THIOCARBONYL COMPOUNDS
CARCINOGENICITY AND STRUCTURE-ACTIVITY
RELATIONSHIPS. OTHER BIOLOGICAL PROPERTIES,
METABOLISM. ENVIRONMENTAL SIGNIFICANCE.
David Y. Lai, Ph. D.
Yin-tak Woo, Ph. D.,
Joseph C. Arcos, D. Sc., and
Mary F. Argus, Ph. D.
Preparation for the Chemical Hazard
Identification Branch "Current
Awareness" Program
-------
Table of Contents;
5.2.2.8 Thiocarbonyl Compounds
5.2.2.8.1 Introduction
5.2.2.8.2 Physical and Chemical Properties. Biological
Effects
5.2.2.8.2.1 Physical and Chemical Properties
5.2.2.8.2.2 Biological Effects Other Than Carcinogenic
5.2.2.8.3 Carcinogenicity
5.2.2.8.3.1 Overview
5.2.2.8.3.2 Thiourea and Related Compounds
5.2.2.8.3.3 Thiouracil and Related Compounds
5.2.2.8.3.4 Thioacetamide and Ethionamide
5.2.2.8.3.5 Modification of Carcinogenesis
5.2.2.8.4 Metabolism and Mechanism of Action
5.2.2.8.4.1 Tissue Distribution and Metabolism
5.2.2.8.4.2 Mechanisms of Action
5.2.2.8.5 Environmental Significance
References
-------
5.2.2.8 Thiocarhonyl Compounds
5.2.2.8.1 Introduction.
Interest in the pharmacological and toxicological studies of this chem-
ical class arose in the early 1940's when the goitrogenic properties of allyl-
thiourea and other related compounds were first noted. In 1941, Kennedy and
Purves (1) and Griesbach (2) found that rats fed a diet containing brassica
seeds developed goiter. Suspecting that a thiourea derivative in the brassica
seeds might be responsible for the goitrogenic effect, Kennedy (3) admini-
stered allylthiourea to rats and noted the induction of goiter in the
animals. Other thiourea related compounds were later shown to possess various
degrees of goitrogenic activity (4-7). These goitrogenic agents produce
thyroid hypertrophy and hyperplasia by inhibiting the synthesis of thyroid
hormones, thereby triggering the secretion of thyroid stimulating hormone
(TSH) from the pituitary gland. A large volume of literature has accumulated
describing the effects of these compounds on the endocrine function of the
thyroid gland. Because of their antithyroid activity, a number of them
including thiouracil (TU), methylthiouracil (MTU) and propylthiouracil (PTU)
were, at one time, considered as drugs of choice for the treatment of hyper-
thyroidism.
Moreover, several thiocarbonyl compounds acquired economic importance
because of their extensive applications in the industry and as pesticides (see
Section 5.2.2.8.5). For instance, in 1945 thiourea and thioacetaiaide (TAA)
were found to be effective fungicides and were used in this capacity in the
prevention of orange decay (8). It was the presence of these chemicals in the
juice of treated oranges that raised great concern of the possible health
hazards to humans. Extenxive toxicological studies have since been carried
1
-------
out on these compounds. Ethylenethiourea (ETU) has been shown to be one of
the major degradation products of ethylene bis-dithiocarbamates, an important
v
and widely used class of fungicides for the treatment of diseases of a large
variety of agricultural crops (cited in ref. 9; see also Section 5.2.1.6). It
has been established in animal bioassays that ETU is carcinogenic, mutagenic
and teratogenic. Because of the potential hazard that ETU represents to human
health, a Rebuttable Presumption Against Registration (better known under the
acronym RPAR) of all pesticide products containing ethylene bis-dithiocarba-
taate was issued by the U.S. Environmental Protection Agency in 1977 (10). The
National Institute for Occupational Safety and Health (NIOSH) also recommended
to minimize occupational exposure to ETU and to handle the compound in the
workplace as if it were a human carcinogen and teratogen (11).
Over half a century ago, Wegelin (12) noted the existence of a link
between endemic goiter and malignant tumor of the thyroid gland. He believed
that benign goitrous hyperplasia predispose to the development of malignant
thyroid neoplasms both in animals and in humans. His view has been substan-
tiated by histopathological studies which reveal that most thyroid cancers of
humans are derived from goitrous glands. Since spontaneous thyroid tumors
rarely occur in laboratory rodents, goitrogen-induced thyroid tumors in rats
and mice have become a valuable model for studies on hormone-dependent tumors
as well as the role of goitrogenic agents in carcinogenesis.
The first study on experimental tumor induction by a thiocarbonyl goitro-
gen dates back to 1944 when Bielschowsky (13) demonstrated the emergence of
benign and malignant tumors in the thyroid gland in rats by the concurrent
feeding of allylthiourea and 2-acetylaminofluorence (2-AAF). Subsequent
studies (14) showed that allylthiourea alone also induces adenomata in the rat
thyroid, although the incidence is lower than that produced by the combined
-------
action of allylthiourea and 2-AAF. 'Since then many investigations have been
carried out on thyroid tumor induction in rodents by antithyroid drugs.
Several thiourea related compounds including trimethylthiourea, diethyl-
thiourea, dlcyclohexylthiourea, dithiobiurea and phenylthiourea have been
tested for carcinogenicity by the U.S. National Cancer Institute, because of
occupational exposure to these compounds in their industrial use. Ethion-
amide, a synthetic antituberculotic drug structurally related to thioacet-
amlde, was also tested by the U.S. National Cancer Institute because it is
often used clinically for extended periods of time.
5.2.2.8.2 Physical and Chemical Properties &.-.. Biological Effects.
5.2.2.8.2.1 PHYSICAL AND CHEMICAL PROPERTIES.
Table I presents the structural formula, molecular weight, solubility and
other physical properties of thiocarbonyl compounds which have been bioassayed
for carcinogenicity. These compounds include thiourea and its aliphatic and
heterocyclic derivatives, which all contain a thioureylene group:
S
\ H s
^ -c - V
In thioacetamide and ethionamide, one of the nitrogen atoms is replaced by a
methyl and a 2-ethylpyridine group, respectively. In these compounds only the
S
H /
thioamide group - C - N is common with other compounds of the class.
Thiourea reacts with various metals under neutral conditions and forms
adducts or complexes. Prolonged heating of thiourea at 170°C yields ammonium
thiocyanate (15). Thioacetamide is relatively stable in neutral aqueous
-------
p. 1 of 3 pp.
Table I. Chemical and Physical PrdHlties of Thiocarbonyl Compounds'
Compound
Structure
M.W.
Physical properties
Solubility
Thiourea
S
II
C -
76 White, glossy crystal; bitter
taste; sp. gr. 1.406; m.p.
176-182°C; sublimes at 150-
160°C under vacuum
Soluble in cold water,
ammonium thiocyanate and
ethanol; sparingly soluble in
ether
Allyl-
chiourea
CH0 = CHCH.
L 2.
S
II
NH - C
- NH,
116 Colorless monoclinic crystal; Soluble in water
o,.
garlic odor; m.p. 74 C
Trimethyl-
chiourea
S
II
(CH ) N - C
- NHCH
118 Prism; m.p. 87-88°C
Soluble in water, ethanol,
and trichloromethane
N,N'-Diethyl-
chiourea
S
II
- C -
132 Crystal; m.p. 68-71°C
Slightly soluble in water;
soluble in methanol, ethanol,
ether, acetone, benzene, and
ethyl acetate
Phenyl-
thiourea
C,HC - NH - C - NH
o j
152 Needle-like crystal; bitter Soluble in water and ethanol
taste; m.p. 152°C
N.N'-Dicyclo-
hexylthio-
urea
C,H, - NH - C - NH - C H ,
oil 611
240 m.p. 182°C
2,5-Dithio-
biurea
S S
II II
H N - C - NH - NH - C - NH
118 m.p. 214 C
-------
ox .i pp.
Table I.
Chemical and Physical Prd^fcties of Thiocarbonyl Compounds'
(contiWed)
Compound
Structure
M.W.
Physical properties
Solubility
Ethylene
thiourea
(ETU)
(see Footnote b)
102 White to green crystal; faint
amine odor; m.p. 199-204°C
Soluble in hot water;
slightly soluble in cold
water, methanol, ethanol,
acetic acid, and naphtha
Thiouracil
(TU)
(see Footnote b)
128 White powder or minute
crystal; bitter taste; melts
with decomposition at about
340°C; combustible
Readily soluble in alkaline
solutions; very slightly
soluble in water, ethanol,
and ether
Methyl-
thiouracil
(MTU)
(see Footnote b)
142 White crystal; bitter taste;
sublimes readily; decomposes
at about 330°C
Soluble in aqueous solutions
of ammonia and alkali
hydroxides; slightly soluble
in ethanol and acetone; very
slightly soluble in cold
water and ether
Propyl-
thiouracil
(PTU)
(see Footnote b)
170 White powdery crystalline
substance; starch-like in
appearance and to touch;
bitter taste; sensitive to
light; m.p. 218-221°C
Soluble in ammonia and alkali
hydroxides; sparingly soluble
in ethanol; very slightly
soluble in water
Thioacetainide
(TAA)
S
II
- C -
75 Colorless leaflet; slight
odor of mercaptans; m.p.
109-114°C
Soluble in water and ethanol;
slightly soluble in water
-------
01 j p|
Table I. Chemical and Physical
erties of Thiocarbonyl Compounds'
Compound
Structure
M.W.
Physical properties
Solubility
Ethionamide
(see Footnote b)
166 Minute yellow crystal; m.p.
164-166 C
Soluble in hot acetone,
dichloroethane and pyridine;
slightly soluble in methanol,
ethanol, propylene glycol;
very sparingly' soluble in
water
Compiled from: Hawley, C.G., (ed)., "The Condensed Chemical Dictionary," 9th ed. Van Nostrand, New York, 1977;
Verschueren, K., "Handbook of Environmental Data on Organic Chemical," Van Nostrand, New York, 1977; Gleason, M.N.,
Gosselin, R.E., Hodge, H.C. and Smith, R.P.: "Clinical Toxicology of Commercial Products," 3rd ed. Williams & Wilkins
Baltimore, 1969; Sax, N.I., "Dangerous Properties of Industrial Materials," 5th ed. Van Nostrand, New York, 1979; Merc
Index, 9th ed., Merck & Co., Rahway, New Jersey, 1976.
'Formulas to Table above:
i
O
\—M
= S
Ethylene thiourea Thiouracil(R=H;TU)
(ETU) MTU(R=CH3)
PTU(R=C3H7)
Ethionamide
-------
solutions. However, if heated at acidic or alkaline pH, it undergoes hydro-
lysis and releases hydrogen sulfide or sulfide ion (16).
5.2.2.8.2,1 Biological Effects Other Than Carcinogenic
Goitrogenicity. The goitrogenic effect of thiourea and related compounds
in rats has been known since the report by Kennedy (3) and the extensive
investigations of Astwood and coworkers (6, 7). These agents act directly on
the thyroid gland to inhibit the synthesis of thyroxine through the inhibition
of a peroxidase which mediates the conversion of iodide ion to iodine
radical. The resulting low level of thyroid hormone in the circulation
triggers an increased secretion (from pituitary glands) of thyroid stimulating
hormone (TSH) which acts on the thyroid epithelium to cause hyperthophy and
hyperplasia. Astwood et al. (7) determined the antithyroid activities of a
large number of compounds in rats. The decrease in thyroid iodine concentra-
tion and increase of weight of the thyroid gland were used as parameters to
compare the activities of the compounds with TU, which was chosen as a stan-
dard of reference and assigned an arbitrary activity of 1. The relative anti-
thyroid activities of some thiocarbonyls were found to be: MTU, 1.00; PTU,
11.00; thiourea, 0.12; triiaethylthiourea, 0.10; diethylthiourea, 0.40; dithio-
biurea, <0.01; thioacetamide, <0.01. The most active compounds are deriva-
tives of TU. Lindsay et_ al_. (17) have also demonstrated that only thiopyrimi-
dines, but not thiopurines, are goitrogenic in rodents. Substitution by alkyl
or aryl groups at position 6 of thiouracil leads to enhanced activity; the
highest activity is with a propyl group. Thyroid glands weighing over 100
times normal weight have been observed in rats administered PTU (18). A
further increase in the length of the side chain results in decreased
activity. Thiourea exhibits only 1/8 to 1/10 activity of TU. Substitution by
-------
methyl groups of up to 3 hydrogens in thiourea does not significantly change
the goitrogenic activity. However, when the substituents on the thiourea
molecule are too large, activity is generally reduced or lost (7).
In humans, thiourea is as goitrogenic as thiouracil. Among the various
6-substituted thiouracils studied, only the methyl derivative was found more
active than the parent compound. Substitution with polar groups at position 6
greatly reduces the activity (19).
Toxicity. Most early toxicity screening studies on this chemical class
were performed in view of its use for the treatment of thyrotoxicosis and as
rodenticides. The toxicity data show that thioureas containing a benzene ring
are much more toxic than other thiourea derivatives (20, 21). For example,
phenylthiourea, administered either orally or by intraperitoneal injection,
exhibits high acute toxicity in rats, mice and rabbits. Most other thiourea
derivatives are relatively non-toxic in rats or mice; few have LD^Q value
lower than 100 mg/kg (Table II).
The manifestations of acute poisoning by thiourea and by its derivatives
are massive pulmonary edema, pleural effusion, and tracheobronchial inflamma-
tion (21, 28). Toxic doses of thiourea and several of its monosubstituted
analogs, including allylthiourea and phenylthiourea, also cause hyperglycemia
and depletion of liver glycogen in rats (24, 29). The disturbances in carbo-
hydrate metabolism are believed to be secondary to the pulmonary effects,which
are the direct cause of death in thiourea toxicity (29).
The side effects of the therapeutic use of TU and its analogs are well
established. Patients treated with TU, MTU, or PTU may develop skin rash,
headache, fever, edema, hepatic necrosis, aplastic anemia and agranulocytosis
(19, 30, 31). Allergic reactions, gastrointestinal disturbances and toxic
-------
Table II. Acute Toxicity of Thiocarbonyl Compounds
Compound
Thiourea
Allylthiourea
Trimethylthiourea
Diethylthiourea
Phenylthiourea
Dicyclohexylthiurea
Dithiobiurea
Ethylenethiourea (ETU)
Thiouracil (TU)
Methylthiouracil (MTU)
Thioacetamide (TAA)
Ethionamide
Species & Route
Rat , oral
i.p.
Mouse, oral
i.p.
Rat , oral
i.p.
Rat , oral
Mouse, oral
Rat , oral
Mouse , oral
i.p.
Rat , oral
i.p.
Mouse, oral
i.p.
Rabbit, oral
Rat, oral
Mouse, i.p.
Rat , oral
Rat , oral
Mouse, i.p.
Rat , oral
Mouse, i.p.
Mouse, oral
i.p.
LD5Q (mg/kg)
125
436
8,500
100
200
500
316
215
316
681
500
3
5
10
25
40
1,500
50
1,832
2,000
200
500
300
1,000
1,350
Reference
(22)
(23)
(22)
(22)
(21)
(24)
(22)
(22)
(22)
(22)
(22)
(21)
(24)
(22)
(22)
(25)
(21)
(22)
(26)
(21)
(22)
(21)
(22)
(27)
(22)
-------
hepatitis have been associated with the use of ethionaraide in the treatment of
tuberculosis (32).
Thioacetamide is a well-known liver necrogenic agent. In rats, toxic
doses of thioacetamide rapidly cause hepatic cell injury which is followed by
centrolobular necrosis (16, 33-35). The most striking early effects in the
hepatic cells are the doubling of nuclear volume, increased nucleolar and cell
volume, mitochondrial swelling, distention of the endoplasmic reticulum
cisternae, detachment of the ribosomes and polysomes from the rough endo-
plasmic reticulura and loss of cytoplasmic azurophilia (34, 36, 37). Chronic
administration to rats produced cirrhosis of the liver characterized by
irregular fibrosis, hyperplasia of the bile ducts, fatty infiltration and
nodular regeneration of the parenchymal cells (33, 38).
Mutagenicity. Both thiourea and thioacetamide were selected for muta-
genesis assay by the U.S. National Cancer Institute for determining the extent
of correlation between carcinogenesis and mutagenesis in several standardized
assay systems (39). In agreement with earlier results of McCann £t_ jil_. (40),
the National Cancer Institute results (41, 42) show that both thiourea and
thioacetamide are not mutagenic in various strains of Salmonella typhimurium,
assayed in the presence and absence of the S-9 mix. However, in the host-
mediated assay, both thiourea and thioacetamide at doses of 125 mg/kg produced
significant increases in the mutation frequencies observed with j^. typhimurium
TA1530 and TA1538, indicating that the two compounds are probably weak muta-
gens (43). Thioacetamide, but not thiourea, is mutagenic in Saccharomyces
cerevisiae D3 (44) and Drosophila melanogaster (45). Both compounds displayed
weak mutagenicity in J3. cerevisiae D6 (46) and a forward mutation system,
galr, in S_. typhinurium (47). Recently, Yamaguchi (48) has also demonstrated
moderate rautagenicity of thiourea in S. typhimurium TA100, a tester strain
-------
previously shown to have no response to the compound (40, 41). Thioacetamide,
on the other hand, did not cause detectable unscheduled DNA synthesis in rat
v
liver (49) or in a primary culture of rat hepatocytes (50).
In a study on the validity of various carcinogen screening techniques,
182 compounds were examined by several short-term assays in Japan between 1973
and 1978 (51). Both thiourea and thioacetamide exhibited negative results in
strains TA98 and TA100 of S. typhimurium, in hamster lung fibroblast cells and
in rat bone-marrow cells (the latter assays tested for chromosomal aberra-
tions). Thioacetamide, but not thiourea, was mutagenic in Bacillus subtilis;
thiourea, but not thioacetamide, however, caused mutations in silk worms.
Extensive research on the mutagenic effects of ethylenethiourea (ETU) has
also been conducted over the past few years, mainly because of its association
with ethylene bis-dithiocarbamate fungicides. The weak mutagenicity of ETU
was demonstrated in several microbial systems: _S^. typhimurium TA98, TA100,
TA1538 (52), TA1535 (52, 53), TA1530 (54, 55), M^G-46 (56), and _S.
cerevisiae D6 (46). Cytogenetic analysis also indicates the weak mutagenicity
of ETU. A dose-dependent increase in the frequency of chromosomal aberrations
was observed in bone marrow cells of mice administered ETU at single or
repeated doses (57). Moreover, Herichova (58) found chromosomal aberrations
and induction of micronuclei in the root tips of Vicia faba treated with ETU.
A number of laboratories were unable to confirm, however, some of the
above findings and failed to detect mutagenic activity of ETU in several other
assays systems. Saffiotti et al. (59) reported that ETU produced no rever-
tants in TA98 and TA100, the two S_. typhimurium tester strains which respond
respectively to frame-shift and to base substitution mutagens. Also, Shirasu
et al. (60) observed no mutagenic activity of ETU in the S. typhimurium
-------
TA1536-8 series, in Escherichia coll WP2 and in Bacillus subtilis. Further-
more, negative results were obtained in mouse dominant lethal tests (54, 57,
61, 62), as well as in cytogenetic studies on cultured cells (62-64) and in
whole animals (54, 55, 57, 62, 63, 65). The frequencies of sex-linked reces-
sive lethals and dominant lethals in Drosophila treated with ETU were also not
significantly higher than those of the controls (66).
Studies on the mutagenicity of other thiocarbonyls are more limited.
Allylthiourea and phenylthiourea displayed moderate mutagenic activities when
tested on _S_. typhimurium TA100 without metabolic activation (48). Assays for
mutagenicity based on the reversion from streptomycin dependence to indepen-
dence in strain Sd-4-73 of E. coli showed that neither TU, MTU, nor PTU were
mutagenic (67). The incidence of thyroid cells containing chromosomal aberra-
tions was also not increased by the administration of PTU to rats for 15 weeks
(68). However, PTU was reported to induce "petite" mutations in _S_. cerevisiae
D6 (46). Testing for mutagenic effects of ethionamide by using _S_. typhimurium
TA98, TA100, JJ. subtilis, Chinese hamster lung fibroblasts (testing for
chromosomal aberrations), human embryo fibroblasts (testing for chromosomal
aberrations and sister chromatid exchanges), bone marrow cells (testing for
chromosomal aberrations), and silk worms, Kawachi ^£^1_. (51) obtained posi-
tive results only with Chinese hamster lung fibroblasts.
Teratogenicity. Since the advent of thiourea and TU, sporadic goiter and
hypothyroidism in fetuses and newborn infants due to the use of these drugs
and their derivatives (e.g., PTU and MTU) by mothers during pregnancy have
been noted (69-76). The effects on infants are transient and usually disap-
pear shortly after birth; however, mental development may be subsequently
retarded in some congenitally hypothyroid children (70, 77). Administration
of thiourea, TU and their derivatives to pregnant rats has been found to cause
8
-------
thyroid hyperplasia and abnormal body development in the offspring (78-83).
Similar effects were observed in weanling rats whose mothers had been treated
with thiourea or TU during the period of lactation (82, 84). Various investi-
gators have shown that both thiourea and TU pass through the placenta to the
fetus (79, 85-91) and are secreted in the milk of humans (92) and animals (85,
90). Presumably, transient fetal or neonatal hypothyroidism results from the
inhibition of thyroxine biosynthesis in the fetus by the drugs, which then
leads to increased production of fetal thyroid-stimulating hormone (TSH) and,
thus, to thyroid hyperplasia. Recently, it has also been suggested that the
effects might be due to the placental transfer of the thyroid-stimulating
immunoglobulin from mother to fetus (75, 76).
Teratogenicity studies in the last decade, carried odt principally by
Khera and his associates, have established that ethylenethiourea (ETU) is a
potent teratogen in the rat (26, 93-98). A wide spectrum of anomalies in the
central nervous, urogenital and skeletal systems as well as in other organs
have been demonstrated in the offspring of rats administered sublethal doses
of ETU during pregnancy. Among the teratogenic effects observed are hydro-
cephalus, microphthalmia, meningoencephalocele, meningorrhagia, meningorrhea,
obliterated neural canal, cleft palate, kyphosis, kinky tail and various
defects of the digits. Studies by administration of single oral doses of ETU
to rats at different gestation stages have revealed that the type of anomalies
and organs affected varies with the treatment stages and coincides with the
onset of organogenesis (94, 98). Ethylenethiourea is one of the most potent
antithyroid agents known (99). Its teratogenic effects in rats are believed
to be due neither to direct action on the affected organs (100) nor to altera-
tion of the maternal thyroid activity, but are caused by direct effect on the
fetal thyroid function (26, 96). A study with various chemicals structurally
-------
similar to ETU established that the 2-ciercaptoimidazolidine structure was
essential for producing the teratogenic effects (101).
Ethylenethiourea has also been reported to be teratogenic in hamsters
(102, 103). However, no prominent evidence was found for teratogenicity of
ETU in rabbits (93), mice (104, 105) or cats (106). Results from pharmaco-
kinetic studies in mice by Ruddick et_£l_. (104, 105) led to the suggestion
that a faster clearance of ETU from the fetal tissues and a more rapid meta-
bolism of ETU to non-teratogenic compounds might be the cause for the lack of
teratogenicity of ETU in this species.
Skeletal malformations have been observed in the offspring of rats admi-
nistered the anti-mycobacterial tuberculostatic drug, ethionamide, on days 6
to 14 of pregnancy (cited in ref. 107). The drug was not found, however, to
exert any significant adverse effect on the development of the human fetus
(108, 109).
5.2.2.8.3 Carcinogenicity.
5.2.2.8.3.1 OVERVIEW.
Spontaneous tumors of the thyroid in rodents are rare (110, 111).
Experimental thyroid tumors have been induced in rats and mice by low-iodine
diet, radioactive iodine, chemicals, ionizing radiation, or by partial
thyroidectomy (reviewed in 112). Thiourea and related compounds, which are
goitrogenic, also induce thyroid adenomas and carcinomas and/or tumors of
other organs after prolonged administration to laboratory animals (Table
III). Hence, thiourea-type goitrogens represent valuable research tools for
the biochemistry and physiology of the thyroid gland, as well as for the
10
-------
i U I
Table III. Carcinogenicit
Thiocarbonyl Compounds
Compound
Thiourea
Allylthiourea
N,N'-Diethylthiourea
Trimethylthioureab
Phenylthiourea
N,N'-Dicyclohexyl-
thiourea
Dithiobiurea
-
Species and Strain
Rat, Wistar, albino
Rat, albino
Rat, Hebrew
University strain
Mouse, A, C57, I,
hybrids, or C3H
Mouse, C3H
Mouse, AKR
Fish, rainbow trout
Rat, Wistar
Rat, Fischer 344
Mouse, B6C3F1
Rat, Fischer 344
Mouse, B6C3F1
Rat, Fischer 344
Mouse, B6C3F1
Rat, Fischer 344
Mouse, B6C3F1
Rat, Fischer 344
Mouse, B6C3F1
Route
Oral
Oral
i.p. and oral
oral
oral
oral
oral
oral
oral
oral
oral
oral
oral
oral
oral
oral
oral
oral
Principal Organs
Affected
Thyroid gland
Liver
Face, eyelid, ear, nose
None
Thyroid gland8
Skull
Liver
Thyroid gland
Thyroid gland
None
Thyroid gland
None
None
None
None
None
None
Nonec
Reference
(113, 114)
(115, 116)
(117, 118)
(119-122)
(123)
(124)
(125)
(14, 126)
(127)
(127)
(128)
(128)
(129)
(129)
(130)
(130)
(131)
(131)
-------
Table III. Carcinogeniclt^Wt Thiocarbonyl Compounds
(continued)
of 3 pp.
Compound
Ethylenethiourea (ETU)
Thiouracil (TU)
Methylthiouracil (MTU)
Species and Strain
Rat, Charles River CD
Rat, —
Mouse, B6C3FJ or B6AKFJ
Hamster, —
Rat, Stanford, Sherman,
or Sprague-Dawley
Mouse, C3H or (C57 X CBA)F1
Mouse, TM
Rat, Wistar, Lister,
albino, or Long-Evans
Rat, albino
Mouse, C3H
Mouse, NZO/B1 or C57d
Mouse , dd
Hamster, Syrian
Hamster, —
Route
oral
oral
oral
oral
oral
oral
oral
oral
oral
oral
oral
oral
oral
oral
Principal Organs
Affected
Thyroid gland
Thyroid gland
Liver, hematopoietic
system
None
Thyroid gland
Liver
None
Thyroid gland
Kidney
Thyroid, liver
Thyroid gland
Pituitary gland
Thyroid gland
Thyroid gland
Reference
(132-135)
(136)
(137)
(136)
(138-141, cited in
ref. 12)
(142-144)
(143)
(145-154)
(155)
(156)
(157, 158)
(159)
(160)
(161)
-------
Table III. Carcinogenicity^|
(continlR
Thiocarbonyl Compounds
'd)
Compound
Species and Strain
Route
Principal Organs
Affected
Reference
Propylthiouracil (PTU) Rat, Wistar, albino,
Long-Evans, or unspecified
Mouse, A
Mouse, C57B1
Hamster, Syrian golden
Guinea pig, —
Rat, albino or Wistar
Mouse, Swiss
Hamster, —
Thioacetamide (TAA)
Ethionamide
Rat, Fischer 344
Mouse, B6C3F1
Mouse, BALB/c/Cb/Se
oral
oral
oral
oral
oral
oral
oral
oral
oral
oral
oral
Thyroid gland
Thyroid and pituitary
glands
Pituitary gland
Thyroid gland
Thyroid gland
Liver, bile duct
Liver
None
None
None
Thyroid gland
(162-168)
(169)
(170)
(171)
(172)
(116, 173, 179)
(180)
(181)
(182)
(182)
(cited in ref. 183)
aOne thyroid adenoma in 1 of 25 C3H male mice that have been castrated.
Administered as a mixture of 85% trimethylthiourea and 15% dimethylthiourea.
cThere was some suggestive evidence that dithiobiurea may induce hepatocellular carcinomas in female mice.
Kept on low-iodine diet.
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elucidation of the mechanism of thyroid gland carcinogenesis. Since humans
are exposed to many of these goitrogens, as pesticide residues or drugs for
the treatment of thyrotoxicosis, their carcinogenicity must be adequately
tested and'their potential hazard to humans assessed. Over the past decades,
a large volume of literature on experimental thyroid tumorigenesis has accumu-
lated. Recently, bioassays of several thiourea related compounds for possible
carcinogenicity have been conducted in rats and mice by the U.S. National
Cancer Institute. Table III summarizes some of the carcinogenicity studies of
thiourea and its analogs, discussed in some detail later in this section.
The development, morphology, histology and metabolism of thyroid gland
tumors have been the subject of several reviews and monographs (112, 155, 183-
187). In both rats and mice, different agents induce a histological variety
of experimental thyroid tumors. Tumor development requires a long induction
period and is often preceded by a hyperplastic stage. Following thyroid
hyperplasia, local areas of proliferating altered cells develop; it is in
these areas that the adenomas and/or carcinomas emerge. It is difficult to
differentiate morphologically between hyperplastic nodules and benign and
malignant forms of thyroid neoplasm. In the hyperplastic state, both the
follicular and parafollicular epithelial cells undergo great variations of
hyperplastic modification. In most cases, these cells become cylindrical with
basophilic cytoplasm and a pale vesicular nucleus. Dalton ji£ jil^. (188)
distinguished two types of hyperplastic modifications: "Type I" represents
cells which are larger in size than normal cells; "Type II" refers to cells
that are similar in size to the normal surrounding cells but have more baso-
philic nuclei. In the adenomas, both cytoplasmic and nuclear changes are
evident and the cellular distribution is more irregular than in hyperplasia
(158). Induced thyroid adenomas and carcinomas are usually papillary or
ii
-------
follicular. Transplantability of these tumors is generally low, which presum-
ably contributes to their low grade of malignancy. However, epidermoid carci-
nomas, which are more malignant than the papillary or follicular tumors, have
also been'observed (183). Thyroid tumors induced by thiourea goitrogens
appear to be more easily transplanted in mice than in rats (112).
5.2.2.8.3.2 THIOUREA AND RELATED COMPOUNDS.
Thiourea. In 1945, Griesbach _et^_al_. (189) reported the induction of
thyroid adenomas in rats by the long-term ingestion of brassica seeds, which
are goitrogenic, in the diet. This observation stimulated a number of inves-
tigations on the carcinogenicity of thiourea and several goitrogenic thiour-
acils. As was expected from the results of the brassica seed experiment,
Purves and Griesbach (113) observed thyroid tumors in most of the male Wistar
rats receiving 0.25% thiourea in the drinking water for a period of nearly two
years. Tumors in 2 of the 30 tumor-bearing animals were considered to be
malignant; the malignant tumors metastasised to the lung and spread invasively
into the blood vessels. This initial study was subsequently extended to
include rats of a local (Norwegian) albino strain of both sexes (114). The
results indicate that there are no significant sex- or strain-dependence in
susceptibility of thyroid tumorigenesis. After a year or more, thyroid
adenomas were found in 22 of 25 rats (88%) given 0.25% thiourea in the
drinking water. Of 13 animals that survived more than 20 months, 7 had
thyroid tumors which showed criteria of malignancy. In addition, 3 thyroid
tumors resembling the "foetal adenoma" of human pathology were encountered.
The observation that the thyroid was the only organ in which neoplasia
developed led Purves and Griesbach (114) to the conclusion that the thyroid
tumors induced are not the result of a direct carcinogenic action of thiourea
but are the consequences of prolonged stimulation by thyrotropic hormone.
12
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In contrast to the above findings, however, a number of reports have
described the induction of tumors by thiourea in other organs. Fitzhugh and
associates (115,, 116) showed that thiourea, administered orally at dose levels
of 0.1, 0.05, 0.025 and 0.01% in the diet for about 2 years, induced hepatic
cell adenomas, without liver cirrhosis, in 14 of 29 albino rats. Levels of
1.0, 0.5 and 0.25% produced marked hyperplasia of the thyroid; however, no
carcinogenic effect of thiourea was seen in these groups due to early death of
all animals. None of the 18 controls surviving the 2-year feeding period
developed tumors. Rosin and Rechmilewitz (117) observed malignant tumors of
the face in 5 of 6 random-bred (Hebrew University strain) rats treated with
thiourea for over 1 year. The treatment consisted of weekly intraperitoneal
injections of 10% aqueous solution of thiourea, in doses of 3.0, 4.0 and 4.0
ml on 3 consecutive days, for a period of 6 months, followed by continued
administration of 0.2% thiourea in the drinking water. Subsequent studies
(118) confirmed the above findings and, in addition, observed tumors on the
eyelids and near the ear ducts in 10 of 12 Hebrew University strain rats
surviving the same treatments for over 1 year. Eighteen out of 19 rats which
received 0.2% thiourea in the drinking water for the entire experimental
period also developed neoplastic lesions after 26 months: 1 had a myxomatous
tumor on the nose and 17 had epidermoid carcinomas near the ears and the orbit
(118).
The chronic effects of thiourea on the microscopic structure of the
thyroid in strains A, I, C57, and hybrid mice were investigated by Gorbman
(119, 120). Administration of 0.2% thiourea in the diet induced hyperplasia
and follicular cysts in the thyroid after 200-300 days of treatment. In 7 of
the 31 strain A mice so treated for more than 500 days, "hyperplastic thyroid
tissue metastases" in the lung were also noted. However, neither the thyroid
13
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changes nor the pulmonary metastases in these mice were considered by the
author to be of neoplastic nature, since all the lesions regressed after the
resumption of a normal diet. These findings were confirmed by Dalton et al.
(121, 122); moreover, these authors found no histological evidence for neo-
plasms in the thyroid of C3H mice treated with 0.25% thiourea in the diet for
up to 63 weeks, despite pulmonary metastasis of thyroid tissue. Casas and
Koppisch (123), on the other hand, observed a thyroid adenoma in 1 of 25 C3H
mice that had been castrated, and received thiourea at the dose level of 0.3%
in the diet for 7 months. Recently,.a significantly increased incidence of
intracranial bone tumor was found in AKR mice treated with a high dose of
thiourea (5g/kg) in the diet (124). Thiourea has also been shown to induce
hepatomas in rainbow trout (125).
Allylthiourea. Whereas 2-acetylaminofluorene did not affect the normal
thyroid tissue, combined treatment of 2-acetylaminofluorene and allylthiourea
induce benign and malignant tumors of the thyroid in rats (13, 145). Subse-
quent studies showed that thyroid neoplasms could be induced in rats given
allylthiourea alone, although the tumorigenic effect was not as pronounced as
with 2-acetylaminofluorene and allylthiourea combined. In one experiment, 5
young female Wistar rats were given 8 mg allylthiourea daily in the diet.
After 9-14 weeks of treatment, 2 of the rats developed a single adenoma of the
thyroid. In another study, a diet containing allylthiourea was fed to 5 male
and 5 female Wistar rats born to and nursed by a mother bearing mammary tumors
induced by 2-acetylaminofluorene. Single adenomas were found in 2 of the 5
males after 25 weeks and 3 of the 5 females after 19 weeks of stimulation by
the goitrogen (14).
Ethylenethiourea (ETU). Data on ETU-induced thyroid carcinogenesis
establish that the compound is clearly carcinogenic in both rats, and mice.
14
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A 1972 preliminary communication by Ulland and associates (132) reported
the dose-related induction of thyroid tumors in Charles River CD rats admini-
stered two doses (175 and 350 ppm) of ETU in the diet for 18 months and
observed for an additional 6 months. At the low dose level, 3 males and 3
females in groups of 26 animals developed thyroid carcinomas; at the high dose
level, 17 males and 8 females had thyroid cancers. In addition, hyperplastic
goiter and thyroid solid-cell adenoma occurred in a few other animals at both
dose levels. The final results of this study have recently been published by
Weisburger et al. (133). The incidences of follicular cell carcinoma of the
thyroid were: 58% in high-dose males, 23% in high-dose females, and 8% in
low-dose groups of either sex. In addition, 3 papillary carcinomas of the
thyroid and 4 hyperplastic nodules of the liver were seen. Ho such neoplastic
lesions were detected in matched or pooled controls.
Similarly, Graham et_^l^ (134, 135) reported that there was a significant
dose-related increase in the thyroid carcinoma incidence in Charles River rats
administered ETU in the diet for 1 or 2 years. After 1-year administration of
ETU , 60% of the rats at the dietary dose of 500 ppm developed thyroid carci-
nomas compared to 0% in the controls (134); in the 2-year study, 23% and 89%
of the rats receiving levels of 250 and 500 ppm, respectively, had thyroid
carcinomas or adenocarcinomas compared to 3% in the controls (135). Adtvtally,
the carcinogenicity of ETU toward the thyroid was already detectable at the
125 ppm dietary level; there was no detectable tumorigenicity at the 25 and 5
ppm levels.
In a life-time carcinogenesis study in rats and hamsters of unspecified
strains, ETU was given at doses of 0, 5, 17, 60 and 200 mg/kg in the diet
(136). The compound was found to be carcinogenic for male and female rats at
60 mg/kg level and 200 mg/kg level, respectively. Again, the target tissue
15
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affected was the thyroid. No tumorigenic effect was observed in hamsters even
at the highest dose level.
Innes et al. (137) have investigated the carcinogenicity of ETU in mice
of 2 hybrid strains (C57B1/6 X C3H/Anf; C57B1/6 X AKR). At 7 days of age, 18
males and females of each strain were given a daily dose of 215 mg/kg ETU,
suspended in 0.5% gelatin, by gavage for 3 weeks. After this initial treat-
ment, the mice were fed ETU at a concentration of 646 ppm in the diet until
necropsied at 82-83 weeks of the experiment. Hepatomas were seen in 14 of the
16 male and in all the 18 female C57B1/6 X C3H/Anf mice versus 8 of the 79
males and none of 87 females in the controls. In the C57B1/6 X AKR mice, 18
of the 18 males and 9 of the 16 females developed liver tumors; the respective
incidences in the controls were: 5/90 (male) and 1/82 (female). Slightly
increased incidence of lymphoma (3/18, male; 4/16, female) was also observed
in mice treated with ETU, compared to the controls (1/90, male; 4/82, female).
Other Thiourea Derivatives. Five thiourea derivatives, diethylthiourea
(127), trimethylthiourea (128), phenylthiourea (129), dicyclohexylthiourea
(130), and dithiobiurea (131) have recently been tested for possible carcino-
genicity by the U.S. National Cancer Institute. The compounds were admini-
stered to groups of 50 Fischer 344 rats and B6C3F, mice at two dietary levels
(approximately 1/2 maximum tolerated dose and maximum tolerated dose) for 77-
109 weeks and observed for an additional period of 1-31 weeks. In the rat,
diethylthiourea (125 or 250 ppm for 103 weeks) was carcinogenic, causing
follicular-cell carcinomas of the thyroid in males (controls 0/18, low-dose
1/45, high dose 11/48) and follicular-cell adenomas or carcinomas of the
thyroid in females (controls 0/18, low-dose 4/46, high dose 17/46). The
increases in tumor incidence were statistically significant in the high-dose
groups. Trimethylthiourea (administered as a mixture of 85% trinethylthiourea
16
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and 15% dimethylthiourea at 250 or 500 ppm for 77 weeks) was also signifi-
cantly carcinogenic (at the high dose) in females, inducing follicular-cell
V
carcinomas of the thyroid (controls 0/17, low-dose 1/38, high-dose 14/47) but
inactive in males. Phenylthiourea (60 or 120 ppm for 78 weeks) and dithio-
biurea (6,000 or 12,000 ppm for 78 weeks) were not carcinogenic to rats at the
doses administered; however, the mortality and weight gain data indicated that
the high doses administered were not sufficiently close to the maximum
tolerated dose. Dicyclohexylthiourea (25,000 or 50,000 ppm for 109 weeks) was
also not carcinogenic in rats despite the fact that the compound induced an
increased incidence of hyperplasia of the follicular cells of the thyroid. In
the mouse, none of the five thiourea derivatives exhibited any significant
carcinogenic effect. There was some suggestive evidence that dithiobiurea
increases the incidence of hepatocellular carcinomas in female mice (controls
2/29, low-dose 8/47, high-dose 9/48); however, the evidence was not considered
sufficient to establish the carcinogenicity of the compound. As in the rat,
dicyclohexylthiourea increased the incidence of hyperplasia of follicular
cells of the thyroid in the mouse.
5.2.2.8.3.3 THIOURACIL AND RELATED COMPOUNDS.
Thiouracil. Several investigators noted that thyroid tumorigenesis by TU
in both rats and mice requires extremely long periods of treatment; the emer-
gence of the tumors is preceded by diffuse hyperplasia and other preneoplastic
changes. Following feeding 0.1% TU in the diet to 111 Stanford albino rats of
both sexes for up to 45 weeks, Laqueur (138) observed diffuse and nodular
thyroid hyperplasia (which was regarded as a benign lesion) in 33% of the
animals. Similar observations were made by Money and Rawson (cited in ref.
112) and by Clausen (141) in Sprague-Dawley rats; adenoiaatous structures and
17
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other types of changes described as neoplastic were found in the thyroid of
the majority of the rats administered TU for 18 to 24 months. Unequivocal
thyroid carcinoma, however, was seen only by Paschkis ^t^ jil_. (139) in 1 of 20
Sherman strain rats following treatment with 0.05 or 0.1% TU in the drinking
water for 884 days. Eleven of the 20 rats so treated over 245 days were
reported to have borne thyroid adenomas. The incidences of thyroid carcinomas
and adenomas were enhanced to 15% and 94%, respectively, when the rats were
also administered 0.03% 2-acetylaminofluorene in the diet (139). A study of
Money .££_£!_• (140) in Sprague-Dawley rats showed that by the 500th day of
treatment with 0.1% TU in the drinking water, all 81 animals developed thyroid
adenomas.
As in many studies with rats, no histological evidence of neoplasia was
found in the thyroid glands of mice ingesting TU at doses of 0.25-0.5% for a
year or more, despite marked hyperplasia and extensive cellular alterations
(120, 122, 188). However, Morris £t^£l_. (190) induced malignant autonomous
thyroid carcinomas by means of serial transplantation of thyroid tissues to
mice ingesting TU. The experiment was so designed as to provide conditions
under which the thyroid tissue was exposed to TU beyond the lifetime of any
one mouse. The study also illustrated the point that a long period of treat-
ment is essential to the induction of thyroid neoplasms in mice by TU.
In studies on the effect of TU on adrenals of castrated C3H mice, Casas
(142) found that almost 100% of the nice fed 0.3% TU in the diet for 10 months
or more bore hepatomas. In subsequent studies with non-castrated C3H mice, a
significantly high incidence of hepatomas was found in mice of both sexes
administered a diet containing 0.3% TU for 18 months (12/13 in males and 14/16
in females) (143). When inbred TM strain mice were used, however, no hepa-
tomas were seen in 44 TU-treated mice and 40 controls (143). The results led
18
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the author to suggest that a genetic predisposition must exist for mice to be
susceptible to hepatocarcinogenesis by TU. Hepatonas were also reported to
occur in 6 of 21 castrated C57 X CBA mice fed 0.2% TU for 11-29 months (144).
Methylthiouracil. The production of thyroid neoplasms in rats, mice and
hamsters following oral administration of methylthiouracil (MTU) has been
repeatedly demonstrated by various investigators.
Hall (145) was the first to observe single adenomas of the thyroid in 3
of 12 Wistar rats receiving 0.01% MTU in the drinking water for 21-42 weeks.
In a subsequent study, the observation period was extended to 94 weeks.
Malignant tumors of the thyroid were found in 2 of 7 rats after 78 or more
weeks of continuous treatment of MTU (146).
A series of studies were conducted by Doniach (147-149) using Lister
rats. Methylthiouracil was given as a saturated solution in drinking water,
prepared by suspending 1 g of the compound in each liter of tap water. In one
experiment, solid and follicle adenomas of the thyroid were present in 10 of
16 rats sacrificed after 12-16 months (147). In two other studies, high
incidences (19/20, 10/14) of thyroid adenomas were also found in rats killed
after 15 months (148, 149).
Of 30 hybrid albino rats given 20 mg MTU in 0.2 ml. water by gavage (5
times a week for up to 100 weeks) 22 animals bore a total of 28 thyroid tumors
(150). Nine of the tumors were malignant. In 24 Long-Evans rats fed 2.5 mg
MTU in a low-iodine diet for 24-33 months, 8 had malignant tumors of the
thyroid (154). Normal thyroid without any signs of hyperplasia was seen only
in 2 rats. In 31 control rats given the low-iodine diet alone, none developed
tumors; 25 of these thyroids were normal.
-------
reported. Castration or thyroidectomy was shown to result in increased tumor
incidence as well as shortened latent period of the tumorigenesis (159).
Only few studies have been conducted in hamsters on thyroid carcino-
genesis by goitrogenic drugs. Thyroid adenomas were noted in hamsters given
MTU at dose levels of 15 mg/100 g body weight (160) or 0.2% in drinking water
(161) after 4-5 months of treatment. By the end of 12 months, 58% of HTU-
treated hamsters developed thyroid adenomas of the papilliferous type; no such
tumors were found in the controls (161). In animals simultaneously admini-
stered MTU and •**!, greater tumor incidence and earlier appearance of
adenomas and carcinomas were noted. On the basis of the high tumor incidence
and short latent period for the development of thyroid neoplasia, it was
suggested that hamsters might also be suitable models for research on thyroid
tumor induction (161).
Propylthiouracil. In view of the turaorigenic effects of other goitro-
genic compounds on the thyroid, the production of thyroid neoplasms in rodents
by prolonged administration of propylthiouracil (PTU) is not unexpected.
Indeed, PTU has been shown to induce thyroid adenomas and carcinomas in the
rat, mouse, hamster and guinea pig.
When 0.2% PTU was administered in the diet to 48 female Wistar rats aged
2-15 months, single adenomas were observed in 24 animals following 2-14 months
of treatment (163). The study indicates that the age of the rats is an impor-
tant factor in the development of thyroid tumors induced by PTU. Older rats
developed thyroid tumors with higher frequency and shorter latent periods than
younger rats.
The induction of thyroid tumors in Wistar strain rats by PTU has been
studied by Willis (162). Propylthiouracil was given in the drinking water for
21
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up to 18 months. The initial dose (0.2%) was reduced to 0.1% at 3 months, to
0.05% at 6 months and to 0.025% at 12 months. In 48 rats surviving for 18
months, 31 developed adenomas and 7 had carcinomas of the thyroid. When PTU
was given'to rats in the drinking water at dose levels adjusted to give an
intake equivalent to human doses (7 mg/kg body weight/day initially, then
reduced to 1 mg/kg/day over a period of 3 months), high incidences of adenomas
(50%) or carcinomas (17%) of the thyroid were observed in 18 rats which
survived to the termination of the experiment at 18 months. Sellers et al.
(164) have detected thyroid tumors in Wistar rats administered low doses of
PTU (0.02%) alone in the diet or treated together with 0.02% sodium iodide or
0.02% dry thyroid powder for 15 months.
When 4 male A strain mice were fed 0.8% PTU in the diet for 18 months,
all were found to have carcinomas of the thyroid and 3 had chromophobe
adenomas of the pituitary gland (169). Multiple adenomas of the pituitary
were also reported to occur in C57B1 mice maintained on a diet containing PTU
levels of 1% or 1.2% for 17 months; the respective tumor incidences were 62%
and 72% (170).
In 102 Syrian golden hamsters receiving 0.2% of PTU in the drinking water
for about 14 months, 32 developed malignant lesions of the thyroid, among
which 10 had netastasizing thyroid neoplasms (171).
Thyroid adenomas were also seen in 3 of 20 guinea pigs given 0.03% PTU in
the drinking water for about 15 months. No tumors of the thyroid occurred in
10 untreated controls (172).
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5.2.2.8.3.4 THIOACETAMIDE AND ETHIONAMIDE.
Thioacetamide. Fitzhugh and Nelson (115) studied the chronic toxicity of
thioacetamide (TAA) in rats and was the first to suggest that TAA night be
hepatocarcinogenic. In a 2-year toxicity test, groups of 10 albino rats were
administered TAA at dietary levels of 0.1, 0.05, 0.025, 0.01, and 0.005%. At
0.01 and 0.005% dose levels, a hepatic cell adenoma in 1 of the 6 survivors
and at 0.05%, a hepatocellular carcinoma was observed.
The development and morphology of the liver tumors in rats following
prolonged feeding of TAA were later described in detail by Gupta (175, 176).
One hundred fifty Wistar rats of both sexes were fed a diet containing 0.032%
TAA. In 36 animals killed between 9-23 weeks, 18 were found to have bile duct
tumors (175). Three hepatomas, one bile duct adenoma, and one hepatoma-
cholangiocarcinoma with metastasis to the ovaries were found in 4 of 5 rats
which ingested TAA for more than 47 weeks (176). Among 32 male rats which
received similar treatment with TAA and survived for mpre than 16 weeks, 29
developed malignant tumors of the liver (173).
Recently, Dasgupta _et_£l_. (179) have detected 4 cases of hepatocellular
carcinoma in 56 Wistar rats fed a diet containing 0.04% TAA. The tumors
appeared after 300 days of TAA treatment and 2 of them metastasized to the
lung.
Several other investigators (174, 177, 178) contributed to the evidence
that TAA is a weak hepatocarcinogeh toward rats. Moreover, Anghileri (178)
and Shetty^jil^ (177) observed in the livers of rats biochemical alterations
usually associated with hepatocarcinogenesis.
Swiss mice are particularly susceptible to the carcinogenic effect of
TAA. Among 47 Swiss mice of both sexes maintained on a diet containing 0.03%
23
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TAA, 6 of 6 males and 6 of 7 females killed 15 months after the beginning of
treatment developed hepatocarcinomas. Some of the tumors were successfully
transplanted in mice of the same strain (180). The progressive morphological,
histological, and biochemical changes in the liver of mice during TAA-induced
hepatocarcinogenesis have been described in some detail (180, 192).
Hamsters appear to be refractory to the carcinogenic effects of TAA. No
liver tumors were found in 10 male and 10 female Syrian golden hamsters given
25 mg TAA by gavage once weekly for 30 weeks (181). The few tumors occuring
in various organs were not considered by the authors to be related to treat-
ment with TAA.
Ethionamide. The tuberculostatic drug, ethionamide, was among the chemi-
cals selected for carcinogenicity bioassay by the U.S. National Cancer
Institute (182). The compound was fed to 35 Fischer 344 rats and 35 B6C3F1
mice of each sex 5 days/week for 78 weeks at the following doses:. 1,500 or
3,000 ppm to rats and 1,000 or 2,000 ppm to mice. The incidences of tumors in
various organs were found not to be significantly different from those of the
untreated controls. However, in a recent review, Biancifiori (183) stated
that papillary and epidermoid carcinomas of the thyroid are obtained in mice
by the administration of ethionamide.
5.2.2.8.3.5 MODIFICATION OF CARCINOGENESIS.
A number of exogenous chemical agents are capable of modifying the
carcinogenicity of thiocarbonyl compounds. Conversely, several thiocarbonyl
compounds have been shown to suppress the spontaneous incidence of mammary
tumors in rodents and inhibit the carcinogenicity of other chemicals.
Examples of such interactions are outlined below.
24
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Bielschowsky (13, 14) reported that combined treatment of 2-acetylamino-
fluorene and allylthiourea induces a significant number of thyroid tumors in
rats. Allylthiourea alone is also carcinogenic, but its effect is much less
pronounced than that of the combined treatment. 2-Acetylaminofluorene alone
has no discernible effect on the thyroid. The results suggest that 2-acetyl-
aminofluorene may potentiate the carcinogenic effect of allylthiourea toward
the thyroid.
As may be expected from the goitrogenic activity of low-iodine diet, the
carcinogenicity of thiocarbonyl compounds toward the thyroid may be poten-
tiated by low-iodine diets or by the administration of radioactive iodide.
Israel and Ellis (158) showed that C57 mice fed a stock diet and given 0.05%
MTU in the drinking water for 480 days do not develop thyroid tumors despite
marked hyperplasia. However, of the 25 MTU-treated mice that were kept on a
low-iodine diet, 11 were found to bear papillary adenomas and one had an
adenocarcinoma of the thyroid. Jemec (156) reported that C3H mice given 0.2-
0.5% MTU in an iodine-poor diet developed a significantly higher incidence of
thyroid tumors (30.7%) than those given 0.1% MTU in drinking water and kept
on an iodine-rich diet (1.2%). The thyroid tumor incidence in the iodine-poor
and iodine-rich controls were 0.7% and 0%, respectively. The results are
suggestive of potentiation of MTU by low-iodine diet; however, it is not known
to what extent the potentiation may have been due to the different dose levels
of MTU used in the iodine-poor and iodine-rich diets.
It is interesting to note that although high-iodine diet may inhibit the
carcinogenic effect of thiocarbonyl compounds toward the thyroid, potassium
iodide may potentiate the carcinogenicity of PTU if the two agents are given
alternatively to produce hyperplasia and involution in repeated cycles.
Zimmerman et_ ai_. (166) showed that in a group of 15 albino rats given 0.1% PTU
25
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in the drinking water for one year, four displayed single adenomas of the
thyroid. However, when rats were maintained alternatively on PTU to produce
hyperplasia, and on potassium iodide (0.01% to involute the thyroid), a much
higher incidence (17/29) of thyroid tumors was encountered. It was hypothe-
sized that nodular goiter is the result of repeated cycles of thyroid hyper-
plasia and involution; apparently, the same treatment also favors tumori-
genesis.
The investigation of Willis (162) indicates that 131I in conjunction with
PTU increases the incidence of thyroid carcinomas and shortens the latent
period. The combined effect of PTU and radioactive iodine was also investi-
gated by Lindsay et al. (167) using weanling male Long-Evans rats. Both PTU
1 O 1
and I appear to play a part in the initiation and promotion of thyroid
carcinogenesis and their effects are additive. Groups of rats were given a
single intraperitoneal injection of -^1 and/or 0.1% PTU in the diet for a.
year. Thyroid adenomas occurred in about 10% of the rats injected with ^Ij;
and in 48% of the rats fed the PTU-containing diet. The incidence of thyroid
101
tumors in the rats treated with the combination of I and PTU was, on the
other hand, 65%. Al-Hindawi et_jil^ (168) did not observe thyroid neoplasms
in rats which received I at the level which was l/100th that used by
Lindsay £t_ ^1_. (167) for 7-9 months. However, Al-Hindawi et_ al^. (168) did
observe a 100% incidence of thyroid tumors in rats treated with the radioac-
tive iodine and PTU (60 ug/ml in drinking water) simultaneously. High inci-
dence (5/18) of invasive carcinomas of the thyroid with metastasis to the lung
1 O 1
were reported in Wistar rats receiving both ijli and PTU and observed for 6-15
months (165).
The suppression of spontaneous mammary tumors by thiourea is illustrated
by the investigations of Morris et_ al_. (193) who reported that only 17% of 42
26
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virgin female C3H mice treated with 0.375-0.5% thiourea in the diet had spon-
taneous mammary tumors, as compared to 94% of 52 untreated animals after 18
i
months. Similarly, Vazquez-Lopez (194) found that 6-raonth administration of
thiourea in drinking water reduced the incidence of spontaneous mammary tumors
in virgin and breeding C3H mice from 40/96 to 5/85. Like thiourea, TU also
inhibits the development of spontaneous mammary tumors in mice; Morris et al.
(195) reported that while 48% of 62 control strain C mice developed mammary
tumors, no neoplasms were found in 62 mice fed 0.375 and 0.5% TU in the diet
until death. Treatment with TU also reduced th.e incidence of spontaneous
mammary tumors in C3H mice from 92% to 19%. Moreover, a lengthening of the
latent period was noted in mice following TU treatment (196). Suppression of
chemically induced mammary carcinogenesis by thiocarbonyls has also been
noted; chronic administration of PTU inhibits the development of mammary
tumors in rats induced by 7, 12-dimethylbenz[a]anthracene (197-199) or
3-methylcholanthrene (200). It was suggested that the suppression of mammary
tumorigenesis by thiocarbonyls is brought about by reduction of the calorie
intake, which has been shown to affect the growth of the mammary gland
(199). An alternative explanation involves the hormonal imbalance resulting
from the hypersecretion of thyrotropic hormone (TSH) in response to the low
thyroxine level caused by thiouracils. Because of the abnormally large
production of TSH, the secretion of hormones related to the development of
mammary tumors would be decreased (193, 196, 198).
The possible synergistic and antagonistic effects of thiocarbonyl com-
pounds with other chemicals in hepatocarcinogenesis have been studied by
various investigators. Deichmann and coworkers (201, 202) fed Osborne-Mendel
rats diets containing 50 or 80 ppm each of thiourea, aramite, methoxychlor and
DDT for 2 years. The incidences of tumors in the rats given the four com-
27
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pounds in the diet were not consistently higher than those in the rats given
the compounds singly. Similar negative results were obtained when groups of
rats were fed the compounds singly, each at a dose representing 50% of their
liver tumor-inducing dose (thiourea, 50 ppm; aramite, 200 ppm; methoxychlor,
1,000 ppm; DDT, 200 ppm), and in combination. These findings led the authors
(202) to conclude that the four compounds do not exert a synergistic or addi-
tive tumorigenic effect in the rat.
The combined effect of TU and 2-acetylaminofluorene (2-AAF) on hepatic
tumorigenesis in rats was studied by Paschkis et al. (139, 203) and by Leathern
and Barken (204). Eighty-four percent of Sherman rats receiving 2-AAF (0.03%
in the diet) alone developed hepatomas, while the incidence of hepatomas was
22% in animals receiving 2-AAF and TU (0.05 or 0.1%, respectively, in drinking
water) in combination (139). In subsequent studies this observation was
confirmed. While hepatomas were induced in 9 out of 9 rats by 2-AAF, only 1
of 16 animals administered 2-AAF + TU developed such tumors (203). Similarly,
the incidence of hepatomas decreased from 50% in rats fed 2-AAF alone to 12.5%
in rats which ingested 2-AAF + TU (204).
Simultaneous administration of TU also protects the rats against liver
tumorigenesis by 4-dimethylaminoazobenzene. Liver tumors were found in 46% of
female Sherman rats given both 4-dimethylaminoazobenzene'and TU, but in 88% of
rats administered 4-dimethylaminoazobenzene alone (139). It has also been
reported that TU treatment significnatly inhibits the development of
cholangiocarcinomas induced by diethylnitrosamine in gerbils (205). Besides
TU, thiourea has been shown to inhibit carcinogenesis by other chemicals.
Gorbman (120) reported that while 30% of strains A and C57 mice developed
local carcinomas and squamous cell carcinomas between 90 and 130 days after a
single subcutaneous injection of 1 rag benzo[ajpyrene, no tumors were found in
28
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mice receiving a mixture of 2% thiourea and 1 mg benzo[a]pyrene in the diet
after 200 days of age. Thiourea is also believed to suppress the carcinogenic
action of concomitantly administered 2-AAF in the mouse (120).
The mechanism of protection by thiocarbonyl compounds against the
carcinogenic action of 2-AAF and other carcinogens is unknown. Paschkis et
al. (203) have shown that the protective effect of TU is not related to the
induced hypothyroidism. The suggestion has also been made that thiouracils
may act upon enzymes in the liver which convert carcinogenic chemicals to
inactive forms (139). They may also act as antimetabolites, interferring with
the uptake of uracil, possibly a nutritional requirement for the emergent
cancer cells induced by 2-AAF (203).
5.2.2.8.4 Metabolism and Mechanism of Action.
5.2.2.8.4.1 TISSUE DISTRIBUTION AND METABOLISM.
Thiourea. Studies conducted in rats and man have shown that thiourea is
rapidly absorbed from the gastrointestinal tract and distributed in whole body
tissues (206). The distribution is not uniform but displays marked variations
in the tissues and body fluids. According to Schulman and Keating (207),
o c
when JJS-thiourea is injected intraperitoneally into rats, notably higher
radioactivity is found in the thyroid and kidney than in other tissues. In
contrast, Maloof and Soodak (208), reported that the thyroid takes up only a
small fraction of the administered S-thlourea, but is very effective in the
metabolism of the compound. The radioactivity in the thyroid is primarily in
O c "} C.
the form of JJS-sulfate. Marked decrease in the metabolism of JJS-thiourea
was noted in the thyroid of hypophysectomized rats, indicating that pituitary
hormones are important in regulating the metabolism of thiourea.
29
-------
Thiourea is excreted rapidly by the kidney (206, 207). Within 48 hours
following its administration, 98% of the radioactivity appeared in the urine
>. o c o c
mainly as undegraded S-thiourea and, to a much lesser extent, as S-sulfate
o c
and S-ethereal sulfate (207). Negligible amounts of radioactivity was
excreted in expired air or in the feces (206, 207).
Phenylthiourea. Following oral administration of S- or C-labeled
phenylthiourea to rabbits, 80-86% and 8-10% of the radioactivity were found in
the urine and in the feces, respectively, within 48 hours. The urinary meta-
bolites were identified as: sulfate (62% of ^S-phenylthiourea), phenyl-
carbamic acid glucuronide (30% of the C-phenylthiourea), p-hydroxyphenyl-
thiourea (16%), _p-hydroxyphenyl urea (14%), phenylthiourea (6%), phenylurea
(4%), phenylcyanamide (1%), aniline (4%), and urea (3%). The metabolism of
phenylthiourea was similar in the rat (25).
Ethylenethiourea (ETU). The distribution, excretion, and metabolism of
ETU have been investigated in the rat (104, 209-214), mouse (104, 215), guinea
*i
pig (213) cat (214), and cow (216). Like thiourea, ETU is readily absorbed
from the gastrointestinal tract and distributed rapidly in various tissues,
and in the fetus (209, 210). The compound is distributed uniformly in most
tissues except the thyroid, which showed significantly higher concentrations
of ETU (209-211, 213). The t^/2 of ETU elimination was about fivefold higher
in the thyroid than in other tissues (211).
Ethylenethiourea was eliminated rapidly through the urine. In rats,
72.8% of the total radioactivity was recovered in the urine within 24 hours
after the administration of 1 C-ETU; at least 95% of the excreted radioac-
tivity was unchanged ETU (209, 211). The metabolites of ETU have been identi-
fied as ethyleneurea (210, 212, 214), imidazolone (212, 214), imidazoline
30
-------
(214), thioimidazole, thiohydantoin, N-methyl-ETU and N-methyl-thioimidazole
(212). Kato £££!_• (210) reported that ETU is also metabolized to C02 via
desulfuration followed by oxidative opening of the imidazolidine ring between
C, and C,-, which is followed by decarboxylation of the 1,3-dicarboxyurea
formed.
While undegraded ETU is the predominant product in the urine of rats,
only 40-50% radioactivity in the urine of mice represents unchanged C-ETU,
indicating that ETU is metabolized to a greater extent in the mouse than in
the rat (104, 215). The desulfurated derivative, ethyleneurea, accounts for
approximately 12% of the radioactivity excreted in the urine of mice (215).
The only other identified metabolite of ETU in mice is 2-imidazolin-2-yl
sulfenate (217).
Of 80% of the radioactive materials excreted in the urine of cats 24
hours after the administration of ^C-ETU, 64% is S-methyl-ETU, 4% is
ethyleneurea, and only 28% represents the undegradated parent compound
(214). In cows, ETU is metabolized to ethyleneurea, ethylenediamine, oxalic
acid, glycine, and urea (216).
In vitro studies on ETU, thiourea and phenylthiourea show that the
compounds are metabolized by the microsomal FAD-containing monooxygenase from
pig liver to sulfinic acid and formamidine sulfinic acid (218).
Thiouracil and Derivatives. These compounds are rapidly absorbed from
the gastrointestinal tract in the rat and in man (219, 220) and can be found
in essentially all tissues and body fluids (219, 220). In vivo studies in
rats with S-labeled thiouracils showed significantly higher accumulation
of 5S-radioactivity in the thyroid than in the plasma (220-223). Compared
to thioureas, thiouracils are less rapidly metabolized in the thyroid and only a
31
-------
small fraction is converted to 35S-sulfate (208, 221). Among the thiouracils,
unmetabolized 35S-PTU accumulates in the thyroid the most and 35S-TU the
least, following administration of equimolar doses (220, 222). In addition to
the intact drugs and 35S-sulfate, other radioactive materials detected in the
thyroid were protein-bound ^S-corapounds and unknown metabolites (221, 223).
The rate of metabolism in other tissues is faster with TU than with PTU
(220). In rats, about 80% of the total radioactivity appeared in the urine
within 24 hours after the administration of ^C-TU (224). In vitro studies
suggest that the metabolism of TU involves initial desulfuration with the
formation of uracil which is then cleaved to produce p-alanine, ammonia, and
C02 (224). Alternatively, TU may be converted to thiouridinemonophosphate and
thiouridinetriphosphate by enzyme systems of liver and thyroid similar to
those involved in the synthesis of UTP from uracil (225-227).
When C-PTU was administered to rats either by gavage or parenterally,
75-90% of the administered radioactivity was excreted in the urine and about
15% in the bile during the first 24 hours (228). Analysis of the 24-hour
urine sample showed that propylthioglucuronide accounts for 40-48% and
intact 14C-PTU for 9-15% of the administered dose (228, 229). Three other
urinary metabolites: sulfate, propyluracil, and S-methyl-PTU were identified
(229). Similar metabolic products were found in the bile (229).
In guinea pig, a urinary metabolite believed to be PTU-disulfide was
identified. Other metabolites in the urine and the bile remain unidentified
(230).
Thioacetamide (TAA). Thioacetamide is rapidly metabolized in rats.
O c
Following subcutaneous injection of S-labeled TAA, more than 80% of the
radioactivity is excreted in the urine within 24 hours, of which approximately
32
-------
25% was unchanged TAA (231). The major metabolic products are inorganic
sulfate (231, 232), TAA-S-oxide (232, 233), and acetamide (234). In vitro
studies indicate that TAA is metabolized by the hepatic mixed-function
oxidases ('235, 236) to TAA-S-oxide, which is then further metabolized by the
inicrosomal monooxygenases or amine oxidase to acetamide and other polar
products (236, 237). The metabqlic pathways of TAA in the rat liver are as
follows (236):
0
S
I, 0 ,NADPH
CH0CNH0 - - — n
3 2 cytochrome
P-450
0 , NADPH
cytochrome
P-450
V"
II
0
II
-r CH3CNH2
N. acetami
Thio-
acetamide
2
Thioacetamide Thioacetamide
S-oxide S-dioxide
polar
metabolite(s)
Ethionamide. The ready biochemical interconversion between ethionainide
and its sulfoxide has been established in mice, rats, dogs (238), and humans
(238-240). The two compounds are rapidly absorbed from the gastrointestinal
tract and considerable amounts of both compounds appear in the blood within 15
minutes, regardless of which compound is administered (238). Only small
amounts of unchanged ethionamide and its sulfoxide are found in the urine of
animals or humans receiving ethionamide. Several urinary metabolites have
been detected. These are 2-ethylisonicotinamide, 2-ethylisonicotinic acid,
and inorganic sulfate (238, 241), as well as metabolic products suspected to
be pyridones (238, 242).
5.2.2.8.4.2 MECHANISMS OF ACTION.
Thiourea, Thiouracil, and Derivatives. The mechanism of carcinogenesis
by thiourea, TU and their derivatives are not clearly understood. It is
33
-------
generally believed that the modes of carcinogenic action of these substances
are identical, at least in thyroid tumorigenesis: they act indirectly by.
causing hormonal imbalance resulting from an altered thyroid-pituitary rela-
tionship. 'As known from their goitrogenic effects, they act by inhibiting the
synthesis of thyroxine; the resulting decrease in the level of thyroxine then
evokes an increased release of thyrotropic hormone (TSH) from the pituitary to
act on the thyroid epithelium, which subsequently becomes hyperplastic and
neoplastic. Several studies have demonstrated that a high level of pituitary
thyrotropic hormone is essential for the formation and growth of thyroid
tumors (126, 167, 168, 189). Thyroid tumors are not found in hypophysec-
tomized animals subjected to carcinogenic goitrogens (243).
The inhibitory effect of thiourea and related compounds on thyroxine
synthesis may be due to direct inhibition of the peroxidation system respon-
sible for the conversion of iodide to iodine, thus inhibiting the iodination
of tyrosine in the production of thyroxine (244, 245). It was also suggested
that these compounds may bind iodine and reduce the level of free iodine
available for reaction with tyrosine (220, 246). However, since 6-amino-TU,
which also reacts with iodine, exhibits little antithyroid activity (247), the
biological activities of thiourea, TU and related compounds appear to be due
to their inhibitory effects on.thyroid peroxidase, rather than to the binding
of iodine. Propylthiouracil also inhibits the monodeiodination of thyroxine
(T4) to triiodothyronine (T3) (248-250).
The metabolism of thiourea by the thyroid of rats is decreased after
hypophysectomy. Yet, thiourea is as effective in inhibiting the formation of
protein bound iodine in the thyroid of hypophysectomized rats as in the
thyroid of non-operated animals, suggesting that metabolism is not essential
for the effect (208). Similarly, the antiperoxidase and antithyroid activ-
34
-------
ities of all known metabolites of TU (227) and PTU (229) are much lower than
those of TU and PTJI themselves indicating that metabolism is unlikely to be
involved in the mechanism of action of these thiouracils.
The relatively high accumulation in and slow disappearance from the
thyroid of unmetabolized TU compounds may explain their selective action on
the thyroid. A correlation between the level of unmetabolized TU, MTU, and
PTU accumulated in the thyroid of rats and their goitrogenic potency has
actually been demonstrated (222). However, in studies of patients treated for
thyrotoxicosis, the concentration of thiouracils in the thyroid was found not
to be associated with the potency of these drugs (220).
The induction of cancer in organs other than the thyroid has led several
workers to suggest that thiourea compounds may also act as direct-acting
carcinogens, although the mechanism is totally unknown (116, 118, 124). The
positive results observed in several mutagenicity assay systems with thiourea
and related compounds (see Section 5.2.2.8.2.2) appear to support this view.
The S-oxygenation of several thioureas to sulfinic acids by microsomal FAD-
containing monooxygenase has been suggested to be an important pathway in
their activation to toxic, reactive metabolites (218). Hollinger and
coworkers (251, 252) have reported the binding of C-thiourea to proteins in
rat tissues; the binding was, however, more extensive in the lung (which is
not a target of the carcinogenic action of thiourea) than in other tissues. A
toxic effect which may be accounted for by this binding is the production of
edema in the lung by thiourea (251).
If hormonal-imbalance is the cause of thyroid cancer induced by thiourea
and its derivatives, conceivably information on the goitrogenic activity of
these compounds is important for the assessment of their carcinogenicity. The
35
-------
relationship between goitrogenic activity and the structure of these chemicals
has been discussed^ (see Section 5.2.2.8.2).
Thioacetamide. Although the liver is the major target organ of thioacet-
amide (TAA) toxicity and carcinogenicity, it does not accumulate higher levels
of the compound than other tissues (231). This observation has led to the
suggestion that the striking specific action of TAA may be due to the hepatic
metabolism of TAA to reactive intermediate^) (34, 35, 231). Comparison of
the toxicity of TAA and of one of its known metabolites, TAA-S-oxide, showed
that the latter produced a more severe hepatic necrosis than equivalent doses
of TAA (235). Thioacetamide-S-oxide has also been stated to exhibit marked
carcinogenic effects (cited in ref. 253). However, TAA-S-oxide is not
considered to be the ultimate carcinogenic metabolite of TAA and it is
believed to undergo further metabolism to reactive metabolite(s) (232, 235,
236). Recently, in vitro binding of TAA-S-oxide to calf thymus DNA in the
presence of rat liver microsomes has been shown (237). The binding reaction
requires NADPH, and is inhibited by CO and by an antibody of rat liver cyto-
chrome P-450, indicating that a microsomal cytochrome P-450 requiring mixed-
function oxidase is involved in the metabolic conversion of TAA-S-oxide to
reactive intermediate^) which then bind to calf thymus DNA. The chemical
nature of the reactive intermediate(s) is not known. The investigations of
Porter et al. (232) indicate that the methyl group and the thiocarbonyl carbon
atom, but not the sulfur atom, of TAA or TAA-S-oxide is directly involved in
the binding with tissue macromolecules (232).
Various cytological and biochemical alterations have been reported to be
associated with TAA hepatocarcinogenesis (35, 177, 254-256). Modification of
the nuclear envelope of liver cells following TAA administration is receiving
increasing attention. The swelling of the nuclei and the increase in phos-
36
-------
phatidylserine in the nuclear envelope have been hypothesized to affect
cellular homeostasis and alter chromatin structure (254, 255).
5.2.2.8.5 Environmental Significance.
Thiocarbonyl compounds have a wide variety of applications; human expo-
sure may occur under various circumstances. Some of these compounds are of
clinical value for the treatment of thyrotoxicosis and in other therapeutic
applications. Others find use in the industry and agriculture while some are
of research or toxicological interest. The production and use of thiourea,
ETU, TU, MTU, and PTU have been extensively reviewed by an International
Agency for Research on Cancer Working Group (155).
Thiourea. Thiourea is commercially available and several synthetic
methods are used in its production. The chemical also occurs naturally in the
seeds of shrubs belonging to the genus Laburnum (257) and as a metabolite of
Verticillium albo-atrum and Bortrylio cinerea (155). Thiourea was or has been
used as an antithyroid agent, fungicide, intermediate in the production of
fireretardant resins for fabrics, as a dye-bath adjuvant of textiles, as
vulcanization accelerator, and as an anti-yellowing agent in the production of
diazo-type coatings for copy paper. The chemical is also useful in the photo-
graphic industry, in cosmetic hair preparations, in the dry-cleaning industry,
and in the synthesis of Pharmaceuticals, insecticides and other organic com-
pounds (cited in ref. 155). Although many of its applications in the industry
are still continuing, the use of thiourea as an antithyroid drug, fungicide,
and food additive has long been discontinued in many countries including the
United States, because of its potential hazard to humans (155). The chemical
decomposes slowly to sulfate and ammonia under the action of microorganisms in
soil and sewage sludge (cited in ref. 258).
37
-------
Ethylenethiourea. Ethylenethiourea is a major degradation product of the
metal salts of ethylenebisdithiocarbamic acid, which are extensively used as
fungicides in the management of diseases of agricultural crops. Amounts up to
14.5% of ethylenethiourea (ETU) have been reported in 28 different commercial
formulations containing maneb, zineb, or mancozeb after 39 days of storage
under controlled conditions of elevated temperature and humidity (259, 260).
Trace levels of ETU residues have also been detected in various vegetables,
fruits, and crops field-sprayed with these formulations (261-265). There is
general agreement that ETU decomposes rapidly in plants, soils and water, and
that accumulation of significant amounts of ETU on the crops or in the
environment is unlikely (266-269). However, home cooking or commecial
processing of certain foods that contain residues of the ethylenebisdithiocar-
bamic acid metal salt fungicides have been reported to cause increases in the
level of ETU in foods (263, 270, 271).
Human exposure to ETU can also be brought about by its applications as
accelerator in the vulcanization of various elastomers, and as intermediate in
the manufacture of dyes, synthetic resins, antioxidants, and Pharmaceuticals
(272). The U.S. National Institute for Occupational Safety and Health esti-
mated that approximately 17,000 employees in 24 different occupations were
potentially exposed to ETU in the workplace between 1972 and 1974. The major
routes of occupational exposure are inhalation and skin contact (272).
Other Thiocarbonyls Compounds. Both diethyl- and trimethylthiourea are
used as vulcanization accelerators in the production of various types of
rubbers. Diethylthiourea is also used as an inhibitor of corrosion in metal
pickling solutions. Other applications of trimethylthiourea include uses such
as inhibitor of ozone fading of polyamide dyes, as a component of adhesives,
and as an intermediate in organic synthesis. Dithiobiurea is used as a fuel
38
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in pyrotechnic disseminating compositions and electroplating baths for
metals. It is also an important compound for the photographic industry.
Phenylthiourea is used occasionally as a rodenticide. The chemical is also
employed as a test agent in medical genetics (273), since the ability to taste
phenylthiourea is an inherited trait. These thiocarbonyl compounds were
produced and marketed in quantities in excess of 1,000 pounds annually in the
United States (cited in refs. 127-129, 131). Dicyclohexylthiourea is
primarily used as a laboratory reagent in biochemical and physiological
research (130).
Thiouracil, MTU, and PTU have been used as antithyroid drugs in human and
veterinary medicine (155). Thiouracil is also effective in the treatment of
angina pectoris and congestive heart failure. In animal husbandry, MTU and
PTU were used for promoting the growth and fattening of animals. However,
these applications of thiouracils appear to have been discontinued in the
United States (155).
Thioacetamide is widely used as a substitute for hydrogen sulfide in the
analytical chemistry of heavy metals (273). Ethionamide is among the drugs of
choice in the treatment of tuberculosis. The drug is administered to adults
at dose level as high as 1 g daily (19).
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Health. Publ. No. 79-109, Cincinnati, Ohio, 1978.
273. Gleason, M.N., Gosselin, R.E., Hodge, H.C. and Smith, R.P.: "Clinical
Toxicology of Commercial Products," 3rd ed., Williams and Wilkins,
Baltimore, 1969.
56
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Notes Added After Completion of Section 5.2.2.8
The presence of an intact sulfur atom [not bearing oxygen atom(s)] in the
molecule of ethylenethiourea is essential not only for its teratogenic action
(see Section 5.2.2.8.2), but also for its mutagenicity. This conclusion is
drawn on the basis of recent observations that the metabolite formed by oxida-
tion of the sulfur in ethylenethiourea does not exhibit any mutagenic activity
in Salmonella typhitnurium TA1950 or in the host-mediated assay in mice (1).
In contrast, thioacetamide-S-oxide, but not thioacetamide itself, shows muta-
genicity in the Ames test without the S9 mix (2).
Hepatocarcinogenesis induced by thioacetamide has been studies in inbred
male ACI rats which do not have a spontaneous liver tumor incidence (3).
Administration of 0.035% thioacetamide in a semipurified diet to the rats for
1 year resulted in the development of primary hepatocarcinomas and cholangio-
carcinomas. The tumor incidence and type were strongly influenced by the
dietary conditions.
Thiobenzamide (CgHc-CSNI^), a thiono compound structurally related to
carcinogenic chemicals of this group, was reported to induce hyperplastic
nodules and tumors in the liver of rats (4). Additional studies (5) suggest
that thiobenzamide acts principally as a promoter in liver carcinogenesis.
References for Section 5.2.2.8 Update
1. Autio, K., von Wright, A., and Pyysalo, H.: Mutation Res. 106, 27
(1982).
2.-- Breau, A.P., Mitchell, W.M., Karkhanis, D.W., and Field, L. : Mutation
Res. 139, 1 (1984).
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j
3. Becker, F.F.: J. Nat. Cancer Inst. 71, 553 (1983).
4. Malvaldi, G.: Bull. Soc. Ital. Biol. Sper. (Italian) ^4, 1027 (1978),
5. Malvaldi, G., Chieli, E., and Saviozzi, M.: Gann 74, 469 (1983).
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