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

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 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

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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

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                                                                                                               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

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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)

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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

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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

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                                                                               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.

-------
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

-------
     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

-------
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

-------
     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

-------
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

-------
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

-------
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

-------
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

-------
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).

-------
     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

-------
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

-------
     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

-------
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

-------
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

-------
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

-------
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

-------
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).
REFERENCES TO SECTION 5.2.2.8









    1.  Kennedy, T.H. and Purves, H.D.:  Br. J. Exp. Pathol. 22, 241 (1941).




    2.  Griesbach, W.E.:   Br. J. Exp. Pathol. 22, 245 (1941).




    3.  Kennedy, T.H.:   Nature (London) 150, 233 (1942).




    4.  Richter, C.P. and Clisby, K.H.:  Arch. Pathol. 33, 46 (1942).






                                      39

-------
 5.  Mackenzie, C.G. and Mackenzie, J.B.:  Endocrinology 32, 185 (1943).


 6.  Astwood, E.B., Sullivan, J., Bissell, A.  and Tyslowitz, R.:
              v

     Endocrinology 32, 210 (1943).


 7.  Astwood, E.B., Bissell, A. and Hughes, A.M.:  Endocrinology 37, 456


     (1945).


 8.  Childs, J.F.L. and Siegler, E.A.:  Science 102, 68 (1945).


 9.  Fishbein, L.:  J. Toxicol. Environ. Hlth. !_, 713 (1976).


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                                    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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