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    THIOPHANATE-METHYL

     First draft prepared by
     J. Taylor and M. Watson
     Pesticides Safety Directorate, Ministry of Agriculture, Fisheries
     and Food
     Mallard House, Kings Pool, York, United Kingdom

    Explanation
    Evaluation for acceptable daily intake
         Biochemical aspects
         Absorption, distribution, and excretion
         Biotransformation
         Effects on enzymes and other biochemical parameters
         Toxicological studies
         Acute toxicity
         Short-term toxicity
         Long-term toxicity and carcinogenicity
         Reproductive toxicity
         Developmental toxicity
         Genotoxicity
         Special studies
              Dermal and ocular irritation and dermal sensitization
              Effects on the thyroid and liver
         Observations in humans
         Comments
         Toxicological evaluation
    References

    Explanation

         Thiophanate-methyl is a systemic fungicide of the benzimidazole
    group. It was evaluated toxicologically by the Joint Meeting in 1973,
    1975, and 1977 (Annex I, references 20, 24, and 28). The ADI of
    0-0.08 mg/kg bw allocated in 1973 was confirmed by the 1975 and 1977
    Joint Meetings. Since that time, additional data have become
    available, and the results of these studies were reviewed at the
    present Meeting. In order to facilitate review of the complete
    database, information presented in the reports of the previous
    monographs (Annex I, references 21, 25, and 29) are included in this
    monograph. Thiophanate-methyl was reviewed by the present Meeting
    within the periodic review programme of the CCPR.

    Evaluation for acceptable daily intake

    1.  Biochemical aspects

    (a)  Absorption, distribution, and excretion

         In a series of studies, an unspecified number of male dd-Y strain
    mice were given a single dose of radiolabelled thiophanate-methyl by
    gavage. Four radiolabelled thiophanate-methyl moieties were used,
    14C-carbonyl thiophanate-methyl, 35S-thiophanate-methyl,
    14C-methyl thiophanate-methyl, and 14C-phenyl thiophanate-methyl.
    Faeces, urine, and expired gas were analysed 3, 6, 12, 24, 48, 60, 72,
    84, and 96 h after administration, and blood samples were analysed
    after 3, 6, 12, 24, 48, and 72 h. Conjugates were determined after
    hydrolysis with hydrochloric acid or -glucuronidase. An unspecified
    number of mice were killed 3, 6, 12, 24, 48, and 72 h after
    administration, and tissue samples were analysed for radiolabel. The
    contents of all moieties in blood and urine declined steadily from
    peak levels within 3 h, and faecal excretion peaked at about 12 h in
    all cases, declining significantly by 48 h. The rate of excretion
    varied slightly between moieties, but total excretion had reached a
    plateau at about 24 h. 14C-Methyl thiophanate-methyl showed a
    different pattern of excretion, suggesting that the methyl group was
    parted from the parent molecule and was rapidly absorbed and partially
    converted to carbon dioxide. Total excretion is summarized in Table 1.

        Table 1.  Percentage of radiolabelled thiophanate-methyl excreted by male
              dd-Y mice after 96 h
                                                                                              

    Compound                            Total radiolabel excreted (%)       Total recovery (%)
                                                                            of radiolabel by 
                                                                            day 4 or 5
                                        Urine     Faeces     Expired gas
                                                                                              

    14C-Carbonyl thiophanate-methyl     78-83     18-20      ND             102 (day 5)
    35S-Thiophanate-methyl              66-86     19-21      ND             105 (day 5)
    14C-Methyl thiophanate-methyl       66-67     16-17      1              ND
    14C-Phenyl thiophanate-methyl       78-89     27-29      ND             117 (day 4)
                                                                                              

    ND, not determined
    
         None of the moieties accumulated in any organ or tissue, and
    radiolabel disappeared relatively rapidly within the 96-h
    investigation period. After three days, 14C-carbonyl thiophanate-
    methyl radiolabel was detectable in liver and blood, 14C-methyl
    thiophanate-methyl radiolabel primarily in liver, kidney, and blood,
    and 14C-phenyl thiophanate-methyl radiolabel in liver, with low
    levels in a number of tissues. 35S-Labelled thiophanate-methyl was
    the only moiety found in bone after 96 h, suggesting that the labelled
    sulfur might be cleaved and hence behave differently. Metabolites were
    investigated in urine, faeces, organs, and tissues by thin-layer
    chromatography. Untreated urine from 14C-phenyl thiophanate-methyl
    produced eight possible metabolites. The parent molecule and three
    metabolites were identified against known standards: carbendazim, an
    intermediate (1-thioureido-2-(3-ethoxycarbonyl-2-thioureido)benzene),
    and a minor metabolite (1,2-bis(3-ethoxycarbonyl-2-ureido)benzene).
    Hydrolysis of the water-soluble fraction with hydrochloric acid or
    -glucuronidase resulted in six spots, the majority of which were
    identified as carbendazim and its hydroxy analogue 5-hydroxy-
    carbendazim, which were found by co-chromatography with authentic
    standards to be glucuronic acid conjugates. 1-Thioureido-2-
    (3-ethoxycarbonyl-2-thioureido)benzene and 1,2-bis(3-ethoxy-
    carbonyl-2-ureido)benzene were also found, but two further metabolites
    were not identified. Faeces, liver, kidney, stomach, and intestine
    were also investigated for metabolites. Thiophanate-methyl,
    carbendazim, 5-hydroxy-carbendazim, and 1-thioureido-2-(3-ethoxy-
    carbonyl-2-thioureido)benzene were identified in faeces; the parent
    molecule, carbendazim, 5-hydroxycarbendazim, and 1,2-bis(3-ethoxy-
    carbonyl-2-ureido)benzene were identified in liver; carbendazim,
    5-hydroxycarbendazim, and 1,2-bis(3-ethoxycarbonyl-2-ureido)benzene
    were identified in kidney; the parent molecule was the main compound
    identified in stomach, with small amounts of carbendazim and
    1,2-bis(3-ethoxycarbonyl-2-ureido)benzene; and the parent molecule and
    carbendazim were identified in the intestine (Noguchi, 1970a, 1971,
    1972; Fujino  et al., 1973).

         An unspecified number of male Wistar rats were given a single
    dose of 14C-thiocarbonyl]-thiophanate-methyl by gavage. Most of the
    radiolabel (83% of the total administered) was excreted within 24 h,
    with 56% in faeces and 28% in urine. By 72 h, 89% of the label had
    been excreted. Table 2 shows the 24-h faecal and urinary metabolites
    that were characterized. Of the 56% radiolabel detected in the faeces,
    1% in the water-soluble phase was uncharacterized and > 4% remained
    in the residue; of the 28% radiolabel in urine, 14% was
    uncharacterized because it was not extractable (Fujino  et al.,
    1973).

        Table 2.  Recovery of 14C-labelled compounds in rat urine and faeces 24 h after
              administration of 14C-labelled thiophanate-methyl
                                                                                              

    Compound                                               Recovery (as % of administered
                                                           14C-labelled thiophanate-methyl)
                                                                                              

                                                           Faeces       Urine
                                                                                              

    Thiophanate-methyl                                     38           1
    4-Hydroxy-thiophanate-methyl                           6            3
    5-Hydroxy-carbendazim                                  2            6
    4-Hydroxy-dimethyl-4,4'-O-phenylene bisallophanate     1            2
    Dimethyl-4,4'-O-phenylene bisallophanate               1            2
    Carbendazim                                            1            1
                                                                                              
    
         An unspecified number of male beagle dogs were given a single
    dose of [14C-carbonyl]-thiophanate-methyl in a capsule. Blood, urine,
    and faeces were collected 8, 15, and 30 min and 1, 2, 3, 6, 12, 24,
    35, 48, 60, 72, and 96 h after administration. Urine contained 74% of
    the total radiolabel and faeces, 14%. Maximal total excretion occurred
    after about 24 h by both routes. The total recovery of radiolabel was
    not specified. Carbendazim was identified by co-chromatography from
    urine samples. Treatment of urine samples with -glucuronidase
    released the 5-hydroxy analogue. No other metabolites were identified
    (Noguchi, 1972; Fujino  et al., 1973).

    (b)  Biotransformation

         Male mice (strain unspecified) received 0.1 g/kg bw of an aqueous
    thiophanate-methyl solution orally, and their urine and faeces were
    collected at 24, 48, and 72 h. Metabolites were extracted into
    ethylacetate; conjugated metabolites were extracted by enzymic
    hydrolysis with -glucuronidase and arylsulfatase. As a proportion of
    the total dose administered, 27% was identified as free metabolites in
    urine, the majority (19%) being carbendazim; other metabolites,
    2-aminobenzimidazole (2.5%), 5-hydroxy-2-aminobenzimidazole (0.4%),
    and 5-hydroxycarbendazim (4.9%), accounted for the other 8% found free
    in urine. Conjugates of glucuronide and/or sulfate were found in urine
    and faeces, but the amounts of each type were not determined and only
    two were identified (representing 8% of total dose), of which 6% was
    5-hydroxycarbendazim and 2% aminobenzimidazole. In faeces, 16% of the
    administered dose was identified as free metabolites, carbendazim
    again being prevalent (11%); 2-amino-benzimidazole (3%), 5-hydroxy-

    2-aminobenzimidazole (0.3%), and 5-hydroxy carbendazim (3%) accounted
    for the other 6% found free in faeces. Of the total dose 8% was
    identified as conjugated metabolites, with similar proportions of the
    two metabolites found in urine. Thin-layer chromatography of faeces
    and urine showed the presence of 10 conjugated metabolites.

         Mouse liver preparations were incubated with 1 mmol/litre
    thiophanate-methyl, and 11 metabolites were identified:

         2-(3-methoxycarbonyl-2-thioureido)aniline,
         1-(3-methoxycarbonyl-2-ureido)-2-(3-methoxycarbaryl-2-thioureido)
         benzene,
         1,2-bis(3-methoxycarbonyl-2-ureido)benzene,
         2-(3-methoxycarbonyl-2-ureido)aniline,
         1-thioureido-2-(3-methoxycarbonyl-2-thioureido)benzene,
         1-(2-ureido)-2-(3-methoxy-2-thioureido)benzene,
         1-(2-ureido)-2-(3-methoxycarbonyl-2-ureido)benzene,
         2-aminobenzimidazole,
         5-hydroxy-2-aminobenzimidazole,
         methylbenzimidazole-2-ylcarbamate (carbendazim), and
         methyl 5-hydroxybenzimidazol-2-ylcarbamate (5-hydroxy-
         carbendazim).

    Incubation with mouse kidney and brain preparations produced similar
    metabolites, with thin-layer chromatography patterns that were
    indistinguishable under fluorescence quenching. Incubation with
    intestinal preparations did not produce cyclized benzimidazole
    derivatives, but 2-(3-methoxycarbaryl-2-thioureido) aniline and
    1-thioureido-2-(3-methoxycarbaryl-2-thioureido)benzene were detected.

         Further experiments were carried out  in vitro with non-cyclized
    compounds (including the 11 thiophanate-methyl metabolites identified)
    as the substrate in mouse liver preparations. The rate of carbendazim
    formation from eight compounds, including thiophanate-methyl, was
    measured. The 11 metabolites were relatively stable but 1-formamido-
    2-(3-methoxy-2-thioureido)benzene and the 2-ureido analogue were
    relatively unstable to nonenzymatic transformation. Formation of
    carbendazim was rapid for substrates with an intact 3-ethoxy-carbonyl-
    3-thioureido constituents, although the formamide and thioformamide
    derivatives showed highest cyclization (the latter being cyclized at
    about 80% of the former). The largest amounts of 2-aminobenzimidazole
    were formed from substrates containing thioureido or ureido
    side-chains. Formation of 2-aminobenzimidazole was 25-50% that of
    carbendazim. Hydrolysis of carbendazim with subsequent cyclization was
    proposed. Additional experiments to investigate the cofactor (NAD,
    NADP, and glucose-6-phosphate) requirements for reactions leading to
    carbendazim suggested that mixed-function oxidase enzymes were
    responsible, as these reactions were inhibited by carbon monoxide and
    SKF 525A (Douch, 1974).

         14C-Thiocarbonyl-labelled thiophanate-methyl was added to liver
    microsome preparations from homogenates obtained from male and female
    Sprague-Dawley rats fed either a commercial diet or a diet containing
    640 ppm thiophanate-methyl for three months. No sex difference and no
    enzyme induction were seen; only one metabolite, carbendazim, was
    identified, by thin-layer co-chromatography (Noguchi, 1970b).

         Male and female Sprague-Dawley rats were fed either a commercial
    diet or a diet containing 640 ppm of 14C-carbonyl thiophanate-
    methyl, 35S-thiophanate-methyl, 14C-methyl thiophanate-methyl, or
    14C-phenyl thiophanate-methyl for three months. No sex difference
    and no enzyme induction was seen. Four metabolites of the 14C
    moieties were identified by autoradiography: dimethyl-4,4'- O-
    phenylenebisallophanate, an intermediate metabolite, carbendazim, and
    5-hydroxycarbendazim; 35S-thiophanate-methyl did not result in
    carbendazim (Noguchi, 1972). In a similar study, 4-hydroxythio-
    phanate-methyl and dimethyl-4,4'- O-phenylene bisallophanate were
    identified  in vitro as metabolites of thiophanate-methyl in rat
    liver microsome preparations (Fujino  et al., 1973).

         A proposed metabolic pathway for thiophanate-methyl is presented
    in Figure 1.

    (c)  Effects on enzymes and other biochemical parameters

         A number of experiments were performed on mice, rats, and rabbits
    to determine the pharmacological properties of thiophanate-methyl
    (purity, > 98%) (Singh & Garg, 1989). The results are summarized in
    Table 3.

    2.  Toxicological studies

    (a)  Acute toxicity

         The acute toxicity of thiophanate-methyl is summarized in Table
    4. It has little acute toxicity. At high oral doses in older studies,
    the active ingredient induced symptoms of toxicity which included
    tremors and tonic and clonic convulsions. In a newer study with
    thiophanate-methyl of 96.55% purity, there were no signs of toxicity
    or deaths at 5000 mg/kg bw orally (Souma & Nishibe, 1990a). After
    acute inhalation of 95.3% pure compound at concentrations close to the
    LC50 (1.7-1.9 mg/litre), the symptoms of toxicity included ataxia,
    decreased motor activity, tremor, and convulsions (Saika & Nishibe,
    1987).

    CHEMICAL STRUCTURE

        Table 3.  Pharmacological effects of thiophanate-methyl
                                                                                                                           

    Parameter                           Species           Dose and route                      Results
                                                                                                                           

    Body temperature                    Wistar rats       380-1500 mg/kg bw orally            No effect
    Analgesic activity                  Mice              500 mg/kg bw intraperitoneally      No effect
    Sedative or hypnotic activity       Rats and mice     100-500 mg/kg bw subcutaneously     No effect
    Cardiovascular and respiratory      Rabbits           20-100 mg/kg bw intravenously       Reduced blood pressure
    effects                                                                                   followed by bradycardia
                                                                                                                           

    Table 4.  Acute toxicity of thiphanate-methyl
                                                                                                             

    Species        Sex              Route               LD50 or LC50       Reference
                                                        (mg/kg bw or
                                                        mg/litre air)
                                                                                                             

    Mouse          Male, female     Orala               3400-3514          Noguchi & Hashimoto (1970a)
    Mouse          Male, female     Intraperitoneal     792-1113           Noguchi & Hashimoto (1970a)
    Mouse          Male, female     Subcutaneous        > 10 000           Noguchi & Hashimoto (1970a)
    Mouse          Male, female     Dermal              > 10 000           Noguchi & Hashimoto (1970a)
    Rat            Male, female     Oral                6640-7500          Noguchi & Hashimoto (1970a)
    Rat            Male, female     Intraperitoneal     140-1640           Noguchi & Hashimoto (1970a)
    Rat            Male, female     Subcutaneous        > 10 000           Noguchi & Hashimoto (1970a)
    Rat            Male, female     Dermal              > 10 000           Noguchi & Hashimoto (1970a)
    Rat            Male, female     Inhalation          1.7-1.9            Nishibe (1987)
    Rat            Male, female     Oralb               > 5000             Souma & Nishibe (1990a)
    Guinea-pig     Male, female     Oral                3640-6700          Noguchi & Hashimoto (1970a)
    Guinea-pig     Male, female     Dermal              > 10 000           Noguchi & Hashimoto (1970a)
    Rabbit         Male, female     Oral                2270-2500          Noguchi & Hashimoto (1970a)
    Rabbit         Male, female     Dermal              > 10 000           Noguchi & Hashimoto (1970a)
    Rabbit         Male, female     Dermalb             > 2000             Souma & Nishibe (1990b)
                                                                                                             

    a  In gum arabic in 5% sodium chloride solution
    b  In distilled water
    c  Mortality rates: 0 at 350 mg/kg bw per day, 2 at 500 mg/kg bw per day, 1 at 650 mg/kg bw per day,
       0 at 800 mg/kg bw per day, 3 at 1100 mg/kg bw per day, and 3 at 1400 mg/kg bw per day
        (b)  Short-term toxicity

    Mice

         Groups of 12 ICR mice of each sex were fed diets containing
    thiophanate-methyl at doses of 0, 12.8, 64, 320, 1600, or 8000 ppm for
    six months. The homogeneity, stability, and concentrations of the diet
    were not reported; clinical signs of toxicity were also not reported.
    There were no treatment-related deaths. The body-weight gain of
    animals of each sex at 8000 ppm was significantly reduced. Erythrocyte
    counts were also reduced in these animals and in males at 1600 ppm,
    and haematocrit values were reduced in animals of each sex at 1600 and
    8000 ppm. No significant differences were seen in the weights of the
    cerebrum, heart, lung, spleen, kidney, or testis, but in animals at
    8000 ppm, liver weights were increased. Histopathological
    investigation revealed a higher incidence of hepatic-cell irregularity
    at the higher doses, with five cases in males and three in females at
    8000 ppm and one case in an animal of each sex at 1600 ppm. Large
    swollen hepatic cells, some with oedematous or granular protoplasm,
    were found to a greater extent at 8000 ppm, especially in the central
    lobules. Constriction of veins by the swollen cells was proposed to
    explain the cloudy appearance of the liver. Sporadic formation of
    acidiphilic bodies from hepatic-cell degeneration was recorded in two
    females and one male at 8000 ppm and one male at 1600 ppm. The NOAEL
    was 320 ppm, equivalent to approximately 48 mg/kg bw per day, on the
    basis of hepatotoxic and haematological effects at 1600 and 8000 ppm
    (Noguchi & Hashimoto, 1970b; Hashimoto  et al., 1973).

    Rats

         Groups of 12 Sprague Dawley rats of each sex were fed diets
    containing thiophanate-methyl at doses of 0, 12.8, 64, 320, 1600, or
    8000 ppm for six months. The homogeneity, stability, and
    concentrations of the diet were not reported, and no clinical signs of
    toxicity were reported. There were no treatment-related deaths. The
    body-weight gain of animals of each sex at 8000 ppm was significantly
    reduced, but food consumption did not differ significantly between
    groups. Reduced erythrocyte counts were seen in animals of each sex at
    8000 ppm. Urinalysis did not show significant treatment-related
    differences. Blood cholesterol levels were raised significantly in
    animals of each sex at 8000 ppm. The thyroid anti liver weights were
    increased in these animals, and the weight of the thymus was increased
    in females. Some histopathological effects on the thyroid were
    reported in animals at this dose: four animals of each sex had small
    thyroid follicles, four males and four females had thickened
    follicular epithelium, and five animals of each sex had decreased
    colloidal substance. There were no treatment-related effects on
    protein-bound iodine levels. The NOAEL was 1600 ppm, equivalent to
    approximately 80 mg/kg bw per day, mainly on the basis of effects on
    thyroid, liver, and body weights (Noguchi & Hashimoto, 1970c;
    Hashimoto et al., 1973).

    Dogs

         Groups of five beagle dogs of each sex (four at the highest dose)
    were fed capsules containing thiophanate-methyl (purity unspecified)
    at doses of 0, 2, 10, 50, or 250 mg/kg bw per day for two years. One
    animal of each sex at each dose was killed and autopsied after 12
    months. There were no deaths or adverse symptoms. Animals of each sex
    at 250 mg/kg bw per day had reduced body weights and body-weight gain.
    Gross pathological effects were not reported, and no effects on the
    eyes or heart were found in periodic ophthalmoscopic and electro-
    cardiographic investigations. There were no treatment-related changes
    in clinical chemical, haematological, or urinary values. After 24
    months, the weight of the thyroid gland was increased in animals of
    each sex at 250 mg/kg bw per day and to a lesser extent at 50 mg/kg bw
    per day. The effects on thyroid tissue at 12 months included altered
    or decreased colloidal substance and slightly taller cuboid follicular
    epithelial cells at 250 mg/kg bw per day. The NOAEL was 10 mg/kg bw
    per day on the basis of effects on thyroid weight in animals of each
    sex at 50 and 250 mg/kg bw per day (Taniguchi, 1972).

         Groups of four male and four female beagle dogs were fed capsules
    containing thiophanate-methyl (purity, 96.55%) for three months at
    doses of 0, 50, 200, or 800 mg/kg bw per day, the last dose being
    lowered to 400 mg/kg bw per day on test day 50 because of severe
    toxicity. One male at the high dose was sacrificed on day 41 because
    of severe toxicity; one male at 50 mg/kg bw per day died on day 36,
    but this death did not appear to be related to treatment. Dose-related
    clinical signs seen in animals at the high dose and to a lesser extent
    at the middle dose included dehydration, thinness, and lethargy.
    Dose-related decreases in body weights and marked decreases in food
    consumption were seen at the middle and high doses. There were no
    treatment-related ophthalmological findings. Slight anaemia, increased
    platelet counts and cholesterol levels, and decreases in serum alanine
    aminotransferase activity and albumin levels were seen at the middle
    and high doses and increased activated partial thromboplastin time at
    the high dose. Thyroid function tests revealed slightly decreased
    triiodothyronine levels in males at the high dose and decreased
    triiodothyronine and thyroxine levels in females at the middle and
    high doses; no clear effects on thyroid-stimulating hormone values
    were apparent. Urinalysis showed no treatment-related findings. At the
    middle and high doses, the weights of the liver (in animals of each
    sex) and thyroid (males only) were increased. Gross examination
     post mortem revealed emaciation in one male at the middle dose and
    three at the high dose. Dose-related histological alterations were
    seen in a number of organs. In thyroids, hypertrophy of the follicular
    epithelium was found in one, three, and four males and one, two, and
    four females at the low, middle, and high doses, respectively, with
    none in controls. The severity of the hypertrophy increased from

    minimal to marked with dose. Hyperplasia of the follicular epithelium
    was found in one male at the middle dose and in most animals at the
    high dose. In two males at the high dose in which both hypertrophy and
    hyperplasia were marked, a large decrease in the quantity of
    intrafollicular colloid was seen. In animals at the middle and high
    doses, dose-related changes were found in the liver (reduction in
    vesiculation of the hepatocellular cytoplasm), gall-bladder
    (intracytoplasmic vacuoles), pancreas (atrophy of acinar cells due to
    a decreased quantity of zymogen granules), spleen (lymphoid-cell
    depletion), thymus (involution), prostate (atrophy), uterus
    (anoestrus), and ovaries (inactive). There was no NOAEL because of the
    presence of follicular-cell hypertrophy in the thyroid of two dogs at
    the low dose (Auletta, 1991).

         Groups of four male and four female beagle dogs were fed capsules
    containing thiophanate-methyl (purity, 96.55%) at doses of 0, 8, 40,
    or 200 mg/kg bw per day for one year. There were no deaths. Tremors
    were seen in all dogs at the high dose 2-4 h after treatment on one or
    more occasions during the initial three weeks of the study but not
    subsequently. The tremors were slight to moderate, except in one
    animal which had severe tremors that progressed to apparent tonic
    convulsions on three occasions. The body-weight gains of animals at
    the high dose were reduced during the study, and body-weight losses
    were noted in the first week of treatment; slight reductions in
    body-weight gain were noted at 40 mg/kg bw per day. The food
    consumption of dogs at the high dose was generally lower than that of
    controls during the early months, but was close to the control value
    thereafter. Ophthalmological examinations and urinalyses showed no
    treatment-related effects. Haematological effects consisted of slight
    decreases in total erythrocyte counts and haemoglobin and haematocrit
    values in males at the high dose. These animals also had decreased
    serum alanine aminotransferase activity, increased alkaline
    phosphatase activity, increased cholesterol levels (also at the middle
    dose), and decreased albumin:globulin ratios (also at the middle
    dose), calcium (also at the middle dose), potassium, and phosphorus
    levels. In females at 200 mg/kg bw per day, serum alanine
    aminotransferase activity was decreased, and alkaline phosphatase
    activity and cholesterol levels were increased. Thyroid function tests
    revealed decreased thyroxine levels in males at the middle and high
    doses but no clear effects on triiodothyronine or thyroid-stimulating
    hormone. Abnormalities seen  post mortem included increased liver
    weights in dogs at the high dose and increased thyroid weights in
    those at the middle and high doses. Microscopic alterations attributed
    to treatment were limited to minimal to moderate hypertrophy and
    slight hyperplasia of the follicular epithelium of the thyroid in the
    dogs at the middle dose (hypertrophy in two females) and the high dose
    (hypertrophy in four males and three females, and hyperplasia in one
    male and one female). The NOAEL was 8 mg/kg bw per day (Auletta,
    1992).

    (c)  Long-term toxicity and carcinogenicity

    Mice

         Groups of 50 ICR mice of each sex were fed diets containing 0,
    10, 40, 160, or 640 ppm thiophanate-methyl (purity, > 94%) for two
    years. The homogeneity, stability, and concentrations of
    thiophanate-methyl in the diets were not reported. The body-weight
    gain of males at 640 ppm was reduced during the study. No
    treatment-related effects on food consumption or on mortality or overt
    signs of toxicity were seen. By 24 months, 74-94% of treated animals
    had died, but the mean survival times were comparable. Organ weights
    and clinical chemical and haematological parameters were not
    investigated. No treatment-related gross macroscopic or histopatho-
    logical alterations were seen, and there was no evidence of
    carcinogenicity. The NOAEL was 160 ppm, equivalent to about 24 mg/kg
    bw per day, on the basis of the reduction in body-weight gain in males
    at 640 ppm (Kosaka, 1973).

         Groups of 60 male and 60 female CD-1 mice were fed diets
    containing 0, 150, 640, 3000, or 7000 ppm thiophanate-methyl (purity,
    95.9-96.6%) for 18 months. Ten animals of each sex in each group were
    necropsied after 39 weeks of treatment, and surviving animals were
    necropsied after 78-79 weeks of treatment; all mice that died or were
    killed  in extremis were also autopsied. The homogeneity, stability,
    and concentrations of thiophanate-methyl were acceptable. Haemato-
    logical and thyroid function were assessed at the interim necropsy and
    before the end of the study. No treatment-related clinical signs were
    observed. The cumulative numbers of deaths up to 18 months of -
    treatment were 10, 11, 14, 16, and 24 males and 12, 13, 15, 17, and 23
    females at 0, 150, 640, 3000, and 7000 ppm, respectively; animals at
    7000 ppm had a significantly higher mortality rate than controls. The
    increased rates in females at 3000 ppm and males and females at
    7000 ppm appeared to be related to amyloid deposition in tissues.
    Body-weight gains were slightly reduced in animals of each sex at
    3000 ppm and to a greater degree at 7000 ppm; food consumption was
    slightly reduced in females at 3000 and 7000 ppm. There was no
    treatment-related effect on ophthalmologic parameters or on the onset
    or number of palpable masses. The only haematological parameter that
    appeared to be altered by treatment was a slightly lower erythrocyte
    count in males at 7000 ppm. At the nine-month interim necropsy,
    enlarged thyroid glands were seen grossly in three of 10 males at
    7000 ppm, the thyroid weights of males and females at 3000 and
    7000 ppm were increased, and the thyroid-stimulating hormone levels
    were higher and the thyroxine levels lower in males at 3000 ppm and
    males and females at 7000 ppm than in controls. The differences in
    thyroid weights and thyroid hormone levels were not seen at 18 months.

    At the nine-month interim necropsy, the liver weights of males and
    females at 3000 and 7000 ppm were significantly increased as a result
    of an increased incidence and severity of hepatocellular centrilobular
    hypertrophy at these two doses; the incidence of hepatocellular
    centrilobular hypertrophy was also increased in females at 640 ppm,
    five out of 10 having minimal hypertrophy. At 18 months, the mean
    liver weights of animals at 7000 ppm were increased as a result of a
    significantly increased number of liver masses, which was also
    significantly increased at 3000 ppm. At the 18-month terminal
    necropsy, an increased incidence of hepatocellular centrilobular
    hypertrophy was seen in males at 3000 and 7000 ppm. Histopathological
    examination revealed that the liver masses seen grossly were adenomas.
    The incidence of hepatocellular adenomas was significantly increased
    in males and females at 3000 and 7000 ppm. The slightly increased
    incidence of hepatocellular adenomas in females at 640 ppm (8.6%) was
    greater than the historical control range (0-2.7%). Two hepatocellular
    carcinomas were seen, one in a male at 640 ppm and the other in a male
    at 7000 ppm; this 2% incidence was similar to the mean incidence for
    historical control males (1.4%; range, 0-6%). One male at 7000 ppm had
    a hepatoblastoma, which is a relatively rare tumour (0.001% in
    historical controls). The incidence of atrial thrombosis was increased
    in females at 3000 ppm and in animals of each sex at 7000 ppm. The
    NOAEL was 150 ppm, equivalent to 29 mg/kg bw per day, in view of the
    possible treatment-related incidence of hepatocellular adenomas in
    females at 640 ppm (Tompkins, 1992).

    Rats

         Groups of 35 Sprague-Dawley rats of each sex (50 of each sex for
    controls) were fed diets containing thiophanate-methyl (purity, >
    94%) at doses of 0, 10, 40, 160, or 640 ppm for two years. Five rats
    of each sex at each dose were killed and autopsied at three and 12
    months. The homogeneity, stability, and concentrations of
    thiophanate-methyl in the diets were not reported. There were no
    treatment-related deaths, although survival was poor in some groups,
    and there were no overt signs of toxicity. Body-weight gain was
    reduced in animals of each sex at 640 ppm, but no significant
    difference in food consumption was observed. No significant treatment-
    related effects were seen on clinical chemical, haematological, or
    urinary values. No significant adverse histopathological effects or
    changes in organ weights were seen at any dose at three or 12 months.
    Necropsy of animals killed after 24 months or which died during
    treatment showed treatment-related histopathological findings in the
    testes and thyroid gland. Males at 640 ppm had an increased incidence
    of decreased colloidal substance and hypertrophy of follicular
    epithelium in thyroid tissue (6/35 compared to 1/50 in controls). Five
    animals at 640 ppm were found to be hypospermatogenic; one died at
    week 91 with a pituitary tumour and was found to have atrophied
    testes. Another male had diminished spermatogenesis at 24 months, and

    another surviving male and one that died during treatment had
    hyperplasia of the testicular interstitium. No other treatment-related
    effects were seen. There was no evidence for carcinogenicity. The
    NOAEL was 160 ppm, equivalent to about 8 mg/kg bw per day, on the
    basis of effects on the testes and thyroid gland and reduced growth at
    640 ppm (Hashimoto, 1972).

         Groups of 60 male and 60 female Fischer 344 rats were fed diets
    containing 0, 75, 200, 1200, or 6000 ppm thiophanate-methyl (purity,
    96.55%) for 24 months. The homogeneity, stability, and concentrations
    of thiophanate-methyl in the diets were acceptable. After 12 months,
    10 rats of each sex in each group (only five males at 6000 ppm) were
    sacrificed. All survivors were sacrificed at the end of treatment. The
    mortality rates are shown in Table 5. Only two males at 6000 ppm
    survived to the end of the study; because of this high rate, no
    statistical analysis was performed for this group at 24 months. The
    main causes of death among these rats were nephropathy (22 rats),
    thyroid follicular-cell tumour (10 rats), and leukaemia (six rats);
    eight males at this dose died or were killed  in extremis during
    weeks 11-12 due to fractures of the nasal bone caused by the feeder
    plate and subsequent dyspnoea (rhinorrhagia). The feeder plates were
    removed.

         No clinical signs attributable to treatment were noted in any
    group during the first 52 weeks. After week 52, dose-related clinical
    signs included pale skin and mucous membranes in males at 6000 ppm,
    alopecia in females at 6000 ppm, and tissue masses on the skin and in
    the subcutis including the lip in males at 1200 and 6000 ppm. These
    changes attained statistical significance at week 77 or later.
    Dose-related, statistically significant depressions in body-weight
    gain were noted in both males and females at 1200 and 6000 ppm.
    Decreased food consumption was seen in animals of each sex at 6000 ppm
    starting at week 76. No dose-related abnormalities were observed in
    ophthalmoscopic parameters at 6, 12, 18, or 24 months. Treatment-
    related signs of anaemia (decreased erythrocyte count, haemoglobin,
    haematocrit, mean corpuscular volume, mean corpuscular haemoglobin,
    and mean corpuscular haemoglobin concentration) were seen in animals
    of each sex (mainly males) at 6000 ppm at 3, 6, 12, and 18 months.
    Increased platelet and leukocyte counts were seen frequently in males
    at 6000 ppm. Clinical chemical investigations revealed increased total
    cholesterol and total protein and decreased albumin:globulin ratios in
    animals of each sex at 1200 and/or 6000 ppm. At 24 months, treated
    males had increased blood urea nitrogen (at 75, 200, and 1200 ppm) and
    creatinine levels (at 1200 ppm), reduced thyroxine and triiod-
    othyronine values, and increased thyroid-stimulating hormone values
    (at 1200 and 6000 ppm, also in females at 6000 ppm). Dose-related
    increases in urinary protein (semi-quantitative analysis) were noted
    in animals of each sex at 6000 ppm at six and/or 12 months.

        Table 5.  Mortality rates in Fischer rats treated with thiophanate-methyl
                                                                                                                           

    Dose       Male                                                   Female                                               
    (ppm)
               Week 52         Week 80            Week 104            Week 52          Week 80          Week 104           

               No.     %       No.      %         No.       %         No.     %        No.     %        No.       %
                                                                                                                           

       0       0/60    0       2/50     4         13/50    26         0/60    0        3/50    6        13/50     26
      75       0/60    0       2/50     4         15/50    30         0/60    0        1/50    2        12/50     24
     200       0/60    0       8/50     16        24/50    48*        0/60    0        1/50    2        8/50      16
    1200       0/60    0       3/50     6         21/50    42         0/60    0        0/50    0        12/50     24
    6000       8/60    13*     18/55    33**      53/55    96**       1/60    2        3/50    6        11/50     22
                                                                                                                           

    Significantly different from control at *P < 0.05; ** P < 0.001 (chi-square test)
        Nephelometry indicated an increase in urinary protein in males at 1200
    and 6000 ppm at various times. Although males at 200 ppm had a
    statistically significant increase in urinary protein at 24 months,
    histopathological examination revealed no treatment-related effect. In
    males at 6000 ppm, ketone bodies, urinary volume, and water
    consumption were increased, and pH and specific gravity were decreased
    significantly at various times. Dose-related increases in the weights
    of the liver, kidney, and thyroid and their body-weight ratios were
    seen in animals of each sex at 1200 and/or 6000 ppm at both interim
    and final sacrifice.

         At interim sacrifice (week 53), treatment-related changes were
    found macroscopically in the liver (brownish-black) and kidneys
    (granular surface, brownish-black) of males and females at 6000 ppm.
    At final sacrifice, males at 1200 ppm had granular kidneys. Females at
    6000 ppm also had enlarged thyroids. In animals at 6000 ppm that died
    during the study, swollen thyroids were seen in animals of each sex
    and white areas in the liver and granular kidney in males. Dose-
    related histopathological changes were found in the thyroid, liver,
    kidney, and adrenal: Thyroid follicular-cell hyperplasia and
    hypertrophy were noted in animals of each sex at 1200 and/or 6000 ppm
    at 12 and 24 months, and the incidence of focal follicular-cell
    hyperplasia was increased. Males at 6000 ppm also had a statistically
    significant increased incidence of thyroid follicular-cell adenomas
    (12/60), and one of two males at 6000 ppm that were killed at 24
    months and 2/53 that were dead or were killed  in extremis had
    thyroid follicular-cell adenocarcinomas, with none in the other groups
    of treated males. The incidence of retinal atrophy was increased in
    females at 6000 ppm at 24 months, and those of parathyroid hypertrophy
    and hyperplasia were increased in males at this dose. Centrilobular
    hepatocellular hypertrophy and lipofuscin deposition were noted in
    animals of each sex at 1200 and/or 6000 ppm at 12 and 24 months and in
    most males and females at 6000 ppm which died during the study.
    Microgranuloma and focal fatty degeneration in the liver were
    increased in incidence in males treated at 6000 ppm. Lipidosis of the
    adrenal cortex was noted in females at 1200 ppm and in animals of each
    sex at 6000 ppm at 12 months. The severity of nephropathy was
    increased in animals of each sex at 6000 ppm at 12 months and in those
    at 1200 and 6000 ppm at 24 months. Most males at 6000 ppm that died or
    were killed  in extremis had severe nephropathy associated with
    hyperplasia of the parathyroid, demineralization of the bone, and
    metastatic calcification in various organs. The NOAEL was 200 ppm,
    equivalent to 8.8 mg/kg bw per day in males and 10.2 mg/kg bw per day
    in females. The increased blood urea nitrogen levels observed in males
    at 75 and 200 ppm and the increased urinary protein levels in males at
    200 ppm at 24 months were not considered to be adverse effects in view
    of the absence of treatment-related pathological findings at these
    doses (Takaori, 1993).

    (d)  Reproductive toxicity

    Mice

         Groups of eight male Swiss-Webster mice were given thiophanate-
    methyl (purity unspecified) as an oral dose of 192 mg/kg bw per day
    for five consecutive days. Twenty-four hours after the final dose
    [1,2-3H]-testosterone was administered intraperitoneally at
    10 Ci/kg bw in order to assess its assimilation by the anterior
    prostate coagulating glands. Animals were killed after 5 min, and the
    anterior prostate glands were removed. Thiophanate-methyl did not
    significantly affect body weights, absolute testicular weights,
    absolute or relative seminal vesicular weights, or total radiolabel
    assimilation by the anterior prostate gland. The absolute prostate and
    adrenal gland weights were significantly increased, and these changes
    were considered by the authors to be due to stress induced by
    thiophanate-methyl. The relative weights of the testes, prostate, and
    adrenal were also increased. Spermatogenesis was not affected, no
    sterile tubules were seen, and the interstitial (Leydig) cells
    appeared to be normal (Thomas, 1974; Thomas & Schein, 1974).

    Rats

         In a three-generation study, groups of 10 male and 20 female
    weaning Sprague-Dawley rats fed diets containing 0, 40 160, or 640 ppm
    thiophanate-methyl (purity unspecified) were mated twice to produce
    the F1a and F1b litters. Ten male and 20 female offspring were
    selected from the F1b litters and mated to produce the F2a and
    F2b litters; 10 male and 20 female F2b offspring were then
    selected and mated to produce the F3a and F3b litters. The test
    diets were fed throughout the study, animals in the F0, F1b, and
    F2b generations being fed for 60 days before mating. In each case,
    mating lasted for 20 days. Animals were re-mated to produce the second
    litters about 10 days after weaning of the first litters. The
    homogeneity, stability, and concentrations of thiophanate-methyl in
    the diets were not reported. In F0 animals, no treatment-related
    effects on mortality, food consumption, or body-weight gain or
    clinical signs of toxicity were seen. No treatment-related effects
    were observed on mating performance, length of gestation, or pregnancy
    rate or at terminal autopsy in F0, F1b, or F2b rats. The total
    litter sizes and weights at birth were slightly reduced in all animals
    at 640 ppm except the F3a litter. Viable litter size and weight at
    weaning were reduced in all generations at 640 ppm; mean pup weight
    was not affected. Viable litter size and weight between birth and
    weaning were slightly reduced in the second F2b litter at all doses,
    the percentage losses increasing with dose; however, average pup

    weights were not affected. The total litter loss and the incidence of
    gross malformations of pups were not treatment-related. No
    dose-related effects were seen on macroscopic appearance, organ
    weight, histopathological parameters, or the skeleton in F3b
    offspring. The NOAEL was 160 ppm, equivalent to about 8 mg/kg bw per
    day, on the basis of effects on litters at 640 ppm (Palmer  et al.,
    1972).

         In a two-generation study, groups of 25 male and 25 female
    Sprague Dawley rats were given diets containing thiophanate-methyl
    (purity, 95.93%) at doses of 0, 200, 630, or 2000 ppm. The
    homogeneity, stability, and concentrations of thiophanate-methyl in
    the diets were acceptable. After 14 weeks of treatment, the parental
    (F0) animals were mated for up to 21 days. The F0 females were
    allowed to litter and to rear their offspring (F1a generation) to
    weaning. After a 14-week maturation period after weaning, the parental
    F1 animals, selected from the F1a offspring, were mated for up to
    21 days and the females were allowed to litter and rear their
    offspring (F2a generation) to weaning. After weaning of the F2a
    pups and review of the results of rearing and weaning, including high
    pup mortality in all groups, the F1 generation was mated again, six
    weeks after weaning of the surviving F2a pups. The F1 animals were
    allowed to mate for a maximum of 21 days with the same partner, as
    during the first mating, and to produce a second litter (F2b). The
    body-weight gain of males of the F0 generation and males and females
    of the F1 generation at 2000 ppm was reduced, but only the
    body-weight gain of male F1 animals at 630 ppm was affected. Food
    consumption was affected by treatment before the first mating and
    during the first and second gestations and first lactation only in
    F1 females at 2000 ppm. At this dose, the males and females of the
    F0 and F1 generations had elevated levels of thyroid-stimulating
    hormone; slightly reduced triiodothyronine levels were noted in F0
    females at necropsy; and males and females of the F0 generation had
    lower thyroxine values at weeks 1 and 8 but not at necropsy. These
    findings are consistent with the histological findings and changes in
    organ weights found at this dose. Thyroid and liver weights were
    increased in male and female animals of the F0 and F1 generations
    at the high dose, and histopathological examinations of these animals
    showed treatment-related centrilobular hepatocyte hypertrophy in the
    liver and follicular-cell hypertrophy and hyperplasia (except in F1
    females) in the thyroid. The weights of male and female F1a, male
    F2a, and male and female F2b pups were reduced at 2000 ppm; at
    630 ppm, only the weights of male and female F2b pups were reduced.
    Very high pup losses, mainly after day 4 of lactation, occurred in all
    animals of the F2a generation, including the controls. No clear
    explanation was found for the pup losses. Those pups that did not die
    had normal postnatal development, except for reduced body-weight gain
    in male pups at the high dose. Such losses were not seen in the F2b

    groups. Functional tests of F1a, F2a, and F2b animals showed no
    treatment-related findings. The NOAEL was 630 ppm for male and female
    F0 animals, 200 ppm for the F1 generation, 630 ppm for the F1a
    and F2a offspring, and 200 ppm for the F2b offspring. The NOAEL
    was 2000 ppm for fertility and general reproductive performance
    (Muller, 1993).

         Since treatment-related histopathological effects were found in
    the thyroid and liver in rats at the high dose in the preceding study,
    organs from all F0 and F1 animals at 200 and 630 ppm were further
    evaluated. The incidence and severity of hepatocyte hypertrophy were
    increased in a dose-related fashion at all doses in F0 males, and
    the incidence and to a lesser extent the severity of thyroid
    follicular-cell hypertrophy and hyperplasia were increased in the same
    animals. F1 males also had increased (but to a lesser extent)
    hepatocyte hypertrophy and thyroid follicular cell hyperplasia at all
    doses. The minimal effect level was thus 200 ppm, equivalent to
    9.7-14.7 mg/kg bw per day (Muller & Singer, 1995).

    (e)  Developmental toxicity

    Mice

         Groups of 20 female ICR mice were given thiophanate-methyl
    (purity, 94%) at doses of 0, 40, 200, 500, or 1000 mg/kg bw per day by
    gavage on days 1-15 of gestation. Animals were killed on day 18 or 19
    (discrepancy between the study report and publication). No deaths,
    abnormalities, or significant differences in body weights were
    recorded. The number of living fetuses per litter was reduced in
    animals at 1000 mg/kg bw per day, partly as a result of increased
    resorptions. No treatment-related effects were observed on
    implantation numbers, litter sizes, fetal body weights, sex ratio, or
    numbers of immature or malformed fetuses. The NOAEL was 1000 mg/kg bw
    per day for maternal toxicity and 500 mg/kg bw per day for embryo- and
    fetotoxicity, on the basis of the reduced number of live fetuses at
    1000 mg/kg bw per day. There was no evidence of teratogenicity
    (Noguchi, 1970c; Makita  et al., 1973).

    Rats

         Groups of 25 female Sprague-Dawley rats were given
    thiophanate-methyl (purity, 97.2%) at doses of 0, 100, 300, or
    1000 mg/kg bw per day by gavage on days 6-19 of gestation. Animals
    were killed on day 20 of gestation. There were five to nine nongravid
    females per group, including the control group. One rat at 100 mg/kg
    bw per day and one at 300 mg/kg bw per day delivered on days 13 and
    11, respectively, and were killed on the day of delivery. The weight
    and development of the offspring were comparable to those of animals
    delivered later, suggesting incorrect assessment of copulation; no

    malformations or maternal abnormalities were seen in these animals. No
    deaths, treatment-related clinical symptoms, or gross pathological
    abnormalities were reported in any dams. Body-weight gain was reduced
    between days 6 and 9 in animals at 1000 mg/kg bw per day. There were
    no treatment-related effects on total implantations, post-implantation
    loss, fetal body weight, fetal sex ratio, or viable fetuses. There
    were no dose-related fetal malformations or developmental
    abnormalities, and the incidences of these parameters were within
    those of historical controls. A positive control study in the same
    strain with a single high dose of vitamin A produced the appropriate
    results. The NOAEL was 300 mg/kg bw per day for maternal toxicity, on
    the basis of the reductions in body-weight gain of dams at 1000 mg/kg
    bw per day during treatment. There was no evidence of teratogenicity
    or embryo- or fetotoxicity (Rodwell  et al., 1981).

    Rabbits

         Groups of 15 female New Zealand white rabbits received
    thiophanate-methyl (purity, 96.2%) at doses of 0, 2, 6, or 20 mg/kg bw
    per day by gavage on days 6-19 of gestation. Samples of test solutions
    were taken during the first and last weeks of treatment, and the
    concentrations of thiophanate-methyl were found to be acceptable. The
    animals were killed on day 29 of gestation. No treatment-related
    maternal mortality or clinical or subsequent maternal effects were
    seen. Dose-related losses in maternal body weight were observed mainly
    at the beginning of treatment (days 6-8 at 6 mg/kg bw per day and days
    6-14 at 20 mg/kg bw per day). The food consumption of animals at
    20 mg/kg bw per day was reduced from the start of treatment, and large
    reductions during days 6-12 were generally associated with subsequent
    abortion or sacrifice  in extremis. Water consumption was unaffected.
    One of 12 animals at 2 mg/kg bw per day, one of 14 at 6 mg/kg bw per
    day, and two of 13 at 0 mg/kg bw per day aborted. One control animal
    and one at 20 mg/kg bw per day suffered total litter loss and
    resorption; the control animal suffered early resorption of its only
    implantation, while the treated animal lost five of five implantations.
    No treatment-related trends were observed in resorptions, pre-
    implantation losses, or fetal or placental weights. The numbers of
    viable young were slightly decreased in animals at 20 mg/kg bw per day
    due to abortions and total litter losses. Gross examination of the
    fetuses showed that in animals at 6 and 20 mg/kg bw per day, there was
    an apparent treatment-related trend in skeletal abnormalities in ribs,
    vertebrae, and pelvis that was generally close to or slightly greater
    than the upper limit of historical controls. Treatment-related effects
    included increased incidences of 13 pairs of ribs, incomplete or
    asymmetric ossification of costal elements of the sacral vertebrae, 27
    presacral vertebrae, and asymmetric pelvises associated with different
    sacral vertebrae. At 20 mg/kg bw per day, the incidences of one or
    more ribs thickened at the costal cartilage were significantly

    increased. The frequencies of these skeletal abnormalities are
    summarized in Table 6. The NOAEL was 2 mg/kg bw per day for maternal
    toxicity, on the basis of the effect on maternal growth rate, and
    2 mg/kg bw per day for developmental toxicity, on the basis of the
    increased incidence of skeletal abnormalities at 6 and 20 mg/kg bw per
    day (Ross  et al., 1986).

        Table 6.  Incidence of selected skeletal abnormalities in fetuses of rabbits
              treated with thiophanate-methyl
                                                                                              

    Observation                   Dose (mg/kg bw per day)       Historical controls           

                                  0     2      6      20        Mean      Range
                                                                                              

    13 pairs of ribs              41    46     63     61        36        12-61
    Thickened ribs                 1     4      2     14         2        0-13
    Incomplete or assymetrical     3     6      7     12         4        0-15
    ossification
    27 presacral vertebrae        16    18     38     43        18        7-44
    Assymetrical pelvis            3     5      7     10         4        0-9
                                                                                              
    
    (f)  Genotoxicity

         The results of studies of the genotoxicity and mutagenicity of
    thiophanate-methyl  in vitro and  in vivo are summarized in Table 7.
    There was no evidence of genotoxicity or mutagenicity. Thiophanate-
    methyl is partially metabolized in mammalian systems to carbendazim,
    which has been shown to act as an aneugen.

         The aneugenic potential of thiophanate-methyl (purity, 95%;
    dispersed in 70% kaolin) was tested in male Swiss albino mice given
    oral doses of 1000 mg/kg bw. Bone-marrow cells were analysed 16, 24,
    36, and 48 h after treatment for micronuclei, chromosomal aberrations,
    hyperdiploidy, and polyploidy. Large micronuclei were significantly
    induced, but the response was relatively weak, and thiophanate-methyl
    was less effective than benomyl or carbendazim. There was no increase
    in the frequency of chromosomal aberrations. At 24 and 36 h, a
    possibly treatment-related increase in the frequency of polyploid and
    hyperdiploid cells was observed, which was of borderline significance
    in view of the very low frequency of changes in ploidy (Barale
     et al., 1993).

        Table 7.  Results of tests for the genttoxicity of thiophanate-methyl
                                                                                                                                              
    End-point                  Test system                   Concentration                 Purity     Results        Reference
                                                             or dose                       (%)
                                                                                                                                              

    In vitro
    Reverse mutation           S. typhimurium TA98,          10, 50, 100,                  97.3       Negativea      Shirasu et al. (1976)
                               TA100, TA1535, TA1537,        500, 1000 g/plate
                               TA1538, E. coli WP2 hcr       dissolved in DMSO
    Reverse mutation           S. typhimurium TA98,          39.1, 78.1, 156.3,            96.55      Negativea      Kanaguchi & Nishibe
                               TA100, TA1535, TA1537,        312.5, 625, 1250,                                       (1990)
                               E. coil WP2 uvrA              2500, 5000 g/plate
                                                             in DMSO
    Host-mediated              S. typhimurium G46            1000, 3000 mg/kg              97.3       Negative       Shirasu et al. (1976)
     mutation                                                bw twice
    Gene mutation              B. subtills H17, M45          20, 100, 200, 500,            97.3       Negative       Shirasu et al. (1976)
                                                             1000, 2000 g,/disk
    Gene mutation              Chinese hamster V79 cells,    6.25, 12.5, 25.0, 50,         96.36      Negativea      Tippins et al. (1984)
                               hprt locus                    100 g/ml in DMSOa
    Chromosomal aberration     Chinese hamster ovary cells   100, 200, 300, 400            95.0       Negative       Murli (1988)
                                                             g/ml in DMSOa
                                                             250, 500, 750,
                                                             1000 g/ml in DMSOb
    Unscheduled DNA            Primary hepatocytes           5, 10, 25, 50, 100,           100        Negative       Myhr (1981)
     synthesis                                               250, 500, 1000 g/ml

    In vivo
    Dominant lethal mutation   ICR mice                      8-500 mg/kg bw                94         Negative       Makita et al. (1973)
                                                             intraperitoneally
    Cytogenicity               Wistar rat bone marrow        62.5-1000 mg/kg bw            94         Negative       Makita et al. (1973)
                               and spermatocytes             intraperitoneally
                                                                                                                                              

    DMSO, dimethyl sulfoxide
    a  Without metabolic activation
    b  With metabolic activation
        (g)  Special studies

    (i)  Dermal and ocular irritation and dermal sensitization

         Thiophanate-methyl (0.5 g; purity, 96.2%) moistened with water
    was applied to the backs of six male New Zealand white rabbits for 4 h
    under an occlusive patch. There were no signs of irritation over a
    72-h observation period (Souma & Nishibe, 1986a).

         Nine male New Zealand white rabbits received 0.1 g
    thiophanate-methyl (purity, 96.2%) in the left eye. The treated eyes
    of three rabbits were washed with water, and those of the others
    remained unwashed. Conjunctival redness was observed in four of the
    unirrigated eyes and in all three of the irrigated eyes after 1 h.
    These symptoms had resolved by 24 h in all animals except one with an
    unwashed eye, in which irritation resolved by 48 h. Thiophanate-methyl
    was considered to be a very mild eye irritant (Souma & Nishibe,
    1986b).

         The skin sensitizing potential of thiophanate-methyl (purity,
    96.2%) was tested in 20 female Hartley guinea-pigs by the
    Magnusson-Kligman technique. Thiophanate-methyl was sensitizing under
    the conditions of the study (Souma & Nishibe, 1989). In a Buehler
    test, thiophanate-methyl (purity, 96.55%) was not sensitizing in 10
    female Hartley guinea-pigs (Mochizuki, 1993).

    (ii)  Effects on the thyroid and liver

         A number of studies were carried out to elucidate the effects of
    thiophanate-methyl on the thyroid and liver (Nishibe & Takaori, 1990).

         Thiophanate-methyl (purity, 96.55%) was administered in the diets
    of six-week-old male Fischer 344 rats for two or eight days at a
    concentration of 6000 ppm. Propylthiouracil, an inhibitor of thyroid
    hormone synthesis, was administered at 1000 ppm for two or eight days,
    and phenobarbital, an inducer of drug-metabolizing enzymes, was
    administered at 500 ppm for eight days, as positive controls.
    Thiophanate-methyl reduced triiodothyronine and thyroxine levels,
    increased thyroid-stimulating hormone activity (at day 8 only),
    increased thyroid weights (at day 8 only), and increased liver
    weights. Propylthiouracil caused similar but more marked changes in
    thyroid hormone levels and weights. Phenobarbital increased liver
    weights. All three chemicals increased serum cholesterol levels by day
    8. Thiophanate-methyl and phenobarbital increased microsomal
    cytochrome P450, cytochrome b5, total protein, and UDP-glucu-
    ronosyltransferase activities.

         Seven-week-old female Fischer 344 rats were given 6000 ppm
    thiophanate-methyl or 500 ppm phenobarbital for eight days. Some were
    killed at eight days, and the others were killed after an eight-day
    recovery. Thyroid weights were increased at day 8.

         Groups of eight-week-old rats (sex not specified) were fed diets
    containing 0 or 6000 ppm thiophanate-methyl, 30 g/kg bw thyroxine, or
    6000 ppm thiophanate-methyl and daily injections of 30 g/kg thyroxine
    subcutaneously, for eight days. Thiophanate-methyl caused hypertrophy
    of the thyroid and liver and increased the level of thyroid-
    stimulating hormone; these changes were suppressed by concomitant
    administration of thyroxine. Thiophanate with and without thyroxine
    also increased serum cholesterol. Thyroxine alone caused none of these
    changes.

         Thiophanate-methyl was less effective than propylthiouracil in
    inhibiting porcine thyroid microsomal peroxidase. The ED50 and ED0
    were 6  10-4 mol/litre and 8  10-5 mol/litre for thiophanate-
    methyl and 2  10-5 mol/litre and 4  10-7 mol/litre for
    propylthiouracil.

         Eleven-week-old male ICR mice and Fischer 344 rats were given
    diets containing 6000 ppm thiophanate-methyl or 500 ppm phenobarbital
    for two or eight days. Liver weights were increased in all treated
    animals. The titre of hepatocyte proliferating cell nuclear antigen,
    the polymerase delta accessory protein, was increased in rats at day 2
    and mice at days 2 and 8 (Nishibe & Takaori, 1990).

    3.  Observations in humans

         In 1969-72, 16 production workers continuously exposed to
    Topsin-E containing thiophanate or Topsin-M containing thiophanate-
    methyl were routinely tested for effects on health. Blood and urine
    samples were taken for analysis every six months: blood was analysed
    for erythrocyte and leukocyte counts, haemoglobin content, and
    specific gravity, and urine for protein, sugar, and urobilinogen.
    Serum alanine and aspartate aminotransferases activities were
    determined twice during the first six months of 1972 only. The initial
    tests were conducted in January 1969, prior to production of Topsin.
    No significant effects were seen in any of the workers for any of the
    parameters investigated (Mori, 1972).

         The same tests reported by Mori (1972) were conducted twice a
    year on 28 production workers exposed to Topsin M and/or Topsin E in
    1973-77, except that leukocyte and erythrocyte counts and haemoglobin
    levels were not tested during 1976 for undescribed 'personal reasons'.
    Serum alanine and aspartate aminotransferase activities were
    determined in all employees in October 1977. More than 13 employees
    were tested each year, but the specific individuals tested changed
    during the study so that only seven individuals were monitored
    continuously. No adverse behavioural, haematological, clinical
    chemical, or urinary findings were made (Ikeda, 1978).

         Between 1985 and 1991, no adverse behavioural symptoms (including
    skin or eye effects) or clinical findings (haematology, clinical
    chemistry, or urinalysis) were observed in workers exposed to
    thiophanate-methyl and thiophanate during production. Health
    examinations were performed twice a year. The workers were involved
    mainly in the production of thiophanate-methyl, usually for 11 months
    of the year (Aizawa, 1991).

    Comments

         Thiophanate-methyl was rapidly absorbed after oral exposure of
    mice, rats, and dogs. Most of the administered radiolabel was
    recovered within 24 h of dosing, predominantly in the urine.
    Carbendazim and 5-hydroxycarbendazim were among the metabolites of
    thiophanate-methyl identified  in vivo and  in vitro.

         Thiophanate-methyl has low acute toxicity in rats, with an oral
    LD50 of about 7000 mg/kg bw. Clinical signs of toxicity after acute
    dosing with thiophanate-methyl were generally non-specific. WHO has
    classified thiophanate-methyl as unlikely to present an acute hazard
    in normal use.

         In a six-month study of toxicity, mice were given dietary doses
    of 0, 13, 64, 320, 1600, or 8000 ppm thiophanate-methyl. The NOAEL was
    320 ppm, equivalent to 48 mg/kg bw per day, on the basis of
    haematological effects and hepatotoxicity. In a six-month study, rats
    were exposed to thiophanate-methyl in the diet at levels of 0, 13, 64,
    320, 1600, or 8000 ppm. Reduced erythrocyte counts, reduced
    body-weight gain, and evidence of hepatotoxicity and thyroid
    stimulation were seen at 8000 ppm. The NOAEL was 1600 ppm, equivalent
    to 80 mg/kg bw per day. Dogs were exposed to thiophanate-methyl by
    administration in capsules at doses of 0, 50, 200, or 400 mg/kg bw per
    day for 13 weeks, 0, 8, 40 or 200 mg/kg bw per day for one year, or 0,
    2, 10, 50, or 250 mg/kg bw per day for two years. Treatment-related
    thyroid hyperplasia was seen in all three studies; the overall NOAEL
    was 10 mg/kg bw per day.

         In a two-year study of carcinogenicity in mice fed dietary levels
    of 0, 10, 40, 160, or 640 ppm, there was no evidence of any
    carcinogenic response; the NOAEL was 160 ppm (equal to 24 mg/kg bw per
    day) on the basis of reduced body-weight gain. In a subsequent
    18-month study in mice fed dietary levels of 0, 150, 640, 3000, or
    7000 ppm, the NOAEL was 150 ppm (equal to 29 mg/kg bw per day) on the
    basis of treatment-related hepatotoxicity and hepatocellular tumours.
    In a two-year study of carcinogenicity in rats fed dietary levels of
    0, 10, 40,160, or 640 ppm, effects were seen on the testes (mainly
    hypospermatogenesis), thyroid gland, and growth rate at 640 ppm; the
    NOAEL was 160 ppm (equivalent to 8 mg/kg bw per day). In a two-year
    study in rats fed dietary levels of 0, 75, 200, 1200, or 6000 ppm, the
    NOAEL was 200 ppm (equal to 9 mg/kg bw per day). Thyroid hyperplasia
    and thyroid tumours were seen at higher doses, together with
    parathyroid hyperplasia, nephrotoxicity, hepatotoxicity, and lipidosis
    of the adrenal cortex.

         In a three-generation study of reproductive toxicity in which
    rats received dietary doses of 0, 40, 160, or 640 ppm, reductions in
    total litter size and weight at birth and at weaning were seen at
    640 ppm; the NOAEL was 160 ppm, equivalent to 8 mg/kg bw per day. In a
    two-generation study in which rats received dietary doses of 0, 200,

    630 or 2000 ppm, there was no evidence of a treatment-related effect
    on reproduction. The lowest dose tested in the study, equivalent to
    10 mg/kg bw per day, was a minimal-effect level on the basis of
    hepatocyte hypertrophy, thyroid follicular-cell hypertrophy, and
    hyperplasia at all treatment levels.

         The teratogenic potential of thiophanate-methyl was investigated
    in mice, rats, and rabbits. In mice exposed by gavage to thiophanate-
    methyl at doses of 0, 40, 200, 500, or 1000 mg/kg bw per day on days
    1-15 of gestation, there was no evidence of teratogenicity or maternal
    toxicity at the highest dose, although embryo- and fetotoxicity were
    seen at this dose. In rats exposed by gavage to thiophanate-methyl at
    doses of 0, 100, 300, or 1000 mg/kg bw per day on days 6-19 of
    gestation, there was no evidence of fetotoxicity or teratogenicity,
    but maternal toxicity, expressed as reduced body-weight gain, was seen
    at the highest dose. In rabbits exposed by gavage to thiophanate-
    methyl at doses of 0, 2, 6, or 20 mg/kg bw per day on days 6-19 of
    gestation, maternal toxicity (reduced growth rate) was seen at 6 mg/kg
    bw per day and above. A dose-related trend of increased fetal skeletal
    abnormalities (ribs, vertebrae, and pelvis) at 6 and 20 mg/kg bw per
    day indicated an NOAEL for developmental toxicity of 2 mg/kg bw per
    day.

         Thiophanate-methyl was adequately tested for genotoxicity in a
    series of assays  in vivo and  in vitro. The only significant
    response was a small increase in the frequency of micronuclei
    characterized by their large size in mouse bone-marrow cells, which
    was not associated with the induction of structural chromosomal
    aberrations. This is indicative of a weak aneugenic effect. The
    Meeting concluded that thiophanate-methyl is not genotoxic.

         Studies on the effects of thiophanate-methyl on the liver and
    thyroid of rats showed liver enzyme induction, reductions in thyroid
    hormones (triiodothyronine and thyroxine), and increases in the level
    of thyroid-stimulating hormone and in thyroid and liver weights.
    Thyroid hypertrophy and increases in thyroid-stimulating hormone
    levels associated with treatment with thiophanate-methyl were
    suppressed by concomitant treatment with thyroxine. Thiophanate-methyl
    was shown to inhibit porcine thyroid microsomal peroxidase, an enzyme
    involved in thyroid hormone synthesis. The thyroidal and hepatic
    changes, including tumours, observed in the studies of toxicity may be
    due to increased hepatocyte turnover, reductions in the thyroid
    hormones triiodothyronine and thyroxine as a result of liver enzyme
    induction, and inhibition of thyroid microsomal peroxidase.

         Workers involved in manufacturing products containing
    thiophanate-methyl showed no treatment-related adverse local (skin or
    eyes) or systemic effects.

         An ADI of 0-0.02 mg/kg bw was established on the basis of the
    NOAEL of 2 mg/kg bw per day for developmental toxicity in rabbits and
    a safety factor of 100.

         The Meeting noted that the use of thiophanate-methyl on crops
    gives rise to residues of carbendazim, although thiophanate-methyl can
    also be detected as part of the residue to which consumers of treated
    produce are exposed. Since the toxicity of thiophanate-methyl is
    qualitatively and quantitatively (when corrected for relative
    molecular mass) different from that of carbendazim, the Meeting
    concluded that the intake of residues in food should initially be
    compared with the ADI for thiophanate-methyl. If further refinement of
    the risk assessment is necessary, the different components of the
    residue (carbendazim and thiophanate-methyl) may be characterized.

    Toxicological evaluation

     Levels that cause no toxic effect

    Mouse:    150 ppm, equal to 29 mg/kg bw per day (18-month study of
              toxicity and carcinogenicity)
              1000 mg/kg bw per day (maternal toxicity and teratogenicity
              in study of reproductive toxicity)
              500 mg/kg bw per day (fetotoxicity in study of developmental
              toxicity)

    Rat:      200 ppm, equal to 9 mg/kg bw per day (two-year study of
              toxicity and carcinogenicity)
              160 ppm, equivalent to 8 mg/kg bw per day (study of
              reproductive toxicity)
              1000 mg/kg bw per day (teratogenicity and fetotoxicity in
              study of developmental toxicity)
              300 mg/kg bw per day (maternal toxicity in a study of
              developmental toxicity)

    Rabbit:   2 mg/kg bw per day (maternal toxicity and teratogenicity in
              study of developmental toxicity)

    Dog:      10 mg/kg bw per day (studies of toxicity up to two years)

     Estimate of acceptable daily intake for humans

         0-0.02 mg/kg bw

     Studies that would provide information useful for continued evaluation
     of the compound

    1.   Comparison of the metabolism of thiophanate-methyl and
         carbendazim in different species, including humans

    2.   Further observations in humans

        Toxicological criteria for setting guidance values for dietary and non-dietary exposure to thiophanate-methyl
                                                                                                                           

    Exposure                  Relevant route, study type, species       Results, remarks
                                                                                                                           

    Short-term (1-7 days)     Oral, toxicity, rat                       LD50 = 7000 mg/kg bw
                              Dermal, toxicity, rat                     LD50 > 10 000 mg/kg bw
                              Dermal, irritation, rabbit                Not irritating
                              Ocular, irritation, rabbit                Mildly irritating
                              Dermal, sensitization, guinea-pig         Sensitizing in maximization test; not sensitizing
                                                                        in Buehler test
                              Inhalation, toxicity, rat                 LC50 = 1.8 mg/litre air

    Mid-term (1-26 weeks)     Oral, reproductive toxicity, rabbit       NOAEL = 2 mg/kg bw per day; maternal and
                                                                        developmental toxicity

    Long-term (> one year)    Dietary, toxicity and carcinogenicity,    NOAEL = 9 mg/kg bw per day; thyroid rumours
                              two years, rat                            and hepatotoxicity
                              Oral, toxicity, two years, dog            NOAEL = 10 mg/kg bw per day; hepatotoxicity and
                                                                        thyroid effects
                                                                                                                           
        References

    Aizawa, T. (1991) Human handling experiences from plant employees
         manufacturing Topsins (3). Unpublished report No. RD-9153 from
         Nippon Soda Co. Ltd. Submitted to WHO by Nippon Soda Co. Ltd,
         Tokyo, Japan.

    Auletta, CS. (1991) A subchronic (3-month) oral toxicity study in the
         dog via capsule administration with thiophanate-methyl.
         Unpublished report Project No. 89-3525 (RE-9119) from
         Bio/dynamics Inc. Submitted to WHO by Nippon Soda Co. Ltd, Tokyo,
         Japan.

    Auletta, C.S. (1992) A chronic (1-year) oral toxicity study in the dog
         via capsule administration with thiophanate-methyl. Unpublished
         report Project No. 89-3526 (RD-9207) from Bio/dynamics Inc.
         Submitted to WHO by Nippon Soda Co. Ltd, Tokyo, Japan.

    Barale, R., Scapoli, C., Meli, C., Casini, D., Minunni, M.,
         Marrazzini, A., Loprieno, N. & Barrai, I. (1993) Cytogenetic
         effects of benzimidazoles in mouse bone marrow.  Mutat. Res.,
         300, 15-28.

    Douch, P.G.C. (1974) The metabolism of some thioureidobenzene
         fungicides in mice and sheep.  Xenobiotica, 4, 457-475.

    Fujino, A., Ohnuma, N., Mori, T., Tanoue, T. & Kamimura, H. (1973) The
         balance and metabolism studies of thiophanate-methyl in animals.
         Unpublished report from the Nisso Institute for Life Science,
         Nippon Soda Co. Ltd, Kanagawa, Japan. Submitted to WHO by Nippon
         Soda Co. Ltd, Tokyo, Japan.

    Hashimoto, Y. (1972) Final report on chronic oral toxicity studies on
         thiophanate-methyl, dimethyl 4,4'-O-phenylene bis 3-thioal-
         lophanate in Sprague-Dawley strain rats. Unpublished report from
         the Nisso Institute for Life Science, Nippon Soda Co. Ltd,
         Kanagawa, Japan. Submitted to WHO by Nippon Soda Co. Ltd, Tokyo,
         Japan.

    Hashimoto, Y., Makita, T., Nishibe, T., Mori, T., Ohnuma, N., Noguchi,
         T. & Ohta, G. (1973) Subacute toxicity of thiophanate-methyl in
         mice and rats.  Pharmacometrics, 7, 929-945.

    Ikeda, K. (1978) Human handling experiences from plant employees
         manufacturing Topsin (2). Unpublished report from Nippon Soda Co.
         Ltd. Submitted to WHO by Nippon Soda Co. Ltd, Tokyo, Japan.

    Kanaguchi, Y. & Nishibe, T. (1990) Thiophanate-methyl reverse mutation
         study on bacteria. Unpublished report No. RD-9065 from Nippon
         Soda Co. Ltd. Submitted to WHO by Nippon Soda Co. Ltd, Tokyo,
         Japan.

    Kosaka, S. (1973) The report on the carcinogenesis studies of
         thiophanate-methyl, dimethyl 4,4'-O-phenylene bis-(3-thioal-
         lophanate) in mice of ICR-SLC strain for 24 months. Unpublished
         report from the Nisso Institute for Life Science, Nippon Soda Co.
         Ltd, Kanagawa, Japan. Submitted to WHO by Nippon Soda Co., Ltd,
         Tokyo, Japan.

    Makita, T., Hashimoto, Y. & Noguchi, T. (1973) Mutagenic, cytogenetic
         and teratogenic studies on thiophanate-methyl.  Toxicol. Appl.
          Pharmacol., 24, 206-215.

    Mochizuki, N. (1993) Topsin M skin sensitization study in guinea pigs.
         Unpublished report No. RD-9347 from Nippon Soda Co. Ltd.
         Submitted to WHO by Nippon Soda Co., Ltd, Tokyo, Japan.

    Mori, H. (1972) Human handling experiences from plant employees
         manufacturing Topsin (1). Unpublished report from the Fine
         Chemical Division, Nippon Soda Co. Ltd. Submitted to WHO by
         Nippon Soda Co. Ltd, Tokyo, Japan.

    Muller, W. (1993) Topsin M two generation oral (dietary
         administration) reproduction toxicity study in the rat.
         Unpublished report HLA Study No. 683-004 (RD-9329) from Hazleton
         Deutschland GmbH. Submitted to WHO by Nippon Soda Co. Ltd, Tokyo,
         Japan.

    Muller, W. & Singer, A. (1995) Topsin M final addendum histopathology
         report and peer review pathology report to MRID 42899101. Two
         generation oral (dietary administration) reproduction toxicity
         study in the rat. Unpublished report HLA Study No. 683-004
         addendum histopathology report (RD-9525) from Hazleton
         Deutschland GmbH. Submitted to WHO by Nippon Soda Co. Ltd, Tokyo,
         Japan.

    Murli, H. (1988) Mutagenicity test on Topsin M technical (thiophanate-
         methyl) in an in-vitro cytogenetic assay measuring chromosomal
         aberration frequencies in Chinese hamster ovary (CHO) cells.
         Unpublished report HLA Study No 10345-0-437 (RD-9120) from
         Hazleton Laboratories America, Inc. Submitted to WHO by Nippon
         Soda Co. Ltd, Tokyo, Japan.

    Myhr B.C. (1981) Primary rat hepatocyte unscheduled DNA synthesis
         assay of thiophanate-methyl. Unpublished report No. LBI Project
         No 21191 (RD-8195) from Litton Bionetics. Submitted to WHO by
         Nippon Soda Co. Ltd, Tokyo, Japan.

    Nishibe, T. (1987) Thiophanate-methyl: Acute inhalation toxicity study
         in rats. Unpublished report No. RD-8711 from the Toxicology
         Institute, Environmental Toxicology Laboratory, Nippon Soda Co.
         Ltd, Kanagawa, Japan. Submitted to WHO by Nippon Soda Co. Ltd,
         Tokyo, Japan.

    Nishibe, T. & Takaori, H. (1990) Summary of mechanistic investigation
         of the effect of thiophanate-methyl on thyroid and liver.
         Unpublished report from the Toxicology Institute, Environmental
         Toxicology Laboratory, Nippon Soda Co. Ltd, Kanagawa, Japan.
         Submitted to WHO by Nippon Soda Co. Ltd, Tokyo, Japan.

    Noguchi, T. (1970a) Toxicological evaluation of thiophanate-methyl
         (V). Some pharmacological properties of a new fungicide,
         thiophanate-methyl. Unpublished report from the Nisso Institute
         for Life Sciences, Kanagawa, Japan. Submitted to WHO by Nippon
         Soda Co. Ltd, Tokyo, Japan.

    Noguchi, T. (1970b) Studies on the biotransformation of thiophanate-
         methyl in animal and plant (Part 1). Unpublished report from
         the Chemical Research Laboratories, Nisso Institute for Life
         Sciences, Kanagawa-ken, Japan. Submitted to WHO by Nippon Soda
         Co. Ltd, Tokyo, Japan.

    Noguchi, T. (1970c) Toxicological evaluation of thiophanate-methyl
         (IV). Studies on the teratogenic effect of thiophanate-methyl
         upon the fetus of ICR strain of mice. Unpublished report from the
         Nisso Institute for Life Sciences, Kanagawa, Japan. Submitted to
         WHO by Nippon Soda Co. Ltd, Tokyo, Japan.

    Noguchi, T. (1971) Studies on the biotransformation of
         thiophanate-methyl in animal, plant and soil (Part II).
         Unpublished report from the Chemical Research Laboratories, Nisso
         Institute for Life Sciences, Kanagawa-ken, Japan. Submitted to
         WHO by Nippon Soda Co. Ltd, Tokyo, Japan.

    Noguchi, T. (1972) Studies on the biotransformation of
         thiophanate-methyl in animal and plant. Unpublished report from
         the Chemical Research Laboratories, Nisso Institute for Life
         Sciences, Kanagawa-ken, Japan. Submitted to WHO by Nippon Soda
         Co. Ltd, Tokyo, Japan.

    Noguchi, T. & Hashimoto, Y. (1970a) Toxicological evaluation of
         thiophanate-methyl (I). Acute and subacute toxicity of
         thiophanate-methyl. Unpublished report from the Nisso Institute
         for Life Sciences, Kanagawa-ken, Japan. Submitted to WHO by
         Nippon Soda Co. Ltd, Tokyo, Japan.

    Noguchi, T. & Hashimoto, Y. (1970b) Toxicological evaluation of
         thiophanate-methyl (II). Studies on the subchronic oral toxicity
         of thiophanate-methyl in mice. Unpublished report from the Nisso
         Institute for Life Sciences, Kanagawa, Japan. Submitted to WHO by
         Nippon Soda Co. Ltd, Tokyo, Japan.

    Noguchi, T. & Hashimoto, Y. (1970c) Toxicological evaluation of
         thiophanate-methyl (III). Studies on the subchronic oral toxicity
         of thiophanate-methyl in rats. Unpublished report from the Nisso
         Institute for Life Sciences, Kanagawa, Japan. Submitted to WHO by
         Nippon Soda Co. Ltd, Tokyo, Japan.

    Palmer, A., Lovell., M. & Newman, A. (1972) Effect of
         thiophanate-methyl on reproductive function of multiple
         generations in the rat. Unpublished report from Huntingdon
         Research Centre, Huntingdon, Cambs, United Kingdom. Submitted to
         WHO by Nippon Soda Co. Ltd, Tokyo, Japan.

    Rodwell, D.E. et al (1981) Teratology study in thiophanate-methyl in
         rats. Unpublished report No. 449-006 (RD-8126) from International
         Research and Development Corporation, Mattawan, Michigan, USA.
         Submitted to WHO by Nippon Soda Co. Ltd, Tokyo, Japan.

    Ross, F.W.  et al. (1986) Thiophanate-methyl: Teratology study in the
         rabbit. Unpublished report No. 86/NIS010/11 (RD-8642) from Life
         Science Research. Submitted to WHO by Nippon Soda Co. Ltd, Tokyo,
         Japan.

    Saike, O. & Nishibe, T. (1987) Thiophanate-methyl: Acute inhalation
         toxicity study in rats. Unpublished report No. 0219 from Nisso
         Institute for Life Sciences, Kanagawa, Japan. Submitted to WHO by
         Nippon Soda Co. Ltd, Tokyo, Japan.

    Shirasu, Y.  et al. (1976) Mutagenicity testing on thiophanate-methyl
         in microbial systems. Unpublished report No. RD-8084 from the
         Institute of Environmental Toxicology. Submitted to WHO by Nippon
         Soda Co. Ltd, Tokyo, Japan.

    Singh, T. & Garg, B. (1989) Effect of thiophanate-methyl on
         cardiovascular system.  Indian Vet. J., 66, 1116-1119.

    Souma, S. & Nishibe, T. (1986a) Thiophanate-methyl primary dermal
         irritation study in rabbits. Unpublished report No. RD-8692 from
         Nisso Institute for Life Sciences, Kanagawa, Japan. Submitted to
         WHO by Nippon Soda Co. Ltd, Tokyo, Japan.

    Souma, S. & Nishibe, T. (1986b) Thiophanate-methyl primary eye
         irritation study in rabbits. Unpublished report No. RD-8691 from
         Nisso Institute for Life Sciences, Kanagawa, Japan. Submitted to
         WHO by Nippon Soda Co. Ltd, Tokyo, Japan.

    Souma, S. & Nishibe, T. (1989) Thiophanate-methyl: Delayed contact
         hypersensitivity study in guinea pigs. Unpublished report No.
         RD-8924 from Nisso Institute for Life Sciences, Kanagawa, Japan.
         Submitted to WHO by Nippon Soda Co. Ltd, Tokyo, Japan.

    Souma, S. & Nishibe, T. (1990a) Thiophanate-methyl acute oral toxicity
         study in rats. Unpublished report No. RD-9083 from Nisso
         Institute for Life Sciences, Kanagawa, Japan. Submitted to WHO by
         Nippon Soda Co. Ltd, Tokyo, Japan.

    Souma, S. & Nishibe, T. (1990b) Thiophanate-methyl acute dermal
         toxicity study in rabbits. Unpublished report No. RD-9084 from
         Nisso Institute for Life Sciences, Kanagawa, Japan. Submitted to
         WHO by Nippon Soda Co. Ltd, Tokyo, Japan.

    Takaori, H. (1993) Thiophanate-methyl combined chronic
         toxicity/oncogenicity study in rats. Unpublished report
         No. RD-9327 from Nisso Institute for Life Sciences, Kanagawa,
         Japan. Submitted to WHO by Nippon Soda Co. Ltd, Tokyo, Japan.

    Taniguchi, T. (1972) Final report on the long-term oral toxicity
         studies of thiophanate-methyl, dimethyl 4,4'-O-phenylene-
         bis(3-thioallophanate) in beagle-dogs for 24 months. Unpublished
         report from the Nisso Institute for Life Sciences, Kanagawa,
         Japan. Submitted to WHO by Nippon Soda Co. Ltd, Tokyo, Japan.

    Thomas, J. (1974) Actions of pesticides and other drugs on the male
         reproductive system (EPA-650/1-74-011), Washington DC, US
         Environmental Protection Agency. Submitted to WHO by Nippon Soda
         Co. Ltd, Tokyo, Japan.

    Thomas, J. & Schein, L. (1974) Effects of thiophanate and thiophanate-
         methyl on the male reproductive system of the mouse.  Toxicol.
          Appl. Pharmacol., 30, 129-133.

    Tippins, R.S.  et al. (1984) Gene mutation in Chinese hamster V79
         cells with thiophanate-methyl. Unpublished LSR-RTC Report
         No. 063013-M-05184 (RD-84109) from Life Science Research.
         Submitted to WHO by Nippon Soda Co. Ltd, Tokyo, Japan.

    Tompkins, E.C. (1992) Topsin-M (thiophanate-methyl) 18-month dietary
         oncogenicity study in mice. Unpublished report No. WIL-75024
         (RD-9328) from WIL Research Laboratory, Inc. Submitted to WHO by
         Nippon Soda Co. Ltd, Tokyo, Japan.
    


    See Also:
       Toxicological Abbreviations
       Thiophanate-methyl (WHO Pesticide Residues Series 3)
       Thiophanate-methyl (WHO Pesticide Residues Series 5)
       Thiophanate-methyl (Pesticide residues in food: 1977 evaluations)