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    PIRIMIPHOS-METHYL

    First draft prepared by D.J. Clegg
    Carp, Ontario, Canada

    EXPLANATION

         Pirimiphos-methyl was previously evaluated by JMPR in 1974 and
    1976 (Annex 1, references 22 and 26). An ADI of 0-0.01 mg/kg bw was
    established in 1976. Relevant data from the previous monograph and
    monograph addendum are incorporated into the present monograph.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    Biochemical Aspects

    Absorption, distribution, and excretion

    Rats

         Oral administration of 0.6 mg 2-14C-ring labelled pirimiphos-
    methyl/kg bw to 5 male rats resulted in a mean urinary excretion of
    80.7% and mean faecal excretion of 7.3% in 24 h, indicating rapid
    absorption. At 96 h, 86.0% and 15.2% of the administered dose had
    been excreted in urine and faeces, respectively. Nine metabolites
    (unidentified) were present in the urine (Bratt & Dudley, 1970).

         Female rats given 7.5 mg 2-14C pirimiphos-methyl/kg bw orally
    were bled (cardiac puncture, 3 rats per time interval) at 0.5, 1, 3,
    5, 7 or 24 h post-dosing. Maximum blood levels (at 0.5 h) were 2-3
    g/ml, declining by 50% 1 h post-dosing. By 24 h, levels of 14C in
    blood were 0.2 to 0.3 g/ml, and of pirimiphos-methyl, 0.01-0.02
    g/ml. Rats treated for 4 days at 7.5 mg 2-14C pirimiphos-
    methyl/kg bw/day and sacrificed at 24 h intervals did not show any
    increase in blood levels with time. Tissue levels of total
    radioactivity in liver, kidney and fat over the 4 day period were
    generally below 2 mg pirimiphos-methyl equivalents/kg tissue (levels
    of unchanged pirimiphos-methyl being less than 0.15 mg/kg tissue).
    There was no evidence of tissue accumulation (Mills, 1976).

         Adult male Wistar rats were intubated with 1 mg 14C
    pirimiphos-methyl/kg bw/day. Four groups of 3 animals were dosed for
    3, 7, 14 or 21 days and sacrificed 24 h after the final dose. A
    further five groups of 3 rats were similarly dosed for 28 days and
    sacrificed 1, 3, 7, 14, or 28 days post-dosing. Each of the 9 groups
    had one rat, undosed, as a control. Following sacrifice, samples of
    liver, kidney, muscle, fat, erythrocytes and plasma were taken for
    analyses. Urine and faeces were collected from 2 rats over a 24 h
    period following the seventh dose. Recovery of 14C from 14C
    pirimiphos-methyl added to control tissues was 96.95.2%. In all
    tissue samples at all time intervals, concentration of radioactivity
    was very low, close to or below detection limits. Levels did not
    increase with repeated dosing. Liver concentrations were fairly
    constant (0.03 ppm) and in some kidney samples, similar levels were
    detected. In other tissues, radioactivity concentration was
    generally below the limits of detection (0.04-0.06 ppm). Three days
    after cessation of dosing one animal had detectable levels of kidney
    radioactivity concentrations. At 7 days and subsequent days, no
    residues were found. Excretion was between 70 and 80% of a single
    dose, following administration of 7 consecutive doses, providing
    evidence of rapid metabolism and elimination rather than poor
    absorption (Hawkins & Moore, 1979).

    Dogs

         Male beagle dogs (1/dose level) given 18.4 or 16.7 mg 2-14C-
    ring labelled pirimiphos-methyl/kg bw by capsule excreted 64.4% or
    82.5% of the administered dose in the urine, and 17.3% or 13.3% in
    the faeces, respectively, in 48 h. Nine metabolites (unidentified)
    were present in urine (Bratt & Dudley, 1970).

    Goats

         In the lactating goat, following a single dose of 0.12 mg 2-
    14C-ring labelled pirimiphos-methyl/kg bw by capsule, 87% and 4%
    were excreted in urine and faeces, respectively, over 8 days. 80.6%
    and 2.7% were excreted in urine and faeces, respectively, during the
    final 48 h. Milk residues during the first 24 h post-dosing
    indicated a total residue of 0.035 ppm, of which only 0.003 ppm was
    parent compound. After 24 h residues declined to 0.004 ppm or less
    (Bowker  et al., 1973).

         A lactating female goat was orally dosed by gelatin capsule,
    twice daily, with 40 mg 14C-pirimiphos-methyl (7.1 x 106g)
    labelled in the 2 position of the pyrimidine ring, for 7 consecutive
    days (equivalent to 45 ppm in the diet). Chemical and radiochemical
    purity exceeded 99%. At sacrifice, 78.1 and 11.3% of total
    administered radioactivity had been excreted in urine and faeces,
    respectively. Residues in milk were approximately 0.2% of the total
    administered dose, the highest residue being 0.208 ppm pirimiphos-
    methyl equivalents at day 2 of dosing. Levels plateaued by day 4 at
    about 0.15 ppm. Sixteen hours after sacrifice, radioactive residues
    in liver, kidney, muscle and fat were 0.31, 0.5, 044, and 0.067 ppm,
    respectively. The major components in fat were unchanged pirimiphos-
    methyl (55.2% of total radioactive residue) and 0-(2-ethylamino-6-
    methylpyrimidine-4-ol)0,0-dimethyl phosphorothioate (17.1%), and
    those in the remaining tissues and in milk were 2-diethylamino-6-
    methylpyrimidine-4-ol,2-ethylamino-6-methylpyrimidine-4-ol, and 2-
    amino-6-methylpyrimidine-4-ol (Skidmore  et al., 1985).

    Cows

         In the cow following a single oral dose of 0.5 mg 2-14C-
    labelled pirimiphos-methyl/kg bw, excretion was similar to that in
    goat, 85% and 14% of the administered dose being excreted in urine
    and faeces, respectively, over 7 days. During the first 3 days post-
    dosing, 0.35% of the labelled was excreted in the milk (residue
    level, 0.04 ppm of which less than 2% was unchanged pirimiphos-
    methyl and phosphorus-containing metabolites). Hydroxypyrimidine
    hydrolyses products or their conjugates constituted the major
    portion of the milk residues (Bullock  et al., 1974).

    Hens

         Hens given a single oral dose of 2, 9 or 20 mg-14C-labelled
    pirimiphos-methyl excreted over 70% of the administered dose within
    24 h. Three major metabolites, namely parent pyrimidine (4.7% of
    dose), 2-ethyl-amino-4-hydroxy-6-methyl pyrimidine (25% of dose) and
    2-amino-4-hydroxy-6-methyl pyrimidine (31%) were identified. When
    fed for 289 days with dietary concentrations of 4 ppm, levels of
    parent compound in white and yolk of eggs never exceed 0.001 ppm
    although total 14C levels increased to 0.032 to 0.038 ppm over 16
    days, and then fell (to equilibrium) to 0.026 to 0.028 ppm, 86-90%
    of which comprised water soluble metabolites. Hens fed dietary
    concentrations of 32 ppm for 7 days yielded eggs containing 0.007
    (whites on day 3) and 0.012 (yolks on day 6) ppm pirimiphos-methyl.
    "Total" pirimiphos-methyl plus equivalents ranged from 0.08 to 0.15
    ppm in whites plus yolk. In muscle, residues of 0.31 and 0.16 ppm
    were present following exposure to 32 and 4 ppm dietary
    concentrations, respectively (Green  et al., 1973).

         Two hens were dosed orally by gelatin capsule with a mean dose
    of 1.52 mg 14C-pirimiphos-methyl (60.75  Ci)/hen for 14
    consecutive days. Radiochemical purity was > 95%. Residues of 14C
    in breast muscle were 0.59% and 0.39 ppm. No unchanged pirimiphos-
    methyl was detected. The majority of the residue (74%) was
    identified as free and conjugated 2-amino-6-methyl-pyrimidin-4-ol.
    Radioactive residues in yolk appeared to plateau about days 7-10,
    but residues in albumen were inconsistent. At least 7 different
    compounds were identified as being present in the eggs, the major
    ones being 2-ethylamino-6-methyl-pyrimidine-4 ol and 2-amino-6-
    methyl-pyrimidine-ol (Hall  et al., 1979).

         14C-Pirimiphos-methyl, labelled at the 2 position of the
    pyrimidine ring was administered to 3 hens by gelatin capsule, twice
    daily for 14 days. The mean dose/day was 2.54 mg pirimiphos-methyl
    (98.4% purity), equivalent to 50 ppm in the diet. An additional bird
    was fed capsules treated similarly to those fed to experimental
    birds, but containing no pirimiphos-methyl. Eggs were collected
    daily and excreta at 24 h intervals. Sixteen hours after the final
    dose, hens were killed, and tissues were taken for analysis. A mean
    value of 97.5% of the administered dose was excreted over the 14 day
    period. At sacrifice, liver, peritoneal fat, subcutaneous fat, leg
    muscle and breast muscle contained 0.2, 0.093, 0.11, 0.67 and 1.3
    ppm pirimiphos-methyl equivalents, respectively. In fat,
    approximately 72% of the radioactive residue was unchanged
    pirimiphos-methyl, which was only found elsewhere (9.5% of the total
    radioactive residue) in egg yolk. In muscle tissue the major residue
    was 2-amino-6-methylpyrimidin-4-ol and 2-ethylamino-6-
    methylpyrimidine-4-ol. These compounds were also found in liver in
    both free and conjugated forms (Skidmore & Tegala, 1985).

    Biotransformation

         The metabolism of pirimiphos-methyl in Wistar rats (5 males)
    given 100 mg 14C-labelled pirimiphos methyl/kg bw and in one
    beagle dog given 20 mg 14C-labelled pirimiphos-methyl/kg bw was
    investigated by TLC separation of urinary metabolites. Twelve
    metabolites were detected in rat, and 11 in dog urine. Five of the
    metabolites were identified. In both species, 2-ethylamino-4-
    hydroxy-6-methyl pyrimidine was the major urinary metabolite (30% of
    dose). The next most predominant metabolite in dog was 4-0(2-
    diethylamino-6-methylpyrimidinyl--D-glucosiduronic acid (11% of
    dose) and in the rat, an unidentified phosphorus-containing product
    thought to be a dealkylated derivative of either pirimiphos-methyl
    or its oxygen analogue (12% of dose). Other identified metabolites
    were 2-amino-4-hydroxy-6-methyl pyrimidine (8% and 5% of dose in rat
    and dog, respectively) (Green  et al., 1973; Bratt & Jones, 1973).

         These studies indicate that the P-O-C bond of pirimiphos-methyl
    is extensively cleaved and that N-de-ethylation and/or conjugation
    are further steps in the metabolism of the pyrimidine leaving group.
    Although the oxygen analogue of pirimiphos-methyl was not detected
    as a urinary metabolite, the fact that cholinesterase inhibition
    occurs  in vivo suggests that the oxygen analogue is also formed
    and may be an intermediate step leading to the identified urinary
    products (Annex 1, reference 23).

    Effects on enzymes and other biochemical parameters

         The only biochemical effects consistently noted in acute or
    chronic toxicity tests was inhibition of cholinesterase. A group of
    36 male rats were given single oral doses of 1450 mg pirimiphos-
    methyl/kg bw. Symptoms were noted and they were sacrificed at
    intervals up to 4 days after dosing for measurements of brain,
    plasma and red cell cholinesterase activity. Few signs were noted at
    6 h after dosing when cholinesterase inhibition was 0, 35 and 51%,
    respectively, for brain, red cell and plasma. Clear signs of
    poisoning only became apparent by 24 h when brain was inhibited by
    46% and red cell and plasma by 70 and 80%, respectively. Recovery of
    cholinesterase activity began to be apparent by 72 h. Plasma
    cholinesterase activity had completely recovered by 96 h but red
    cell and brain remained 47 and 30% inhibited, respectively, at this
    time (Clark, 1970). From these studies it appears that inhibition of
    brain cholinesterase by 40% or more results in obvious signs of
    toxicity (Annex 1, reference 23).

         A group of 25 male (probably Wistar CFT, but not specified)
    were dosed with 1000 mg/kg bw. Twenty-five additional rats served as
    controls. Five rats/group were sacrificed at 4, 8, 24, 48 or 72 h
    post-dosing and plasma and brain cholinesterase and non-specific
    carboxylesterase activities were measured. Plasma cholinesterase
    inhibition was rapid (60% inhibition by 4 h) whereas brain

    cholinesterase inhibition was slower (36% by 8 h). Both attained
    maximum inhibition by 24 h (93% for plasma and 61% for brain).
    Recovery was apparent at 48-72 h in both enzymes, but that for brain
    was slower. Non-specific esterase (NSE) activity was inhibited,
    attaining maximum inhibition (plasma 80%, and brain 47%) at 24 h.
    Inhibition of NSE was less than for cholinesterase, and recovery in
    plasma was more rapid. In brain, NSE and cholinesterase activity
    recovery were comparable (Rajini & Krishnakumari, 1988a).

         Pirimiphos-methyl, 90.5% purity, was fed at dietary
    concentrations of 0, 1000 or 1500 ppm to groups of 30 male Wistar
    rats for 28 days. Five rats/group were necropsied 7, 14, 21 or 28
    days post-initiation of exposure, and 5 rats/treated group were
    sacrificed at 35 days (i.e. after 7 days withdrawal from pirimiphos-
    methyl exposure). The fate of the remaining 5 rats is not reported.
    Brain and erythrocyte cholinesterase inhibition showed significant
    dose-related depression at all time intervals during exposure.
    Erythrocyte cholinesterase was consistent during exposure, but brain
    cholinesterase did not achieve consistent levels until 14-21 days.
    Post-exposure recovery occurred in all groups but in brain,
    cholinesterase activity was still biologically significantly
    depressed (26 and 28% at 1000 and 1500 ppm, respectively). Plasma
    cholinesterase activity was variable, but was depressed 17-44% over
    the various time intervals, the least depression being at the
    highest dose. Recovery was complete 7 days after cessation of
    dosing. Non-specific brain carboxylesterase activity was depressed
    at 1500 ppm at all time intervals, but only after 14 days at 1000
    ppm. Recovery was rapid and complete at both dose levels following
    withdrawal. Plasma non-specific carboxylesterase activity was
    markedly depressed at all time intervals, but was still
    significantly depressed following 7 days withdrawal. Renal non-
    specific carboxylesterase activity was slightly reduced only at 1500
    ppm after 14 days treatment and recovered rapidly upon cessation of
    dosing (Rajini  et al., 1989).

    Toxicological studies

    Acute toxicity studies

    Table 1.  Acute toxicity of pirimiphos-methyl
                                                                      
    Animal      Sex  Route  LD50 (mg/kg)      Reference
                                                                      

    Mouse       M    oral   1180 (1030-1360)  Clark, 1970

    Rat         F    oral   2050 (1840-2260)  Clark, 1970

    Rat         M    oral   1861 (1266-2928)  Rajini & Krishnakumari
                                              (1988a)

    Table 1 (cont'd)
                                                                      
    Animal      Sex  Route  LD50 (mg/kg)      Reference
                                                                      

    Rat         F    oral   1667 (1187-2284)  Rajini & Krishnakumari
                                              (1988a)

    Guinea-pig  F    oral   1000-2000         Clark, 1970

    Rabbit      M    oral   1150-2300         Clark, 1970

    Cat         F    oral   575-1150          Clark, 1970

    Dog         M    oral   > 1500            Gage, 1972

    Hen         F    oral   30-60             Clark, 1970

    Quail       F    oral   approx. 140       Gage, 1971a
                                                                      

         Test material was 90-94% purity, with at least 14 days post-
    dosing observation. Toxic signs were typical of those resulting from
    cholinesterase inhibition.

        Table 2. Acute toxicity of metabolites
                                                                             
    Compound                      Animal   Route  LD50 (mg/kg)  Reference
                                                                             

    2-diethylamino-4-hydroxy-6-   Rat      oral   800-1600      Gage, 1971b
    methylpyrimidine

    2-ethylanino-4-hydroxy-6-     Rat (F)  oral   2093 (1841-   Parkinson,
    methylpyrimidine                              2380)         1974

    2-amino-4-hydroxy-6-methyl-   Rat (F)  oral   > 4000        Parkinson,
    pyrimidine                                                  1974
                                                                             
             Toxic signs reported for 2-ethylamino-4-hydroxy-6-methyl
    pyrimidine comprise urinary incontinence and salivation. No toxic
    signs were observed with 2-amino-4-hydroxy-6-methylpyrimidine.

    Short-term toxicity studies

    Rats

         Repeated and daily dosing of 10 rats/sex/dose, 5 times weekly
    for 2 weeks at 200 mg/kg bw/day produced mild signs of poisoning

    (fibrillation and urinary incontinence) noted only after 7 doses and
    not increasing in intensity. Body-weight gain was depressed;
    haemoglobin levels were depressed about 9% and reticulocyte counts
    were increased - marked aniscytosis and some anisochromia were noted
    in erythrocytes as well as occasional nucleated erythrocytes and
    some Howell-Jolly bodies; histopathological changes comprised
    splenic haematopoiesis and in 1/8 animals, haemosiderosis. A second
    identical study at 400 mg/kg bw/day resulted in signs of poisoning
    in 2 days, increasing in severity and resulting in 9/10 deaths in
    males, and 3/10 deaths in females. Haematology and histopathology
    were similar to the effects noted at 200 mg/kg bw/day (Clarke,
    1970).

         Four groups of 25 Alderley Park SPF rats/sex/dose were fed
    diets containing 0, 8, 80 or 360 ppm 93.1% purity pirimiphos-methyl
    for 90 days. Twenty/sex/dose were sacrificed at termination of
    dosing, and the remainder, 28 days later. Body-weight gain in
    females was reduced 18 and 21%, compared to controls, at 80 and 360
    ppm, but food intake in these groups was slightly increased. Plasma
    cholinesterase was depressed in males (41-72%) and females (56-88%)
    during weeks 2-12 at 80 and 360 ppm. Recovery to normal activity was
    observed one week after withdrawal of pirimiphos-methyl. Erythrocyte
    cholinesterase was depressed in males (39-52%) and females (43-71%)
    at 360 ppm with incomplete recovery occurring by week one (40%
    inhibition in males, and 30% inhibition in females). Brain
    cholinesterase was depressed (mainly in females) at 80 and 360 ppm.
    Recovery did not occur within the 4-week post-dosing period.

         No effects were observed on haematological parameters
    (haemoglobin concentration, PCV, MCHC, reticulocyte counts, total
    and differential white cell counts, platelet counts, mean
    corpuscular diameter and Kaolin-cephalin prothrombin time) or in the
    incidence of gross or histopathological lesions relative to those
    observed in controls. The NOAEL was 8 ppm, equivalent to 0.4 mg/kg
    bw/day (Clapp and Conning, 1970).

         Five groups of 12 young male rats (probably Wistar CFT strain,
    but not specified) were fed dietary concentrations of 0, 10, 250,
    500 or 1000 ppm pirimiphos-methyl (90.5% purity) for 28 days. There
    were no effects on body-weight gain or food intake. Compound intake
    was calculated to be 0, 4, 100, 200 or 400 mg/kg bw/day (data not
    given). A slight increase in liver weight was reported at 1000 ppm.
    No treatment-related pathological changes were observed in liver,
    brain, lung, heart, adrenal, kidney, spleen or testes. Increased
    serum transaminases (1000 ppm) and increased alkaline phosphatase
    (500-1000 ppm) were noted. Hepatic transaminases (-glucoranidase
    and alkaline phosphatase) were unaffected. Cholinesterase activity
    (plasma and brain) were inhibited at 250 ppm and above. The NOAEL
    appears to be 10 ppm (stated to be equal to 4 mg/kg bw) (Rajini &
    Krishnakumari, 1988a).

         Four groups of male Wistar CFT rats were fed dietary
    concentrations of 0, 500, 1000 or 1500 ppm pirimiphos-methyl for 28
    days. Four rats/group were sacrificed at 7, 14, 21 or 28 days.
    Mortality, growth rate and food consumption were stated to be
    unaffected at any dose level. Blood glucose levels in treated rats
    were consistently below control levels. The authors stated that
    "decrease in blood glucose level was evident at all dosages during
    the second week and, at all intervals in 1500 ppm group". This
    statement is questionable since, when converted to percentage of
    control values, blood glucose level decrease was greatest in week 1
    at 1000 (58%) and 1500 (61%) ppm. Dose-effect relationships are
    difficult to determine and control values were high (125-138 mg/100
    ml). Although standard deviations are given (3.06-9.20), in the
    absence of individual data, interpretation is equivocal. Blood urea
    levels were consistently elevated above control levels, but the
    effect was generally greatest at the mid-dose. Dose/effect
    relationships do not appear to be present, based on the data
    available for evaluation. Similarly data on urinary excretion of
    urea was sporadic and usually non-dose related. Protein excretion in
    urine was generally increased but, except in week 4, dose/effect
    relationships are questionable. Creatinine and creatine excretion is
    equally difficult to interpret (Rajini & Krishnakumari, 1988b).

         Four groups of 20 male Wistar CFT rats were administered 0
    (coconut oil), 50, 100 or 200 mg 90.5% purity pirimiphos-methyl/kg
    bw/day, five times weekly for 4 weeks. Four rats/group were
    sacrificed on days 7, 14, 21 or 28. Signs of toxicity were seen only
    in week 4 at 200 mg/kg bw/day. Body-weight and food intake were
    comparable in all groups. Total RBC were depressed at all time
    intervals and all doses except at 100 ppm and 3 weeks, which were
    fractionally above control counts. Dose-effect relationships were
    virtually non-existent despite statistically significant decreases
    in mean values. A wide variation in control values (9.95 x 10-6/l
    - 8.05 x 10-6/l) also render interpretation difficult, especially
    with only 4 rats/group.

         Similar problems existed in total white cell counts (15 275 -
    28 500/l in controls over the 4 week period) and differential
    counts (12 800 - 23 400 for lymphocytes, 1800-4000 for neutrophils).
    The only consistent effects seem to be the increased clotting time
    and prothrombin time and decreased platelet counts seen at all dose
    levels after week 2 (Rajini  et al., 1987).

         An additional short-term study in rats was reviewed. The
    purpose of this study was to alleviate concerns that the rats used
    in the long-term study were rather older (based on their body-weight)
    than those normally used in such a study. Hence possible effects
    (especially on cholinesterase activity and inhibition) may have
    occurred in young rats. To ensure this was not the case, the
    following study was conducted.

         Groups of 12 rats/sex/dose were fed diets containing 0, 5, 8,
    10 or 50 ppm 97% purity pirimiphos-methyl for 28 days. The rats were
    approximately 6 weeks old at receipt and were placed on study over a
    3-week period - females during the first week and males during the
    third week. Plasma and erythrocyte cholinesterase activity were
    measured on groups of 5 rats/sex/dietary concentration on days -14,
    -7, 1, 3, 7, 14, 21 and 28 days, and brain cholinesterase was
    measured on 5 rats/sex/dietary concentration on day 28. There were
    no effects of pirimiphos-methyl on body-weight gain (animals weighed
    weekly) or on clinical conditions and behaviour. Food consumption
    (measured on groups of 3 rats/week) was reduced, males at 5 ppm
    (statistically significant) and 8 ppm (not statistically
    significant). At 5 ppm, this reduced food intake was associated with
    a slight (non-statistically significant) decrease in body-weight
    gain (ca. 5%). Food utilization was comparable in all groups. At
    termination, gross pathology of rat(s)/sex/group did not reveal any
    lesions attributable to pirimiphos-methyl. Plasma cholinesterase
    depression consistently exceeded 20% in the 50 ppm group. Sporadic
    inhibition was noted at 8 and 10 ppm, as was sporadic elevation.
    Erythrocyte cholinesterase inhibition was unaffected by pirimiphos-
    methyl even at 50 ppm. Brain choline-sterase inhibition exceeded 10%
    in both sexes at 50 ppm but was not significantly affected at lower
    dietary concentrations (Berry & Gore, 1975).

    Dogs

         Four groups of beagle dogs/sex were fed gelatin capsules at
    pirimiphos-methyl doses of 0, 2, 10, or 25 mg/kg bw/day, for 13
    weeks. Two dogs/sex/dose were sacrificed at 13 weeks, and the
    remainder at 17 weeks. Clinical signs (dry skin, dull coat during
    weeks 2 and 3, and increased incidence of vomiting during the first
    5-6 weeks) were observed at 25 mg/kg bw/day. An increased frequency
    of liquid stools was noted at 10 and 25 mg/kg bw/day. Body-weight
    gain was reduced in both sexes at 25 mg/kg bw/day and in females at
    10 mg/kg bw/day. At 2 mg/kg bw/day, female body-weight gain
    reduction was of borderline significance. Reduced food intake (dose-
    related) occurred in all female test groups and in males at 25 mg/kg
    bw/day. At 2 and 10 mg/kg bw/day male food intake was inconsistent.
    Heart rate was reduced (both sexes) at 25 mg/kg bw/day. Plasma
    cholinesterase was depressed at all dose levels (more than 20%) but
    recovery was rapid following cessation of dosing. Erythrocyte
    cholinesterase activity was depressed in a dose-related pattern, the
    onset being late (10-12 weeks) at 2 mg/kg bw/day. At 10 and 25 mg/kg
    bw/day, inhibition increased with time. Terminal brain
    cholinesterase activity at 13 and 17 weeks was comparable to
    controls in all groups. Two dogs showed high ALAT and SAP levels
    after 3 months dosing, and a third, a less marked ALAT increase at
    25 mg/kg bw/day. Bile duct proliferation and portal cirrhosis
    occurred in 1/2 males at 25 mg/kg bw/day, and bile duct
    proliferation only in 1/2 males at 10 mg/kg bw/day at 13 weeks.
    After withdrawal, minimal bile duct proliferation occurred in 1/2

    dogs of each sex at 25 mg/kg bw/day. The NOAEL was 2 mg/kg bw/day,
    based on reduced body weight gain in females at 10 mg/kg bw/day
    (Noel  et al., 1970).

         Four groups of 4 beagle dogs/sex were dosed with 0, 0.5, 2 or
    10 mg/kg bw/day via gelatin capsules for 2 years ( corn oil
    solution). A 7-day break in dosing occurred at 140 days (due to
    problems with liquefying the test material). Brain cholinesterase
    activity was 81, 78 and 44% of control values at 0.5, 2 and 10 mg/kg
    bw/day, respectively. Erythrocyte cholinesterase activity was
    depressed with respect to pre-dosing levels at 2 mg/kg bw/day
    (greater than 20% from week 12 onwards) and 10 mg/kg bw/day. Plasma
    cholinesterase activity depression was 21-33% almost consistently
    throughout the study at 10 mg/kg bw/day, 21-33% from week 4 and
    frequently thereafter at 2 mg/kg bw/day, and infrequently (weeks 38,
    77, and 102) by 20-25% at 0.5 mg/kg bw/day. One female dog died on
    day 401 after showing few clinical signs of intoxication. At 10
    mg/kg bw/day, reduced food intake and body-weight gain were noted.
    Electrocardiogram records and ophthalmoscopy were normal in all
    groups as were haematological parameters (erythrocyte counts, Hb,
    PCV, HCHE, MCV, reticulocyte counts, total and differential white
    cell counts, platelet counts and prothrombin index), and clinical
    chemistry (urea, glucose, protein, SAP, K+ and Na+) except for a
    slight increase in ALAT at 12, 26 and 38 weeks in some dogs at 10
    mg/kg bw/day. Histopathological changes were comparable in all
    groups although absolute and relative (to body-weight ) liver
    weights were increased at 10 mg/kg bw/day (Rivett  et al., 1973).
    The 1974 JMPR (Annex 1, reference 23) did not appear to consider the
    19% depression of brain cholinesterase activity at 0.5 mg/kg bw/day
    to be toxicologically significant.

         Subsequent data have been submitted (ICI, 1988) which comprise
    historical control data on dog studies performed between 1970 and
    1975. These data clearly indicate that the apparent brain
    cholinesterase inhibition at 0.5 and 2 mg/kg bw/day are well within
    historical control values, based on 8 studies from the same
    laboratory, using the same measurement techniques. Concurrent
    controls in the pirimiphos-methyl study were, in 2 males and 3
    females, abnormally high. The Meeting concluded that the NOAEL was 2
    mg/kg bw/day, based on reduced cholinesterase activity and reduced
    body weight gain at 10 mg/kg bw/day.

         The potential hepatic effect of pirimiphos-methyl on dog liver
    was investigated using 8 male dogs dosed by capsule at 25 mg/kg
    bw/day, and 2 male dogs dosed at 25 mg/kg bw/day for 8 weeks and
    then increasing to 35 mg/kg bw/day (14 days), 45 mg/kg bw/day (7
    days), and finally 50 mg/kg bw/day (17 days). Six control animals
    received corn oil only. Four out of eight dogs at 25 mg/kg were
    withdrawn from treatment for 2 days after week 3 because of toxic
    signs. During week 4, 3/8 dogs were sacrificed (due to weight loss)
    and during week 12, 1/2 dogs, after 7 days at 50 mg/kg bw/day was

    taken off treatment for 5 days. Slight bile-duct proliferation was
    seen in 2 dogs receiving 25 mg/kg bw/day for 13 weeks but not in any
    other animals, even at 50 mg/kg bw/day. Investigation of plasma
    alkaline phosphatase, plasma alanine aminotransferase, aspartate
    aminotransferase, and plasma glutamate dehydrogenase showed
    elevation of these enzymes in almost all test animals on occasions,
    throughout the study. No correlation with histopathology was
    apparent. Individual sensitivity was extremely variable (Garuti  et
     al., 1976).

    Long-term toxicity/carcinogenicity studies

    Mice

         Three groups of 52 CFLP mice/sex were fed dietary
    concentrations of 0, 5 or 250 ppm 97.8% purity pirimiphos-methyl for
    80 weeks. A fourth group was fed 300 ppm increasing weekly by 50 ppm
    to a final concentration of 500 ppm. Satellite groups of 12 mice/sex
    fed 0, 5 and 500 ppm were utilized for blood cholinesterase studies.
    Mortality was not significantly affected. Body-weight gain was not
    affected overall, although food consumption was slightly reduced in
    females at 500 ppm over the 80 week period. Water consumption was
    unaffected. Erythrocyte cholinesterase activity (measured in weeks
    0, 12, 36 and 80) was depressed in males at 5 ppm in week 36 only,
    and in both sexes at all time intervals at 500 ppm. Plasma
    cholinesterase activity was significantly decreased (> 20%) at all
    time intervals at 500 ppm, and at 5 ppm in males at weeks 12 and 36,
    and in females at week 80. Histopathological changes (hepatocytic
    vacuolation hepatic nodular hypoplasia in females only, renal foci
    of lymphocytic infiltration, perivascular or peribronchial lymphoid
    aggregations in the lung and abscesses and small haemorrhages in the
    ovaries were not deemed to be of toxicological significance since
    they occurred in all groups and no dose-relationship was apparent.
    There was no evidence of tumour induction at dose levels up to 500
    ppm pirimiphos-methyl. The NOAEL was 5 ppm, equal to 0.5 mg/kg
    bw/day in male mice, and 0.6 mg/kg bw/day in female mice (Hunter  et
     al., 1976).

    Rats

         Four groups of 49 Wistar SPF rats/sex were fed pirimiphos-
    methyl at dietary concentrations of 0, 10, 50 or 300 ppm for 2
    years. An additional 24 rats/sex/group were fed the same diets and
    were sacrificed (8/sex/group) at 12, 26 and 52 weeks for brain
    cholinesterase studies. At termination of dosing, 8 rats/sex/group
    were fed control diet for 4-8 weeks prior to sacrifice, all other
    rats were sacrificed at 2 years. Survival (33-56%) was unaffected by
    pirimiphos-methyl at dose levels up to 300 ppm. Two outbreaks of
    infection were reported - one at 16 weeks, when "several animals of
    either sex developed swellings (lasting no more than 72 h) in the
    area of the salivary glands". No changes in behaviour, appetite or

    condition were observed. The condition, distributed between groups,
    did not recur. The second outbreak, occurring in the latter weeks of
    the test, was a respiratory disease, resulting in several deaths.
    All surviving rats were treated with 18 mg oxytetracycline/kg bw/day
    for 5 consecutive days during week 86.

         Body-weight gain and food intake were comparable in all groups.
    At 300 ppm, marked plasma cholinesterase inhibition (50-80%) and
    some inhibition of erythrocyte and brain cholinesterase (20-40%)
    were noted. The only other adverse effect at this dietary
    concentration was slight anaemia in female rats. Only female rat
    plasma cholinesterase was consistently inhibited (50-65%) at the 50
    ppm dietary level. Brain cholinesterase was reduced (> 20%) in
    males at weeks 26 and 104 at 50 ppm. Slight concurrent plasma
    cholinesterase inhibition occurred in females at 10 ppm. Brain
    erythrocyte cholinesterase was not affected at this dietary
    concentration. Recovery of depressed cholinesterase activity was
    usually complete following the 8-week withdrawal period. Organ
    weights and weight ratios, and incidence of gross or
    histopathological changes were comparable between groups. Tumour
    incidences in treated groups were generally comparable to incidences
    in control groups. The NOAEL was 10 ppm, equivalent to 0.5 mg/kg
    bw/day (Gore  et al., 1974a).

    Reproduction studies

    Rats

         A multigeneration study (3 generations, 2 litters/generation
    using b litters for parental animals) using groups of 24 female and
    12 male Charles River CD rats fed diets containing 0, 20 or 200 ppm
    nominal concentrations was performed. Since dietary analysis
    indicated levels of only 3-8.7 ppm were, in fact, present in the
    diet of the F0 generation (nominal level 20 ppm), the study at
    this dose level was extended to produce a 4th generation (a and b
    litters).

         No adverse effects on parental mortality, body-weight, or gross
    pathology were seen in any test group. Neither were toxic signs
    observed. At 200 ppm, mating performance and pregnancy rate were
    reduced in the production of F2-F3 litters and in F3 litters
    at 20 ppm. The decreased pregnancy rate was dose-related. Litter
    data (total litter losses, litter size, litter and mean pup weights,
    pup mortality, and length of gestation) were comparable between
    groups. Examination of the ultimate litters (F3b at 200 ppm and
    F4b at 20 ppm) by organ weight analyses, skeletal staining of
    10/pups/sex from each group, and histopathology of 10 pups/sex from
    0 and 200 ppm dose levels did not reveal any compound-related
    effects. A NOAEL was not demonstrated in this study (Palmer & James,
    1972).

         The testes of the F1b and F2b male rats were examined
    histopathologically in an attempt to find an explanation for their
    reduced mating performance. However, in the testes examined from
    rats of the control and both treatment groups, the degree of
    activity and maturity of the process of spermatogenesis were
    comparable (Annex 1, reference 23).

         Groups of 20 SPF CF strain rats/dose level were fed dietary
    concentrations of 0, 5, 10 or 100 ppm continuously for 3 generations
    (1 litter/generation). There were no consistent effects on parental
    animals as assessed by toxic signs, mortality, food consumption,
    body-weight, mating perfornance, pregnancy rate or duration of
    gestation. Parental plasma and erythrocyte cholinesterase depression
    (> 20%) was recorded in one or both sexes at 100 ppm after 7-9
    weeks on diet (i.e. during the premating period). No effects were
    observed in pups as determined by litter size, pup mortality, litter
    or mean pup weights, gross pathology of F3a or incidence of
    anomalies. The NOAEL was 100 ppm, equivalent to 5 mg/kg bw/day
    (Palmer & Hill, 1976).

    Special studies on potentiation

         Combined administration of half LD50 dose of pirimiphos-
    methyl with either gamma-BHC or dichlorvos to rats did not indicate
    potentiation (Annex 1, reference 23). However, similar studies with
    bioresmithrin indicated some possible potentiation but to an extent
    which does not appear to be sufficient to be of practical
    significance (Annex 1, reference 27).

    Special study on mammalian cell transformation

         An  in vitro study utilizing Syrian hamster kidney cells
    (fibroblast morphology, cell line BHK21/c13) exposed to doses
    ranging from 0.23 to 2300 g/ml, according to the method of Styles,
    1977) failed to induce cell transformation (Trueman, 1983).

    Special studies on teratogenicity

    Rats

         Three groups of 18-21 pregnant Alderby Park SPF rats were fed
    diets of 0, 10 or 200 ppm from gestation days 1 through 20 (day of
    insemination: day 0). Pups were removed by caesarean section on day
    20. Maternal body-weight gain was slightly reduced on day 7 of
    gestation, but was normal by day 20. No differences in food
    consumption or gross pathology of the dams were observed.
    Implants/dam, resorption incidence, sex ratio, and incidence of
    malformations were within the normal range of distribution At 200
    ppm, mean fetal weight was decreased, but this was probably a
    consequence of a concomitant increase in litter size (Hodge and
    Moore, 1972).

         A second rat study used 24 Alpk:AP Wistar derived rats/dose
    level. Rats were administered 0, 1.5, 15 or 150 mg pirimiphos-methyl
    (purity 88.5% w/w, doses corrected for 90.9% purity) in corn oil/kg
    bw/day by gavage on gestation days 7-16 inclusive (day 1 being the
    day of confirmation of mating) indicated maternal toxicity (changes
    in chemical signs and one death), decreased body-weight gain and
    food intake were observed at 150 mg/kg bw/day. In the 1.5 mg/kg
    bw/day pre-implantation losses were increased with concomitant
    effects on numbers of live pups and mean gravid uterine and litter
    weights. Values were, however, stated to be within normal ranges for
    the strain. It is improbable that this observation is compound-
    related because of the absence of similar findings at higher doses -
    and the minimal exposure which may have occurred prior to
    implantation. No compound- or dose-related effects on pup weight,
     in utero survival or incidence of malformations/variants were
    noted except for slight evidence of delayed ossification in  pes
    scores at 150 mg/kg bw/day. NOAELs for maternal toxicity (15 mg/kg
    bw/day), embryotoxicity (15 mg/kg bw/day, based on reduced  pes
    scores) and teratogenicity (<150 mg/kg bw/day) were identified
    (ICI, 1985).

    Rabbits

         Three groups of 16 or 17 artificially inseminated Dutch rabbits
    were orally administered gelatin capsules containing corn-oil
    solutions of pirimiphos-methyl to give 0, 1 or 16 mg/kg bw/day from
    gestation days 1 to 28 inclusive. Three does died during the study;
    two in the 1 mg/kg bw/day group, and one in the 16 mg/kg bw/day
    group. Litter data, however, were only available in summary format,
    on 10, 10 and 11 does at 0, 1 and 16 mg/kg bw/day respectively,
    despite 16, 15 and 15 does surviving to term. Whether this
    discrepancy is due to unsuccessful fertilization or pre-implantation
    loss or total resorption of litters cannot be ascertained. Based on
    the available data, mean number of implants/reported litter were
    similar in all groups, and resorption rates were highest in the
    control group. Litter size was increased at 16 mg/kg bw/day, and
    mean litter weight was slightly depressed at this dose level. Sex
    ratio (M/F) appears to increase with dose, but are stated (no data)
    to be within normal ranges. One high-dose fetus, with placental
    thrombosis and necrosis, showed multiple malformations. A dose-
    related increase in the occurrence of 14 caudal vertebrae was noted.
    Cholinesterase depression occurred in erythrocytes at 1 (23%) and 16
    (32-60%) mg/kg bw/day and in plasma at 16 mg/kg bw/day (23%). The
    NOAEL for embryofetal toxicity and teratogenicity was 16 mg/kg
    bw/day. A NOAEL for maternal toxicity was not demonstrated since at
    1 mg/kg bw/day, erythrocyte cholinesterase inhibition exceeded 20%
    (Gore  et al., 1974b).

    Special studies on neurotoxicity

    Hens

         In a study designed to assess acute delayed neurotoxicity,
    pirimiphos-methyl was administered by oral intubation as a solution
    in arachid oil to groups of five Light-sussex adult hens at dose
    levels of 20, 30, 40, 50 or 60 mg/kg bw. The LD50 was established
    at approximately 79 mg/kg bw. Over an observation period of 21 days
    post-dosing none of the animals showed signs of neurotoxicity. At
    day 26 the surviving birds in the two highest dose groups (4/5 at 50
    mg/kg bw and 2/5 at 60 mg/kg bw) were re-dosed after being protected
    with intramuscular injection of atropine (10 mg/kg bw) and 2-PAM (50
    mg/kg bw) and observed for a further 21 days. Group sizes were made
    up to 5 with "new" protected birds which received a single dose of
    pirimiphos-methyl. A further group of 5 birds were dosed with 500 mg
    TOCP/kg bw as a positive control. Clinical observations were
    confined to two birds in the 50 mg/kg bw dose group which showed
    sporadic doubtful signs of incoordination. Surviving birds (2/5) in
    60 mg/kg bw dose group showed no signs of neuropathological changes.
    All birds in the positive control group showed signs of ataxia and
    significant neuropathological changes. It is concluded that
    pirimiphos-methyl does not cause delayed neurotoxicity following
    acute doses up to 60 mg/kg bw (Annex I, ref. 27; Ross  et al.,
    1975).

         In a study designed to examine the potential of pirimiphos-
    methyl to cause delayed neurotoxicity after subchronic exposure,
    groups of ten adult hens were dosed at 0 (untreated control), 0
    (corn oil control), 0.5, 1.0, 2.5, 5.0 or 10 mg pirimiphos-methyl
    (93.5% purity) kg bw/day by oral intubation for 90 days. A further
    group of ten birds received 90 daily doses of 7.5 mg TOCP/kg bw as a
    positive control. A further 2 groups of 10 hens received 90 doses of
    either 5 or 10 mg pirimiphos-methyl/kg bw/day followed by a recovery
    period of 90 days when the birds were not dosed. Dosing was
    discontinued for birds in the two recovery groups when signs of
    toxicity were severe and restarted when signs of recovery were
    noted. The final recovery period was started when a bird had
    received a total of 90 doses.

         Analysis of test solutions indicated concentrations were within
    8% of nominal values, and were stable up to 10 days. Mortalities
    were 1/10, 3/10 and 4/10 in the main groups at 1.0, 2.5, 5.0 and 10
    mg/kg bw/day; 1/10 and 4/10 in the 5 and 10 mg/kg bw/day ancillary
    groups during treatment. Post-treatment deaths, during the recovery
    period were 4/9 and 1/6 at 5 and 10 mg/kg bw/day, respectively.
    Signs of toxicity, incidence and severity increasing with dose, were
    observed at 1.0 mg/kg bw/day and above. There were no signs of
    ataxia occurring as a result of delayed neurotoxicity except in the
    positive control group (7/10 birds). Food intake was variable during
    dosing, but overall indicated a dose-related decrease at 10 mg/kg

    bw/day, and in the positive control group. Food intake increased in
    the recovery groups after termination of dosing. Mean body-weight
    values were decreased in one group at 5 mg/kg bw/day and both groups
    at 10 mg/kg bw/day of dosing. Degenerative damages in nerve tissues
    at histopathological examination were only observed in the positive
    control group. The study did not indicate any evidence of delayed
    neurotoxicity due to exposure of adult hens to toxic doses of
    pirimiphos-methyl (Roberts  et al., 1983).

         A pilot study using 3 groups of 6 ISA hens protected with 50
    mg/kg bw 2-PAM i.p. and 10 mg atropine sulphate/kg bw i.p. at dose
    levels of 0, 100 or 117 mg pirimiphos-methyl/kg bw/p.o. with 14 day
    post-dosing observation determined a dose of 100 mg pirimiphos-
    methyl/kg bw to be a suitable dose for protected hens in a delayed
    neuropathy assessment study.

         Enzyme activity (NTE and AChE) were determined in hens given
    corn oil (4 birds), TOCP (2 birds), protected hens given 100 mg
    pirimiphos-methyl/kg bw (10 birds), p.o. with sacrifice at 24 h (2
    control, 1 TOCP and 3 treated birds) and 48 h (similar numbers). NTE
    was unaffected in control and pirimiphos-methyl dosed birds, but
    markedly inhibited in those given TOCP at both 24 and 48 h.
    Conversely, TOCP had minimal effect on brain or spinal cord
    acetylcholinesterase, which was markedly inhibited by pirimiphos-
    methyl.

         In the main study, 10 hens were given corn oil, 10 TOCP, and 40
    received 100 mg pirimiphos-methyl in corn oil/kg bw p.o., the dose
    being repeated after 21 days in control and pirimiphos-methyl
    treated birds. TOCP birds were sacrificed at 21 days, and the others
    at 42 days. Delayed ataxia was only seen in TOCP treated birds.
    Control and pirimiphos-methyl treated birds showed similar spinal
    cord and peripheral nerve histopathology, whereas TOCP showed
    disruption, fragmentation and distortion of some spinal cord axon
    with some damage to the myelin shealt or peripheral nerves as well
    as axonal degeneration (Lock & Johnson, 1990; Hawkin  et al.,
    1989).

    Special studies on genotoxicity

         The results of genotoxicity studies on pirimiphos-methyl are
    summarized in Table 3.


    
    Table 3. Results of genotoxicity assays on pirimiphos-methyl
                                                                                                                   
    Test                          Dose                          Result                      Reference
                                                                                                                   

    Male mouse dominated          15, 80 or 150 mg/kg bw/day    Pregnancy frequency         McGregor, 1975
    lethal                        for 5 days prior to pairing   reduced at 150 mg/kg bw

    Salmonella typhimurium        ditto                         +                           Hanna & Dyer, 1975
    (No metabolic activation)

    Escherichia coli              ditto                         -                           Hanna & Dyer, 1975
    (No metabolic activation)

    Micronucleus                  ditto                         -                           Seiber (1975)

    Micronucleus (mouse)          100, 200 and 400 mg/kg bw     -                           Rajini  et al. (1986)
    Sperm-morphology              100, 200 and 400 mg/kg bw     -                           Rajini  et al. (1986)
    (mouse)

    Bone marrow cytogenetic       Single dose                   -                           Done & McGregor (1980)
    study (rat)                   32, 102, 320 mg/kg bw

    Bone marrow cytogenetic       5 consecutive daily           - ve at 32 and 102 mg/kg    Done & McGregor (1980)
    study (rat)                   32, 102, 320 mg/kg bw         bw. At 320 mg/kg
                                                                bw/day, chromatid gap
                                                                incidence was increased
                                                                and one chromosome gap
                                                                was recorded

    Salmonella typhimurium        1.6-5000 g/plate             -                           Callander (1984)
    TA1535, TA1537, TA1538,
    TA98 and TA100 (with
    and without metabolic
    activation)

    Table 3 (Cont'd)
                                                                                                                   
    Test                          Dose                          Result                      Reference
                                                                                                                   

    Mouse lymphoma cell           12.5-200 g/ml                -                           Cross (1986)
    assay (L5178Y cells) (with
    and without metabolic
    activation

    Human lymphocyte
    cytogenetics (with and
    without metabolic                                           -                           Wildgoose  et al. (1986)
    activation)                                                 -
    DONOR 1                       12, 58, 116 g/ml**
    DONOR 2                       12, 29, 58 g/ml**

    Hamster sister chromatid
    exchange in lung
    fibroblasts                                                 +- ***                      Howard  et al. (1986)
    with metabolic activation     0.14, 0.29, 1.4, 2.9, 14, 29  +-****
    without metabolic             0.14, 0.29, 1.4, 2.9, 14, 29,
    activation                    145 g/ml
                                                                                                                   

    *    The doses in this study tended to be low and metaphase chromosome spread was variable, resulting in
    reduced numbers of cells available for scoring.

    **   Maximum tolerated doses for the different donors.

    ***  Positive results (dose-related) were noted at 14, 29 and 145 g/ml. At 145 g/ml, the assessment was
    based on a single cellulite at a dose which exhibited marked toxicity.

    **** Positive results were noted at all dose levels except 2.9 g/ml. Dose/effect relationships were not
    clearly established.


    
    Observations in humans

         A dose of 0.25 mg 97.8% purity pirimiphos-methyl/kg bw/day was
    taken orally for 28 days by 5 healthy males (59.5-73 kg bw, 25-45
    years old). Blood samples were taken on days -14, -7, 1, 3, 7, 14,
    21 and 28. One subject showed plasma cholinesterase inhibition
    (21.5%) on day 28. Otherwise variations, both above and below pre-
    dosing values, were within 12%. Four of five subjects had red-cell
    cholinesterase activity values slightly below the pre-exposure
    values during the last 2 weeks of the study. However, the group
    means for each time interval did not differ significantly and the
    variations noted were within the range of variations found by others
    for normal untreated subjects (Chart  et al, 1974).

         Three male (62-73 kg, 22-27 years old) and 4 females (44-60 kg,
    21-49 years old) were given 0.25 mg 97.8% purity pirimiphos-
    methyl/kg bw/day by capsule for 56 days. Blood samples were taken
    twice prior to initiation of dosing, and on days 7, 14, 21, 28, 35,
    42, 49 and 56 of the study and also in the recovery period 7, 14, 21
    and 29 post-treatment. Controls comprised 2 females (44 and 46 kg,
    aged 29 and 30). No compound-related effects were observed on liver
    function (alanine aminotransferase, aspartate aminotransferase,
    alkaline phosphatase and glutanyl transpeptidase in plasma),
    haematology (Hb, PCV, MCHC, total and differential WBC, platelets,
    ESR) or erythrocyte cholinesterase. Plasma cholinesterase was
    depressed about 20% in 2/4 females on days 14, 21 and 28 and in one
    female on days 28 and 35. The effect did not increase with time. All
    values were normal during the withdrawal period (Howard & Gore,
    1976).

         Two groups of spray workers (5/group) together with 1 mixer-
    loader per group applied actellic 50 EC to grain using a compression
    sprayer, hand held, for 3 consecutive days (4-5 h/day). The
    formulation, containing 50% pirimiphos-methyl, was sprayed at a
    concentration of 5 g/l with an application rate of 0.158 a.i./m2.
    A second study, using similar groups applied actellic 40 EC
    containing 40% pirimiphos-methyl sprayed at a concentration of 46
    g/l and application rate of 2.0 a.i./m2 in unoccupied dwellings
    for mosquito control. In the two studies, spray workers in one group
    were in normal clothing, in the second group, protective clothing
    (cotton overalls, caps, canvas shoes and safety glasses, and in the
    case of the dwelling spray workers, cotton masks). Mixer-loaders
    wore rubber boots, gloves, caps, cotton jackets, cotton masks and
    safety glasses. Cholinesterase (blood and plasma) was measured on 3
    consecutive days prior to spraying, at lunchtime and the evening on
    the days of spraying, and on days 1, 2, 3 and 10 post-spraying.
    Clinical examinations were performed by registered practitioners and
    comprised case histories and a general medical examination with
    emphasis on gastrointestinal, neuromuscular, cardiorespiratory,
    visual and psychological effects and peripheral and central nervous
    systems prior to, during, and subsequent to spraying. Cholinesterase

    depression occurred on a group basis in all groups over the spray
    period, with full recovery within 24 h post-dosing. The greatest
    depression occurred in workers applying the 40 EC formulation. Ten
    of the 24 exposed individuals showed > 15% depression of
    erythrocyte cholinesterase, the maximum depression being 23%. Levels
    of plasma cholinesterase inhibition were inconsistent, and less than
    those seen for erythrocyte cholinesterase inhibition. There were no
    clinical manifestations of toxicity (Chester & Hart, 1986).

    COMMENTS

         Following oral administration of pirimiphos-methyl to male
    rats, 80.7% and 7.3% of the administered dose were excreted via
    urine and faeces, respectively, within 24 h. In the dog, 48 h after
    dosing with either 18.4 or 16.7 mg/kg bw, urinary excretion was
    64.4% or 82.5% and faecal excretion 17.3% or 13.3%.

         Metabolic data indicated that the P-O-C bond of pirimiphos-
    methyl was readily cleaved and that N-de-ethylation and/or
    conjugation were further steps in the metabolism of the pyrimidine
    leaving group. Although the oxygen analogue of pirimiphos-methyl was
    not detected as a urinary metabolite, the fact that cholinesterase
    inhibition occurred  in vivo suggests that the oxygen analogue was
    also formed and may be an intermediate step leading to the
    identified urinary products.

         In rats and dogs 2-ethylamino-4-hydroxy-6-methylpyrimidine was
    the major metabolite (30% of the administered dose).

         The oral toxicity of pirimiphos-methyl is low. WHO has
    classified the compound as slightly hazardous (WHO, 1992).

         The only biochemical effect consistently observed with
    pirimiphos-methyl in acute short-term or long-term studies was
    cholinesterase inhibition.

         In a series of short-term rat studies at dose levels of 0, 8,
    80 or 360 ppm for three months, 0, 10, 250, 500 or 1000 ppm for 28
    days, 200 mg/kg bw five times weekly for 14 days, and 0, 5, 8, 10 or
    50 ppm for 28 days (young rats) the overall NOAEL was 10 ppm
    (equivalent to 0.5 mg/kg bw/day) with effects on erythrocyte
    cholinesterase and brain acetylcholinesterase at 80 ppm. At high
    dose levels (200 mg/kg bw, five times weekly for two weeks)
    erythrocyte morphology was affected. The NOAEL in young rats was
    also 10 ppm, with brain (but not erythrocyte) acetylcholinesterase
    depressed at 50 ppm after 28 days.

         In two dog studies (13 weeks at dose levels of 0, 2, 10 or 25
    mg/kg bw/day via capsule and 0, 0.5, 2 or 10 mg/kg bw/day for two
    years by capsules) the NOAEL was 2 mg/kg bw/day, based on brain
    acetylcholinesterase inhibition.

         In an 80-week study in mice at dietary concentrations of 0, 5,
    250 or 500 ppm, a NOAEL based on blood cholinesterase depression was
    5 ppm (equal to 0.5 mg/kg bw/day) (blood cholinesterase was not
    measured at 250 ppm, only at 5 and 500 ppm). Pirimiphos-methyl was
    not carcinogenic in mice.

         In a two-year study in rats at dietary concentrations of 0, 10,
    50 or 300 ppm, tumour incidence was comparable to controls. The

    NOAEL was 10 ppm (equivalent to 0.5 mg/kg bw/day) with brain
    acetylcholinesterase inhibition occurring at higher levels.
    Pirimiphos-methyl was not carcinogenic in rats.

         In a four-generation reproduction study in rats at nominal
    dietary concentrations of 0, 20 or 200 ppm, dose-related reduction
    of pregnancy rates and reduced mating performance at 200 ppm were
    noted. Dietary analyses indicated that the 20 ppm diet only
    contained 9 ppm pirimiphos-methyl. No NOAEL was demonstrated in this
    study.

         A repeat study at dietary concentrations of 0, 5, 10 or 100 ppm
    for three-generations (1 litter/generation) did not show any adverse
    effects on reproductive parameters at any dose level. The NOAEL was
    100 ppm (equivalent to 5 mg/kg bw/day) for reproductive effects.

         In two rat teratology studies, one at dietary concentrations of
    0, 10, or 200 ppm and the second at dose levels of 0, 1.5, 15, or
    150 mg/kg bw/day, dosing extending over or beyond the period of
    embryogenesis did not demonstrate any evidence of teratogenicity.
    Fetotoxicity was observed at 200 ppm (equivalent to 10 mg/kg bw/day)
    and 150 mg/kg bw/day. NOAELs for maternal toxicity (15 mg/kg
    bw/day), embryotoxicity (15 mg/kg bw/day) and teratogenicity
    (< 150 mg/kg bw/day) were identified.

         A rabbit teratogenicity study at doses of 0, 1 or 16 mg/kg
    bw/day administered from days 1-28 of gestation did not show any
    evidence of teratogenic effects. The NOAEL for fetotoxicity and
    teratogenicity was 16 mg/kg bw/day.

         Four studies in hens indicated that pirimiphos-methyl does not
    cause delayed neurotoxicity.

         After considering the available  in vitro and  in vivo
    genotoxicity data, the Meeting concluded that pirimiphos-methyl was
    not genotoxic.

         In two experimental studies with human volunteers of 28 and 56
    days, the highest dose tested in both studies (0.25 mg/kg bw/day)
    failed to induced erythrocyte cholinesterase inhibition in either
    study.

         In determining the ADI the first multigeneration study in rats
    was discarded because the dietary concentrations were uncertain, and
    the adverse effects noted (decreased pregnancy rate and mating
    performance) were atypical of those normally seen in reproduction
    studies with organophosphorous esters (decreased pup weight gain and
    pup mortality during early lactation). A clear NOAEL of 100 ppm
    (equivalent to 5 mg/kg bw/day, the highest dose tested) was
    demonstrated in the repeat study.

         Studies with mice, rats, and dogs, showed NOAELs of 0.5 mg/kg
    bw/day or above. In human studies, no cholinesterase inhibition was
    seen at 0.25 mg/kg bw/day (the highest dose tested). On this basis,
    the Meeting revised the ADI to 0.03 mg/kg bw/day by applying a 10-
    fold safety factor to the NOAEL in the human studies.

    TOXICOLOGICAL EVALUATION

    Level causing no toxicological effect

         Mouse:    5 ppm, equal to 0.5 mg/kg bw/day (80-week study)

         Rat:      10 ppm, equivalent to 0.5 mg/kg bw/day (two-year
                        study)

                   100 ppm, equivalent to 5 mg/kg bw/day (three-
                        generation reproduction study)

         Dog:      2 mg/kg bw/day (two-year study)

         Humans:   0.25 mg/kg bw/day

    Estimate of acceptable daily intake for humans

         0-0.03 mg/kg bw

    Studies which will provide information valuable in the continued
    evaluation of the compound

         Further observations in humans.

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    See Also:
       Toxicological Abbreviations
       Pirimiphos-methyl (WHO Pesticide Residues Series 4)
       Pirimiphos-methyl (Pesticide residues in food: 1976 evaluations)
       Pirimiphos-methyl (Pesticide residues in food: 1977 evaluations)
       Pirimiphos-methyl (Pesticide residues in food: 1979 evaluations)
       Pirimiphos-methyl (Pesticide residues in food: 1983 evaluations)