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    TOLCLOFOS-METHYL

    First draft prepared by
    A. Moretto,
    Institute of Occupational Medicine,
    University of Padua, Padua, Italy

         Explanation
         Evaluation for acceptable daily intake
              Biochemical aspects
                   Absorption, distribution and excretion
                   Biotransformation
              Toxicological studies
                   Acute toxicity
                   Short-term toxicity
                   Long-term toxicity and carcinogenicity
                   Reproductive toxicity
                   Embryotoxicity and teratogenicity
                   Genotoxicity
                   Special studies
                        Skin and eye irritation and ski201
                        Delayed neuropathy
              Observations in humans
              Comments
              Toxicological evaluation
         References

    Explanation

         Tolclofos-methyl is an organophosphorus fungicide that is
    effective in controlling soil-borne diseases caused by infection of
    Basidiomycetes fungi such as  Rhizoctonia solani and  Corticium
     rolfsii. It was evaluated for the first time by the present JMPR.

    Evaluation for acceptable daily intake

    1.  Biochemical aspects

    (a)  Absorption, distribution and excretion

    Mice

         Male and female ICR mice, eight weeks of age, were given an
    oral dose of 5 mg/kg bw of [14C-4-methyl]-tolclofos-methyl
    (radiochemical purity, 99%) dissolved in corn oil, and radiocarbon
    was monitored in urine, faeces and expired air for seven days after
    administration. Within 24 h, 69-76% of the administered radiolabel
    was excreted in the urine, 4-6% in the faeces and less than 1% in
    the expired air. Total radiocarbon residues in the whole body
    represented less than 1% of the dose seven days after administration
    (Mihara  et al., 1981).

    Rats

         Six-week old Sprague-Dawley rats were given an oral dose of 5
    mg/kg bw of [14C-4-methyl]-tolclofos-methyl (radiochemical purity,
    99%) dissolved in corn oil, and radiocarbon was monitored in urine,
    faeces and expired air for seven days after administration. Within
    24 h, 62-67% of the administered dose was excreted in the urine,
    16-21% in the faeces and less than 1% in the expired air. Total
    radiocarbon residues in the whole body represented less than 1% of
    the dose seven days after administration. Whole-body autoradiography
    performed 1 and 6 h after treatment showed the highest accumulation
    of radiolabel in stomach and intestines, followed by kidney and
    liver (Mihara  et al., 1981).

         Male and female Sprague-Dawley rats received single oral doses
    of 5 or 200 mg/kg bw of tolclofos-methyl labelled uniformly with
    14C in the benzene ring (radiochemical purity, > 99%). Another
    group of animals was treated orally for 14 consecutive days with
    unlabelled tolclofos-methyl at 5 mg/kg bw per day and then with a
    single oral dose of [14C-phenyl]-tolclofos-methyl at 5 mg/kg bw.
    The administered radiocarbon was readily excreted, more than 95% of
    the dose being eliminated in the urine and faeces within 48 h. The
    amount excreted in urine after seven days was 85-91%; elimination in
    faeces at that time was: consecutive dose group, 9.3% in males and
    12% in females; low-dose group, 20% in males and 19% in females; and
    high-dose group, 20% in males and 12% in females. Excretion as
    14C-carbon dioxide accounted for < 0.1% of the dose in all
    groups. The concentration of 14C reached a peak within 2 h in

    almost all tissues. After administration of the low dose, the
    highest concentrations were found in the kidney; expressed in
    tolclofos-methyl equivalents, the levels were 4700 ng/g tissue in
    males and 3450 ng/g tissue in females; the levels in plasma were
    1140 ng/ml in males and 1270 ng/ml in females. Those in the liver
    were 1240 ng/g tissue in males and 1220 ng/g tissue in females, and
    those in blood were 736 ng/ml in males and 835 ng/ml in females. The
    concentrations of 14C in various organs 72 h after administration
    were < 5% of the respective peak concentrations. After seven
    days, radioactive residues accounted for less than 1% of the
    administered dose (Krautter  et al., 1987, 1988; Esumi & Yokoshima,
    1989).

         In male and female rats with bile-duct cannulas, cumulative
    excretion of 14C over 48 h was 5.8-12% of the dose in bile, 47-59%
    in urine and 42-24% in faeces (Esumi & Yokoshima, 1989).

    (b)  Biotransformation

         Male ICR mice were given 5 mg/kg bw [14C-4-methyl]-tolclofos-
    methyl orally; metabolites were isolated from the faeces and urine
    by chromatography and identified by co-chromatography with authentic
    standards and/or spectroanalysis. The following major metabolites
    were detected in the excreta: 2,6-dichloro-4-methylphenol (see
    Figure 1; 9% of administered label),  O,O-dimethyl- O-(2,6-
    dichloro-4-carboxyphenyl)phosphate (11%),  O-methyl- O-hydrogen-
     O-(2,6-dichloro-4-carboxyphenyl)phosphate (12%), 3,5-dichloro-4-
    hydroxybenzoic acid (12%) and 3,5- O-dichloro-4-hydroxybenzyl
    glycine (13%). The major biotransformation reactions are oxidative
    desulfuration to oxon and related derivatives, oxidation of the
    4-methyl group to alcohols and acids, cleavage of P-O-aryl and
    P-O-methyl linkages and conjugation of the resultant acid with
    glycine. The metabolites found in mice are similar to those seen in
    rats, except for 3,5- O-dichloro-4-hydroxybenzylglycine (Mihara  et
     al., 1981).

         Male and female Sprague-Dawley rats were given an oral dose of
    5 or 200 mg/kg bw tolclofos-methyl labelled with 14C either in the
    4-methyl group or uniformly in the phenyl ring, with or without
    pretreatment with unlabelled tolclofos-methyl at 5 mg/kg bw per day
    for 14 consecutive days. Metabolites were isolated from the faeces,
    urine, bile and major tissues by chromatography and identified by
    co-chromatography with authentic standards and/or spectroanalysis.
    More than 10 metabolites were detected in the excreta. No marked
    differences were seen in relation to sex or dose. The major
    metabolites detected in the excreta were  O-methyl  O-hydrogen-

     O-(2,6-dichloro-4-methylphenyl)phosphate ((see Figure 1; 10-26% of
    urinary 14C),  O-methyl- O-hydrogen- O-(2,6-dichloro-4-
    hydroxymethylphenyl) phosphorothioate (12-25%),  O-methyl
     O-hydrogen- O-(2,6-dichloro-4-carboxyphenyl)-phosphorothioate
    (11-35%) and  O-methyl- O-hydrogen- O-(2,6-dichloro-4-
    methylphenyl)-phosphorothioate (12-44%). In rats with bile cannulas,
    most of the radiolabel excreted into the bile within 24 h after
    administration was associated with polar metabolites; the major
    metabolites in the bile were  O-methyl- O-hydrogen- O-(2,6-
    dichloro-4-hydroxymethylphenyl)-phosphorothioate and 2,6-dichloro-4-
    methylphenol glucuronides. Radiocarbon excreted into the faeces
    within 24 h after administration was associated only with the parent
    compound (Esumi & Yokoshima, 1989).

         Two hours after oral administration, the major metabolites in
    blood, liver and kidney were  O,O-dimethyl- O-(2,6-dichloro-4-
    carboxyphenyl) phosphorothioate ((see Figure 1), 3,5-dichloro-4-
    hydroxybenzaldehyde,  O-methyl- O-hydrogen  O-(2,6-dichloro-4-
    methylphenyl)-phosphorothioate and  O-methyl- O-hydrogen- O-(2,6-
    dichloro-4-hydroxymethylphenyl)-phosphorothioate. Only a small
    amount of the parent compound was detected in the liver. The major
    biotransformation reactions were oxidative desulfuration to oxon and
    related derivatives, oxidation of the 4-methyl group to alcohols and
    acids, cleavage of the P-O-aryl and P-O-methyl linkages and
    conjugation of the resultant acids and phenols with glucoronic acids
    (Mihara  et al., 1981; Krautter  et al., 1987, 1988; Esumi &
    Yokoshima, 1989).

         The general pattern of metabolism of tolclofos-methyl is shown
    in Figure 1.

    FIGURE 01

    2.  Toxicological studies

    (a)  Acute toxicity

         Data on the acute toxicity of tolclofos-methyl (technical-grade
    and formulations) in laboratory animals are summarized in Table 1.
    Animals showed decreased spontaneous motor activity, dyspnoea,
    piloerection, urinary incontinence and ataxia. Recovery was complete
    by day 10. Brain cholinesterase activity was lower 16 days after
    treatment in dogs given 1000 mg/kg bw than in animals given lower
    doses; however, as no control data were reported, conclusions cannot
    be drawn. No treatment-related gross changes were seen at necropsy
    in any species.
        Table 1.  Acute toxicity of technical-grade and formulated tolclofos-methyl in laboratory animals
                                                                                                 
    Species  Strain  Sex   Route            Formulation        LD50          Reference
                                            (purity)           (mg/kg bw)
                                                                                                 

    Mouse    dd      M     Oral             Technical (97%)    3500          Segawa, 1978
                     F                                         3600
    Mouse    ICR     M&F   Oral             50% wettable       > 5000        Segawa, 1981a
                                             powder (98%)
    Mouse    ICR     M&F   Oral             10% dust (98.8%)   > 5000        Segawa, 1981b
    Mouse    dd      M&F   Dermal           Technical (97%)    > 5000        Segawa, 1978
    Mouse    ICR     M&F   Dermal           50% wettable       > 5000        Segawa, 1981c
                                             powder (98%)
    Mouse    ICR     M&F   Dermal           10% dust (98.8%)   > 5000        Segawa, 1981d
    Mouse    dd      M     Intraperitoneal  Technical (97%)    1070          Segawa, 1978
                     F                                         1260
    Mouse    dd      M&F   Subcutaneous     Technical (97%)    > 5000        Segawa, 1978
    Rat      SD      M&F   Oral             Technical (97%)    approx. 5000  Segawa, 1978
    Rat      SD      M&F   Oral             50% wettable       > 5000        Segawa, 1981e
                                             powder (98%)
    Rat      SD      M&F   Oral             10% dust (98.8%)   > 5000        Segawa, 1981f
    Rat      SD      M&F   Oral             50% flowable       > 5000        Hiromori  et al.,
                                                                             1989a
    Rat      SD      M&F   Dermal           Technical (97%)    > 5000        Segawa, 1978
    Rat      SD      M&F   Dermal           50% wettable       > 5000        Segawa, 1981g
                                             powder (98%)
    Rat      SD      M&F   Dermal           10% dust (98.8%)   > 5000        Segawa, 1981h
    Rat      NR      M&F   Dermal           50% flowable       > 2000        Hiromori  et al.,
                                                                             1989b

    Table 1 (contd)
                                                                                                 
    Species  Strain  Sex   Route            Formulation        LD50          Reference
                                            (purity)           (mg/kg bw)
                                                                                                 

    Rat      SD      M     Intraperitoneal  Technical (97%)    5000          Segawa, 1978
                     F                                         4900
    Rat      SD      M&F   Subcutaneous     Technical (97%)    > 5000        Segawa, 1978
    Rat      Wistar  M&F   Inhalation       Technical (97.7%)  > 3.32 mg/la  Hardy  et al., 1986
    Rat      SD      M&F   Inhalation       50% wettable       > 1.9 mg/l    Eschbach &
                                             powder (98%)                    Hogan, 1981a
    Rat      SD      M&F   Inhalation       10% dust (98.8%)   > 1.8 mg/l    Eschbach &
                                                                             Hogan, 1981b
    Rat      SD      M&F   Inhalation       50% flowable       > 1.07 mg/la  Jackson  et al., 1990
    Dog      NR      M&F   Oral             Technical (98.7%)  > 1000        Pence  et al., 1978
                                                                                                 

    SD, Sprague-Dawley; M, male; F, female; NR, not reported
    a 4-h whole-body exposure
        (b)  Short-term toxicity

    Mice

         Groups of 15 male and 15 female ddY mice were fed diets
    containing tolclofos-methyl (purity, 97%) at 0, 10, 30, 100 or 3000
    ppm, equal to 1.2, 3.8, 12 or 510 mg/kg bw per day for males and
    1.4, 4.1, 14 or 560 mg/kg bw per day for females, for nine months.
    Three satellite groups of five males and five females each were
    similarly treated and killed at 2, 4 and 13 weeks for determination
    of cholinesterase activity. Animals were observed daily for
    mortality and signs of toxicity; at the end of exposure, an
    ophthalmological examination and clinical chemical analyses
    (including plasma and erythrocyte cholinesterase) were undertaken,
    and brain cholinesterase activity was measured. Most organs were
    weighed and examined microscopically. There were no overt signs of
    toxicity and no deaths. Body weights were reduced by about 20% and
    body-weight gain by about 35% in animals of each sex at 3000 ppm,
    but at this dose food consumption was increased in males and females
    and water intake was increased in males. No treatment-related
    ophthalmological changes were observed, and haematological
    parameters were unaffected by treatment. The cholesterol level was
    increased by 35% in females at 3000 ppm. A dose-related decrease in
    plasma cholinesterase activity was found in animals of each sex,
    with activities that were 63-94% of the control values in mice at 30
    ppm, 30-56% at 100 ppm and 4-35% (61% in one case) at 3000 ppm. At
    the high dose, the activity reached a plateau after four weeks.
    Erythrocyte cholinesterase activity was decreased to 39-46% of the
    control level in males at 3000 ppm at weeks 13 and 40 and to 57-75%
    of the control level in females at this dose at all time points.

    Brain cholinesterase activity was unaffected by treatment in females
    but was reduced by about 24% in males at 3000 ppm at weeks 13 and
    40. Organ weights in the 3000-ppm group were reduced, but neither
    gross nor histopathological changes attributable to treatment were
    observed. The NOAEL was 100 ppm, equal to 12 mg/kg per day, on the
    basis of inhibition of brain cholinesterase activity and an effect
    on body weight at the highest dose (Suzuki  et al., 1978).

    Rats

         Groups of 10 male and 10 female Sprague-Dawley rats were fed
    diets containing tolclofos-methyl (purity, 97.9%) at 0, 200, 1000,
    5000 or 20 000 ppm for 32-34 days. Animals were observed twice daily
    for mortality and clinical signs, and food consumption and body
    weight were recorded weekly. Ophthalmoscopic examination, standard
    haematological tests and clinical chemical analyses were performed
    at week 4, and certain tests were repeated at termination. Plasma
    and erythrocyte cholinesterase activities were determined in the
    control and high-dose groups at week 4 and in all animals at
    termination, when brain cholinesterase activity was also measured.
    At terminal sacrifice, relevant organs were weighed and most organs
    were examined microscopically. There were no treatment-related
    deaths. Reductions in food consumption (-16% in males and -6% in
    females) and body-weight gain (-45% in males and -37% in females)
    were observed at 20 000 ppm. Body-weight gain was slightly reduced
    in animals of each sex at 5000 ppm during week 1. Haematological
    parameters were unaffected by treatment. Cholesterol levels were
    increased (by 116-138%) in animals of each sex at 20 000 ppm, and
    total protein and albumin levels were slightly higher in males in
    this group. Other differences between treated and untreated animals
    were detected, but the values were within the normal range. Plasma
    cholinesterase activity was reduced by 14% in males and by 50% in
    females at 20 000 ppm. Erythrocyte cholinesterase activity was
    reduced by 9-19% in animals of each sex at 5000 and 20 000 ppm;
    however, no clear dose-response relationship was seen. Brain
    cholinesterase activity was lower (by 12-31%) than that in controls
    in males in all treated groups and in females (by 20%) at 5000 and
    20 000 ppm. Although the reduction was statistically significant in
    all male groups, no clear dose-response relationship was found, and
    the activity in controls was slightly higher than usual. There were
    no ophthalmoscopic alterations or gross changes at necropsy. The
    relative weights of livers were increased (by 12%) in females at
    5000 ppm and in males and females at 20 000 ppm (by 27% in males and
    39% in females). Hypertrophy of hepatocytes was observed in animals
    of each sex at 20 000 ppm. The relative weights of the kidneys were
    slightly increased in animals of each sex at 5000 and 20 000 ppm,
    but no treatment-related histopathological changes were seen. The
    NOAEL was 1000 ppm, equal to 79 mg/kg bw per day, on the basis of
    increased relative kidney weights and brain cholinesterase activity
    at higher doses (Colley  et al., 1982).

         Groups of 12 male and 12 female Sprague-Dawley rats were fed
    diets containing tolclofos-methyl (96.6% pure) at 0, 100, 1000 or 10
    000 ppm, equal to 6.5, 66 or 653 mg/kg bw per day for males and 7.1,
    71 or 696 mg/kg bw per day for females, for 13 weeks. Animals were
    observed daily for mortality and clinical signs, and body weight and
    food and water consumption were determined weekly. Ophthalmological
    analysis, urinalysis, standard haematological tests, clinical
    chemical analyses and measurement of plasma, erythrocyte and brain
    cholinesterase activities were undertaken at termination, when
    relevant organs were weighed and most organs were examined
    microscopically. No deaths and no significant abnormal clinical or
    ophthalmological signs were noted during the study. Depression of
    body-weight gain of males (-20%) and females (-15%) at 10 000 ppm
    was seen throughout the treatment period and was associated with
    similar decreases in food and water consumption. A slight decrease
    in food consumption was noted in females at 1000 ppm during week 1.
    Decreased activities of plasma (16-53%), brain (8-9%) and
    erythrocyte (19-20%) cholinesterase were noted in animals of each
    sex at 10 000 ppm; a minimal decrease in erythrocyte cholinesterase
    activity (10%) was also seen in females at 1000 ppm. Liver weight
    was increased in animals of each sex at 10 000 ppm, and this was
    found at histopathological examination to be associated with
    hypertrophy of hepatocytes. Changes in some clinical chemical
    parameters were observed at 10 000 ppm, which included increased
    levels of cholesterol (48% in males, 102% in females) and
    phospholipids (37% in males, 64% in females) and slightly increased
    alpha2- and -globulin levels in animals of each sex. Other
    changes, although statistically significant, were minor and of
    questionable biological significance. At 10 000 ppm, increased
    relative kidney weights were seen in animals of each sex, increased
    blood urea nitrogen levels were seen in males only and decreased
    urinary pH was seen in males and females. At 1000 ppm, relative
    kidney weights were slightly increased in females and relative liver
    weights in males. The Meeting considered that the increased weight
    ratios observed at 1000 ppm were not biologically relevant and that
    the NOAEL was 1000 ppm, equal to 66 mg/kg bw per day (Kimura  et
     al., 1990).

         Groups of 15 male and 15 female Sprague-Dawley rats were fed
    diets containing tolclofos-methyl (purity, 97%) at 0, 300, 1000,
    3000 or 10 000 ppm, equal to 16, 51, 164 or 540 mg/kg bw per day in
    males and 18, 65, 184 or 623 mg/kg bw per day in females, for 28
    weeks. Animals were observed daily for clinical signs, and body
    weight and food consumption were measured weekly. Ophthalmologic
    examinations were carried out after three months and at termination;
    standard haematological tests and clinical chemical analyses were
    performed at termination. Urinalysis was carried out in 12 animals
    per group after three months and at termination. Plasma and
    erythrocyte cholinesterase activities were determined after 2, 4 and
    13 weeks, and at termination; brain cholinesterase was determined

    only at termination, when relevant organs were weighed and several
    were examined histologically.

         There were no overt signs of toxicity and no treatment-related
    deaths. Although the body weights of animals of each sex at 10 000
    ppm were lower than those of controls throughout the study, their
    initial body weights were also statistically significantly lower.
    Body-weight gain was significantly reduced (by 18%) in females at 10
    000 ppm. Food consumption was unaffected by treatment. Treatment did
    not affect ophthalmologic, urinary, haematological or clinical
    chemical parameters. Plasma cholinesterase activity was lower (by
    23-41%) in females at 10 000 ppm than in controls at weeks 2, 4, 13
    and 28. Erythrocyte cholinesterase activity was highly variable and
    was reduced by 15-19% in comparison with controls in males at 10 000
    ppm at weeks 2, 4 and 28. Brain cholinesterase activity was
    unaffected by treatment. Kidney and liver weights were slightly
    increased in animals of each sex at 10 000 ppm and in females at
    1000 and 3000 ppm, but the biological significance of these findings
    was questionable. No gross abnormalities were seen at necropsy.
    Bile-duct proliferation was found in the livers of 3/15 females at
    10 000 ppm and oval-cell proliferation was seen in 2/15 females at
    3000 ppm and 3/15 at 10 000 ppm. No histopathological changes were
    seen in the kidneys. The NOAEL was 1000 ppm, equal to 65 mg/kg bw
    per day, on the basis of histopathological changes in the livers of
    females at higher doses (Hiromori  et al., 1978).

    Rabbits

         Groups of five male and five female New Zealand white rabbits
    received tolclofos-methyl (purity, 97.7%) in acetone on the skin at
    doses of 0, 30, 300 or 1000 mg/kg bw per day for 6 h per day, on
    five days per week for 21 days. There were no deaths and no
    treatment-related clinical signs; neither body weight nor food
    consumption was affected by treatment. Dermal irritation (slight
    erythema) was seen in some animals in all treated groups from day 6.
    Eosinophil counts were increased in males at 1000 mg/kg bw per day,
    and the level of inorganic phosphorus was decreased (by 14-20%) in
    animals of each sex at this dose. Brain cholinesterase activity was
    about 15% lower than that in controls in males and about 15% higher
    in females at 300 and 1000 mg/kg bw per day. These changes were
    considered not to be of biological significance. Erythrocyte
    cholinesterase activity was lower than in controls in males at the
    same two doses, but there was no dose-effect relationship. Plasma
    cholinesterase activity was lower (by 22-29%) than in controls in
    animals of each sex at 300 and 1000 mg/kg bw per day. The relative
    weights of the kidneys were increased (by 20%) in females at 1000
    mg/kg bw per day. Macroscopic findings consisted of accumulation of
    the compound on treated skin; microscopic examination revealed
    hyperkeratosis, acanthosis and subepidermal pleocellular
    infiltration of the skin (Gargus, 1986).

    Dogs

         Groups of six male and six female beagle dogs were fed diets
    containing tolclofos-methyl (purity, 98.7%) at 0, 200, 600 or 2000
    ppm, equal to 7.4, 23 or 69 mg/kg bw per day in males and 4.1, 21 or
    65 mg/kg bw per day in females, for 26 weeks. Animals were observed
    daily for mortality and weekly for clinical signs, body weight and
    food consumption. Ophthalmoscopic examinations were performed before
    treatment and at termination of the study. Standard haematological
    tests and clinical chemical analyses were performed at regular
    intervals until termination. Erythrocyte and plasma cholinesterase
    activities were determined at weeks 2, 4, 8, 13 and 26. Brain
    cholinesterase was determined at termination, when relevant organs
    were weighed and most were examined histologically. There were no
    deaths and no overt signs of toxicity. Body-weight gain was reduced
    by 54% in males and by 46% in females at 2000 ppm, although food
    consumption was unaffected by treatment. There were no
    treatment-related ocular changes. Although the haematocrit was
    within the normal range, decreased values in comparison with
    controls (-11% in males and -10% in females) were observed at 2000
    ppm. The mean corpuscular volume and mean corpuscular haemoglobin
    concentration were not affected by treatment. Alkaline phosphatase
    activity was increased by 139-295% in animals of each sex at 2000
    ppm throughout the study. Total bilirubin was not increased in
    treated animals. At 2000 ppm, plasma cholinesterase activity was
    decreased by 19-26% in females throughout the study, but no
    significant decreases were seen in erythrocyte cholinesterase
    activity in animals of each sex or in plasma cholinesterase activity
    in males. Brain cholinesterase activity was unaffected by treatment.
    Urinalysis gave unremarkable results, and there were no gross
    changes at necropsy. Liver weights were increased (by 59% in males
    and 43% in females) at 2000 ppm, but there were no concomitant
    histological changes, and there were no treatment-related
    histopathological changes. The NOAEL was 600 ppm, equal to 21 mg/kg
    bw per day, on the basis of increased liver weights, reduced
    body-weight gain and increased alkaline phosphatase activity at 2000
    ppm (Pence  et al., 1979a).

         Groups of six male and six female beagle dogs were fed diets
    containing tolclofos-methyl (purity, 96.7%) at 0, 80, 400 or 2000
    ppm, for 52 weeks. Animals were observed daily for mortality and
    weekly for clinical signs. Ophthalmoscopic examinations were
    performed before treatment and at weeks 26 and 52. Standard
    haematological tests, clinical chemical analyses and determinations
    of plasma and erythrocyte cholinesterase activities were performed
    one week before treatment and at weeks 13, 26 and 52. Brain
    cholinesterase was determined at termination, when relevant organs
    were weighed and most were examined histologically. No clinical
    signs of toxicity were observed. Decreased food consumption (-9% in
    males and -16% in females) and body-weight gain (-32% in males and
    -54% in females), slightly lower erythrocyte counts (-8% in males

    and -20% in females), haematocrit value (-7% in males and -17% in
    females) and haemoglobin concentration (-8% in males and -7% in
    females), slightly decreased albumin and total protein levels
    (significantly at some time points) and elevated alkaline
    phosphatase levels (> 200%) were found at 2000 ppm.

         Plasma cholinesterase activity was slightly, but not
    significantly lower than that in controls in females at the highest
    dose; erythrocyte and brain cholinesterase activities were not
    affected by the treatment. No treatment-related ophthalmoscopic
    alterations were observed. Increases in mean liver weights, both
    absolute (+32% in males and +61% in females) and relative (+52% in
    males and +74% in females), were seen in dogs at 2000 ppm.
    Microscopic examination of the livers revealed increased incidences
    of hepatocytic hypertrophy and intracytoplasmic homogeneous material
    and an increased amount of hepatocytic pigment in these animals. The
    amount of this pigment was also increased at 400 ppm, and it was
    present in control animals. Its nature has not been elucidated, but
    it does not contain iron or bilirubin. These dogs also had decreased
    absolute (-46%) and relative (-40%) weights of the prostate, and the
    absolute and relative weights of the pancreas were increased by
    16-27% and 28-49%, respectively. The NOAEL was 400 ppm, equal to 11
    mg/kg bw per day, on the basis of reduced body-weight gain,
    increased liver weights (with hepatic hypertrophy) and slight
    anaemia at 2000 ppm (Cox  et al., 1988; Moore, 1993).

    (c)  Long-term toxicity and carcinogenicity

    Mice

         Groups of 70 male and 70 female B6C3F1 mice were fed diets
    containing tolclofos-methyl (purity, 94.3%) at 0, 10, 50, 250 or
    1000 ppm, equal to 1.3, 6.5, 32 or 134 mg/kg bw per day in males and
    1.3, 6.8, 34 or 137 mg/kg bw per day in females, for up to 104
    weeks. Animals were observed twice daily for clinical symptoms and
    mortality. Body weight was measured weekly during the first 13 weeks
    and then once every four weeks. Food intake was measured weekly.
    Standard urinalysis was performed in 10 animals from each group at
    6, 12 and 18 months and in all survivors at termination of the
    study. Standard haematological tests were performed in 10 animals
    from each group at 12 months and in all survivors at termination.
    Standard clinical chemical analyses and measurements of serum,
    erythrocyte and brain cholinesterase activities were done in 10
    animals from each group at 6 and 12 months and in all animals at
    termination. Animals in the control and 1000-ppm groups were
    subjected to ophthalmologic examination at 12 and 24 months. Ten
    animals from each group were necropsied at 6 and at 12 months, at
    termination or when found dead or moribund. Relevant organs were
    weighed and examined microscopically.

         There were no treatment-related effects on mortality and no
    overt signs of toxicity, including ophthalmologic changes.
    Body-weight gain was slightly reduced in females at 1000 ppm up to
    week 52; food consumption was also slightly decreased in this group
    after 52 and 104 weeks. Food conversion efficiency, water intake,
    urinary parameters and the haematological profile were unaffected by
    treatment. Serum cholinesterase activity was decreased in animals of
    each sex at 250 ppm (by 25-52%) and at 1000 ppm (by 43-81%) at both
    times, and erythrocyte cholinesterase activity was decreased by
    11-28% at 250 ppm and by 13-47% at 1000 ppm; inhibition of both
    enzymes was greater at termination. A dose-related decrease was
    found in brain cholinesterase activity in females (by 7-24% at 250
    ppm and 9-33% at 1000 ppm) and males (by 10-13% at 250 ppm and
    17-26% at 1000 ppm). Serum and brain, but not erythrocyte,
    cholinesterase activities were lower (by 12 and 18%, respectively)
    in females at 50 ppm at week 28 than in controls, which had higher
    levels than at week 52 and at termination. Glucose levels were
    increased in males and were slightly increased in females at 1000
    ppm at week 104. Absolute and/or relative kidney weights were
    increased in animals of each sex at 1000 ppm and in males at 250 ppm
    at week 52, and in females at 250 ppm at week 104. The weights of
    the thymus were decreased in females at 1000 ppm at weeks 52 and
    104, and pituitary weights were increased in females at this dose at
    week 104. No treatment-related changes were seen at necropsy or on
    histopathological examination at any time. Tolclofos-methyl was not
    found to be carcinogenic. The NOAEL was 50 ppm, equal to 6.5 mg/kg
    bw per day, on the basis of reduced brain cholinesterase activity
    and increased absolute and relative kidney weights at higher doses
    (Satoh  et al., 1983).

    Rats

         Groups of 65 male and 65 female Fischer 344 rats were fed diets
    containing tolclofos-methyl (purity, 94.9-98.7%) at 0, 100, 300 or
    1000 ppm for 122 weeks (males) or 129 weeks (females). At week 52,
    10 males and 10 females from each group were killed. Animals were
    observed twice a day for mortality and signs of morbidity. Body
    weight and food consumption were recorded weekly (weeks 0-26), every
    two weeks (weeks 27-52) or every four weeks until termination.
    Ophthalmoscopic examinations were performed every 26 weeks and at
    termination. Standard haematological tests, clinical chemical
    analyses and measurements of erythrocyte and plasma cholinesterase
    activities were conducted in 10 animals of each sex in each group
    one week before treatment, at weeks 4, 13, 26, 52, 78 and 104 and at
    termination. Brain cholinesterase activity was determined in 10
    animals of each sex per group at week 52 and at termination. All
    animals killed or found dead were necropsied, and relevant organs
    were weighed; most organs were then examined histologically.

         There were no treatment-related effects on mortality and no
    overt signs of toxicity. A slight decrease in body-weight gain was

    observed in males at 1000 ppm. Food consumption and food conversion
    efficiency were unaffected by treatment, as was the haematological
    profile. There was a dose-related trend to decreased alkaline
    phosphatase activity (by 9-46%) in all treated groups: The activity
    was significantly decreased in males at 1000 ppm throughout the
    study, in males at 300 ppm from week 13, and occasionally in the
    other treated groups. Brain cholinesterase activity was decreased in
    all treated groups at the time of the interim sacrifice, but there
    was no dose-response relationship. At the time of terminal
    sacrifice, brain cholinesterase activity was reduced in males at the
    middle dose (by 52%) and in females at the low (by 57%) and middle
    doses (by 49%), but not in animals at the high dose. Erythrocyte and
    plasma cholinesterase activities were also inconsistently lower in
    treated groups, although no dose-response relationship was seen.
    Urinalysis revealed no remarkable findings, organ weights were
    unaffected by treatment, and there were no treatment-related gross
    changes at necropsy. Toclofos-methyl was not carcinogenic in this
    study. No NOAEL could be identified, given the variability of the
    data on cholinesterase activity (Pence  et al., 1982).

         Groups of 30 male and 30 female Fischer 344 rats were fed diets
    containing tolclofos-methyl (purity, 98.3-97.3%) at 0, 100, 300 or
    1000 ppm for 104 weeks, and brain, erythrocyte and plasma
    cholinesterase activities were assessed. Ten males and 10 females
    from each group were sacrificed at week 52. Animals were observed
    daily for mortality, and they were examined for body weight, food
    consumption and gross signs of toxicity weekly until week 26, every
    two weeks during weeks 26-52 and every four weeks during weeks
    52-104. Blood samples were taken for determination of plasma and
    erythrocyte cholinesterase activities at initiation of the study, at
    weeks 5, 14, 27, 53, 79 and at termination. Brain cholinesterase
    activity was determined at week 52 and at termination.

         There were no treatment-related effects on survival, and the
    incidences of clinical signs and palpable tissue masses were
    comparable among the groups. Mean plasma cholinesterase activities
    were slightly decreased (by < 25%) in males when compared with
    controls at 300 and 1000 ppm at weeks 27 (statistically
    significant), 53, 79 and 105. Mean erythrocyte and brain
    cholinesterase activities were comparable among the groups
    throughout the study. Plasma cholinesterase activity was reduced (by
    < 20%) at certain times in treated animals but no dose-response
    relationship was found. Gross pathological findings were unrelated
    to treatment. Doses < 1000 ppm did not depress erythrocyte or
    brain cholinesterase activity or induce gross pathological effects
    (Miyamoto, 1985; Pence  et al., 1985a).

         The combined NOAEL in these two studies was 1000 ppm, equal to
    41 mg/kg bw per day, on the basis of the absence of any significant
    findings.

    (d)  Reproductive toxicity

    Rats

         A three-generation study of reproductive toxicity was performed
    in Sprague-Dawley rats fed diets containing tolclofos-methyl
    (purity, 97.9-98.7% pure) at 0, 100, 300 or 1000 ppm, equivalent to
    10, 30 or 100 mg/kg bw per day, with two litters per generation. The
    F0 generation consisted of 30 rats of each sex per group. The
    F1a, F2a and F3a generations were sacrificed at weaning and
    underwent gross necropsy. Five rats of each sex in each group were
    selected at weaning from the F1b, F2b and Fn3b litters for
    histopathological examination, and 25 rats of each sex from each
    group were selected from the F1b and F2b litters to breed the
    following generation. A 15-week growth period was allowed before
    mating of the 'a' litters, and a minimal 10-day rest period was
    allowed between weaning of the 'a' litters and mating to produce the
    'b' litters. After birth of the 'b' litter, all parental animals
    were necropsied.

         There were no treatment-related deaths or overt signs of
    toxicity. Pregnancy rates were low at times in all groups, but the
    pattern was not related to dose. The lowest pregnancy rate (52%) was
    observed in the F1b generation at the middle dose. Pregnancy rates
    were below 70% in three of the six generations in the controls, in
    two generations at the low dose, in four generations at the middle
    dose and in one generation at the high dose. The body weights of
    F2a and F2b pups at 300 and 1000 ppm were lower than those of
    controls at the start of the growth period; however, these
    reductions were not dose-related, and the growth rates and body
    weights in other groups were comparable throughout the study. There
    were no treatment-related gross changes at necropsy in any group.
    The weights of the ovaries were increased in non-pregnant adult
    F2b females at 1000 ppm, but this was not associated with
    histomorphological alterations. There was an increased incidence
    (not statistically significant) of fine cytoplasmic vacuolation of
    the adrenal cortex in adult F2b females at 1000 ppm that became
    pregnant at both matings. The etiology and toxicological
    significance of this finding, given its occurrence in the controls,
    were unknown. There was no effect on reproductive parameters or on
    offspring survival and development in any litter, and there were no
    treatment-related gross or histopathological findings in the
    litters. The NOAEL for reproductive toxicity was 1000 ppm
    (equivalent to 100 mg/kg bw per day) on the basis of the absence of
    significant findings at any dose level (Pence  et al., 1985b).

    (e)  Embryotoxicity and teratogenicity

    Rats

         Groups of 21-26 pregnant Fischer 344 rats were administered 0,
    5, 15 or 50 mg/kg bw per day tolclofos-methyl (purity, 94.9%; dose
    adjusted to 100%) in 0.5% w/v methylcellulose by gavage on days 6-15
    of gestation. There were no overt signs of toxicity, and body-weight
    gain and food consumption were unaffected by treatment. There were
    no treatment-related gross lesions in the dams at necropsy.
    Pregnancy rates were slightly higher in all treated groups (77-87%)
    than in controls (70%). There were no treatment-related changes in
    the number of fetal deaths, fetal viability or size or the
    incidences of visceral or skeletal anomalies or variations.
    Tolclofos-methyl was not embryotoxic, fetotoxic or teratogenic in
    rats at doses up to and including 50 mg/kg bw per day. It should be
    noted, however, that the highest dose was not maternally toxic
    (Pence  et al., 1979b).

         Groups of 23 pregnant Sprague-Dawley rats were administered 0,
    100, 300 or 1000 mg/kg bw per day tolclofos-methyl (purity, 96.7%)
    in 0.5% methyl cellulose orally on days 6-15 of gestation. On day 20
    of gestation, all animals were sacrificed and caesarean section was
    performed. Rats were observed daily for mortality and clinical signs
    and weighed. At termination, dams were observed for gross visceral
    abnormalities, and their uteri were weighed and examined for
    implantations and resorptions. Live fetuses were weighed, sexed and
    examined for external, visceral and skeletal abnormalities. All dams
    survived to the day of scheduled sacrifice with no clinical signs of
    toxicity. Mean body-weight gain in animals at 100 and 1000 mg/kg bw
    per day was significantly less than in controls on days 6-11. Both
    mean body-weight gain (days 6-16) and mean net body-weight gain
    (days 0-20) in rats at 1000 mg/kg bw per day were lower (by 10 and
    14%, respectively) than the corresponding control value. In
    addition, mean food consumption in animals at 1000 mg/kg bw per day
    on days 6-16 and 16-20 was slightly below the control value. There
    were no treatment-related differences in implantation efficiencies,
    and mean fetal viability, sex ratio and fetal body weight were
    similar in all groups. The number of fetuses with unossified fifth
    and/or sixth sternebrae was significantly greater in the 1000-mg/kg
    bw per day group than in the control group; however, the incidence
    of total fetal skeletal variations was similar in all groups. Other
    variations in development were not related to dose. Two to three
    malformed fetuses were found in each group, but neither the type nor
    the frequency of malformations indicated a teratogenic or
    embryotoxic response. Tolclofos-methyl was found to be neither
    teratogenic nor embryotoxic in this study at doses up to and
    including 1000 mg/kg bw per day. This dose was slightly toxic to the
    dams, as indicated by the lower body-weight gain. The NOAEL for
    maternal toxicity was 300 mg/kg bw per day (Morseth  et al., 1987).

    Rabbits

         Groups of 13-17 pregnant New Zealand white rabbits were
    administered 0, 300, 1000 or 3000 mg/kg bw per day tolclofos-methyl
    (purity, 98.7% pure) in 5% carboxymethylcellulose orally on days
    6-18 of gestation. The animals were observed daily for mortality and
    clinical signs and were weighed. At termination, dams were observed
    for gross visceral abnormalities, and their uteri were weighed and
    examined for implantations and resorptions. Live fetuses were
    weighed, sexed and examined for external, visceral and skeletal
    abnormalities. One rabbit at 3000 mg/kg bw per day died on day 14 of
    gestation of an undetermined cause. There were no overt signs of
    toxicity in any group. Spontaneous abortion occurred in one dam at
    1000 mg/kg bw per day and in two at 3000 mg/kg bw per day on or
    after day 21 of gestation. The mean body weights of animals in the
    treated groups were significantly lower than the control values
    throughout the study (including day 0). Body-weight gain was reduced
    by 76% in animals at 3000 mg/kg bw per day; and at termination,
    weight gain (days 0-29) was 19% lower than in controls. At 1000
    mg/kg bw per day, body-weight gain was reduced by 56% during
    treatment, but it was similar to that of controls at termination.
    Food consumption was decreased by up to 38% in animals at 1000 and
    3000 mg/kg bw per day during treatment. There were no
    treatment-related changes in maternal organ weights. One animal at
    3000 mg/kg bw per day resorbed her entire litter. There were no
    other treatment-related changes in implantation efficiency, mean
    fetal viability, size, sex ratio, fetal body weight or external,
    visceral or skeletal development. Tolclofos-methyl was not
    teratogenic in this study at doses up to and including 3000 mg/kg bw
    per day, which was toxic to dams. The NOAEL for maternal toxicity
    was 300 mg/kg bw per day (Motoyama  et al., 1991).

    (f)  Genotoxicity

         The results of tests for the genotoxicity of tolclofos-methyl
    are summarized in Table 2.

    (g)  Special studies

     (i)  Skin and eye irritation and skin sensitization

         Six male albino Japanese rabbits received 500 mg of
    tolclofos-methyl (purity, 97%) on clipped intact or abraded dorsal
    skin for 4 h under an occlusive dressing. No signs of irritation
    were seen at any of the application sites seven days after treatment
    (Matsubara  et al., 1978).


        Table 2.  Results of tests for the genotoxicity of tolclofos-methyl
                                                                                                                   
    End-point        Test system             Concentration              Purity   Results       Reference
                                             of tolclofos-methyl        (%)
                                                                                                                   

    In vitro
    Reverse          S. typhimurium TA98,    10, 100, 500, 1000, 2000   97.0     Negativea,b   Suzuki & Miyamoto,
     mutation         100, 1535, 1537, 1538  g/plate

    Reverse          S. typhimurium TA98,    10, 50, 100, 500, 1000,    98.7     Negativea,c   Moriya  et al., 1981
     mutation         100, 1535, 1537, 1538  5000 g/plate
                      E. coli WP2uvrA

    Reverse          B. subtilis H17 rec+,   1, 10, 100, 1000 g/disc   97.0     Negatived     Suzuki & Miyamoto,
     mutation         M45 rec-                                                                 1978

    Reverse          B. subtilis             20, 50, 100, 200, 500,     98.7     Negativee     Moriya  et al., 1981
     mutation         H17 rec+, M45 rec-     1000, 2000, 5000 g/disc

    Host-mediated    S. typhimurium in       870, 1750 mg/kg            97.0     Negativef     Suzuki & Miyamoto,
     assay             male ICR mice                                                           1978

    Chromosomal      Chinese hamster         10, 20, 40 g/ml           96.6     Negativea,g   Kogiso  et al., 1990
     aberration       K1 ovary cells         37.5, 75, 150 g/ml

    Unscheduled      Male Sprague-Dawley     0.3, 1, 3, 10, 20, 40      96.6     Negativea,i   Hara  et al., 1990
     DNA synthesis    rat hepatocytes        g/mlh

     Unscheduled     Human carcinoma         0.3, 3, 30, 300 g/ml      NR       Negativej     Monaco & Nunziata,
     DNA synthesis    cells (HeLa)                                                             1981

    Gene mutation    Chinese hamster         1.5, 15, 150, 1500 g/ml   NR       Negativea,k   Monaco & Nunziata,
                      V79 lung cells                                                           1981

    Table 2 (contd)
                                                                                                                   
    End-point        Test system             Concentration              Purity   Results       Reference
                                             of tolclofos-methyl        (%)
                                                                                                                   

    In vivo
    Chromosomal      Male ICR mice           500, 1000, 2000,           99.8     Negativei     Hara & Suzuki, 1981
     aberration                              4000 mg/kg i.p.h

    Dominant         Male Sprague-           62.5, 208.3, 625.0         NR       Negativej     Brusick, 1981
     lethal           Dawley rats            mg/kg orally
     mutation
                                                                                                                   

    NR, not reported
    a With and without metabolic activation.
    b Positive controls ( N-methyl- N'-nitro- N-nitrosoguanidine and 2-acetylaminofluorene) yielded expected positive
      results.
    c Several positive controls yielded expected positive results.
    d Positive control ( N-methyl- N'-nitro- N-nitrosoguanidine) yielded expected positive results.
    e Positive (mitomycin C) and negative (Kanamycin) controls yielded expected results.
    f Positive control ( N-nitrosodimethylamine) yielded expected positive results.
    g Positive controls (mitomycin C and cyclophosphamide) yielded expected positive results.
    h At 40 mg/ml, cell viability was less than 20%.
    i Positive control (2-acetylaminofluorene) yielded expected positive results.
    j Positive controls (methyl methanesulfonate and urethane) yield expected positive results.
    k Positive controls (methyl methanesulfonate and  N-nitrosodimethylamine) yielded expected positive results.
    l At 2000 and 4000 mg/kg bw there was > 50% mortality at 48 h.
    m Positive control (cyclophosphamide) yielded expected positive results.
    n Positive control (triethylenemelamine) yielded expected positive results.

    

         Groups of six male New Zealand white rabbits received 500 mg of
    either a '50% wettable power' or '10% dust' moistened with saline on
    1-inch2 (6.5-cm2) sites on the clipped dorsal intact or abraded
    skin for 24 h under an occlusive dressing. No irritation, such as
    erythema and oedema, was observed (Hara  et al., 1981a,b).

         Two male and one female New Zealand white rabbits received 0.5
    ml of '50% flowable' tolclofos-methyl on 1 inch2 (6.5 cm2) of
    clipped dorsal intact or abraded skin for 4 h under an occlusive
    dressing. No irritation was observed (Nakanishi  et al., 1989).

         Each of eight male albino Japanese rabbits received 50 mg of
    tolclofos-methyl (purity, 97%) in one eye. Five minutes after the
    application, the treated eyes of five animals were flushed with 300
    ml saline for 2 min. The treated eyes of the remaining animals were
    similarly flushed 24 h after treatment. There were no corneal,
    conjunctival or iridal effects up to seven days after treatment
    (Matsubara  et al., 1978).

         Groups of nine male New Zealand white rabbits received 100 mg
    of either a '50% wettable power' or '10% dust' in one eye; 30 s
    after the application, the treated eyes of three animals per group
    were flushed with 300 ml lukewarm water for 1 min. Slight congestion
    of the iris was observed 24 h after application. Slight to moderate
    hyperaemia and slight chemosis and/or discharge in conjunctiva were
    also observed 1-48 h after application of the '50% wettable power'
    to unwashed eyes. These changes had disappeared by 72 h after
    application in all animals. No ocular lesions were found in the
    washed eyes. The irritation potency of this formulation was judged
    to be mild. Slight conjunctival hyperaemia and/or chemosis were
    observed in animals with unwashed eyes and in one with washed eyes
    1-24 h after application of the '10% dust'. There were no other
    signs of irritation at any time. The formulation was classified as
    minimally irritating to eyes (Hara  et al., 1981a,b).

         One male and two female New Zealand white rabbits received 0.1
    ml of '50% flowable' tolclofos-methyl in one eye. Slight redness was
    observed in conjunctiva after application, which disappeared within
    24 h (Nakanishi  et al., 1989).

         The skin-sensitizing potential of tolclofos-methyl (purity,
    97%) was assessed in guinea-pigs by the Landsteiner-Draize method.
    Groups of 10 male Hartley guinea-pigs were given 10 intradermal
    injections of 1 or 5% tolclofos-methyl (0.05 or 0.1 ml) in corn oil
    at intervals of two to three days. Two weeks after the final
    induction, the animals were challenged at a fresh site with 0.05-ml
    intradermal injections of tolclofos-methyl at the same
    concentrations used for induction. Negative control animals for both
    dose groups were given the challenge injection only. Positive
    control animals were treated with 0.05% 2,4-dinitrochlorobenzene
    three times before challenge. Slight erythema and swelling were

    observed in two animals after challenge treatment with 1%
    tolclofos-methyl, in three after challenge with 5%, and in one
    animal in each negative control group. Moderate erythema and/or
    swelling was observed in the animals treated with
    2,4-dinitrochlorobenzene. It was concluded that tolclofos-methyl did
    not sensitize skin in this study (Matsubara  et al., 1980).

         The skin sensitizing potential of a '50% wettable powder' and a
    '10% dust' of tolchlofos-methyl was tested in guinea-pigs by the
    Buehler method. Groups of 10 male Hartley guinea-pigs received 500
    mg of one formulation, slightly moistened with water, on clipped
    dorsal skin under an occlusive dressing for 24 h. Induction was
    performed 10 times at two- to three-day intervals.Positive control
    animals were similarly treated with 0.5% 2,4-dinitro-chlorobenzene.
    Negative controls were not subjected to the induction treatment. Two
    weeks after the final induction, the test animals and positive
    controls were challenged as in the sensitizing treatment. No skin
    reactions were observed in the negative control or treated animals.
    Slight to severe erythema and swelling were observed in the positive
    controls. The formulations were considered not to sensitize skin
    (Hara  et al., 1981c,d).

         The skin sensitizing potential of a '50% flowable formulation'
    was tested in guinea-pigs by the Buehler method. Ten male Hartley
    guinea-pigs received 0.5 ml of the formulation for 6 h once a week
    for three weeks on the clipped dorsal skin under an occlusive
    dressing. Positive control animals were similarly treated with 0.5%
    2,4-dinitrochlorobenzene. Negative control animals were not
    subjected to the induction treatment. Two weeks after the final
    induction, animals were challenged as in the sensitizing treatment.
    No skin reactions were observed in the negative control or treated
    animals. Slight to severe erythema and swelling were observed in the
    positive controls. The formulation was considered not to sensitize
    skin (Nakanishi  et al., 1990).

     (ii)  Delayed neuropathy

         Groups of 10 Leghorn hens were administered 0 or 8000 mg/kg bw
    tolclofos-methyl (purity, 97%) or 500 mg/kg bw tri- ortho-cresyl
    phosphate orally in corn oil. After a 21-day observation period, a
    second dose of vehicle or tolclofos-methyl was administered. Animals
    were sacrificed 21 days after the second dose. There were no deaths
    in the groups given the vehicle or tolclofos-methyl. Plasma
    cholinesterase activity in the group treated with tolclofos-methyl
    was decreased by nearly 50% eight days after the first dose but had
    recovered to the pre-treatment level 21 days after dosing. Hens
    treated with tolclofos-methyl had no signs of leg weakness or
    paralysis and no histopathological changes in the nervous tissues.
    Birds treated with tri- ortho-cresyl phosphate had the typical
    clinical and histopathological signs of delayed polyneuropathy
    (Okuno  et al., 1982).

    3.  Observations in humans

         The medical records of 20 workers in Japan were reviewed. All
    workers had been engaged continuously in packaging operations since
    manufacture of technical-grade tolclofos-methyl began in 1988, for
    an average of 4 h/day. No occupation-related problems were observed
    or reported. Plasma and erythrocyte cholinesterase activities were
    not measured (Murayama, 1991).

    Comments

         Tolclofos-methyl is excreted rapidly in rats and mice,
    predominantly in the urine; less than 1% of the dose was retained in
    tissues after seven days. In both species, metabolism occurred
    mainly by oxidation of P=S to P=O, oxidation of the 4-methyl group
    and cleavage of the P-O-aryl and P-O-methyl linkages. There are four
    main metabolites in mice, one of which is a glycine conjugate, and
    four in rats, which are excreted as glucuronides.

         Tolclofos-methyl had low acute toxicity when administered by
    the oral, dermal, subcutaneous or intraperitoneal route. The overt
    signs of acute toxicity are not typical of an anticholinesterase, as
    no chromodaccryorrhoea, lachrymation or fasciculation was seen,
    although some inhibition of plasma, erythrocyte and brain
    cholinesterase activities was observed. WHO (1992) has classified
    tolclofos-methyl as unlikely to present an acute hazard in normal
    use.

         In a nine-month study of toxicity in which mice were fed
    tolclofos-methyl in the diet at 0, 10, 30, 100 or 3000 ppm, the
    NOAEL was 100 ppm, equal to 12 mg/kg bw per day, on the basis of
    inhibition of brain cholinesterase activity and effects on body
    weight at 3000 ppm.

         In a 32-34-day study of toxicity in which rats were fed diets
    containing 0, 200, 1000, 5000 or 20 000 ppm, the NOAEL was 1000 ppm,
    equal to 79 mg/kg bw per day, on the basis of inhibition of brain
    cholinesterase activity and increased relative kidney weight at 5000
    ppm. In a 13-week study of toxicity in which rats were fed diets
    containing 0, 100, 1000 or 10 000 ppm, the NOAEL was again 1000 ppm,
    equal to 66 mg/kg bw per day, on the basis of effects on body, liver
    and kidney weights at 10 000 ppm. In a 28-week study of toxicity in
    which rats were fed dietary levels of 0, 300, 1000, 3000 or 10 000
    ppm, the NOAEL was also 1000 ppm, equal to 65 mg/kg bw per day, on
    the basis of histopathological changes in the livers of females at
    3000 ppm.

         In a 26-week dietary study in dogs fed levels of 0, 200, 600 or
    2000 ppm, the NOAEL was 600 ppm, equal to 21 mg/kg bw per day, on
    the basis of reduced body-weight gain, an increased serum level of
    alkaline phosphatase and increased liver weight at 2000 ppm.

         In a 52-week study of toxicity in dogs given dietary
    concentrations of 0, 80, 400 or 2000 ppm, the NOAEL was 400 ppm,
    equal to 11 mg/kg bw per day, on the basis of increased liver weight
    (with hepatocytic hypertrophy), reduced body-weight gain and slight
    anaemia at 2000 ppm.

         In a 104-week study of toxicity and carcinogenicity in which
    mice were given dietary concentrations of 0, 10, 50, 250 or 1000

    ppm, the NOAEL was 50 ppm, equal to 6.5 mg/kg bw per day, on the
    basis of reduced brain cholinesterase activity and increased
    absolute and relative kidney weights at higher levels. There was no
    evidence of carcinogenicity.

         A 122-129-week study of toxicity and carcinogenicity was
    performed in which rats were given dietary concentrations of 0, 100,
    300 or 1000 ppm. The NOAEL was 1000 ppm, equal to 41 mg/kg bw per
    day, on the basis of the absence of any significant finding. There
    was no evidence of carcinogenicity.

         In a three-generation study (two litters per generation) in
    rats, tolclofos-methyl was given at dietary levels of 0, 100, 300 or
    1000 ppm. The NOAEL was > 1000 ppm, equivalent to 100 mg/kg bw per
    day, on the basis of the absence of any significant findings.

         In a study of teratogenicity in which rats were given 0, 5, 15
    or 50 mg/kg bw per day of tolclofos-methyl by gavage, the NOAEL was
    50 mg/kg bw per day on the basis of the absence of any significant
    findings. The study was not considered to be fully adequate because
    the highest dose tested was not maternally toxic. A similar study
    was conducted at levels of 0, 100, 300 or 1000 mg/kg bw per day. The
    NOAEL was 300 mg/kg bw per day on the basis of reduced body-weight
    gain in dams at 1000 mg/kg bw per day. There was no evidence of
    teratogenicity.

         A study of teratogenicity was conducted in rabbits given 0,
    300, 1000 or 3000 mg/kg bw per day orally. The NOAEL for maternal
    toxicity was 300 mg/kg bw per day on the basis of reduced
    body-weight gain in dams at 1000 mg/kg bw per day. There was no
    evidence of teratogenicity.

         Tolclofos-methyl was studied in a wide range of tests for
    genotoxicity  in vivo and  in vitro. The Meeting concluded that
    the compound is not genotoxic.

         Tolclofos-methyl did not cause delayed neuropathy in chickens.

         The available observations in humans were considered by the
    Meeting but did not directly contribute to an estimation of an ADI.

         An ADI was established on the basis of a NOAEL of 50 ppm, equal
    to 6.5 mg/kg bw per day, in the 104-week study of toxicity and
    carcinogenicity study in mice, and a safety factor of 100.

    Toxicological evaluation

    Levels that cause no toxic effect

         Mouse:    50 ppm, equal to 6.5 mg/kg bw per day (104-week study
                   of toxicity and carcinogenicity)

         Rat:      1000 ppm, equal to 41 mg/kg bw per day (122/129-week
                   study of toxicity and carcinogenicity)

         Rabbit:   300 mg/kg bw per day (maternal toxicity in a study of
                   teratogenicity)

         Dog:      400 ppm, equal to 11 mg/kg bw per day (52-week study
                   of toxicity)

    Estimate of acceptable daily intake for humans

         0-0.07 mg/kg bw

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

         Further observations in humans

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    See Also:
       Toxicological Abbreviations