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    METHIOCARB        JMPR 1998

    First draft prepared by
    T.C. Marrs
    Department of Health
    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 studies of toxicity 
                   Long-term studies of toxicity and carcinogenicity
                   Genotoxicity 
                   Reproductive toxicity 
                        Multigeneration reproductive toxicity
                        Developmental toxicity 
                   Special studies 
                        Neurotoxicity 
                        Antidotes 
                        Interaction with other pesticides 
              Observations in humans  
         Comments 
         Toxicological evaluation 
         References 


    Explanation

         Methiocarb was evaluated for toxic effects by Joint Meetings in
    1981, 1983, 1984, 1985, and 1987 (Annex 1, references 36, 40, 42, 44,
    and 50). An ADI of 0-0.001 mg/kg bw was allocated in 1981,which was
    extended at subsequent meetings. Methiocarb was evaluated by the
    present Meeting within the CCPR periodic review programme. This
    monograph summarizes the new data and relevant data from the previous
    monographs and monograph addenda on methiocarb (Annex 1, references
    37, 41, and 46).

    Evaluation for Acceptable Daily Intake

    1.  Biochemical aspects

     (a)  Absorption, distribution, and excretion

         [ring-1-14C]-Methiocarb (4.8 mCi/mmol; purity, > 97%) was
    administered to three female rats by gavage at a dose of 20 mg/kg bw.
    Similarly labelled methiocarb (14.5 mCi/mmol; purity, > 97%) was
    administered to three male and three female rats by gavage at a dose

    of 0.25 mg/kg bw. Organic extracts of 48-h urine samples collected
    from each rat were subjected to one-dimensional or two-dimensional
    thin-layer chromatography on plates pre-coated with silica gel. More
    than 90% of the radiolabel administered at the higher dose was
    excreted in the urine. Similar results were obtained with the lower
    dose, but a somewhat smaller proportion of the radiolabel (73-86%) was
    excreted over 48 h; at this dose, there was no major difference
    between males and females (Stanley & Johnson, 1976).

     (b)  Biotransformation

         In the urine samples collected by Stanley and Johnson (1976), the
    major components of the chloroform extract at the higher dose were
    methiocarb phenol, methiocarb sulfoxide phenol, and an unknown
    component which may correspond to  N-hydroxymethyl methiocarb
    sulfoxide. A trace of methiocarb sulfoxide was detected. After
    incubation of the aqueous phase of the urine samples with maltase,
    about half of the radiolabel present was rendered organosoluble, the
    major component being methiocarb sulfoxide phenol; methiocarb phenol
    and methiocarb sulfone phenol were also present. The results were
    similar at the lower dose, at which there was no major difference
    between males and females; however, methiocarb sulfone phenol was
    found in the chloroform extract. 

         These results differ from those of an earlier study in rats with
    [carbonyl-14C]- and [methyl-3H]-methiocarb, which was not available
    for evaluation. It was reported that methiocarb phenol was the primary
    metabolite in the earlier study, whereas in the study of Stanley and
    Johnson (1976), methiocarb sulfoxide phenol was the metabolite at
    highest concentration in the urine. The report of the latter study
    also cites an earlier study in dogs in which the only metabolites seen
    were methiocarb sulfoxide phenol and methiocarb sulfone phenol.

         In studies of the metabolism of methiocarb  in vitro (Strother,
    1970, 1972; Menzie, 1974) with 14C-methiocarb, human liver fractions
    were slightly less metabolically active than fractions from
    Sprague-Dawley rats. The major metabolite was methiocarb sulfoxide. In
    a comparative study of the metabolism of methiocarb (and Zectran, a
    related compound) in rat and dog liver and kidney homogenates and
    various blood fractions  in vitro, the metabolic pathway seemed to be
    similar in the two species, sulfoxidation of the sulfur atom being the
    main route.  N-Methyl hydroxylation was also found to occur (Wheeler
    & Strother, 1971).

         Flavin adenine dinucleotide-dependent monooxygenases from pig
    liver microsomes had some activity in the sulfoxidation of methiocarb
    (Haijar & Hodgson, 1982). Methiocarb is reported to cross the placenta
    into the fetus (Salama et al., 1993).

         The main products of metabolism of methiocarb in plants were
    conjugates of the phenol, sulfoxide phenol, and sulfone phenol;
    methiocarb sulfone was also seen, while methiocarb sulfoxide occurred
    in some plant products (Murphy et al., 1982).

         Figure 1 shows the proposed metabolic pathways for methiocarbin
    various species and media.

     (c)  Effects on enzymes and other biochemical parameters

         Single doses of 1, 10, 25, or 50 mg/kg bw methiocarb were
    administered by gavage to groups of five Wistar rats of each sex,
    while five rats of each sex acted as controls. Plasma and erythrocyte
    cholinesterase activity was measured after 20 min, 2 h, and 5 h, and,
    in males at the highest dose, additionally at 1.5 and 3 h; brain
    acetylcholinesterase activity was measured at 30-min intervals up to
    5 h. Cholinergic signs, starting immediately after treatment and
    abating within 2 h, were seen at > 10 mg/kg bw. Male rats receiving
    the highest dose died after 2-3 h. Maximum depression of plasma
    cholinesterase and erythrocyte acetylcholinesterase activity was
    observed after 20 min at < 25 mg/kg bw and after 20 min to 2 h at
    the highest dose. In a separate study, methiocarb was given at a dose
    of 10 or 20 mg/kg bw, and brain acetylcholinesterase activity was
    determined at 30 min and 1, 2, 3, and 5 h. Inhibition was maximal at
    2 h. 

         In a four-week study, 10 rats of each sex received methiocarb at
    doses of 1, 3, or 10 mg/kg bw per day by gavage. Plasma and
    erythrocyte cholinesterase activity was determined in three rats of
    each sex 20 min after administration of the test material on days 4,
    8, 14, 21, and 28; brain acetylcholinesterase activity was determined
    in five rats of each sex 2 h after the last dose. At the highest dose,
    cholinergic signs were observed briefly. Plasma, erythrocyte, and
    brain cholinesterase activity was depressed at 10 mg/kg bw per day.
    The NOAEL for cholinesterase depression was thus 3 mg/kg bw per day
    (Eben & Kimmerle, 1973).

         Technical-grade methiocarb (purity, 97%) and methiocarb sulfoxide
    (purity, 95.2%) were administered by gavage to groups of 15 female
    Sprague-Dawley-derived rats at a dose of 0.5 or 2 mg/kg bw per day on
    five days per week for four weeks, while 15 controls received the
    vehicle (Carbowax). The rats were observed daily for general
    appearance and, in addition, for cholinergic signs at 0.5, 1, and 4 h
    after dosing for the first five days of treatment. For determination
    of cholinesterase activity, each group was subdivided into three
    subgroups of five. Blood was collected from the first subgroup before
    and 30 min after dosing on days 0, 7, 14, 21, and 28; blood was
    collected similarly from the second group but 4 h after dosing on days
    4, 11, 18, and 25. The third subgroup was held in reserve in case
    anaemia developed. Depression of cholinesterase activity was
    calculated by comparison with activity before treatment. 

         Tremors were seen during the first five days of the study in some
    rats receiving the sulfoxide at the higher dose; no clinical signs
    were seen in other groups. A 20% depression in plasma cholinesterase
    activity was found in controls 4 h after dosing on day 25, and
    depressions of 1-21% were found 30 min after dosing with methiocarb at

    FIGURE 1

    0.5 mg/kg bw; 4 h after dosing at 0.5 mg/kg bw, depressions of < 10%
    were noted. At the higher dose of methiocarb, depressions of 19-41%
    were seen 30 min after dosing and 0.2-19% 4 h after dosing. With the
    sulfoxide, plasma cholinesterase activity was inhibited by 21-39% at
    the low dose at 30 min but was generally not inhibited at 4 h. At the
    high dose, 39-62% inhibition was seen at 0.5 h and 10-25% inhibition
    at 4 h. Erythrocyte acetylcholinesterase activity was depressed by
    5-12% 30 min after dosing at 0.5 mg/kg bw and by < 10% 4 h after
    dosing. At the higher dose of methiocarb, erythrocyte
    acetylcholinesterase activity was depressed by 15-29% 30 min after
    dosing and by 1-13% 4 h after dosing. With the sulfoxide, erythrocyte
    acetylcholinesterase activity was inhibited by 13-31% at the low dose
    at 30 min but was generally not inhibited at 4 h. At the high dose,
    32-46% inhibition was seen at 0.5 h and 10-21% at 4 h. The NOAEL for
    erythrocyte acetylcholinesterase inhibition was therefore
    approximately 0.5 mg/kg bw per day for methiocarb, but there was no
    NOAEL for the sulfoxide (Hixson, 1981).

         Technical-grade methiocarb (purity, 97%) and methiocarb sulfoxide
    (purity, 95.2%) were administered orally in gelatine capsules to
    groups of two adult beagle dogs and two bitches at a dose of 0.05 or
    0.5 mg/kg bw per day for 29 days; two control animals of each sex
    received empty capsules. The animals were observed twice daily, and
    blood was taken for measurement of plasma and erythrocyte
    cholinesterase activity before the first dose, 1, 3, 6, and 24 h after
    the first dose, and 3 h after the third dose. During the second week,
    blood was taken before the first dose, 2, 6, and 24 h later, and 2 h
    after the second dose. During weeks 3 and 4, blood was taken before
    the first dose, 2, 6, and 24 h later, and 2 h after the third dose. In
    week 5, blood was again taken before the first dose and 2, 6, and 24 h
    later; dosing was then stopped, and another blood sample was taken on
    the third day. 

         Clinical signs of toxicity (salivation and vomiting) were
    observed at the higher dose of each test material and in animals of
    each sex; additionally, slight salivation was observed in one bitch
    given the sulfoxide at the lower dose. Depression of cholinesterase
    activity was calculated by comparison with the level measured just
    before the first dose each week: inhibition was variable, but peak
    inhibition occurred 0-3 h after dosing; considerable inhibition was
    seen with both materials at the higher dose in animals of each sex. At
    the lower doses, smaller depressions of both plasma and erythrocyte
    cholinesterase activity, of up to about 20%, were sometimes seen with
    both materials. Cholinesterase activity was generally normal by 6 h
    (Hayes, 1981). 

         At equimolar concentrations, methiocarb was the least effective
    of four carbamates in inhibiting bovine erythrocyte
    acetycholinesterase and equine plasma cholinesterase activity
    (Barthová et al., 1989). 

         Methiocarb was reported not affect liver function in rabbits at a
    single oral dose of 25 mg/kg bw (Kimmerle, 1960).

    2.  Toxicological studies

     (a)  Acute toxicity

         The results of studies of the acute toxicity of methiocarb and
    putative metabolites of methiocarb are shown in Table 1.

         No erythema or oedema was seen when technical-grade methiocarb
    (purity unspecified) was applied to abraded and unabraded skin of six
    New Zealand white rabbits. Application to the eyes of six rabbits did
    not produce ocular irritation (Crawford & Anderson, 1970). 

         A study of the skin-sensitizing potential of methiocarb (purity,
    97.8% pure) was conducted by the Magnusson and Kligman technique. A
    group of 20 guinea-pigs (strain BOR:DHPW) each received methiocarb
    intradermally at a concentration of 1% and topically at a
    concentration of 25%. There were two control groups of 10 guinea-pigs.
    The test animals were first challenged with methiocarb at a
    concentration of 25% and then at 12.5%. Although there was a small
    excess in the number of animals that reacted positively after the
    second challenge, the overall result was considered to be negative
    (Mihail, 1984).

         In a test for dermal sensitization by the Buehler technique,
    technical-grade methiocarb (purity, 99.2%) was applied to 15 Hartley
    guinea-pigs once weekly for three weeks, with a challenge dose a
    fortnight later. The material was applied as a single dose to five
    previously unexposed animals at the time of the challenge dose.
    Dinitrochlorobenzene, used as the positive control, was applied in the
    same way to a further group of five guinea-pigs, weekly for three
    weeks, with a challenge dose two weeks later, and to a further group
    of five as a single dose at the time of the challenge dose. Methiocarb
    was not considered to be a dermal sensitizer (David, 1988).

         A case of allergic contact dermatitis was reported in a man who
    grew carnations. He developed acute severe eczema of the hand and
    showed a positive reaction in a patch test to 0.5% methiocarb (Willems
    et al., 1997).

     (b)  Short-term studies of toxicity

     Rats

         Methiocarb in aqueous tragacanth suspension was given by gavage
    to albino rats daily. The dose was 2 mg/kg bw per day for the first
    three days and 4 mg/kg bw per day for the next 24 days. Two groups of
    three animals were killed every week for determination of
    cholinesterase activity. The activity had fallen to 80% of the control
    values after 14 days and to 50% by the end of the study. No abnormal
    clinical signs were observed; the animals gained weight normally
    (Kimmerle, 1960).


        Table 1. Acute toxicity of methiocarb and putative metabolites

                                                                                                                                           

    Species       Strain            Sex     Route                LD50 (95% CI or       Purity             Reference 
                                                                 range) (mg/kg bw)     (%)
                                                                                                                                           

    Methiocarb

    Rats          Sprague-Dawley    M       Oral                 130                   Technical          DuBois & Raymund (1962) 
                                    F                            140
    Rats          Sprague-Dawley    F       Oral                 100                   Recrystallized     DuBois & Raymund (1962)
    Rats          NR                NR      Oral                 67                    NR                 Kimmerle (1966a)
    Rats          Sprague-Dawley    M       Oral                 30 (20-45)            99                 Crawford & Anderson (1973)
                                    F                            30 (20-45)
    Rats          Sprague-Dawley    M       Oral (non-fasting)   46 (38-56)            99                 Crawford & Anderson (1973)
                                    F                            47 (36-63)
    Rats          Sprague-Dawley    M       Oral                 15 (9-26)             Technical          Lamb & Matzkanin (1976a)
                                    F                            31 (18-54)
    Rats          Sprague-Dawley    M       Oral                 13 (9-17)             Technical          Lamb & Matzkanin (1976b)
                                    F                            32 (24-44)
    Rats          Sprague-Dawley    M       Oral                 14 (12-16)            Technical          Lamb & Matzkanin (1977)
                                    F                            16 (13-20)
    Rats          Sprague-Dawley    M       Oral (non-           51 (45-58)            Technical          Lamb & Matzkanin (1977)
                                    F                            79 (65-96)
    Rats          NR                M       Oral                 22 (19-25)            98.5               Flucke (1978) 
                                    F                            24 (21-28)
    Rats          NR                M       Oral                 22.1 (18-27)          98.3               Flucke (1980) 
    Rats          Sprague-Dawley-   M       Oral                 33 (22-50)            98                 Nelson (1979)
                  derived           F                            47 (31-70)
    Rats          NR                M       Oral                 17 (16-19)            98.6               Heimann (1983)
    Rats          NR                M       Oral                 19 (16-23)            98.2               Flucke (1988)
                                    F                            26 (19-36)
    Rats          Albino            M       Oral                 100                   NR                 Kimmerle (1960)
    Rats          Wistar-CFN        M       Oral                 87                    NR                 Klimmer (1963)
    Rats          NR                M       Oral                 33 (29-38)            98.4               Thyssen (1977a)
                                                                 35 (29-42)            98.3 
                                                                 35 (30-42)            98.2               
                                                                 31 (26-35)            97.8               28 (24-33)97.4

    Table 1. (continued)

                                                                                                                                           

    Species       Strain            Sex     Route                LD50 (95% CI or       Purity             Reference 
                                                                 range) (mg/kg bw)     (%)
                                                                                                                                           

    Rats          Sprague-Dawley    M       Intraperitoneal      35                    Technical          DuBois & Raymund (1961a)
                                    F                            30
    Rats          Sprague-Dawley    F       Intraperitoneal      25                    Recrystallized     DuBois & Raymund (1962)
    Rats          Wistar-CFN        M       Intraperitoneal      43                    NR                 Klimmer (1963)
    Rats          Sprague-Dawley    M       Dermal               > 200                 Technical          DuBois & Raymund (1961a)
    Rats          Sprague-Dawley    F       Dermal               > 300                 Recrystallized     DuBois & Raymund (1962)
    Rats          Albino            M       Dermal               > 1000                NR                 Kimmerle (1960)
    Rats          Wistar-CFN        M       Dermal               350-400               NR                 Klimmer (1963)
    Rats          NR                M       Dermal               > 500                 99.2               Solmecke (1969)
    Rats          Wistar            M       Dermal               > 5 g                 98.1               Thyssen (1977b)
                                    F                            > 5 
    Rats          Sprague-Dawley    M       Inhalation (1 h)     1200 mg/m3 a          98.8               Shiotsuka (1987a)
                                                                 (780-1700)
                                    F                            1100 mg/m3 a
                                                                 (740-1600)
    Rats          NR                M       Inhalation           540 g/L3              NR                 Kimmerle (1966b)
    Rats          Sprague-Dawley    M       Inhalation (1 h)     > 20 000 g/L          Technical          Crawford & Anderson (1972a)
                                    F                            > 20 000 g/L
    Rats          Sprague-Dawley    M       Inhalation (4 h)     580 mg/m3 (340-700)   98.8               Shiotsuka (1987b)
                                    F                            430 mg/m3 (290-580)
    Rats          Wistar            M       Inhalation (4 h)     > 320 mg/m3           97.9               Thyssen (1982)
                                    F                            > 320 mg/m3
    Rats          Wistar            M       Inhalation           > 300 mg/m3           97.9               Thyssen (1982)
                                    F       (5 x 6 h)            > 300 mg/m3

    Mice          NR                M       Oral                 25 (21-30)            NR                 Kohgo (1970)
    Mice          NR                M       Oral                 52                    98.2               Kimmerle (1972)
    Mice          NR                M       Subcutaneous         940 (760-1200)        NR                 Kohgo (1970)
    Mice          Carworth Farm     M       Intraperitoneal      6                     Technical          DuBois & Raymund (1961a)
                                    F                            5.5
    Mice          Dierolf Farm      F       Intraperitoneal      16                    Technical          Baron et al. (1964)

    Table 1. (continued)

                                                                                                                                           

    Species       Strain            Sex     Route                LD50 (95% CI or       Purity             Reference 
                                                                 range) (mg/kg bw)     (%)
                                                                                                                                           

    Rabbits       New Zealand       M       Dermal               > 2000                99                 Crawford & Anderson
                  white             F                            > 2000                                   (1972b)

    Guinea-pigs   NR                M       Oral                 40                    Technical          DuBois & Raymund (1961a)
    Guinea-pigs   NR                F       Oral                 (50-100)              99.2               Kimmerle (1969a)
    Guinea-pigs   Albino            F       Oral                 14 (7.9-25)           Technical          Crawford & Anderson (1972a)
    Guinea-pigs   NR                M       Intraperitoneal      17                    Technical          DuBois & Raymund (1961a)

    Dogs          Beagle            F       Oral                 (10-25)               99.2               Kimmerle (1969b)
    Dog           Mongrel           M       Oral                 ~ 25                  Technical          Lamb & Matzkanin (1975)
                                    F                            ~ 25

    Chicken       NR                F       Oral                 175                   Technical          DuBois (1962)
    Chicken       White Leghorn     F       Oral                 380 (300-490)         98.5               Thyssen & Schilde (1978)

    Methiocarb phenol

    Rats          NR                M       Oral                 > 1000                NR                 DuBois (1964)
    Rats          NR                M       Dermal               > 1000                NR                 DuBois (1964)
    Rats          NR                NR      Oral                 > 1000                NR                 Solmecke (1970)

    Methiocarb phenol sulfoxide

    Rats          NR                M       Oral                 > 1000                NR                 DuBois (1964)
    Rats          NR                NR      Oral                 > 1000                NR                 Solmecke (1970)
    Rats          NR                M       Dermal               > 1000                NR                 DuBois (1964)

    Methiocarb phenol sulfone

    Rats          NR                M       Oral                 > 1000                NR                 DuBois (1964)
    Rats          NR                NR      Oral                 > 1000                NR                 Solmecke (1970)
    Rats          NR                M       Dermal               > 1000                NR                 DuBois (1964)

    Table 1. (continued)

                                                                                                                                           

    Species       Strain            Sex     Route                LD50 (95% CI or       Purity             Reference 
                                                                 range) (mg/kg bw)     (%)
                                                                                                                                           

    Methiocarb sulfoxide

    Rats          NR                NR      Oral                 43 (37-50)            NR                 Solmecke (1970)
    Rats          Sprague-Dawley    M       Oral                 9 (7-13)              NR                 Lamb & Matzkanin (1976a)
                                    F                            7
    Rats          Sprague-Dawley    M       Oral                 6 (5-8)               NR                 Lamb & Matzkanin (1976a)
                                    F                            8 (6-10)

    Methiocarb sulfone

    Rats          NR                NR      Oral                 > 1000                NR                 Solmecke (1970)

    Hydroxymethyl methiocarb

    Rats          NR                M       Oral                 > 110                 NR                 Nelson (1979)
                                    F                            > 110

    Hydroxymethyl methiocarb sulfone

    Rats          NR                M       Oral                 > 110                 NR                 Nelson (1979)
                                    F                            > 110

    Hydroxymethyl methiocarb sulfoxide

    Rats          NR                M       Oral                 > 160                 NR                 Nelson (1979)
                                    F                            > 160
                                                                                                                                           

    Technical, purity unstated; NR, not reported
    a There was a large descrepancy between nominal and measured concentrations owing to deposition in the chamber; 
      these figures were measured gravimetrically.
    

         Methiocarb was administered by gavage to groups of 25 male
    Wistar-CFN rats at a dose of 2.5 or 5 mg/day on six days per week for
    six months. Three deaths occurred at the higher dose due to
    bronchopneumonia, but weight gain was the same in the two treated
    groups and in 20 control animals; no abnormal clinical signs were seen
    (Klimmer, 1963). 

         Groups of 12 Sprague-Dawley rats of each sex were fed diets
    containing methiocarb (purity unspecified) at concentrations of 0, 5,
    10, or 50 ppm for 16 weeks. Five animals of each sex from each group
    were examined histopathologically, and five of each sex were used for
    measurements of cholinesterase activity in blood, brain, and
    submaxillary glands by a manometric method. No effect on growth rate,
    food intake, or mortality rates was observed at any dose. In males at
    the highest dose, serum, erythrocyte, brain, and submaxillary gland
    cholinesterase activity was inhibited by 21, 14, 12, and 7%,
    respectively. In females at the highest dose, serum cholinesterase
    activity was reduced by 28% and erythrocyte acetylcholinesterase
    activity by 15%, all by comparison with concurrent controls. Brain
    acetylcholinesterase activity was reduced by 5% in females, and
    inhibition of submaxillary gland enzyme was seen in all test groups. A
    NOAEL could not be identified in this study (Doull et al., 1962).

         Groups of 10 Wistar rats of each sex were exposed to an aerosol
    of methiocarb (purity, 97.9%) of a mass median diameter of 2.1-2.5.
    The groups were exposed to either air, solvent, methiocarb at 6 mg/m3
    in solvent, methiocarb at 23 mg/m3 in solvent, or methiocarb at 96
    mg/m3 in solvent; exposure was for 6 h/day, five days per week for
    three weeks. The animals were examined daily and weighed weekly. Blood
    was taken from five animals per group at the end of the study and was
    used for haematological and clinical chemical tests; urinary analyses
    were also carried out. Plasma and erythrocyte cholinesterase activity
    was determined before treatment and after 5, 10, and 15 exposures for
    all groups except the air controls. At the end of the study, the
    animals were sacrificed and autopsied, selected organs were weighed
    and processed for histopathological examination, and brain
    acetylcholinesterase activity was measured. 

         No deaths were observed in any group. Animals at the highest
    concentration showed clinical signs of compound-related effects
    (tremor); no clinical signs were seen at lower concentrations. There
    body weight of males at the highest concentration was reduced by
    comparison with the air controls but not with the solvent controls. No
    change in haematological or clinical chemical parameters was seen that
    was attributable to the test material, with the exception of
    inhibition of cholinesterases. Plasma and brain cholinesterase
    activity was decreased at the highest concentration, brain
    acetylcholinesterase activity being 61 and 74 % of that in concurrent
    solvent controls in males and females, respectively. Some inhibition
    of plasma cholinesterase activity was seen at the intermediate
    concentration, and males at this concentration had an associated
    decrease in brain acetylcholinesterase activity (65% of concurrent
    control value). Erythrocyte acetylcholinesterase activity was less

    strongly affected than plasma enzyme, but marginal inhibition was
    observed in males at the highest concentration at week 1 (82% of
    concurrent solvent control value). There were no toxicologically
    significant alterations in organ weights, and no compound-related
    findings were noted on histopathological examination. The NOAEL was
    6 mg/m3 on the basis of reduced brain acetylcholinesterase activity
    in males (Thyssen & Mohr, 1983). 

     Rabbits

         Technical-grade methiocarb (purity, 99.2%) was applied to five
    male and five female chinchilla rabbits, daily for two weeks, at a
    dose of 500 mg; five males and five females served as controls. The
    material was applied for 24 h/day, and new material was applied at the
    end of each 24-h period. After the two-week application period, the
    animals were observed for a further fortnight. The animals were
    inspected daily, and haematological and biochemical studies were
    carried out before treatment, at the end of treatment, and two weeks
    later. Clinical chemistry was restricted to liver function tests and
    urinary analysis; cholinesterase activity was not measured. No
    abnormal clinical signs were seen, and there was no effect on
    body-weight gain or any perturbation in haematological or clinical
    chemical variables (Kimmerle, 1969c).

         Methiocarb (purity, 99.3%) was applied at doses of 0, 60, 150, or
    375 mg/kg bw per day for 6 h/day to the skin of groups of five male
    and five female New Zealand white rabbits, and the site of application
    was occluded. The animals were examined twice daily, and any signs of
    skin irritation were scored. The rabbits were weighed twice weekly;
    food consumption was measured three times weekly until the last week,
    when it was measured four times. Blood was taken for haematological
    and clinical chemical analysis before treatment and pre-terminally.
    Plasma and erythrocyte cholinesterase activity was measured at the end
    of the 6-h exposure period on days 1, 7, 14, and 21; additionally,
    blood was taken 16 h after the end of exposure on these days from the
    group at the high dose. Animals were sacrificed on the day after the
    last treatment, at which time the brain was taken for measurement of
    acetylcholinesterase activity in a homogenate of the entire left half
    of the brain. Selected tissues were weighed, examined grossly,
    processed, and examined histopathologically. 

         Two animals at the low dose did not survive to the end of the
    study. No clinical signs related to the test material were seen, and
    it was not irritating. There was no differences between the groups in
    body weight, but food consumption appeared to be reduced in animals of
    each sex at the high dose, and particularly in males. Clinical
    chemical parameters did not differ between the groups, and, although
    one or two differences in haematological measurements were seen, none
    appeared to be related to treatment. Plasma cholinesterase activity
    appeared to be reduced in males at the high dose at 14 and 21 days.
    The measurements made 6 h after the end of exposure suggested that the
    inhibition was not reversed overnight. No intergroup differences in
    plasma cholinesterase activity were observed among females.

    Erythrocyte acetylcholinesterase activity was very variable but did
    not appear to be inhibited in a dose-related fashion. Intergroup
    differences were not observed in brain acetylcholinesterase activity.
    No gross or microscopic abnormality was observed that was attributable
    to the test material. The NOAEL was 150 mg/kg bw per day on the basis
    of reduced food consumption (Procter, 1988).

         Methiocarb (purity, 97.5%) was applied to the skin of five male
    and five female New Zealand white rabbits at a single dose of 500
    mg/kg bw per day for 6 h/day under occlusion. Five controls of each
    sex received saline. The animals were examined twice daily, and any
    signs of skin irritation were scored. They were weighed twice weekly,
    while food consumption was measured every two days. Blood was taken
    for haematological and clinical chemical tests before treatment and
    preterminally. Plasma and erythrocyte cholinesterase activity was
    measured at the end of the 6-h exposure period on days 1, 7, 14, and
    21 and, in the test animals, 16 h later. Animals were sacrificed on
    the day after the last treatment, at which time the left half of the
    brain was taken for measurement of cholinesterase activity. Selected
    tissues were weighed, examined, processed, and examined
    histopathologically. 

         Two test animals removed their dressings and presumably ingested
    the material; these animals developed clinical signs of cholinergic
    poisoning, which disappeared within a few hours. No other
    compound-related clinical signs were seen, and no animal died before
    completion of the study. Treated females were lighter than controls
    throughout the study, and the food consumption of animals of each sex
    was reduced. A reduction in serum calcium and increased activities of
    alanine and aspartate aminotransferases were seen in females in
    comparison with concurrent controls. Plasma cholinesterase activity
    was lower than that before treatment in both males and females, but
    only inconsistently in comparison with concurrent controls.
    Erythrocyte acetylcholinesterase activity was reduced in males on day
    1, at both 6 and 16 h, in comparison with pretreatment levels, but
    inconsistently in comparison with concurrent controls; it was
    concluded that compound-related inhibition of erythrocyte
    acetylcholinesterase activity had not occurred in either males or
    females. Brain acetylcholinesterase activity was not inhibited. No
    abnormality was seen at autopsy or on histopathological examination.
    The design of this study was not appropriate for identifying a NOAEL
    (Procter, 1989).

     Cats

         Methiocarb at a dose of 5 mg/kg bw per day, given daily by gavage
    to cats, was reported to have no adverse effects (Kimmerle, 1960).

     Dogs

         Methiocarb was incorporated into the diet of groups of two male
    and two female beagles at a concentration of 0, 50, 100, or 250 ppm,
    equal to 0, 1.25, 2.5, or 6.25 mg/kg bw per day. The animals were

    examined daily and weighed fortnightly; plasma and erythrocyte
    cholinesterase activity was measured weekly. No clinical effects were
    seen, and body-weight gain was unaffected by treatment. There was no
    clear difference in plasma or erythrocyte cholinesterase activity
    between the test groups and concurrent controls. The small group size
    renders this study inappropriate for identifying a NOAEL (Root et al.,
    1963).

     Chickens

         Chickens  (Gallus gallus Babcock 300) were fed diets containing
    a 9:1 mixture of methiocarb and methiocarb sulfoxide (based on studies
    of plant metabolism) at a dose of 20, 60, 120, or 360 ppm, equal to
    2.5, 7.5, 15, and 45 mg/kg bw per day, over 28 days. Treatment
    decreased feed consumption in a dose-related fashion, and the body
    weights of birds at the two highest doses were decreased. Egg
    production was unaffected. Plasma cholinesterase activity was
    decreased by 40-50 % in comparison with concurrent controls at the
    three highest doses but was unaffected at the lowest dose (Strankowski
    & Minor, 1976).

     (c)  Long-term studies of toxicity and carcinogenicity

     Mice

         Groups of 50 male and 50 female BOR:CFW1 mice received diets
    containing methiocarb (purity, 98.5%) at concentrations of 0, 67, 200,
    or 600 ppm, equal to 0, 15, 43, and 130 mg/kg bw per day in males and
    0, 20, 57, and 170 mg/kg bw per day in females. Haematological and
    clinical chemical tests were performed on five animals of each sex per
    dose at 12 months and 10 animals of each sex per dose at 24 months,
    and on satellite groups of 15 mice of each sex per dose, which were
    sacrificed after one year and necropsied. Cholinesterase activity was
    determined in the plasma of five animals of each sex per dose at 1 and
    12 months and on 10 animals of each sex per dose (or the survivors if
    fewer than 10) at the end of the study. Brain cholinesterase activity
    was determined at the end of the study. Animals that died or became
    moribund were autopsied, as were 10 mice of each sex per group at 12
    months. All surviving animals were sacrificed at the end of the study
    and autopsied, and selected organs were weighed, examined, and
    processed for histopathological examination. 

         The appearance, behaviour, mortality rate, and food consumption
    of the animals were not affected at any dose, except for a slight
    decrease in body weight at the highest dose, throughout the study in
    males and up to week 30 in females. Overall survival in this study was
    not good: survival at 18 months was 74% of male controls, 47% males at
    the low dose, 59% males at the intermediate dose, 66% males at the
    high dose, 69% of female controls, 53% at the high dose, 64% at the
    intermediate dose, and 69% at the high dose. Intergroup differences in
    mortality rates did not appear to be compound-related, as mortality
    was higher in males receiving 67 or 200 ppm than in those given the
    high dose. Moreover, survival of females up to the end of the study

    was poorest for controls. All treated male mice had higher mean
    corpuscular haemoglobin concentrations than controls at 12 months, and
    males at the highest dose had higher mean corpuscular haemoglobin
    values; similar changes were not seen in females. At 24 months, the
    mean corpuscular haemoglobin concentration of males at 200 and 600 ppm
    was decreased and the mean corpuscular haemoglobin value at 600 ppm,
    again, with no similar change in the females. Higher leukocyte counts
    were observed in all treated females at 24 months, but the authors
    ascribed this finding to high individual values; no perturbution of
    the differential count was seen. 

         No clinical chemical abnormalities were found at 12 months, but
    at 24 months the activity of alanine aminotransferase (ALAT) was
    increased in animals at 200 and 600 ppm. At one month, males at 200
    and 600 ppm had reduced plasma cholinesterase activity (by 51 and 34%
    in comparison with concurrent controls), while reductions of 5% or
    less were seen at 12 months and 5-11% at 24 months. In females, plasma
    cholinesterase activity was inhibited by 24, 43, and 34% at the low,
    intermediate, and high doses, respectively, at one month; smaller
    reductions (< 12%) were observed at 12 months, and no cholinesterase
    inhibition was observed at 24 months. Erythrocyte acetylcholinesterase
    activity was not measured. Small reductions in brain
    acetylcholinesterase activity were observed, by 5, 11, and 10% in
    males at the low, intermediate, and high doses and by 3, 9, and 3% in
    females at the three doses, respectively. No compound-related change
    in organ weights was seen, nor was there any abnormality in gross of
    histological appearance that was related to treatment. Methiocarb was
    not tumorigenic. There was no NOAEL because of haematological changes
    at all doses in males at 12 months and in females at all doses at 24
    months (Krötlinger & Janda, 1983; Krötlinger, 1989).

     Rats

         Methiocarb (purity, 98.9%) was admixed with the diet of groups of
    60 Wistar TNO W.74 rats for two years at concentrations of 0, 67, 200,
    or 600 ppm, equal to 0, 3.3, 9.3, and 29 mg/kg bw per day for males
    and 0, 5, 14, and 42 mg/kg bw per day for females. The rats were
    inspected daily, and body weights were determined weekly for the first
    26 weeks and then at fortnightly intervals. Haematological, clinical
    chemical, and urinary measurements were made in 10 animals of each sex
    per dose at 3, 6, 12, and 24 months. Cholinesterase activity in plasma
    and erythrocytes was determined one and two days after the start of
    the study and at 1, 2, 4, 8, 13, 26, 52, 78, and 105 weeks in 10
    animals of each sex per dose. Brain acetylcholinesterase activity was
    determined at the end of the experiment on 10 animals of each sex per
    dose. Animals that died and those sacrificed  in extremis were
    examined grossly and necropsied; when possible, tissues were taken for
    histopathological examination. The survivors at two years were also
    examined grossly and necropsied, and tissues were taken for weighing
    and histopathological examination. 

         None of the doses had any effect on the appearance, behaviour, or
    mortality rates of rats of either sex. The mortality rates at two
    years were 10-20% for males and 23-32% for females. There was no
    significant intergroup difference in food consumption. Weight gain was
    not depressed at 67 or 200 ppm in comparison with the controls; at 600
    ppm, weight gain was slightly but consistently depressed throughout
    the period of administration of methiocarb, and at termination total
    body weight was reduced in the group at the high dose. At three
    months, an increased leukocyte count was seen in females at the
    highest dose and an increased reticulocyte count in females at the two
    higher doses. At six months, the mean corpuscular haemoglobin
    concentration was reduced in males at the highest dose, and some
    elevation in leukocyte count was found in males at 67 and 200 ppm. Red
    blood cell counts and haemoglobin and haematocrit values were
    decreased in females at the two higher doses, and reticulocytosis was
    observed in these groups. At 12 months, the mean corpuscular
    haemoglobin concentration was decreased in males at the intermediate
    dose and was increased in females at the highest dose; an increased
    reticulocyte count was seen in females at the high dose. At 24 months,
    no compound-related changes in haematological variables were seen. 

         Although a few intergroup differences were observed in
    biochemical tests, the only ones that appeared to be related to
    treatment were total protein concentration and cholinesterase
    activity. Total protein concentrations were raised in females at the
    two higher doses at six months and in males at the three highest doses
    at 12 months; at 24 months, the total protein concentration was
    similar in treated and control groups. ALAT activity in the blood was
    elevated in all three groups of treated females at 12 months and in
    females at the highest dose at two years, but at no time interval in
    males. Increased urea was found in plasma from males at the highest
    dose at three and 12 months and in females at 12 and 24 months. Plasma
    cholinesterase activity was depressed at the high dose at one day and
    from eight weeks onwards in males (except in the 52-week assay) and in
    females at one day and 1, 2, 4, and 13 weeks. The measurements of
    erythrocyte acetylcholinesterase activity were difficult to interpret:
    a statistically significant depression was found only at the low dose
    in males at 105 weeks and females at 78 weeks, and the degree of
    depression was small (5 and 6%, respectively). Males at the
    intermediate dose had significantly depressed activities at 8, 78, and
    105 weeks, with 5, 7, and 8% depression, respectively. In females at
    this dose, significant depression was seen at four and 78 weeks (8 and
    13% depression, respectively). In males at the highest dose,
    significant depressions were seen at 8, 13, 78, and 105 weeks (7, 7,
    9, and 7%, respectively), and in females at this dose depression was
    seen at two days and 4 and 78 weeks (6, 8, and 11%, respectively). The
    Committee considered that none of these depressions was biologically
    significant. No depression of brain acetylcholinesterase activity was
    seen. 

         Males at the highest dose had decreased absolute weights of the
    thyroid, heart, lung, liver, spleen, and adrenals, but these were
    reflected only in reductions in the relative weights of the spleen and
    therefore probably reflect reduced body weight. In the females, only
    the absolute weight of the spleen was reduced, while the absolute
    weight of the thyroid was increased. As this finding was due to a
    single animal, it is unlikely to be attributable to the test material.
    No gross or histopathological abnormality related to treatment was
    seen at any dose. Methiocarb was not tumorigenic. The NOAEL was 67
    ppm, equal to 3.3 mg/kg bw per day, on the basis of haematological
    changes at 3, 6, and 12 months (Krötlinger et al., 1981; Krötlinger,
    1990).

     Dogs

         Groups of four beagle dogs and four bitches were fed methiocarb
    (purity, 98.4%) in the diet at 0, 5, 60, or 240 ppm; the group given 5
    ppm group had been given 15 ppm for the first 15 days. These dietary
    concentrations were equivalent to daily doses of 0, 0.12, 1.5, and 6
    mg/kg bw per day, ignoring the first 15 days of treatment of animals
    at the lowest dose. The animals were examined daily, and food
    consumption was recorded daily and body weight measured weekly.
    Clinical examinations including ophthalmoscopy were undertaken, and
    haematological and biochemical variables were measured in blood at
    weeks 0, 14, 27, 40, 53, 66, 79, 92, and 104; urine was also analysed.
    Erythrocyte and plasma cholinesterase activity was measured before the
    start of treatment and at weeks 2, 3, 4, 7, 10, 13, 27, 40, 53, 66,
    79, 92, and 104, before feeding and 2 h afterwards.
    Acetylcholinesterase activity on the olfactory bulb of the brain was
    measured at sacrifice. Animals were examined grossly  post mortem, 
    and selected tissues were examined histologically. 

         One death occurred, of an animal at 5 ppm, which was considered
    not to be related to treatment. The only clinical findings were mild
    weakness of the hind limbs, trembling, reduced alertness, and some
    vomiting at the highest dose during the first 14 weeks of the study.
    The results of tests for reflexes and ophthalmic parameters were
    normal. Food intake was reduced in animals of each sex at the highest
    dose and in bitches at the intermediate dose, but the body weights
    were not significantly affected. Haematological and biochemical
    parameters were unaffected, apart from cholinesterase activity. Plasma
    cholinesterase activity was depressed at doses of 15 ppm and higher,
    and it was for this reason that this dose was reduced to 5 ppm.
    Depression of plasma cholinesterase activity was not seen at 5 ppm but
    occurred at the two higher doses. Erythrocyte and brain
    acetylcholinesterase activity was not consistently inhibited at any
    dose; the maximum inhibition of erythrocyte acetylcholinesterase
    activity was in animals at the high dose (17% for each sex) and at the
    intermediate dose (10% in dogs and 5% in bitches). Organ weights were
    unaffected, and no organ-specific toxicity observed. The NOAEL was 60
    ppm, equivalent to 1.5 mg/kg bw per day, on the basis of clinical
    signs. The reduced food intake of bitches at the intermediate dose was
    not considered relevant (Hoffman & Schilde, 1980).

     (d)  Genotoxicity

         The results of assays for the genotoxicity of methiocarb are
    shown in Table 2.

     (e)  Reproductive toxicity

    (i)   Multigeneration reproductive toxicity

     Rats

         Groups of 10 male and 10 female FB 30 rats (Elberfield breed)
    received technical-grade methiocarb (purity, 99%) admixed with the
    diet at concentrations of 0, 30, 100, or 300 ppm, equivalent to 3, 10,
    and 30 mg/kg bw per day. The rats were weighed weekly. The F0
    generation was mated twice, first after 70 days of treatment and again
    after 149 days, to produce the F1a litters, which were sacrificed,
    and the F1b litters. Ten males and 10 females from each group of F1b
    rats were used to produce the next generation and were again mated
    twice. The resultant F2a rats were sacrificed, while 10 male and 10
    female F2b rats were mated twice to produce the F3a and F3b
    generations, which were sacrificed. The rats were weighed weekly, and
    the body weights of the offspring were measured at birth, five days
    after birth, one week after birth, and then weekly. The pups were
    examined grossly for malformations immediately after birth and during
    lactation. The F3a young were killed four weeks after birth and the
    F3b rats at three weeks. Blood samples were taken for analysis from
    the F0 generation at the end of the preliminary treatment period and
    after rearing of the second little (Löser, 1969). 

         In the F0 generation, there was no significant difference in
    weight gain between the groups, and no treatment-related changes were
    seen in haematological parameters at either sampling time. Although
    ALAT and aspartate aminotransferase activities were higher in animals
    at the highest dose at the earlier sampling time, they were stated to
    be within the normal range for the laboratory; similar increases were
    not seen at the later sampling time. The gestation rate was lower at
    the highest dietary concentration, but was still within the normal
    range. The weight gain of F1a pups was similar in all groups, and no
    malformations were seen at sacrifice. After the second mating, there
    were no dose-related changes in litter size or pup weight and weight
    gain. No significant differences were seen between the groups of F1b
    pups. Their weight gain after weaning was similar in all groups. After
    the first mating, the gestation rate was lower at the highest dietary
    concentration but was still within the normal range. The weight gain
    of F2a pups was similar in all groups, and no malformations were seen
    at sacrifice. After the second mating, the mean weight of the group at
    300 ppm was significantly lower than that of controls at the end of
    the four-week lactation period; however, there were no significant
    intergroup differences in body weight at sacrifice of the F2b
    animals. After both matings of these rats, the gestation rate was
    similar and the litter size and pup weights were comparable in all


        Table 2. Results of assays for the genotoxicity of methiocarb

                                                                                                                       

    End-point               Test object              Concentration             Purity     Result       Reference
                                                                               (%)
                                                                                                                       

    In vitro 

    Reverse mutationa       S. typhimurium           4-2500 µg/plate           98.5       Negative     Herbold (1978)
                            TA1535, TA1537,
                            TA98, TA100

    Reverse mutationa       S. typhimurium           20-12 500 µg/plate        98.4       Negative     Herbold (1986)
                            TA1535, TA1537,
                            TA98, TA100

    DNA damagea             E. coli pol A+ and       625-10000 µg/plate        98.6       Negative     Herbold (1983)
                            pol A-                                             

    Gene mutation           Chinese hamster          1.25-60 µg/ml             99.3       Negative     Lehn (1989)
                            ovary cells, hprt

    Unscheduled DNA         Rat primary              0.1-100 µg/ml             98.8       Negative     Curren (1988)
    synthesis               hepatocytes

    Chromosomal             Chinese hamster          4.92-497 µg/ml            99.4       Positive     Murli (1990)
    aberrationa             ovary cells

    Sister chromatid        Chinese hamster          2-40 µg/ml                98.2       Negative     Putman (1986)
    exchangea               ovary cells

    Table 2. (continued)

                                                                                                                       

    End-point               Test object              Concentration             Purity     Result       Reference
                                                                               (%)
                                                                                                                       

    In vivo

    Micronucleus            Mice                     5,10, and 20              98.5       Negative     Herbold (1979a)
    formation                                        mg/kg bw
                                                     twice orally

    Dominant lethal         Mice                     6 mg/kg bw                98.5       Negative     Herbold (1979b)
    mutation
                                                                                                                       

    a With and without metabolic activation
    

    groups. The body weights of the F3b, but not the F3a, groups at 100
    and 300 ppm during lactation were lower than those of controls. This
    finding may be due to larger litter size. No abnormalities were found
    at birth or sacrifice in F3a or F3b pups, and no abnormalities were
    found at autopsy of the F0, F1b, and F2b generations. The NOAEL was
    300 ppm, equivalent to 30 mg/kg bw per day, the highest dose tested
    (Löser (1970). 

    (ii)   Developmental toxicity

     Rats

         Groups of 19-20 fertilized FB 30 rats received 10 oral doses of
    0, 1, 3, or 10 mg/kg bw per day methiocarb (purity, 98.9%) by gavage
    on days 6-15 of gestation. The highest dose reduced weight gain, but
    no other effects were observed. No effects were seen on the number of
    implantations or resorptions or the weights of fetuses or placentas.
    No teratogenic effects were seen, nor was fetotoxicity observed. The
    NOAEL for maternal toxicity was 3 mg/kg bw per day, and that for fetal
    toxicity was 10 mg/kg bw per day, the highest dose tested (Lorke,
    1971; see also Renhof, 1988).

     Rabbits

         Methiocarb (purity, 97.3%) was administered by gavage to groups
    of 17 pregnant New Zealand white rabbits at doses of 1, 3, or 10 mg/kg
    bw per day on days 6-18 of gestation. A group of 19 rabbits were used
    as controls. On day 29 of gestation, the rabbits were sacrificed and
    examined; the uterus was weighed, and the numbers of corpora lutea,
    implantation sites, and resorption sites, and the number and
    distribution of live and dead fetuses were noted. The weight and sex
    of the fetuses and placental weights were recorded and the fetuses
    examined for abnormalities. Rabbits receiving the highest dose showed
    clinical signs of cholinergic poisoning and initial marked weight
    loss. Weight gain was also reduced at 3 mg/kg bw per day towards the
    end of the study, but this was attributable to a single animal. Weight
    gain was unaffected at the lowest dose. The litter parameters were
    similar in all groups, and there was no indication of teratological
    effects. The NOAEL for maternal toxicity (clinical effects and weight
    loss at the highest dose) was 3 mg/kg bw per day . The NOAEL for
    fetotoxicity was 10 mg/kg bw per day, the highest dose tested (Tesh et
    al., 1981).

         Methiocarb (purity, 99.4-99.6%) was applied under occlusion for
    6 h/day to about 10% of the body area of groups of 16 mated chinchilla
    rabbits at doses of 0, 10, 50, or 250 mg/kg bw per day on days 6-18
    post coitum. The animals were sacrificed after 28 days and the fetuses
    removed. One animal at the high dose was sacrificed because of a
    broken femur. No clinical signs were observed at any dose, nor was
    irritation of the skin noted. At the highest dose, reduced food
    consumption was observed between 11 and 15 days  post coitum and
    body-weight loss between 6 and 8 days  post coitum, whereas at the
    lower doses,no such effect was seen. The numbers of corpora lutea,

    implantations, pre- and post-implantation losses, and live fetuses
    were not affected by treatment. There was a slight decrease in mean
    fetal weight at the highest dose (4%), and slight retardation of
    skeletal development was noted at this dose, as shown by incomplete or
    no ossification of the phalangeal nuclei. No effects on the fetuses
    were seen at lower doses. The NOAEL for both maternal and fetal
    toxicity was 50 mg/kg bw per day (Dotti & Biedermann, 1992). 

     (f)  Special studies

    (i)   Dermal and ocular irritation and dermal sensitization

    (ii)   Neurotoxicity

         In a poorly described study, no persistent paralytic effects were
    produced in hens  (Gallus gallus) of unstated strain given a single
    dose of methiocarb by gavage and observed for 14 days (DuBois, 1962).

         Methiocarb (purity, 98.5%) was administered twice at a dose of
    380 mg/kg bw (approximately the LD50) to 20 white Leghorn laying
    hens, aged 15-20 months, after an intramuscular injection of 50 mg/kg
    bw atropine sulfate. After an interval of 21 days, the 18 surviving
    hens were treated again, with the same doses of atropine and
    methiocarb. The 16 survivors of the second treatment were sacrificed
    three weeks later, and brain, spinal cord, and portions of peripheral
    nerve were excised and processed for histological examination. Five
    positive controls were given tri- ortho-cresyl phosphate as a single
    oral dose and sacrificed three weeks later. The hens treated with
    methiocarb exhibited brief cholinergic signs and were lethargic, but
    none showed ataxia or paralysis. At  examination,  post mortem there
    were no histopathological changes indicative of polyneuropathy in
    either the central or peripheral nervous system, whereas the hens
    treated with tri- ortho-cresyl phosphate showed the classical signs
    of delayed polyneuropathy and mild but characteristic signs of delayed
    polyneuropathy on histopathological examination (Thyssen & Schilde,
    1978). 

    (iii)   Antidotes

         A study was carried out in which the effect of antidotes to an
    oral LD50 of methiocarb was investigated in rats. The regimens used
    were atropine at 50 mg/kg bw, pralidoxime (salt unstated) at 50 mg/kg
    bw, obidoxime at 20 mg/kg bw, atropine and pralidoxime at 50 mg/kg bw
    each, or atropine at 50 mg/kg bw with obidoxime at 20 mg/kg bw. All
    the antidotes were given intraperitoneally just before clinical signs
    became evident, i.e. 1-2 min after administration of methiocarb. The
    LD50 values were 67 mg/kg bw without antidotes, 470 mg/kg bw with
    atropine, 190 mg/kg bw with pralidoxime, 220 mg/kg bw with obidoxime,
    500 mg/kg bw with pralidoxime and atropine, and 510 mg/kg bw with
    obidoxime and atropine. Thus, pralidoxime and obidoxime added little
    to the antidotal effect of atropine (Kimmerle, 1966a). 

         In mice, 10 mg/kg bw of atropine increased the LD50 of
    methiocarb by sevenfold, while pralidoxime methiodide at 100 mg/kg bw
    increased the LD50 1.3 times. Administration of the two antidotes
    together increased the LD50 by 21 times. Obidoxime at 50 mg/kg bw
    increased the LD50 3.1 times, while obidoxime plus atropine increased
    it by 9.1 times (Kohgo, 1970).

         Atropine sulfate at 50 mg/kg bw administered within 10 min of
    methiocarb intraperitoneally increased the LD50 of methiocarb given
    orally to rats by more than sixfold, and tetraethyl ammonium chloride
    given by the same route increased it by approximately fourfold;
    however, the combined effect of the two antidotes was less than that
    of atropine alone (Kimmerle, 1971).

         In the study of Thyssen and Schilde (1978) in hens at
    approximately the LD50 (see above), atropine appeared to have a
    powerful antidotal effect.

    (iv)   Interaction with other pesticides

         When an intraperitoneal dose equal to 50% of the LD50 of
    methiocarb was given to female Sprague-Dawley rats in combination with
    another pesticide at a dose equal to 50% of the LD50, the combined
    mortality was less than 50% in all cases. The highest combined
    mortality rates (> 40 to < 50%) were seen with EPN, Guthion,
    mevinphos, and carbaryl. Combined mortality rates of > 20 to < 40%
    were found with parathion, parathion-methyl, disulfoton, malathion,
    carbophenothion, tributyl phosphorotrithioite, and ethion. The
    mortality rates seen with the remaining insecticides (demeton,
    dioxathion, and schradan) were 5-20% (DuBois & Raymund, 1961b). In
    another study of similar design, the combined mortality rates were 55%
    with trichlorfon, 50% with propoxur, 35% with coumaphos, 30% with
    oxydemeton-methyl, and 20% with fenthion (DuBois & Raymund, 1961c).

    3.  Observations in humans

         The 250 workers in two plants manufacturing methiocarb over about
    20 years were subjected to annual medical examinations and assays of
    cholinesterase activity in whole blood. No adverse health effects or
    changes in laboratory parameters were observed (Faul, 1993).

    Comments

         Methiocarb appeared to have been well absorbed in a small study
    of absorption, distribution, metabolism, and excretion of the
    radiolabelled compound in rats. More than 70% of the administered
    radiolabel was excreted within 48 h, mostly in the urine. Methiocarb
    was extensively metabolized. The main route of metabolism in both
    animals and plants appears to be to methiocarb phenol, methiocarb
    phenol sulfoxide, and methiocarb phenol sulfone. In some studies,
    methiocarb sulfoxide was also found.

         In single-dose and short-term studies in rats, methiocarb
    administered by gavage inhibited plasma and erythrocyte cholinesterase
    activity. In the short-term study, plasma, erythrocyte, and brain
    cholinesterase activities were depressed at 10 mg/kg bw per day; the
    NOAEL for this effect was 3 mg/kg bw per day. In a short-term study of
    the ability of methiocarb or its sulfoxide to inhibit cholinesterase
    activity in rats, the NOAEL for inhibition of erythrocyte
    acetylcholinesterase activity was 0.5 mg/kg bw per day for methiocarb,
    but a NOAEL was not identified for the sulfoxide. In a 29-day study in
    which dogs were given methiocarb or methiocarb sulfoxide in gelatine
    capsules at 0.05 or 0.5 mg/kg bw per day, both plasma and erythrocyte
    cholinesterase activities were inhibited by both treatments.

         The acute toxicity of methiocarb given by a number of routes has
    been measured in a number of species. The oral LD50 in fasting rats
    ranged from 13 to 130 mg/kg bw. The oral LD50 values for methiocarb
    phenol, methiocarb phenol sulfoxide, and methiocarb phenol sulfone in
    rats are all greater than 1 g/kg bw, as is that of methiocarb sulfone,
    but that of methiocarb sulfoxide is between 6 and 43 mg/kg bw.

         WHO has classified methiocarb as moderately hazardous (WHO,
    1996).

         The short-term toxicity of methiocarb has been tested in rats,
    rabbits, cats, dogs, and chickens. Few of these studies were
    appropriate for identifying NOAEL values and, of those that were, a
    study in rats was carried out by inhalation and that in rabbits by
    dermal application. In the study in rats, which were exposed by
    inhalation, for five days per week for three weeks, no findings
    related to treatment were seen on histopathological examination but
    depressed brain acetyl-cholinesterase activity was observed at the
    highest dose in animals of each sex and in males at the intermediate
    dose. Consequently, the NOAEL was 6 mg/m3 per day. In a 21-day study
    of the dermal toxicity of methiocarb in rabbits, the substance was
    applied for 6 h/day at 0, 60, 150, or 375 mg/kg bw per day. Plasma
    cholinesterase activity was reduced in males at the highest dose, but
    no significant differences were seen between groups in the activities
    of erythrocyte and brain acetylcholinesterase. The NOAEL was 150 mg/kg
    bw per day on the basis of reduced food consumption at the highest
    dose.

         In a long-term study of toxicity in mice, methiocarb was
    administered at dietary concentrations of 0, 67, 200, or 600 ppm. A
    NOAEL was not identified because haematological changes were observed
    in all treated males at 12 months and in all treated females at 24
    months. The LOAEL was 67 ppm, equal to 15 mg/kg bw per day, on the
    basis of minor haematological changes. Methiocarb was not carcinogenic
    in mice.

         In a two-year study of toxicity, rats received methiocarb at
    dietary concentrations of 0, 67, 200, or 600 ppm. The NOAEL was 67
    ppm, equal to 3.3 mg/kg bw per day, on the basis of haematological
    changes at 3, 6, and 12 months. Methiocarb was not carcinogenic.

         In a two-year study of toxicity, dogs were fed methiocarb in the
    diet at 0, 5, 60, or 240 ppm. The NOAEL was 60 ppm, equivalent to 1.5
    mg/kg bw per day, on the basis of reversible clinical signs at the
    next highest dose, which were not observed after 15 weeks.

         Three studies of developmental toxicity were available, one in
    rats and two in rabbits. Fertilized rats received methiocarb by gavage
    on days 6-15 of gestation at 0, 1, 3, or 10 mg/kg bw per day. The
    NOAEL for maternal toxicity was 3 mg/kg bw per day on the basis of
    reduced body-weight gain at the highest dose. As no fetotoxicity was
    observed, the NOAEL for this end-point was 10 mg/kg bw per day, the
    highest dose tested. In a study of developmental toxicity in rabbits,
    pregnant animals received methiocarb at 0, 1, 3, or 10 mg/kg bw per
    day by gavage on days 6-18 of gestation. The NOAEL was 3 mg/kg bw per
    day for maternal toxicity on the basis of clinical effects and weight
    loss at the highest dose. As no fetotoxicity or teratogenicity was
    observed, the NOAEL for these end-points was 10 mg/kg bw per day, the
    highest dose tested. In a further study, rabbits received methiocarb
    by dermal application at 0, 10, 50, or 250 mg/kg bw per day for
    6 h/day on days 6-18 of gestation. The NOAEL for both maternal and
    fetal toxicity was 50 mg/kg bw per day on the basis of reduced
    maternal food consumption and some decrease in mean fetal weight at
    the highest dose; slight retardation of fetal development was also
    observed. A multigeneration study of reproductive toxicity was
    undertaken in rats given methiocarb at dietary concentrations of 0,
    30, 100, or 300 ppm. The NOAEL was 300 ppm, equivalent to 30 mg/kg bw
    per day, as no effect clearly related to treatment was observed. No
    teratogenic effect was observed in any of these studies.

         Methiocarb has been tested for genotoxicity in an adequate
    battery of tests  in vitro and  in vivo. The Meeting concluded that
    methiocarb is not genotoxic.

         Methiocarb did not cause skin sensitization in studies conducted
    by either the Magnusson and Kligman or the Beuhler technique.

         In an early study of neurotoxicity in hens, methiocarb did not
    cause delayed polyneuropathy of the organophosphorus type. Atropine
    has consistently been shown to be an effective antidote for
    methiocarb, while the effects of pyridinium oximes were somewhat
    inconsistent.

         The Meeting established an ADI of 0-0.02 mg/kg bw on the basis of
    the NOAEL of 1.5 mg/kg bw per day in the two-year study of toxicity in
    dogs and a safety factor of 100. This ADI results in a further safety
    factor of 10 on the LOAEL in the long-term study of toxicity in mice.

         An acute RfD was allocated on the basis of the NOAEL of 1.5 mg/kg
    bw per day in the two-year study in dogs, because the signs observed
    were acute, and a safety factor of 100. Of the shorter studies in
    dogs, neither the 29-day nor the 12-week study was considered by the
    Meeting to be adequate for the purpose of establishing a NOAEL.

    Toxicological evaluation

     Levels that cause no toxic effect

         Mouse:         No NOAEL; LOAEL: 67 ppm, equal to 15 mg/kg bw per
                        day (long-term study of toxicity)

         Rat:           67 ppm, equal to 3.3 mg/kg bw per day (long-term
                        study of toxicity)
                        300 ppm, equivalent to 30 mg/kg bw per day
                        (maternal and fetal toxicity in a study of
                        reproductive toxicity)
                        3 mg/kg bw per day (maternal toxicity in a study
                        of developmental toxicity)
                        10 mg/kg bw per day (developmental toxicity)

         Rabbit:        3 mg/kg bw per day (maternal toxicity in a study
                        of developmental toxicity)
                        10 mg/kg bw per day (developmental toxicity)

         Dog:           60 ppm, equivalent to 1.5 mg/kg bw per day (two-
                        year study of toxicity)

     Estimate of acceptable daily intake for humans

              0-0.02 mg/kg bw 

     Estimate of acute reference dose 

              0.02 mg/kg bw

     Studies that would be useful for continued evaluation of the 
     compound

         1.   A modern study of absorption, distribution, metabolism, and
              excretion

         2.   A modern multigeneration study of reproductive toxicity

         3.   Further observations in humans

        List of end points relevant for setting guidance values for dietary and non-dietary exposure
                                                                                                 

    Absorption, distribution, excretion, and metabolism in mammals

    Rate and extent of oral absorption        No data
    Dermal absorption                         No data
    Distribution                              No data
    Potential for accumulation                Not likely to accumulate
    Rate and extent of excretion              73 to > 90% within 48 h
    Metabolism in animals                     Sulfoxidation and loss of carbamate side-chain 
    Toxicologically significant compounds     Parent compound and methiocarb sulfoxide
    (animals, plants and environment)

    Acute toxicity

    Rat: LD50, oral                           13-135 mg/kg bw
    Rat: LD50, dermal                         350 to > 5000 mg/kg bw
    Rat: LC50 inhalation                      > 300 mg/m3
    Skin irritation                           Not irritating
    Eye irritation                            Not irritating
    Skin sensitization                        Not sensitizing

    Short-term toxicity

    Target/critical effect                    Clinical signs
    Lowest relevant oral NOAEL                Dog: 1.5 mg/kg bw per day
    Lowest relevant dermal NOAEL              Rabbit: 150 mg/kg bw per day
    Lowest relevant inhalation NOAEL          Rat: 6 mg/m3

    Genotoxicity                              Not genotoxic

    Long-term toxicity and carcinogenicity

    Target/critical effect                    Clinical signs
    Lowest relevant NOAEL                     Dog: 1.5 mg/kg bw per day
    Carcinogenicity                           Not carcinogenic 

    Reproductive toxicity

    Reproductive target/critical effect       No effect
    Lowest relevant reproductive NOAEL        Rat: 30 mg/kg bw per day
    Developmental target/critical effect      Maternal toxicity: reduced weight gain; no 
                                              fetotoxicity observed
    Lowest relevant developmental NOAEL       Rabbit: 3 mg/kg bw per day

    Neurotoxicity/Delayed neurotoxicity       Does not cause delayed polyneuropathy

    Other toxicological studies               No data

    Medical data                              No data

    Summary                  Value                 Study               Safety factor
    ADI                      0-0.02 mg/kg bw       Dog, 2 years        100
    Acute reference dose     0.02 mg/kg bw         Dog, 2 years        100
                                                                                                 
    
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    See Also:
       Toxicological Abbreviations
       Methiocarb (ICSC)
       Methiocarb (Pesticide residues in food: 1981 evaluations)
       Methiocarb (Pesticide residues in food: 1983 evaluations)