidence of androgenic
stimulation. All males that developed hepatomas had stimulated seminal ves-
icles and virtually all females bearing hepatomas had evidence of androgenic
stimulat-ion of the kidney and submaxillary glands. On the other hand, reticu-
lar tissue neoplasms were associated with estrogenic stimulation. There was
no relationship between occurrence of lung tumors and specific type of sex hor-
mone stimulation (195) In dd mice, the development of urethan-induced
thymic lymphoma occurred much earlier and faster in females than in males.
The testes were reported to exert an inhibitory influence on the thymus which
could account for the later development of thymic lymphomas in male mice
(201, 202). The effect of gonadectomy on the induction by urethan of hepatomas
in 7-day-old (C57 X C3H)F mice has been studied by Vesselinovitch and Mihail-
ovich (197). In control (sham-operated) mice the incidences of hepatomas_ were
96% in males and 20% in females. The orchidectemized males had a signifi-
cantly lower (62%), and the ovariectomized group a higher (67%), incidence and
multiplicity of hepatomas than their corresponding non-castrated groups. Thus,
gonadectomy practically abolished the sex difference in the induction of hepatomas
by urethan
Immune response. Impairment of host immune competence is often as-
sociated with an enhancement of tumor development Urethan itself is an im-
munodepressant (albeit weak), as demonstrated by the impairment of allograft
or homograft rejection response in urethan-treated mice (203-205). Th€ im-
munodepres sant activity is more pronounced in newborn than in adult mice
-------
625
(203). It JLS not known whether the immunodepressant effect of urethan is a
component factor of its carcinogenic activity.
The host immune system appears to play an important role in modifying
the development of urethan-mduced lung tumors. Trainin and associates have
shown Sie immunological impairment in Swiss mice as a result of neonatal
thymectomy (206), thymectomy, and treatment with antilymphocyte serum (207)
or autoimmune lymphocytes (208) invariably led to an increase in the incidence
of urethan-mduced lung tumors. Even a short duration of antilymphocyte ser-
um treatment, permitting later immunologic recovery, was sufficient to en-
hance the host carcinogenic susceptibility if the carcinogen was administered
during the period of immunodepres sion, suggesting that the early stage of ure-
than carcinogenesis was affected by immune manipulation (207). Innoculation
\
of neonatally thymectomized mice with incompletely syngenic immunocompetent
lymphoid cells, which further impaired the host immune competence via a
"graft-versus-host" reaction, further enhanced the pulmonary susceptibility
(209). On the other hand, repeated implantation of syngenic thymuses into neo-
natally thymectomized mice restored the >rnmun<«>competence and normalized
the host response to urethan (207). The increased pulmonary susceptibility to
urethan after neonatal thymectomy has also been shown by various other inves-
tigators using the same or different strains of mice (204, 210-212) In agree-
ment with the above findings, Menard et al. (213) found that immunodepression
by cortisone treatment enhances the pulmonary carcinogenicity of uretlian in
mice. On the other hand, pretreatment of mice with BCG (living bacillus
-------
626
i
Calmette-Guerm) and trehalose-6, 6-dimycolate ("cord factor") enhances the
immunocompetence and reduces the number of urethan-mduced lung tumors
(214). Kraskovskn and Kagan (215) lowered the pulmonary carcinogenicity of
urethan-by innoculating the mice with immunocompetent embryo spleen (but not
embryo liver) lymphoid cells.
In contrast to the above findings some negative correlations between im-
munodepression and enhancement of susceptibility have been reported Delia
Porta e± aj^ (212) did not find any significant effect of splenectomy or cortisone
treatment on the pulmonary carcinogenicity of urethan. Unlike thymectomized
mice,' genetically thymusless mice (BALB/c nu/nu) were not more susceptible
to the pulmonary carcinogenic action of urethan after either prenatal (184) or
postnatal exposure (216).
Diet. The modifying role of diet on the carcinogenicity of urethan has not
been adequately explored, there are only a few and somewhat conflicting re-
ports. Rogers (187) in 1951 reported that fasting of mice 19 hrs before ure-
than treatment had no significant effect on the incidence of lung tumors. On
the other hand, Klarner and Klarner (217) showed that mice on a high-casein
diet developed more lung tumors in response to urethan than those on a low-
-casein diet Newberne _et a_L_ (218) found that dietary choline deficiency (in
spite of inducing liver cirrhosis) has a slightly inhibitory effect on the weak car-
cinogenic action of urethan in the rat. In a recent study by French (219), diets
containing added nicotmamide, or choline, or myo-inositol significantly re-
duce the number of lung tumors induced by urethan in mice. The nicotmamide
-------
627
effect appeared to be specific and could not be replaced by nicotinic acid. French
(219) suggested that the carcinogenic effect of nicotmamide may, at least in
part, be due to its ability to inhibit tumor-derived tRNA methylase. The pos-
sible mechanisms of action of chohne and myo-mositol have not been explored.
Physical trauma Physical trauma is known to elicit unscheduled cell
proliferation which in turn can promote chemical carcinogenesis. As discussed
in Section 5.2.1. 6. 3. 2, urethan has, in general, a weak or no carcinogenic ac-
tion on the liver of adult animals, after partial hepatectomy, however, high in-
cidences of liver tumors have been observed by various investigators. Hol-
lander and Bentvelzen (220) reported that injection of 25 mg urethan into 2-month-
-old C3H/HeA male mice led to the induction of hepatomas in 11/36 (31%) ani-
mals. Partial hepatectomy one week after urethan treatment increased slight-
i
ly the incidence to 50% (18/36) whereas when the partial hepatectomy preceeded
urethan injection by 4 days, it strongly enhanced the incidence of hepatomas
(81% of the animals developed one or more tumors). The incidence of lung tu-
mors was not affected by partial hepatectomy. Similar results have been ob-
served by Lane et al. (199) using BALB/c mice, Chernozemski and Warwick
(221) using B6AF mice and Pound and Lawson (200) using Hall strain mice.
In addition, significant sex difference was noted, the enhancing effect was sub-
stantially higher in male than in female mice (199, 200) Furthermore, the
enhancing effect was positively correlated with the extent of partial hepatec-
tomy and the rmtotic activity of the liver thus produced (199, 200). Metabolic
studies in intact and partially hepatectomized mice (200, 222) showed no
-------
6Z8
major difference, strongly supporting the concept that rapid cell proliferation
and not the change in the metabolic rate of urethan accounts for the enhance-
ment of susceptibility of liver to the carcinogenic action of urethan
Exogenous Chemicals. A variety of exogenous chemical agents are capa-
ble of modifying the carcinogenicity of urethan. On the bther hand, a number
of carbamatES and related compounds y^y modify the carcinogenicity of other
chemicals. Some oi lUe examples of such interactions are briefly outlined
below.
The carcinogenic effects of combined treatment of urethan and a number
of other chemical carcinogens have been investigated. Kawamoto jit al (1 91)
were the first to note that urethan potentiates the leukemogenic action of 3-meth-
ylcholanthrene in DBA mice This synergism, however, could not be demon-
strated in the pulmonary carcinogenesis in strain A/J mice, instead, Yarria-
(^with,; " "-
moto et al (Z23) found that the combined treatmentpg 3-methylcholanthrene
and urethan (inducing an average of 27. 4 nodules in all mice) was less"carcin-
ogenic than 3-methylcholanthrene alone (inducing 92 9 nodules in all mice).
Bojan et al (224) have shown a clear syncarcinogenic effect between urethan
and diethylstilbestrol in the induction of lymphoma in CFLP mice, the syner-
gistic effect was the greatest if diethylstilbestrol was given 14 days after ure-
than treatment The finding is in accord with that of Kawamoto et al. (191) who
reported that combined treatment of urethan and estradiol to castrated C57
mice induced leukemia in 59.4% of the mice compared to 6.6% for estradiol or
(subsequent)
0% for urethan alone In aVr-erriMT^abstract presented by Yoshimura et al. (225),
-------
629
urethan was reported to act synergistically with carcinogens present in engine
exhaust gas. Female ICR-JCL mice exposed to the exhaust gas by inhalation
UoJ
andjurethan via drinking water.developed more malignant lung tumors than those
exposed-to exhaust gas or urethan alone. The combined effect of urethan and
aflatoxin in the induction of liver tumors in the rat was tested by Newberne et
al. (218). There was no evidence of synergism, in fact, slightly fewer liver
tumors were observed after combined treatment than after aflatoxin alone.
The effect of combined treatment of urethan and some of its homologs
has also been investigated. Thus, butyl and isoamyl carbamates have no sig-
nificant effect on the pulmonary carcinogenic effect of urethan {.Lacpa^nJi oi
ggg((226). Pound (121) did not show any modifying effect of methyl-, ji-propyl-
and ji-butyl-carbamates and several N-alkyl urethans -on the induction of tumors
I .
in the skin, liver or lung by urethan in "Hall" strain mice. In contrast, Garcia
and Guerrero (141) bhowed that simultaneous treatment of C3H mice with ure-
than and ri-butyl carbamate lead to higher incidence of mammary tumors than
when any of the three carbamates was administered separately
Disulfiram has received much attention in recent years as a potential
chemo-preventive agent of chemical carcinogenesis. Disulfiram inhibits the
carcinogenic action of the following agents of 1, 2-dimethylhydrazine and to
a lesser extent of azoxymethanol toward the colon of CF mice (227), of brack-
en fern on the intestines of albino rats (228), of 4-hydroxybutylbutylnitrosamine
on the urinary bladder of Wistar rats (229), of 3-methyl-4-dimethylami5oazo-
benzene on the liver of Sprague-Dawley rats (230), and oi benzo(a)pyrene on the
-------
630
forestomach of ICR/Ha mice (231). The mechanism of the inhibitory action of
Disalfiram has been shown or postulated to be due to inhibition of the metab-
olic activation of carcinogens (227, 229, 232, 233), inhibition of the binding of
carcinogens to macromolecules (231, 233), or to some other as yet unknown
mechanism(s). It should be noted, however, that the carcinogenesis-inhibitory
activity of Disulfiram is not a general phenomenon. Administration of Disul-
firam to Sprague-Dawley rats inhibits the hepatocarcinogenic action of diethyl-
nitrosamine but significantly enhances the induction of esophageal tumors by
the agent Similarly, the principal carcinogenicity target organ of dimethyl-
nitrosamine is shifted from the liver to the paranasal sinus by treatment with
Disulfiram (169). Moreover, an unusual case of synergism has been reported
(234) Feeding Sprague-Dawley rats with Disulfiram and ethylenedibromide
resulted in a significant increase in the induction of hemangiosarcoma _of the
liver, spleen, omentum and kidney, and mammaiy adenocarcinoma At the
doses administered (20 ppm ethylenedibromide in air, 0 05% Disulfiram in
diet), neither agent alone was carcinogneic (234).
In addition to Disulfiram, a number of other carbamates and related com-
pounds have been tested for their activity to modify the carcinogenic action of
chemical agents. Like Disulfiram, sodium diethyldithiocarbamate and Maneb
are effective inhibitors of colon carcinogenesis by 1, 2-dimethylhydrazine,
whereas Chloropropham is ineffective. The structural requirements for effec-
tive inhibition have been discussed V^- rlv. A aval's (227, 233). On the other hand,
Carbaryl has been found to enhance the induction by benzo(a)pyrene of tumors
-------
631
in the forestomach of ICR/Ha mice or in the lung of A/J mice The enhancing
effect was attributed to 335 increase of Elaaiitt. aryl hydrocarbon hydroxylase
activity.which is believed to be involved in the metabolic activation of polycy-
clic aromatic hydrocarbons (250)
The effect of a variety of inducers and inhibitors of microsomal mixed-
-function oxidase (MFO) on the carcinogenicity of urethan has been
tested in an attempt to delineate role of the MFO system in the activation or de-
toxification of the carcinogen. Phenobarbital, in particular, has been studied
by several groups of investigators (223, 235-237) and was found consistently to
inhibit urethan-mduced lung carcinogenesis in various strains of mice. Other
MFO inducers such as chlordane (223), p-naphthoflavone (223), chlordiazepox-
ide (238), and nikethamide (238) have been found to have the same effect. In
contrast, phenothiazine, a MFO inducer under some conditions, was without
effect (223). Diethylaminoethyl diphenyl valerate (SKF-525A), a well knovvn
inhibitor of MFO, also failed to affect the pulmonary carcinogenic yield of ure-
than in Swiss (239) as well as A/J mice (223).
Consistent with the finding of the enhancement of urethan carcinogenicity
by partial hepatectomy, two chemicals known to stimulate cell proliferation
also display similar activity. Witschi et al. (240) showed that butylated hydroxy-
toluene (BHT), an antioxidant used as food additive, produced proliferation of
alveolar cells of the lung of Swiss-Webster and A/J mice Repeated stimulation
of cell proliferation by BHT beginning 7 days after urethan treatment significantly
enhanced the yield of lung tumors The level of BHT (equivalent to 300 mg/kg
-------
632
body weight) used was reported to be 100-10, 000 times higher than that per-
mitted in the food. The enhancing effect of BHT has been confirmed by Bojan
et ai._ (241) using CFLP mice. However, in contrast to the above study, the
BHT effect was significant only if administered 6 or 7 days prior to urethan
treatment. The reason for this discrepancy is not known. In addition to BHT,
the herbicide, Paraquat (1, 1' -dimethyl-4, 4l-dipyridylium dichloride), also
stimulates the proliferation of lung cells and enhances urethan-induced lung
tumorigenesis (241).
A number of general inhibitors of nucleic acid and protein synthesis have
been investigated regarding their potential role in modifying the carcinogenicity
of urethan. Shimkin e_t al. (242) found that treatment of A/Jax mice with actin-
omycin D, puromycin, cytosine arabinoside or 5-fluorouracil has no effect on
the incidence of urethan-induced lung tumors. The mean number of tumors per
mouse was slightly reduced by actinomycin D, puromycin and cytosine arabin-
oside and enhanced by 5-fluorouracil, but none of these differences was stat-
istically significant The lack of modifying effect of actinomycin D and puromycin
was confirmed by Yamamoto et al (223) who, in addition, showed that cyclohex-
imide, an inhibitor of cytoplasmic protein synthesis, was ineffective. In con-
trast, chloramphenicol, an inhibitor of mitochondrial protein synthesis, was
reported to inhibit the induction of lung tumors in BALB/c and strain A mice
by urethan (243) It is not known if the two effects are related . It
was sugges-ted (243) that the protective action of chloramphenicol against
urethan could be mediated via its influence on enzyme systems or on binding
of the carcinogen to cellular macromolecules.
-------
633
Caffeine has recently gained wide recognition as an inhibitor of error-
-prone post-replication DNA repair synthesis. Nomura (60, 244) reported un-
published data that caffeine given after urethan-treatment suppresses the induc-
tion of lung tumors in ICR-JCL mice.'' The suppressing effect was attributed
s
to the inhibition of post-replication DNA repan, thus resulting in increased
*s
cell death. Presumably, some of these cells could have produced a tumor if
they had survived. The suppressing effect of caffeine has been confirmed by
Theiss and Shimkin (245) using strain A mice However, the most pronounced
effect was observed when caffeine was given in two doses 3 hr. before arid 3 hr.
after urethan treatment. The spontaneous incidence of lung tumors in this
strain was also suppressed by caffeine The authors (245) suggested that the
anticarcinogenic effect of caffeine against urethan was likely to be due to a-gen-
i
eral suppression of lung DNA synthesis (rather than post-replication DNA re-
pair). Alternatively, caffeine could exert protective effect by binding to ure-
than, thus decreasing the likelikhood of binding to lung DNA.
Colchicine, a well known mitotic inhibitor, when given one week or one
day before urethan treatment has no effect on the induction of lung tumors in
Swiss and strains A and C mice (187) In a more recent study colchicine was
administered to ICR mice 24, 9 or 5 hr prior to urethan initiation and TPA
promotion Be._ve.ubimn *»rl Aviriul.IV (246) . Significantly higher total incidences
of skin tumors and a greater number of malignant tumors were found only in
the group receiving colchicine 9 hr before urethan, corresponding to the peak
of metaphase arrest It was postulated (246) that the most sensitive
-------
634
period of tumor initiatioruoccurred during the M phase of the cell cycle (see
Suppletory Note ) and that colchicine treatment caused the accumulation of
cells at metaphase, thus enhancing the incidence of initiation and the eventual
development of skin tumors. It should be noted that the exact phase of cell
cycle when initiation takes place is a question of great controversy Conflict-
ing results have been presented by various investigators, other phases such
as Q , S, and G -S boundary have all been proposed as the most sensitive
£ I
period.
In addition to the above compounds, the effect of butylated hydroxyanisole
(BHA), an antioxidant food additive, on the carcinogenicity of urethan has been
tested in A/Hej mice (247). Unlike BHT, BHA was found to reduce the lung tu-
mor yield, the average number of tumors per mouse was reduced from 12.-3 to
2. 7 after BHA treatment (247). The mechanism of action has not been explored.
Sulfanilamide is another compound that has been shown to exert an inhibitory
effect on urethan-induced lung tumors Both the incidences and multiplicity
of lung tumors in Swiss mice were substantially reduced by the agent (235).
When tested under similar conditions, p-aminobenzoic acid was without effect
(235).
Viral Interaction. Viral infection may play an important role in the al-
teration of host susceptibility to chemical carcinogens The viral effect is ap-
parently variable and may be specific for the type of virus involved. Imagawa
et al^ (248) reported in a 1957 abstract that mice exposed to urethan i ~p and
influenza A (PRS strain) virus intranasally developed more lung tumors than
-------
635
control animals receiving^either agent alone. Casazza et al. (249) were, how-
ever, unable to detect any synergistic action between influenza A, virus and
I*
urethan Similarly, Stomskaya (250) showed that the incidence of lung tumors
in mice-that received a combined treatment of myeloid chloroleukemia virus
and urethan was not significantly different from the group receiving urethan
only. Germ-free BALB/c mice were reported to develop fewer urethan-m-
duced lung tumors than did conventional BALB/c mice (251). The most strik-
ing difference between the two sub-colonies of mice was the frequent viral in-
fection (particularly pneumonia virus of mouse and Sendai virus) of the con-
ventional mice and the lack of such infection in the germ-free mice There
is no evidence, however, to directly link the viral infection to the greater sus-
ceptibility to urethan. A suppression of urethan-induced lung carcmogenes-is
t
was observed when strain A/He mice were treated with the Maloney strain of
munne sarcoma virus (252). The suppressive effect of the virus could be_dim -
inished by increasing the urethan dose The investigators (252) attributed the
viral effect to its immunostimulatory activity. A somewhat more complex pat-
tern of interaction between reovirus type 3 and urethan has been demonstrated
by Theiss e_t al. (253) using strain A/St mice When mice were subjected to a
single exposure of an aerosol containing the virus either 6 days before, on the
same day as, or 14 days after urethan treatment, suppression of lung carcino-
genesis of the order of 30-60% was observed. However, when mice were re-
peatedly exposed to the virus, a slight enhancement of urethan carcmogpenicity
was observed The authors (253) hypothesized that a single viral exposure was
-------
636
immunostimulatory through a "rebound" effect, whereas continuous exposure
led to imrnunosuppressionfthus inhibiting and enhancing the carcinogemcity of
urethan, respectively.
Radiation and Other Physical Factors. The interaction of X-radiation
and urethan is quite complex. Depending on the dosage and the target organ
concerned, X-radiation may exert either an inhibitory, an enhancing, or no ef-
fect on the carcinogemcity of urethan. In general, when given at high doses
which inhibit cell division in the lung, X-radiation usually inhibits the develop-
ment of urethan-induced lung carcinogenesis in mice (see rev ref. 254) For
adult (C57L X A)F mice, the inhibitory doses required range from 500-900?
(255) The inhibitory effect was abolished if the thorax was shielded during ir-
radiation (256). In contrast to the inhibition to lung carcinogenesis, X-rad-ia-
tion increases the leukemogenic action of urethan in mice, the enhancing ef-
fect is particularly evident at low X-ray doses. Kawamoto et^ a_L_ (191) obo_erved
substantial increase in the incidence of leukemia after exposing mice to ure-
than and to low doses (11 x 40 r or 4x 90 r ) of X-ray. Berenblum and Traimn
(257) demonstrated that the enhancing effect of combined treatment with-X-ray
and urethan could be observed only if urethan was given after irradiation. On
the other hand, Foley and Cole (256), observed a synergistic action whether
urethan was given before or after X-ray treatment. Vesselinovitch ^t al. (258)
treated infant mice with low doses of urethan and observed that such treatment
enhanced the leukemogenic action of X-radiation given on the 42nd day of life
More recently, Myers (127) studied the interaction of X-ray (5 x l65r ) and
-------
637
urethan (5 x 0. 9 g/kg) in 3 strains (Sprague-Dawley, Long-Evans, and Collip)
of rats At the dosages used, the overall effect of the combined treatment in
the induction of mammary, skin, lymphatic and various types of tumors was
less than the sum of their separate effects. Clearly additive effect was found
only in the induction of lymphatic neoplasms in Collip rats.
239
Related to X-radiation, inhalation of plutonium dioxide ( PuO_ , an
o(-emitter) 2 weeks before or after urethan treatment to A2G mice reduced the
yield of lung tumors (259) The inhibitory effect was attributed to the cell
damaged caused by Q(-irradiation.
Atmospheric pressure and oxygen concentrations may also affect the car-
cinogenicity of urethan. Mori-Chavez (260, 261) compared the incidences of
urethan-induced lung tumors in mice kept at high altitudes and at sea level"..
i
High incidences were associated with mice kept at high altitudes, he concluded
that lower atmospheric pressure enhanced the susceptibility to lung carcino-
genesis. This observation could not be confirmed by Ellis et al. (262) who sim-
ulated high altitudes by keeping mice in decompression chambers The treat-
ment reduced rather than enhanced the susceptibility of strain A and A/Grb
mice to the carcinogenic action of urethan. The C57B1 mice were unaffected
DiPaolo (1 78) investigated the effect of oxygen concentration on the transplacental
carcinogenicity of urethan in strain A mice. Offspring from mothers kept at
hyperoxic (100% oxygen) and hypoxid (10% oxygen) conditions developed more
lung tumors than those from mothers kept in room air. t
5. 2 1 6.4 Metabolism and Mechanism of Action.
U re than Despite decades of research and the relatively simple chemic-
al structure, the metabolic activation and the mechanism of carcinogenic action
-------
638
of urethan remain obscure. It is generally accepted that cellular metabolism
plays a dual role in determining the biological action of uiethan, by both catab-
olizmg or activating the compound. The literature on the catabolism of ure-
than has-been thoroughly reviewed by Mirvish (Zl) in 1968, no major findings
have been reported since then. Essentially, hydrolysis seems to be the major^
route of urethan catabolism with the release of ethanol, carbon dioxide and
ammonia (289)
NH COOC H esterases y C H OH + CO + NH
tt £4 j * L* D £t 3
Hepatic microsomal esterases appear to be the principal enzyme system in-
volved (21, 290). The rate of catabolism apparently determines the length of
time urethan remains in the body which may be a critical factor in determining
j.i.5" cai Ciuogenic potential. The rate of catabolism of urethan in newborn S.WR
mice, for example, was shown (194) to be 1/10 of that of adults The rate" in-
creased slowly for the first 10 days of postnatal life and then sharply'between
the 15th and 20th day. As it has been discussed in Sections 6. 2. 1 6 3.-2 and
6.2.1.6.3.9, newborn and infant animals are much mo-re susceptible to the
carcinogenic action of urethan than the adults; the lower catabolic rate is most
likely a major factor The rate of urethan elimination (which includes catab-
olism and urinary excretion) from the blood of adult Swiss mice (291, 292),
rats (293), rabbits (294) and humans (295) was estimated to be around 60, 50,
25 and 4 mg/ml blood/hr, respectively These data could suggest that urethan
might have a longer retention time and possibly greater carcinogenic action in
-------
639
humans, however, this projection must at best be considered a matter of con-
jecture in the absence of supportive inter-species correlation studies.
There is general consensus that the carcinogenic action of urethan is me-
diated vra an active metabolite (rev. 296). The identity of the active metabol-
ite ("ultimate carcinogen") has, however, remained elusive. Research efforts
in the mid-1960's (rev. 21) were mainly directed at investigating the role of
r\
N-hydroxy urethan in the metabolic activation of urethan. N-Hydroxyurethan
has a carcinogenic potency comparable to that of urethan ( s ee Table
is chemically more reactive than urethan (Zl) and has been identified as a mi-
nor j_n vrvo metabolite of urethan (297, 298) In analogy to the well established
role of N-hydroxy lation in the activation of carcinogenic aromatic amines (see
Volume IIB), N-hydroxylation was also suggested to be the initial step in the
i
metabolic activation of urethan (17, 288, 298). This attractive hypothesis was,
however, not supported by the investigations of Mirvish (299,300) which re-
vealed that the reverse was probably true, u e^ , N-hydroxy urethan more like-
ly acquires carcinogenic properties via conversion to urethan. N-Hydroxy iire-
than is readily reduced to urethan by an enzyme system which is inhibited by
SKF-525A, a well known inhibitor of the rmcrosomal mixed-function oxidase
<-\
(MFO) system In newborn mice, N-hydroxy urethan is also readily converted
to urethan, whereas the reverse conveision was not detected (284). Further-
more, Kaye and Trainin (239) showed that the carcinogenic action of N-hydroxy-
urethan (but not urethan) could be inhibited by treatment of mice with SKF-525A,
supporting the conclusion of Mirvish (299, 300) that N-hydroxy urethan must
first be converted to urethan to exert carcinogenic action.
-------
640
The 3earck-fo~f~"the actuerl-active metabolite of urethan was not
pursued until recently. Intrigued by reports ofrpotent carcinogenicity of vinyl
chloride (see Section 5 2.2.1) andjfdemonstration of incorporation of the ra-
14 18
diolabelrf romj labeled ethyl group {but not from C-carbonyl or O-ethoxy) of
{intoj
urethanWcellular macromolecules (140, 301), Dahl et al. (1 07) suggested that
fi*
vinyl carbamate might be a proximate carcinogen of urethan. They proposed ' fr7
<
(Fig. 30) that vinyl carbamate could be metabolically formed by dehydrogenation
The proximate carcinogen thus formed may undergo epoxidation to form a reac-
tive electrophilic ultimate carcinogen This hypothesis is supported by the
finding that vinyl carbamate is more carcinogenic and mutagenic than urethan.
Vinyl carbamate has been shown to be 10-50 times more active in the initiation
of skin tumors and in the induction of lung tumors in mice. The compound
f
is mutagenic inTSalmonella test in the presence of activation system, whereas
urethan is not (see Section 5. 2. 1. 6. 2. 2). The activation of vinyl carbama_te
can be inhibited by typical MFO inhibitors such as DPEA or SKF-525A,although
it is not inducible by phenobarbital or 3-methylcholanthrene pretreatment The
hypothesis is, however, not supported by the failure of various attempts to de-
tect vinyl carbamate as an in vivo metabolite of urethan. It is possible that
vinyl carbamate may be formed at an _in vivo enzymatic site and immediately
further metabolized. Alternatively, vinyl carbamate may not be a metabolite
of urethan, instead, both compounds may be metabolized by a common pathway
to an unknown ultimate carcinogenic form ("X") that binds covalently to-macro-
molecules
-------
H2N
0
-C-O-CH
Vinyl
corbomate
dehydrogenation
0
NH2-C-0-CH2-CH3
Urethane
NH2-C-0-CH-CH2
Epoxyethyl
carbamate
covalent
binding to
critical
cellular
macromolec
Figure 30
-------
LEGEND TO FIGURE 30
Fig. 30. Proposed possible mechanism of metabolic activation of urethan.
[Adapted from G A. Dahl, J.A. Miller and E.G. Miller, Cancer Res. 38, 3793
(1978).]
-------
641
It is now generally Relieved that the great majority of chemical carcino-
gens (or their active metabolites) initiate carcinogene sis by binding covalently
to critical cellular macromolecules. The in vivo covalent binding of urethan
metabokte(s) to macromolecules in target tissues has been demonstrated by
various investigators (107, 140, 200, 273, 301-310). Studies by Law son and Pound
ififs. demonstrated that covalent binding of urethan metabolite to liver DNA oc-
curres to a greater extent than that to RNA or cell protein (305) Partial he-
patectomy, which is known to enhance the susceptibility of liver to the carcin-
ogenic action of urethan, increases the level of binding to liver DNA, without
significantly affecting the binding to lung or epidermal DNA (200, 306, 307).
The greater susceptibility of male mice to urethan-induced liver carcinogene-
sis M'ulJ is reflected by the higher level of binding, no such
sex difference is observed in the binding to lung or epidermal DNA (200). Com-
parison of the binding of urethan and its homologs or N-alkyl derivatives (which
are either weaker carcinogens or inactive) to liver epidermal DNA indicate/
that urethan is the most active compound and urethan binding persists significantly
longer than other carbamates (273) The persistence of urethan binding to epi-
dermal DNA is even further prolonged by croton oil treatment, which is known
to promote fg=e skin carcinogenesis by urethan (273)
The possible role of RNA binding in the initiation of urethan carcinogene-
sis has been investigated by Williams and associates (296, 302, 310). Adminis-
tration of radioactively labeled urethan to mice brought about substantial incor-
poration of the radioactivity to liver RNA Most of the radioactivity was present
-------
642
in a single compound, ethyl cytosme-5-carboxylate. No such preferential in-
corporation occurred in control animals receiving radioactively labeled ethan-
ol or sodium bicarbonate (302). The extent of this preferential incorporation
parallels the activity of RNA synthesis (310). Based on some suggestive evi-
dence that the carcinogenic activity is greatest if administered during the peak
of RNA synthesis, Williams and coworkers (296, 310) hypothesized that the pre-
sence of ethyl cytosme-5-carboxylate in RNA could play a role in urethan car-
cinogenesis. This hypothesis was, however, considerably weakened by the dem-
onstration (310) that in vivo labeling of RNA also occurred with noncarcinogenic
methyl carbamate Furthermore, it is not known whether the formation of
LtheJ
ethyl cytosine-5-carboxylate occurred as a result of direct binding oflurethan
metabolite to RNA or indirect incorporation of pre-formed nucleoside analog
into RNA.
As might be expected from the inadequate knowledge of the ultimate-car-
cinogenic form of urethan, the nature of urethan binding to macromolecules has
not been clearly elucidated The isolation of labeled ethyl cytosme-5-carbox-
1 4
ylate from liver RNA of mice treated with urethan, labeled with C at the carb-
oxy (or carbonyl) position, indicated that the entire ethoxycarbonyl residue may
be bound to RNA (302), however, it could not be ascertained whether the for-
mation of the adduct occurs as a result of direct binding of urethan metabolite
(of)
to RNA (310). The presence! some unidentified radioactively labeled components
_ 14 3
in DNA and RNA of rats g-VloS administered of [l_- C ]-urethan or"[2- H]-ure-
than suggested that labeling of the ethyl residue alone was sufficient to demonstrate
-------
643
the binding (303). Lawsorx and Pound (301) compared the binding of NH -CO-
-O-14CH -CH and NH -14CO-O-CH -CH to mouse liver DNA and concluded
L, J L, L, J
that the carbonyl (or carboxyl) carbon was not involved in the binding. The con-
clusive evidence that only the ethyl residue is involved, was furnished in the
18 18
study using NH -CO- O-CH -CH . No O enrichment was detected in the
Ct C* -J
oxygen of liver DNA, indicating that only the ethyl residue, not the ethoxy res-
idue, was involved in the binding (140). Chromatographic analysis of acid hy-
14 "
drolysates of liver DNA from mice treated with [l- C]urethan provided no '
evidence of alkylation of purine or pynmidine bases. Various hydrolytic and
14
en-zymatic treatments revealed that C was bound in an alkyl group as an es-
ter to a phosphate group in the DNA chain Enzymatic release of the alkyl
group yielded a volatile compound identified as ethanol (140). Thus, it app-ears
l
that only the ethyl residue is involved in the binding and the site of binding is
mainly at the phosphate backbone. A different conclusion, however, has b_een
recently reached by Dahl £t aj_ (107). These investigators compared.the level
314
of binding of H and C to hepatic DNA, rRNA and protein of mice given
[l - C]urethan and[l,2- H]urethan and found that the H/ C ratio of the ad-
QheJ
duct was substantially lower than that oiyurethan administered. They postulated
that the ethyl residue must be dehydrogenated in vivo to vinyl carbamate (thus
losing its tritium label) before binding to macromolecules possibly in the form
of an epoxide.
In addition to nucleic acid binding, modification of cell proteins by urethan
has also been reported A recent study by Gronow and Lewis (311) indicated
-------
644
that a protein fraction of non-histone protein of mouse liver may be modified
(as measured by isoelectric focusing electrophoresis) after a single carcinogen-
ic dose of urethan to suckling mice. This finding may be of potential signifi-
cance IH urethan carcinogenesis in view of the increasing evidence that non-
-histone proteins play a crucial role in the control of gene transcription.
Diaryl Acetylenic Carbamates. A number of diaryl acetylenic carbarmtes
have been shown to be potent carcinogens (see Section 5. 2. 1 6. 3. 3). Based on
structura 1 considerations, these compounds could conceivably alkylate tissue
nucleophiles directly by substitution reactions involving loss of carbamate an-
lon Such an electrophilic activity was indeed demonstrated by Sharpe et al.
(25) using methionme or guanosine as nucleophiles Incubation of several acetyl-
3 14 -
enic carbamates with H-labeled methionme or C-labeled guanosine yielded re-
action products. 1, l-Diphenyl-2-butynyl N-cyclohexylcarbamate and l_-phenyl-
-1 -(3, 4-xylyl)-2-propynyl N-cyclohexyl carbamate were the most reactive com-
pounds in this assay. Mass spectral analysis of a major product between 1, 1-di-
phenyl-2-butynyl N-cyclohexyl carbamate and methionme was consistent with
the formation of 1-methylmercapto-1, 1 -diphenyl-2-butyne. The direct-acting
alkylating activity of these compounds is consistent with their direct-acting mu-
tagenic activity (see Section 5. 2. 1. 6 2.2) Tumor induction may, however, oc-
cur at gite(s) distant from the site of administration..
Carbamate Pesticides The metabolism of carbamate pesticides has been
a subject of tatengive research in the last decade, a number of excellent re-
views on this topic have been published in recent years (3, 312-316) Essen-
tially, oxidation, conjugation and hydrolysis are the three principal types of
-------
645
metabolic reactions that carbamate pesticides may undergo in living organisms.
Oxidation, generally involving microsomal mixed-function, oxidase (MFO), is
the most important route. Depending on the functional groups present in the
molecule,-a variety of reactions, including aliphatic hydroxylation, aromatic
hydroxylation, sulfoxidation, and N-, O- and possibly S-dealkylation (as^de- ^~i» ? •/
picted in Fig. 31) may occur. Carbamate pesticides and metabolites contain-
ing such functional groups as epoxide, hydroxyl, ammo, carboxyl or sulfhydryl
may be readily conjugated, predominantly with the formation of sulfates, glucuron-
ides and mercaptunc acids (the latter derived from glutathione conjugates). Carba-
mate pesticides may also be metabolized by esterase-catalyzed hydrolysis; how-
ever, depending on the compound and animal species involved, the hydrolytic
route may be only of minor importance. With some exceptions, most of the above-
\
-mentioned metabolic pathways are detoxifying in nature, 11 the following paragraphs,
only the reactions that are of potential significance in the generation of mutagenic
or carcinogenic intermediates are discussed.
Carbaryl is the most extensively studied carbamate insecticide. The me-
tabolism of this compound has been reviewed (3, 312-314), the proposed metab-
-*r-
olic pathways are shown in Fig. 32. Although not actually identified, the for-
mation of 3,4- or 5,6-epoxides as intermediates in the metabolism of carbaryl
is implicit from the known end products. These epoxides are highly reactive
electrophiles and can conceivably bind covalently to nucleophilic sites in cell
macromolecules. The in vitro covalent binding of carbaryl was indeed ^demon-
strated by Oonithan and Casida (317). Incubation of rat liver microsomes with
-------
HD
0-deolkylotion
-
Rv /
X,,,/ t N-deolkylotion
/
\\
- I - sulfoxidation_ ' n sulfoxidation
\J O \J " ~ "" ^~ ^^ O \y
I I
R R
sulfone
su If oxide
Figure 31-
-------
LEGEND TO FIGURE 31
Fig. 31 Possible sites of oxidation on a hypothetical N-methyl-
aromatic carbamate pesticide. [Modified from R.J. Kuhr and H.W. Borough,
"Carbamate Insecticides Chemistry, Biochemistry and Toxicology", CRC Press,
Cleveland, Ohio, 1976.]
-------
OH
Conjuqotes
O-C-W-C^OH
Coniugotes
1-Nophlhol
HZ°
Cwiugotes
8
O-C-NHCHj
N Hydro.
0
0-C-
NH-CHj
Conjugotes
O-C-NH-
0-C-NK-CHj
SCHjCHCOOH
NHCOCHj
S-(5-Hydraxy-l- Mercnptunc acid con|ugole
nophthyl) cysteine
0
0-C-^H-CHj
CHj
Corboryl34 epoxide
4-Hydroxycorboryl
,
Connotes
5,6-dihydroxy-5,6-dihydro
corboryl
OH
H OH
l-Hydroxy-5,6-dihydro
5i6-dihyoroxynophlholene
0-C-NH-CH,
SCHjCHCOOH
NHCOCH,
OH
SCHjCHCOOH
Metaptunc ocid conjugate S-(4-Hydroxy 1 naphthyl)
Cysteine
Figure 32
-------
LEGEND TO FIGURE 32
Fig. 32. Proposed metabolic pathways of Carbaryl.
-------
646
1 4
naphthyl- C carbaryl in-the presence of NADPH led to significant incorpora-
tion of radioactivity into microsomal proteins This finding has recently been
14
confirmed by Miller £t ah_ (31 8) Carbaryl with C-labeled N-methyl groap
did not "bind to microsomes indicating that only the aromatic ring was invol\ed
in the binding. The binding reaction is catalyzed by microsomal MFO (indicated
by the dependence on NAPDH and oxygen; inhibition by nitrogen, carbon monox-
jandj
ide, SKF-525A7)^timalation by phenobarbital or 3-methylcholanthrene pretreat-
ment) and is inhibited by glutathione or cystein The biological significance
of this finding remains to be elucidated.
Zectran (Mexacarbate) is one of the very few carbamate pesticides for
which there is some (although not convincing) evidence of potential carcino-
genicity (see Section 5.2.1. 6. 3. 6). Very little information is available on the
metabolism of Zectran in mammals (3, 313, 315), the principal metabolites are
•^—" f"i<9 3 3
shown in Fig 33. Zectran (i) is readily metabolized, predommantly^by hy- J ' •
14 "
drolysis, when administered orally to mice (319). Almost 70% of C from
14 14
C-carbonyl-labeled Zectran is expired as CO within 6 hours (319) The
the '
nature ofpradioactively labeled metabolites in the urine of dogs given rmg-3-
14
C-methyl-labeled Zectran was studied (320) The major metabolites were
4-dimethylamino-3, 5-dimethylphenol (11) and its conjugates, small amounts of
conjugated 2, 6-dimethylhydroquinone (111) were also detected. As many as 9
metabolites were detected in in vitro metabolic studies using rats or human
liver microsomes (317, 321-324) The two major metabolites are 4-dimeth-
ylamino-3, 5-dimethylphenyl N-hydroxymethylcarbamate (iv) and 4-methylami-
no-3, 5-dimethylphenyl N-methylcarbamate (v), the minor metabolites include
-------
Conjugates
t
OH
Conjugates
t
OH
H3C
CH3 H3C
CH,
In Vivo (dog)/
(ll)
OH
(in)
0
0-C-NH-CH3
CH
0
II
0-C-NH-CH2OH
Rat or
human liver
microsomes, NADPH
0
0-C-NH-CH3
H,C
0-C-NH-CH3
(vi)
Figure 33
-------
LEGEND TO FIGURE 33
Fig 33 Some in vivo and in vitro metabolites of Zectran. The designa-
tions used are T, = Zectran, ^^ = 4-dimethylamino-3,5-dimethylphenol, ^^^ =
2,6-dimethylhydroquinone, ^v = 4-dimethylamino-3,5-dimethylphenyl N-hydro-
xymethylcarbamate, V = 4-methylamino-3,5-dimethylphenyl N-methyl carbamate,
VI = 4-methylformamido-3,5-dimethylphenyl N-methylcarbamate, U^^ = 4-amino-
3,5-dimethylphenyl N-methylcarbamate.
-------
647
j
4-methylformamido-3, 5-dimethylphenyl N-methylcarbamate (vi) and 4-amino-3j 5-di-
methylphenyi' N-methylcarbamate (vn). The possible mutagenicity or carcm-
ogemcity, if any, of the above metabolites ha& not been explored It is possi-
H-C O H ,'C CELOH
3 \t 3 \ / 2
ble that unstable reactive intermediates (e_ £ , N— N ) may
/ I
H3C
be formed in the process of N-demethylation of the 4-dimethylamino group
Such intermediates were thought to be the reactive intermediates of carcino-
genic compounds such as hexamethylphosphoramide (see Section 52141)
4-Methylformamido-3, 5-dimethylphenyl N-methylcarbamate may also be a potential-
ly active metabolite (it is structurally similar to the carcinogen N-methyl-N-
-formyl-hydrazine) (Section 5. 2 1.3 3 2 5)
S-Chloroallyl Thiocarbamates Three S-chloroallyl thiocarbamates (Di-
allate, Sulfallate, and Inallate) have been shown to be mutagenic (Section
521622) Of these, ' Diallate and Sulfallate are carcinogenic in ro-
dents, whereas the carcinogenicity of Tnallate does not appeal
to have been studied Mutagenicity studies indicate that metabolic activation is
required for consistently demonstrable activity The mechanism of metabolic
activation of Diallate has been investigated by Schuphan £t aj_ (105) Rats given
14 14
an oral dose of C-cai bonyl-labeled Diallate expired CO (20% of the dose) and
Lj
excreted labeled meicapturic acid conjugate (62% of the dose) and other metab-
olites The fate of the chloroallyl portion of Diallate was examined by using
14
C-allyl-labeled Diallate. Dichloroallylsulfonic acid was the major metabolite
14
in. both-in^yitro and in vivo studies, whereas 2- C -chloroacrolem appears to
-------
648
have been detected only injin vitro studies. Eased on these results, the auth-
ors (105) proposed that Diallate was first sulfoxidized to Diallate sulfoxide
(which is highly reactive and cannot be isolated from the metabolites). Most
of the su-lfcocide thus formed is then detoxified by reacting with glutathione to
yield mercapturic acid conjugate and dichloroallylsulfonic acid as the final me-
tabolites, whereas a portion may undergo spontaneous 2, 3-s igmatropic rear-
rangement followed by 1, 2-elimination to yield dusopropylcarbamoylsulfenyl
chloride and 2-chloroacrolem (Fig 34) The latter pathway has been proposed
as the activation mechanism of Diallate. Both 2-chloroacrolem and synthetic
Diallate sulfoxide have been shown to be mutagenic without activation The
cis-isomer of Diallate, which yields more 2-chloroacrolein than its trans-iso-
mer, also displays greater mutagenicity after metabolism (105) It remains to
be investigated whether 2-chloroacrolein is carcinogenic The metabolic ac-
tivation of Triallate and Sulfallate probably follows a similar mechanism a_s
Diallate (105).
DtaIkyIdithiocarbamates With a few exceptions, dialkyldithiocarbamates
have not been shown to be carcinogenic, although there is some evidence that a
variety of dimethyldithiocarbamates (but not their ethyl analogs) are mutagen-
ic without metabolic activation It has not been clearly established whether the
carcinogenic action of the active dialkyldithiocarbamates is due to the dialkyldi-
thiocarbamate anion or to the metal ion. The metabolic pathways of dialkyldithio
carbamates (Fig 35) appear to be applicable to both dimethyl (325) as w^ll as
-------
C-S-CH2-CCI=CHCI
Dial late
R=-CH(CHj)2
Mojor
dttoiiticolion x
route
'+6SH
-NHCHjCOOH
NHj
Dnsopropylcorbomoyl-GSH conjugate
R. 9 VCOOH
^N-C-S-CHjCH
R NNHCCH3
Diisopropylcorbamoyl mercaptunc
acid conjugate
'" R? 1
SN-C-S-CH2-CCI=CHCI
R" J
Dial late sulfoxide
^
H-S-CH2-CCI=CHCI
Dichloroallyl sulfenic
acid
^
HO-S-CH2-CCI=CHCI
0
Dichloroollyl sulfonic
acid
S-0-Allylsulfenate
ester
R °
V I'
1J-C-S-CI
Diisopropylcorbamoyl
sulfenyl chloride
CHj=CCI-CHO
2-chloroocrolein
Figure 3k
-------
LEGEND TO FIGURE 34
Fig. 34. Proposed metabolic pathways of Diallate. [Modified from
I. Schuphan, J.D. Rosen and J.E. Casida, Science 205, 1013 (1979).]
-------
*< expired air
CS2
^ / _'
R.
R
\ \\
l-C-S-Glucuronide
R
Pigvire 35
-------
LEGEND TO FIGURE 35
Fig. 35 Metabolic pathways of dialkyldithiocarbamates,
-------
649
diethvldithiocarbamates [326). None of the metabolites are more reactive than
the parent compound Thus, it is possible that the active dialkyldithiocarba- -
mates may exert carcinogenic or mutagenic action by acting directly on tar-
get motecules This is supported by the demonstration (45, 46) that dimethyl-
dithiocarbamates are mutagenic as such One possible mechanism of action is
the chelation of metals of key enzymes or proteins involved in the control of
cell differentiation or gene transcription
Tetraalkylthturam Bisulfides retraaLkylthiuram disulfides (£ g., Thiram,
Disulfiram) are metabolized in a manner essentially similar to that of their
corresponding dialkyldithiocarbamates (3, 327, 328). The first step involves
the conversion of the parent compound, either by free radical process or by
nucleophilic substitution reaction on sulfhydryl groups,to form a new mixed
sulfide plus a dialkyldithiocarbamate
RSSR SRRS.
\ II II / II / V II
N-C-S-S-C-N -f R'-SH V R'-S-S-C-N + N-C-SH
/ \ \ /
R R R R -
The dithiocarbamate thus formed is then metabolized, -as summarized in Fig
35. Thus, the mutagenic or carcinogenic action of tetraalkylthiuram disulfides
may also proceed puutnlily via direct action on target molecules This is in
fact the case for Thiram which induces reversion of base-substitution mutants
(TA 100 or 1535) in the Salmonella test without metabolic activation, however,"
Disulfiram does not appear to be mutagenic. Interestingly the expression of
mutagenic activity of Ihiram in frame-shift mutagens (TA 98 or 1538) does re-
quire metabolic activation (48). It is possible that some active metabolites of
-------
650
Thiram have yet to be identified. One hypothetical mechanism of action of tet-
raalkylthiuram disulfide is to act as a cross-linking agent, linking two peptide
chains containing free SH groups by disulfide budge by the substitution reac-
tion dep-icted above.
Ethyle neb is dithiocar hamate s. The metabolism of sodium, manganese,
and zinc salts of ethylenebisdithiocarbamic acid (Nabam, Maneb, Zmeb) has
been extensively studied and this topic has been exhaustively reviewed in 1976
(23, 312, 316) A variety of new reports have appeared since then (329-332).
The metabolic pathways are depicted in Fig 36 Ethylenebisdithiocarbamate
may be metabolized by (a) oxidation to ethylenethiuiam disulfide (ETD), pos-
sibly mediated in vivo by cytochrome c, (b) degradation of the molecule to re-
lease ethylenediamme (EDM) and carbon disulfide, and (c) formation after
splitting off hydrogen sulfide of ethylenebisisocyanato sulfide (EBIS),
ethylenebisisothiocyanate (EDIT) and ethylenethiuram monosulfide (ETM)._ The
latter yields ethylenethiourea (ETU) which, in turn, may be oxidized ^to ethyl-
eneurea (EU). Among these metabolites, ETU has been shown to be carcino-
genic and has been considered to be responsible for the carcinogenic action of
Maneb and Zmeb in some studies, the details of the carcmogenicity of ethylene-
thiourea will be further discussed in Section 5. 2. 2. 6. It should be noted that,
in addition to the metabolites shown in Fig 36, the presence of large amounts
of unidentified polar metabolites has been noted (332, 333), the biological activ-
ity of these polar metabolites has yet to be elucidated
52165 Environmental Significance. Urethan has been a compound
of considerable environmental concern Possible exposure to this carcinogen
-------
CHo-NH-C-S
CHp-NH-C-S
* II
/ S
/ ETD
S
II r
CH2-NH-C-SH -CS2
CH2-NH-C-SH
6 II
!„!
CH2-NH2 -CS^CH2-NH2
CH2-NH-C-SH CH2-NH2
-
K>S
T l
HCT __h-N=c's 1^2u
VV»X"s<» /- 1
C C=S CHo-NH-C-SH
s-s I
CHz-'
11
1 S J EDM
:-H2S
'
W2
CH2-N=C=S
L S
EBIS /
/ ' S
:H2-N=C=S CH2-NH-C,
1 >
;H2-N=C=S CH?-NH-C
II
CH2-NH. CH2-NHV
| >S — | >0
CH2-NH^ CH2-NHX
EBIT ETM ETU EU
Figure 36
-------
LEGEND TO FIGURE 36
Fig. 36 Proposed metabolic pathways of ethylenebisdithiocarbamates.
The chemical names of the metabolites are ETD = ethylenethiuram disulfide,
EDM = ethylenediamine; EBIS = ethylenebisisocyanato sulfide, EBIT = ethylene-
bisisothiocyanate, ETM = ethylenethiuram monosulfide, ETU = ethylenethiourea,
EU = ethyleneurea
-------
651
may occur from pharmaceuticals, industrial chemical mtermediates.as well as
fermented beverages and foods. Urethan was formerly used in human medi-
cine as an antineoplastic agent (particularly in the treatment of chronic leuke-
mia and -multiple myeloma), as a hypnotic or sedative, as a component of a
sclerosing solution (together with quinine) for varicose veins, as an antidote
to strychnine poisoning, as an adjunct to sulfonamide therapy, and as a topic-
al bactericide (108, 334, 355). There is no evidence that urethan is still being
used in the U.S. in human medicine, but its veterinary usage probably still con-
tinues. In a 1975 publication of Nomura (336), urethan was reported to be pres'
ent (as a co-solvent) in four Japanese pharmaceutical products intended for
human parenteral use One of these products, "Grelan Injection", when admin-
istered to ICR-JCL, mice, actually induced lung tumors It is possible thatat
^
the present time inventories and stocks | drugs may still carry products con-
taining urethan, as they may have been manufactured before the discontinua-
tion of the use of urethan in human medicine.
Industrially, urethan has not been used in any large amounts in recent
years. The annual production in 1977 was estimated to be greater than 1, 000
Ibs. (337) Urethan has been mainly used as a chemical intermediate in the
preparation of ammo resins, and as a solubilizer and co-solvent for fumigants,
pesticides, and cosmetics The major present use of urethan is believed to
be as a chemical intermediate, primarily for reaction with formaldehyde to
produce N-hydroxymethyl derivatives useful as cross-linking agents in textile
treatment (108, 338)
-------
652
Urethan has been reported to occur as a result of the reaction of ammonia with
diethylpyrocarbonate, an antimicrobial food additive for beverage. By isotope
dilution technique, Lofroth and Gejvall (339) estimated that as much as 2 6
mg/1 urethan could be produced by the addition of 500 mg/1 diethylpyrocarbonate
to a white wine at pH 3. 4 and having an estimated ammonia content of 5 mg/1.
The urethan yield in beer and orange juice treated with the food additive was
of the order of 1. 3 and 0. 17-0 58 mg/1, respectively (339). This has been
confirmed by other investigators, but a much lower level (as much as 100 times
less) of urethan was found (340-343) In 1972, the U.S. Food and Drug Admin-
istration rescinded the permission for the use of diethylpyrocarbonate as an
SH3 additive in beverages (344). Ough (342) has recently developed a new anal-
ytical method for the determination of low levels of urethan in beverages and
loods. Most fermented foods and beverages were found to contain urethan
ranging from a trace to 6 0 (Jg/l. A sample of Japanese sake tested con-
tained an exceptionally high level of urethan (154-192 ^g/1) No urethan was
detected in unfermented food products. The urethan was appar-
(jindj
ently from natural sources] was postulated to arise as a result of the
[of ethanolj
reaction\with carbamyl phosphate, the latter could be synthesized from ATP,
ammonia and carbon dioxide by yeast. Ough (343) further re-evaluated the ex-
tent of urethan formation in commercial wine by the addition of 50-100 mg/1
diethylpyrocarbonate and found that the additional amounts of urethan formed
were generally less than 1 ^tg/1. The author called for a re-evaluation of FDA's
ban of the additive
-------
653
It should be noted that the carcmogemcity of urethan is well established.
•
A dose-response study by Schmahl et al. (345)indicates that a daily intake as
low as 100 fig/kg urethan may induce tumors in mice. Furthermore, the demon
stration-by Goerttler and Lohrke (124) (Section 5.2.1.6 3 8), that low doses of
^thej
urethan may cross the placental barrier and initiate tumongenesis in/fetus,
as well as the many examples of synergism and enhancement of urethan car-
cinogenesis by various chemicals (Section 5 2. 1 6. 3. 9) underscore the impor-
tance of urethan as a poteitial environmental carcinogen for humans.
prcaont authoro1 viow, tho finding of Ough (342, 343) ohould raioo the quootion
rather thatr bo consider tho-rotn-otafaomont of tho U3O of tho controvoroial food
addfrfetve Although "Seemingly' impo&e-t'blei—natuially occurring carcinog?>. r-eg-uiattoti.
The environmental significance of other carbamates, thiocarbamates,
and substituted urea compounds, ha~s not been critically evaluated. Many of these
are used in very large amounts agriculturally and industrially, and could con-
ceivably be a major concern Fortunately, most of these compounds have a
relatively low tendency to accumulate in the environment and relatively few
have been unequivocally proven to be carcinogenic The major uses and the
annual production or consumption data of some of the more important carba- ' P*
^andj -*- .
mates (7 related compounds are tabulated in Table CXXX. Essentially, hu-
man exposure to these compounds may occur via occupational handling^ phar-
maceutical use, aid ingestion of pesticide residues in food products The
-------
Table CXXX
p 1 of 2 pp
Major Uses and Annual Production or Consumption of Some Important
Carbamate, Thiocarbamate and Substituted Urea Compounds
Compound
Majoi Uses
Annual Production/Consumption
Zectran (Mexacarbate)
Propoxur (Baygon)
Carbaryl (Sevin)
Carbofuran (Furadan)
Propham (IPC)
Chloropropham (CIPC)
Benomyl (Benlate)
Aldicaib (Temik)
Diallate (Avadex)
Sodium diethyldi-
thiocarbamate
Ziram
Ineecticide, molluskicide
Insecticide
Insecticide, acancide, molluskicide
Insecticide, nematocide
Herbicide
Herbicide
Fungicide
Insecticide, miticide, nematocide
Pre-emergence herbicide
Chelating agent, rubber
vulcanization accelerator,
metal poisoning therapy
Fungicide, rubber vulcanization
accelerator
x£.l,0001b (discontinued in 1975)
3 x 105 Ib. (1974)
26 x 106 Ib. (1974)
12 x 106 Ib (1974)
2 x 105 Ib (1975)
1 1 x 106 Ib (1975)
j
3 x 106 Ib. (1975)
1.6 x 106 Ib (1974)
1-5 x 10 kg (world-wide)
1x10 kg (world-wide)
1. 89 x 10 Ib (1976)
-------
Table CXXX, continued
p 2 of 2 pp
Ethyl zimate
Butyl zimate
Ferbam
Thiram
Disulfiram
Maneb
Zineb
Carbromal
Monuron
Rubber vulcanization accelerator,
heat stabilizer for polyethylene
Accelerator for latex dispersion and
cement, ultraaccelerator
for lubricating oil additive
Fungicide, rubber vulcanization
accelerator
^vulcanization J
Rubberjaccelerator, fungicide,
animal repellant
^vulcanization J
Rubberjaccelerator, aversion
therapy for chronic alcoholism
Contact fungicide
Fungicide
Sedative, hypnotic
Herbicide on non-crop land
0.927 x 10 Ib. (1975)
2 92 x 10 Ib. (1976)
8.0 x 10 Ib (1968)
5.8 x 10 kg (1975)
5. 1-5 5x10 kg
5. 5 x 10 Ib. (1972)
1.4x 106 Ib (1968)
2 3-4.0 x 10 kg (1973)
Except where specified, the production/consumption data are those of the U.S. Numbers in
parentheses indicate the year the data were obtained Source of information SRI, "A Study of Industrial
Data on Candidate Chemicals for Testing", EPA 560/5-77-006, Office of Toxic Substances, U.S. Environ-
mental Protection Agency, Washington, DC, 1 977, IARC Mo nog No 12, Intei national A gency for Research
on Cancer, Lyon, France, 1976, and information dossiers prepared for the U.S. Environmental Protection
Agency, Washington, DC.
-------
654
threshold limit values (TLVs) for occupational exposure adopted by the Amer-
ican Conference of Governmental and Industrial Hygienists for a number of
carbamatesY' related compounds are as follows Propoxur, 0. 5 mg/m ,
Carbary-1,-5 mg/m , Carbofuran, 0 1 mg/m , Benomyl, 10 mg/m (proposed),
3 3
Thiram, 5 mg/m , Disulfiram, 2 mg/m (157) As might be expected from the
heavy use of carbamates and related compounds as pesticides in crop protec-
tion, the presence of residual amounts of these compounds in harvested foods
appears to be inevitable although there is a paucity of quantitative data Many
recent articles have discussed the problems of measurement, degradation,
distribution, and bioavailability of carbamate and thiocarbamate pesticide
residues in food and plant products for human consumption (£ £ , 3, 22, 23,
312, 346-348) A detailed discussion of the subject is out of the scope of this
Section. The residue tolerance level permitted in harvested plant food is 7
mg/kg for Ziram, Thiram, and Ferbam whereas that of Maneb is 2-10 mg/kg
(312) The World Health Organization (349) has provisionally recommended
0-0.005 mg/kg body weight as the "acceptable" daily intake A rebuttable pre-
sumption against the registration and continued registration of pesticide prod-
ucts containing Diallate has been forwarded by the U.S. Environmental Protec-
tion Agency, because of the increasing evidence of carcinogenicity and mutagen-
icity of the compound (l6l) Based on structural and metabolic consideration,
the whole class of S-chloroallyl thiocarbamates may probably pose a potential
carcinogenic risk to exposed humans As of 1973, the use of Monuron~m agricul-
tural crop protection is no longer registered (350), Monuron is now mainly used
in non-crop applications, such as landscaping
-------
SUPPLETORY NOTE FOR SECTION 5.2.1.6
The cell cycle of a growing cell is the interval from one
cell division to the next. A series of highly programmed, time
sequence, biochemical events that are common to almost all cell
types are known to occur in the interphase, a period between cell
division and the onset of the following mitosis. The terminology
of the cell cycle was introduced in 1953 by Howard and Pelc (351)
and has since been broadly adopted. This terminology formalizes
the time sequence of cell division with respect to DNA synthesis.
Studies using microspectrophotometry (352) or autoradiography
(351) have demonstrated that DNA replication takes place only
during a certain part of the interphase and not in mitosis.
Using DNA synthesis as a marker, the interphase can be subdivided
i
into G^, S and G>2 periods; S denotes the period of DNA synthesis
while G, (for Gap 1) and 62 (for Gap 2) designate the pre and
post intervals in the interphase during which no nuclear T)NA is
synthesized. Early stages of chromosome condensation, synthesis
and assembly of the mitotic apparatus are presumed to occur in
G2» Following G2 begins the period of cell division called the D
phase (for division) or more frequently the M phase (for
mitosis).
Owing to the improvement of cell culture techniques during
the past decades, the duration of each period of the cell cycle
can be determined. This can be accomplished by autoradiographic
assay for isotope in the chromosomes of cells in mitosis at vari-
ous time intervals after a single pulse exposure of the cells to
-------
O JO
H-thynudine. Alternatively, it can also be determined by analyz-
ing the cell growth curve following addition and subsequent
removal of an inhibitor of DNA synthesis ($_•%_•, 5-fluorode-
oxyundine) or of mitosis (_£_•.£[/ i colchicine). The biology of the
cell cycle and detailed methods of cell cycle analysis have been
described (353-357). In general, the duration of S* G2 and M are
relatively constant and quite similar in most mammalian cells: S
about 6 to 9 hours, G2 about 2 to 5 hours, and M 0.2 to 1.0 hour
(356). Conversely, great variation occurs in the length of G^,
which may be unmeasurably short or may last days or even weeks
depending on the cell type (354, 356). Accordingly, the length
of the cell cycle of any cell type is largely affected by the
length of G,. Moreover, it has been shown that changes in
generation time of cells in culture, due to alteration of culture
conditions, also stem predominantly from prolongation or
shortening of G, with little or no changes of S, G2 or M (358).
While S and M are relatively well define'd, information about
the biochemical events that occur in G^ and G2, as well as their
roles in the progress of the state of the cell within the cycle
is still meager. However, once a cell enters S from G^, it will
automatically transit to G2/ undergo mitosis, and then divide.
The initiation of DNA synthesis appears, therefore, to be a
critical step in the regulation of cell proliferation. Studies
with inhibitors have shown that both RNA synthesis and protein
synthesis are required for the transition of G, to S. Based on
the observations that new daughter cells with smaller mass
-------
synthesize more protein during the G, period and also have a
longer length of G, than those with larger mass, it was suggested
that a certain quantity of protein must be synthesized before DNA
replication can be initiated (359). In accord with these obser-
vations are the findings that if protein synthesis is blocked for
a certain time during G^, the transition to S is delayed for an
equally long period (360). Little is known at present about the
nature of proteins involved in the control of DNA replication in
the eukaryotes. Enzymes mediating DNA synthesis such as DNA
polymerase, thymidylate synthetase, kinase etc. have been shown
to sharply increase in liver and kidney cells immediately before
DNA replication (361, 362). However, these enzymes in other cell
types were found present throughout the G, phase and have no
significant increase with respect to the onset of DNA synthesis
(363, 364).
Protein synthesis is required not only for the initiation
but also for the successful completion of replication of DNA in
eukaryotes (365, 366). Although the role of histone in the
structure and function of the chromatin is not yet clearly
understood, there is ample evidence that histone synthesis occurs
concurrently with DNA synthesis (e.g., 367, 368). Moreover, the
phosphorylation of histone HI appears to be intimately involved
in the modification of chromatin structure during the entire cell
cycle (369, 370).
-------
658
REFERENCES TO SECTION 5216
1 Holmstedt, B. The Ordeal of Old Calabar The Pageant of Physotig-
ma Venenosum in Medicine I_n "Plants in the Development of Modern
Medicine" (T Swain, ed ) Harvard Univ. Press, Cambridge, Mass ,
1972
2 Thorn, G D , and Ludwig, R.A. "The Dithiocarbamates and Related
Compounds" Elsevier, Amsterdam, 1962
3. Kuhr, R J , and Borough, H. W. "Carbamate Ins ecticides Chemistry,
Biochemistry and Toxicology" CRC Press, Cleveland, Ohio, 1976
4 Seller, J P Mutation Res _58_, 353 (1978)
5 Clark, A J. "Applied Pharmacology" 7th edn , Churchill, 1940, p
235
6 Nettleship, A , Henshaw, P S , and Meyer, H L .J. Natl Cancer Inst
4, 309 (1943).
7 Graffi, A , Vlamynck, E , Hoffmann, F , and Schulz, I. Arch" Gesch-
wulstforsch 5_, 110 (1953).
8 Salaman, M. H , and Roc. F J C. Br J Cancer 7, 472 (1953)
9. Berenblum, 1 , and Haran-Ghera, N Br J Cancer 11, 77(1957)
10 Tannenbaum, A. A eta Un int. Cancr. 1_7_, 72(1961)
11 Tannenbaum, A NCI Monog 14, 341 (1964)
12 Tannenbaum, A., and Silvers tone, H. Cancer Res 18, 1225(1958)
13 Tannenbaum, A , and Maltotii, C , Cancer Res 22, 1105 (1962) "
-------
14 Tannenbaum, A , Vesselinovitch, S. D , Maltoni, C , and Mitchell, D S.
•
Cancer Res. 22. 1362(1962).
15 Larsen, C. D J Natl Cancer Inst 8, 99(1947)
16, Larsen, C. D J Natl Cancer Inst 9. 35(1948)
17. Berenblum, I , Ben-Ishai, D. , Haran-Ghera, N , Lapidot, A , Simon,
E. , and Trainin, N. Biochem Pharmacol. 2, 168(1959).
18. NTIS Evaluation of Carcinogenic, leratogenic and Mutagenic Activities
of Selected Pesticides and Industrial Chemicals, Vol I, Carcinogenic
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683b
SOURCE BOOKS AND MAJOR REVIEWS FOR SECTION 5.2.1.6
1. Kuhr, R.J., and Borough, H.W.: "Carbamate Insecticides:
Chemistry, Biochemistry and Toxicology" CRC Press, Cleveland,
Ohio, 1976, 301 pp.
2. National Technical Information Service, "Evaluation of
Carcinogenic, Teratogenic and Mutagenic Activities of Selected
Pesticides and Industrial Chemicals, Vol. I, Carcinogenic Study"
PB-223159, Springfield, Va., 1968, 92pp.
3. Adams, P., and Baron, F.A.: Chem. Rev. 65, 567-602 (1965).
4. Mirvish, S.S.: Adv. Cancer Res. 11, 1-42 (1968).
5. Fishbein, L.: J. Toxicol. Environ. Health 1, 713-735 (1976).
6. International Agency for Research on Cancer: "Some Ant.i-Thyroid
and Related Substances, Nitrofurans and Industrial Chemicals"
IARC Monographs on the Evaluation of Carcinogenic Risk- of
Chemicals to Man, Vol. 7, Lyon, France, 1974, 326 pp.
7. International Agency for Research on Cancer: "Some Carbamates,
Thiocarbamates and Carbazides" IARC Monographs on the Evaluation
of Carcinogenic Risk of Chemicals to Man, Vol. 12, Lyon, France,
1976, 282 pp.
8. Fukuto, T.R.: Drug Metab. Rev. 1, 117-151 (1973).
9. Ryan, A.J.: CRC Crit. Rev. Toxicol. 1, 33-54 (1973).
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NOTES ADDED AFTER COMPLETION OF SECTION 5.2.1.6
Hedenscedt £t_£l_. (1) tested the mutagenicity of 12 thiram and dithiocar-
bamate compounds in the Ames test. Thiram was found to be a much more potent
mutagen than its monosulfide derivative (tetramethylthiram monosulfide),
inducing reversions in the base-substitution- and possibly frameshift-
sensitive strains. Disulfiram, the ethyl homolog of thiram, was inactive.
Among the 9 dithiocarbamates tested, four (tellurium diethyl-, zinc dibutyl-,
nickel dibutyl-, piperidinum pentamethylene-) are inactive. The mutagenic
compounds (mostly towards base-substitution mutants with no requirement of
metabolic activation) include, in this decreasing drder of potency: zinc
dimethyl- (ziram), cadmium diethyl-, zinc diethyl-, zinc ethylphenyl-, and
copper dimethyldithiocarbamates. Comparison of the bacterial mutagenicity
with in vitro alkylating activity [reaction with 4-(p-nitrobenzyl)-pyridine or
deoxyguanosine] shows no correlation (2). Koga jet_£l_. (3) have demonstrated
that, in contrast to the lack of mutagenicity of ethyl carbamate in the Ames
test, alkyl N-hydroxycarbamates exhibited weak but significant mutagenic
activity. The relative mutagenic potency of alkyl N-hydroxycarbamates follows
the order: ethyl » propyl > methyl. Acylation of ethyl N-hydroxycarbamate
markedly enhances its mutagenic activity. Ethyl N-benzoyloxycarbamate, the
most active compound, is about 260 times more potent than ethyl N-hydroxycar-
bamate (based on comparison of revertants/nmole using strain TA 100). Further
N-acylation, yielding N-acyloxy-N-acylcarbamates does not bring about addi-
tional increases in mutagenicity. Another closely related compound, methyl
N-nethylolcarbamate (which is employed to obtain a flame-retardant finish on
cotton) has been tested by MacGregor _et__al_« (4) and found to be inactive in
the Ames test. Douglas £t_^l_. (5) reported that Diallate and Triallate
exhibited mutagenic activity towards both base-substitution and frameshift
mutants.
-------
In other mutagenicity tests, Zdzienicka et_£l_. (6) found that thiram was
positive in prophage induction, Salmonella typhimunum repair and Aspergillus
nidulans_ forward-nutation tests. Benomyl was negative in gene-conversion
tests using Saccharomyces cerevisiae or Aspergillus nidulans (7). Carbaryl
was negative in the nicronucleus test (8)0 Diallate and Triallate caused
cnromosome damage or sister-chronatid exchanges (SCE) in cultured mammalian
cells (5). Both vinyl carbamate and urethan (ethyl carbamate) induced SCE in
cultured human lymphocytes; however, the activity of the former was enhanced
while that of the latter was reduced by S-9 mix (9). Cheng _et__al_. (10)
compared the SCE-inducing ability of four alkyl carbamates, the relative
potency: ethyl > ethyl N-hydroxy- > isopropyl > methyl (inactive) is in good
agreement with their carcinogenic potency.
/
The carcinogenicity of vinyl carbamate has been further tested by Dahl e
al. (11) and compared to urethan and several other carbamates. When admini-
stered during the first 3.5 or 5 weeks after birth, both vinyl carbamate and
urethan induced liver tumors, thymomas, lung adenomas, and Harderian gland
tumors in C57BL/6J x C3H/HeJ mice or ear duct and hepatic carcinomas and
neurofibrosarcomas of the ear lobe in Fischer rats. Vinyl carbamate was
significantly more active than urethan in the induction of various types of
tumors. In mouse (strain A/J) lung adenoma assay, deuterated (CDoCD2-)
urethan was as active as unsubstituted (CH-jCIL,-) urethan whereas ^-butyl
carbamate was inactive. The final analysis of NCI carcinogenicity data on
Diallate (Avadex) and potassium bis-(2-hydroxyeth"yl)-dithiocarbamate has
recently been completed (12). In contrast to some previous reports (see
Section 5.2.1.6.3), it appears that both compounds do not have statistically
significant carcinogenic effects in Charles River CD rats.
-------
A variety of agents have been tested as potential modifiers of carcino-
genesis by carbamate compounds. High doses of the mixed-function oxidase
inhibitor 2-(2,4-dichloro-6-phenyl)-phenoxyethylamine inhibited the induction
of lung adenomas by ethyl N-hydroxycarbamae but had no effect on vinyl carba-
mate or urethan. On the other hand, caffeine inhibited the lung adenoma
induction by urethan, but had no consistent effect on vinyl carfaamate or ethyl
N-hydroxycarbanate (11). The lung adenoma assay has also been utilized by
Theiss jJt_£l_e (13) who investigated the potential of commercial saccharin as a
promoter or co-carcinogen of urethan. At the low (0.1 g/kg) dose of urethan,
saccharin had no effect. At the high (1 g/kg) dose, however, saccharin signi-
ficantly increased the number of adenomas per mouse over the number found in
mice treated with urethan alone. The carcinogenic effect of combined treat-
ment of CFLP mice with urethan and diethylstilbestrol (DES) was studied by
Ferenc .et^j*!/ (14). The incidence of lymphoma was 0% in controls, 0% with
urethan only, 4.1% with DES only, 18.1% with DES followed by urethan 14 days
later, 32% with urethan and DES given simultaneously, and 44% with urethan
followed by DES 14 days later. All urethan-treated mice developed lung tumors
regardless of DES treatment. In a preliminary communication, Witschi (15)
reported that butylated hydroxytoluene (BHT) enhanced the lung tumor-inducing
effect of urethan in Swiss-Webster mice if administered after urethan. Viral
infection has been shown to suppress the pulmonary adenoma-inducing effect of
urethan in mice, a brief review of this topic has been presented by Nettesheim
jjt_^l_. (16). The development of lung tumors was^jobserved in Kid:CFLP mice
treated intragastrically with diethyl pyrocarbonate (an antimicrobial agent
used as a perservative of beverages and food) and ammonia. The authors (17)
suggested that the carcinogenic effect may result from the in vivo formation
of urethan from the two precursors.
-------
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