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    PYRAZOPHOS

    First draft prepared by S. Caroldi
    Istituto di Medicina del Lavoro
    Padova, Italy

    EXPLANATION

         Pyrazophos is an organophosphorus systemic fungicide used on a
    wide range of crops and cereals in the control of powdery mildew.
    Pyrazophos was scheduled for evaluation at the 1985 Joint Meeting
    but the data base available at that time was insufficient for the
    estimation of an ADI (Annex 1, reference 44).

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOLOGICAL DATA

    Biochemical aspects

    Absorption, distribution, and excretion

    Rats

         Eight Wistar rats/sex were intubated with a single 2 mg dose of
    14C-pyrazophos (3a-14C) in 1 ml sesame oil. Radioactivity was
    measured at different times in blood, plasma, urine, exhaled air,
    faeces and after 168 hours post-dosing, in several tissues and
    organs.

         Blood and plasma levels of radioactivity peaked between 4 and 6
    h post-dosing in both sexes; the calculated half-life was
    approximately 5 h. Over a period of 24 h, approximately 71% of the
    original dose was eliminated via urine and 24% via faeces.
    Radioactivity was completely eliminated within 168 h (73% via urine
    and 27% via faeces). A negligible amount of radioactivity (less than
    0.01%) was found in exhaled air within 24 h and in tissues and
    organs at 168 h. Metabolites were detected in urine and faeces
    collected during the first 24 h when approximately 95% of total
    radioactivity was eliminated. Pyrazophos was not detected in urine
    but it was the main substance found in the faeces (66% of
    radioactivity in faeces and 16% of total radioactivity). Four
    metabolites were identified in urine; the most concentrated was a
    pyrazophos P-O hydrolysis product present in urine as free compound
    or sulphate and glucoronide conjugates (Lachmann, 1986).

         Eleven Wistar rats/sex were intubated with a single dose of 0.4
    mg 14C-pyrazophos (3a-14C) in 1 ml sesame oil. A further 10
    Wistar rats/sex received 14 daily doses of 0.4 mg of pyrazophos
    followed by a single dose of 0.4 mg 14C-pyrazophos. Radioactivity
    was measured at different times in blood, plasma, urine, exhaled
    air, faeces and at 168 h post-test substance in several tissues and
    organs. Peak blood and plasma radioactivity levels were measured
    within 4 h post-dosing. The calculated half-life was about 5 h. Over
    the initial 24 h, 75% of the original dose was excreted via urine
    and 18% via faeces. No differences between single or repeated doses
    were detected. Negligible amounts of radioactivity were present in
    tissues and organs at 168 h from the last pyrazophos administration.
    Six different metabolites were found in urine. The most important
    was the product of a double hydrolysis 2-hydroxy-5-methyl-
    pyrazolo(1,5-a)pyrimidine-6-carboxylic acid. Other metabolites were
    O-pyrazophos and the products of hydrolysis either at the phosphate
    or carbonate bond. Two metabolites remained unknown. Pyrazophos was
    the only radioactive component present in faeces (Lachmann,1987).

    Toxicological studies

    Acute toxicity studies

         Results of acute toxicity tests with pyrazophos are given in
    Table 1.
        Table 1.  Acute toxicity of pyrazophos
                                                                                      
    Species  Sex   Route        LD50        LC50       Reference
                                (mg/kg bw)  (mg/m3)
                                                                                      

    Rats     M     oral         242-778a               Hollander & Weigand (1978a,b,c)
             F                  151-468                Otaka  et al. (1981a,b) 
                                                       Hollander & Weigand (1977f)
                                                       Scholz & Weigand (1972a)
             M     i.p.         280                    Hollander & Weigand (1977a,b)
             F                  305
             M                  172                    Otaka  et al. (1981e,f)
             F                  135
             M     s.c.         202                    Otaka  et al. (1981c,d)
             F                  193
             M                  221                    Hollander & Weigand (1977g,h)
             F                  266
             F     dermal       > 2000                 Hollander & Weigand (1981)
                   (24 h exp)
             M&F   inhalation               > 1220     Hollander & Weigand (1982)
                   (4 h exp)

    Mice     M     oral         214                    Hollander & Weigand (1977d,e)
             F                  205
             M                  413                    Otaka  et al. (1981g,h)
             F                  321
             M     i.p.         581                    Scholz & Weigand (1972b)
             F                  438
             M                  167                    Otaka  et al. (1981m,n)
             F                  146
             M     s.c.         339                    Otaka  et al. (1981i,l)
             F                  348
             M                  181                    Hollander & Weigand (1977i,l)
             F                  199

    Dogs     M&F   oral         > 500b                 Hollander & Weigand (1978d)
             M&F                > 100c                 Hollander & Weigand (1979b)
                                                                                      

    a  Symptoms were mostly of the cholinergic type. Pathology showed atrophy of spleen
       and haemorrhage of intestine. Histopathology was negative.

    Table 1 (continued)

    b  Clear cholinergic symptoms were observed from 200 mg/kg bw, no animals died up to
       the highest tested dose. At 500 mg/kg bw, emesis occured within 2 h from dosing which
       likely reduced the absorbed dose.

    c  Clear cholinergic symptoms were observed from 50 mg/kg bw, no animal died up to
       the highest tested dose. At 100 mg/kg bw, emesis occured within 2 h from dosing which
       could have reduced the absorbed dose.
    
    Guinea-pigs

         Pyrazophos produced no indications of any allergenic properties
    in the sensitization test in the guinea-pig according to the Buehler
    test (Leist & Weigand, 1982).

    Rabbits

         Undiluted pyrazophos was non-irritant to the rabbit skin.
    Undiluted pyrazophos and a 10% dilution of pyrazophos in sesame oil
    resulted in slight irritantion to the eye. A 1% dilution of
    pyrazophos in sesame oil resulted in no irritantion to the eye
    (Hollander & Weigand, 1977c)

    Short-term toxicity studies

    Mice

         Five groups of ten SPF mice/sex were treated with pyrazophos
    for 28 days at dietary concentrations of 0, 1, 5, 25 and 125 ppm
    equal to 0, 0.2, 0.9, 4.7, 23.3 mg/kg/day and 0, 0.2, 0.9, 5.0, and
    22.2 mg/kg bw/day for males and females, respectively. No signs of
    toxicity were observed; body-weight gain and food intake were not
    affected by pyrazophos administration. Dose-related inhibition of
    both plasma (from 5 ppm) and erythrocyte (from 25 ppm)
    cholinesterase was measured in animals of both sexes. Marginal
    inhibition (approximately 20%) of brain acetylcholinesterase
    activity was detected after 28 days of treatment at 125 ppm in male
    mice only. The NOAEL in this study was 25 ppm equal to 4.7 mg/kg
    bw/day and 5.0 mg/kg bw/day for male and female mice, respectively
    (Hollander & Weigand, 1978e).

         Four groups of twenty Charles River CD-1 mice/sex were treated
    with pyrazophos technical at dietary concentrations of 0, 1, 5, or

    25 ppm for 28 days. No signs of toxicity were observed; body-weight
    gain was not affected by pyrazophos administration. Inhibition of
    both plasma and erythrocyte cholinesterase was observed in animals
    of both sexes from the 1 ppm dose level. Brain cholinesterase
    activity was not reduced by pyrazophos administration up to the
    highest dose. The NOAEL in this study was 25 ppm equivalent to 4
    mg/kg bw/day (Estes, 1979).

    Rats

         Seven groups of ten albino rats/sex from the CIVO-colony
    (Wistar-derived) were treated with pyrazophos (technical) at dietary
    concentrations of 0, 1, 2.5, 5, 15, 45 or 150 ppm for 28 days in a
    range-finding toxicity study. Ten additional rats per sex at 0 and
    150 ppm dietary concentrations were discontinued from the feeding of
    pyrazophos for 21 days to examine recovery of cholinesterase
    activities. No signs of toxicity were observed; body-weight gain and
    food and water intake were not affected by pyrazophos
    administration. Haematology and pathology did not show any adverse
    effects. Dose-related inhibition of both plasma and erythrocyte
    cholinesterase was observed at 15 ppm and higher in both sexes.
    Plasma cholinesterase activity returned to normal levels in a week
    after discontinuing pyrazophos administration at 150 ppm. Inhibition
    (approx. 20%) of brain acetylcholinesterase activity was detected
    after 28 days of treatment at 150 ppm in female rats only. The NOAEL
    in this study was 45 ppm equivalent to 4.5 mg/kg bw/day in both
    sexes (Til  et al., 1978)

         Five groups of ten weanling Wistar-derived rats/sex were fed
    pyrazophos (technical substance, purity 90%) at dietary
    concentrations of 0, 5, 8, 10 or 50 ppm for 14 weeks. The diets were
    prepared every two weeks and stored at room temperature. Data on
    stability and homegeneity of test substance in diets are not
    reported. Growth, symptoms, food consumption, haematology, blood
    chemistry (whole blood cholinesterases included), urinalyses, organ
    weights and pathology were the parameters checked in this study. No
    mortality occurred throughout the duration of the study. Food and
    water intake and body-weight were not affected by pyrazophos
    administration. Scattered differences of no biological relevance
    between groups were observed on haematology, biochemical blood
    values, urinalysis and pathology. Dose-related inhibition of whole
    blood cholinesterase activity was observed at 10 and 50 ppm
    pyrazophos in both sexes. The NOAEL in this study was 8 ppm of
    pyrazophos equivalent to 0.8 mg/kg bw/day (deKnecht-vanEekelen &
    Dreef-vanderMeulen, 1978).

         Four groups of thirty-six F344 Charles River rats/sex were
    dosed with technical pyrazophos (92.8% purity) for 13 weeks at
    dietary concentrations of 0, 2.5, 50 or 1000 ppm equal to 0, 0.21,
    4.2 or 90 mg/kg bw/day and to 0, 0.21, 4.0 or 100 mg/kg bw/day for
    males and females, respectively. Twelve rats sex/group were killed
    at the end of the exposure period, the remaining 24 rats/sex/dose
    were killed, 12 after two weeks and 12 after four weeks of recovery.
    The diets were prepared every three weeks and stored at 4 C.
    Homogeneity and accuracy of the test diets were checked (analytical
    results not reported). Survival was not affected by pyrazophos
    administration. Clinical symptoms of the cholinergic type were
    observed during the first 4 weeks of treatment at the highest dose
    level in both sexes. Body-weight gain was decreased at the highest
    dose level during the first 7 weeks of treatment in males and
    throughout the duration of the study (recovery period included) in
    females. Scattered differences of no biological relevance in food
    intake, food efficiency and water intake were observed throughout
    the study in all groups. Reduced erythrocyte counts, haematocrit and
    haemoglobin were measured at the highest dose level at the interim
    examination and at the end of the exposure (also an increased number
    of reticulocytes was measured at this time) but these parameters
    recovered to normal level within the 4 weeks of recovery period.

         Cholesterol and albumin concentrations in blood were also
    reduced during pyrazophos administration at 1000 ppm and returned to
    normal level after cessation of exposure. Dose-related inhibition of
    plasma and erythrocyte cholinesterase was measured at 50 and 1000
    ppm dose levels. At the end of the exposure period at 1000 ppm,
    brain cholinesterase were inhibited by 64% and 93% in males and
    females, respectively. Brain cholinesterase was also marginally
    affected (22% inhibition) at 50 ppm in female rats but not in males.
    Inhibition of brain cholinesterase was detectable at 1000 ppm 4
    weeks after the end of exposure. Several organ weights of the 1000
    ppm group differed from control values: the absolute and relative
    weights of adrenals, spleen and liver (only the relative weight of
    liver was increased in females) were increased in both sexes, testis
    and pituitary (females only) weights were decreased. Most of the
    differences in organ weights disappeared during the 4 weeks of
    recovery. Proliferation of large mononuclear cells of spleen and fat
    deposition in fascicular zone of adrenals were considered as dose-
    related, but they disappeared after the 2 weeks recovery period. The
    NOAEL in this study was 2.5 ppm, equal to 0.21 mg/kg bw/day, in both
    sexes, based on marginal brain cholinesterase inhibition measured at
    the termination of the study in females at 4.0 mg/kg bw/day (Otaka
     et al., 1981o)

         Four groups of forty F344 rats/sex were fed technical
    pyrazophos (purity 92.9%) for 52 weeks at dietary concentrations of
    0, 2, 20 or 200 ppm equal to 0.1, 1.0 or 11 mg/kg bw/day and 0.1,
    1.4 or 14 mg/kg bw/day in males and females, respectively. Diets
    were prepared every three weeks and were stored at 4 C until use.
    Homogeneity and accuracy of the test diets were checked just before
    starting the study and thereafter every three months. Actual
    concentrations of pyrazophos (mean values) were within  10% of
    nominal concentrations. General conditions, body-weight, food and
    water intake were recorded throughout the study. Parameters
    investigated were: haematology, blood biochemistry, PSP and BSP
    test, in plasma in erythrocytes and brain cholinesterase activity
    and urinalysis. Pathology (organ weights) and histopathology
    (incomplete) were performed at the end of the study. Moreover the
    concentration of unmetabolized pyrazophos was measured in plasma,
    brain, liver, kidneys and fat at weeks 13, 26 and 52 (detection
    limit: 0.05 ppm).

         No clinical signs of toxicity or excess mortality related to
    administration of the test compound were detected. No differences in
    food and water intake and body-weight gain were observed among
    different groups. Scattered differences of some parameters were
    observed throughout the duration of the study. Among them, increased
    erythrocyte counts and haematocrit and decreased cholesterol
    concentration in blood were observed at the highest pyrazophos level
    in both sexes. Urinalysis and the excretory function for PSP and BSP
    did not reveal toxic effects. Plasma and erythrocyte cholinesterase
    activities were reduced in both sexes at 20 and 200 ppm pyrazophos.
    A slight inhibition of brain cholinesterase activity (30%) was noted
    at 200 ppm of pyrazophos in females only. Pathology (organ weights)
    and histopathology did not show biologically relevant changes.
    Pyrazophos was measured only in fat tissue of male and female rats
    fed 200 ppm. Pyrazophos concentration in fat was higher in females
    than in males and it was higher in both sexes at the end of the
    study than at 13 week and 26 week determinations. The NOAEL in this
    study was 20 ppm equal to 1.0 and 1.4 mg/kg bw/day in males and
    females, respectively. The NOAEL was based on 30% inhibition of
    brain cholinesterase activity detected in the brain of female rats
    fed dietary concentrations of 200 ppm. The results of this study
    were obtained at the interim sacrifice of a carcinogenicity study
    discontinued because of the high mortality rate occuring in all
    groups, including controls (Otaka, 1983).

    Dogs

         Four groups of four pedigree English beagle dogs/sex were fed
    pyrazophos at dietary concentrations of 0, 0.5, 2.0, 5.0 or

    10/125/320 ppm for 92 days. The highest dose level of 10 ppm was
    increased to 125 ppm on day 60 from the beginning of the study and
    further increased to 320 ppm on day 75. Two animals/sex/dose (except
    0.5 ppm group) were observed for approximately 6 weeks after the end
    of pyrazophos administration and cholinesterase activities were
    estimated.

         The following parameters were investigated: clinical
    observations (general conditions, body-weight, behaviour, reflex
    excitability, eye examinations, visible mucosae, dentition),
    laboratory examinations (haematology, cholinesterase activity in
    serum and in erythrocytes, other serum enzymes, urinalyses) and
    pathology (organ weights, gross pathology, histopathology). Slight
    reduction of food intake, impairment of general conditions, tetanic
    cramps of the cervical muscles, disturbances of motility and slight
    miosis were observed in the highest dose group when the dietary
    concentration was increased to 320 ppm. Toxic effects were not
    observed in animals of the other groups. Slight anaemia was found at
    the highest dose level after the dose of pyrazophos was raised to
    320 ppm; other clinical chemistry tests did not show any adverse
    effects. A dose-related inhibition of serum cholinesterase activity
    was observed at 5 ppm and higher in both sexes. At 5 ppm
    approximately 20% inhibition of erythrocyte cholinesterase activity
    was measured (range of 6 determinations 15%-25%) in females only. At
    the highest dose, erythrocyte cholinesterase activity was reduced in
    both sexes. Increased mean weight of the pancreas was noted in all
    treated groups which did not correspond to any microscopic
    abnormalities. The NOAEL in this study was 5 ppm, equivalent to 0.4
    mg/kg bw/day based on marginal inhibition of erythrocyte
    cholinesterase activity measured at the next highest doses (Scholz &
    Brunk, 1973).

         Four groups of eight English beagle dogs/sex, 13 months old at
    the beginning of the study, were fed pyrazophos (92.9% purity grade)
    at dietary concentrations of 0, 1.2, 18 or 320 ppm for 6 months.
    Diets were prepared daily, immediately before feeding. The following
    parameters were investigated: clinical observations (general
    conditions, body-weight, behaviour, reflex excitability,
    ophthalmoscopic examinations, hearing test, visible mucosae,
    dentition), laboratory examinations (haematology, clinical
    chemistry, cholinesterase determinations, hepatic and renal function
    tests, urinalyses) and pathology (organ weights, gross pathology,
    histopathology). Clinical symptoms of the cholinergic type were

    observed at 320 ppm as occasional diarrhoea and tetanic spasm. Some
    dogs of this group developed atrophy of temporal muscles. Decline of
    general health conditions, reduction of both food intake and body-
    weight were also detected. One dog in poor nutritional state died on
    day 107 of treatment, possibly due to respiratory muscle failure
    resulting from extreme cholinesterase reduction. Animals showed
    slight anaemia and increase of ASAT, ALAT and alkaline phosphatase
    in serum. Apart from occasional diarrhoea, no other toxic symptoms
    were observed at 18 ppm and dogs in the 1.2 ppm group were
    comparable to controls. Serum and erythrocyte cholinesterase
    activities were reduced in animals at 18 and 320 ppm throughout the
    duration of the study. At the end of the study, dose-related
    inhibition of brain cholinesterase was observed at 18 and 320 ppm
    (20% and 76%, respectively). The relative weights of several organs
    differed from control values at the 320 ppm dose level only.
    Pathology did not reveal compound related organic lesions. The NOAEL
    in this study was 1.2 ppm equivalent to 0.09 mg/kg bw/day, based on
    occasional diarrhoea and marginal brain cholinesterase inhibition
    observed at 18 ppm (Brunk  et al., 1982).

         Four groups of four pedigree English beagle dogs/sex (13 months
    old at the beginning of the study) were fed pyrazophos (purity grade
    92%) at dietary concentrations of 0, 2, 5 or 10/125/320 ppm for 2
    years. The highest dose level of 10 ppm was increased to 125 ppm on
    day 14 from the beginning of the study and further increased to 320
    ppm on day 28. The test substance was mixed with food and given
    daily during the mid-day meal.

         The following parameters were investigated: clinical
    observation (general conditions, body-weight, behaviour, reflex
    excitability, eye examinations, hearing test, visible mucosae,
    dentition), laboratory examinations (haematology, cholinesterase
    activities, serum enzymes, urinalysis), pathology (organ weights,
    gross pathology, histopathology).

         One male dog in the 320 ppm group died of a chronic suppurating
    pancreatitis and a chronic ascending pericholangitis after 462
    administrations of pyrazophos. Several parameters were affected in
    this animal but since the cause of death was not dependent on
    pyrazophos administration they were not considered. All remaining
    dogs survived till the end of the study. Food intake was not
    affected by administration of the test compound. At the end of the
    study, body-weight gain of the animals of the 320 ppm group was

    approximately 50% less than that in the remaining groups. Behaviour,
    reflex responses, ophthalmic findings, hearing test, dentition and
    visible mucosae were not affected. Slight anaemia and increased
    alkaline phosphatase were observed in animals at 320 ppm dose level.
    Dose-dependent decreases of serum cholinesterase activity were
    observed at 5 and 320 ppm. Erythrocyte cholinesterase activity was
    reduced only at 320 ppm. Pathology and organ weights did not show
    significant difference between groups. Histopathology was comparable
    to controls in the 2 and 5 ppm groups. At 320 ppm, calcifications in
    some regions of the basement membranes of the Bowman's capsules of
    numerous renal glomeruli, in tubular basement membranes and in the
    interstitium between some renal tubules were observed.

         The NOAEL in this study was 5 ppm, equivalent to 0.4 mg/kg
    bw/day based on erythrocyte cholinesterase inhibition, reduced body-
    weight gain and histopathological abnormalities in kidneys observed
    in dogs fed 320 ppm (Brunk  et al., 1976)

    Long-term toxicity/carcinogenicity studies

    Mice

         Four groups of 70 Charles River CD-1 mice/sex, 4 weeks of age
    were treated with pyrazophos technical (92.8% purity grade) at
    dietary concentrations of 0, 1, 5 or 25 ppm, equal to 0, 0.14, 0.7
    or 3.5 mg/kg bw/day for males and 0, 0.16, 0.8 or 4 for females. Ten
    animals/sex/dose were sacrificed after 12 months of treatment. The
    remaining animals were sacrificed on week 92 (females) and on week
    96 (males). Pyrazophos was stored under refrigeration until used;
    test substance was offered to mice after being mixed with basal
    laboratory diet. Due to difficulties in obtaining homogeneous
    mixture, dietary preparation procedure was changed during the study.
    Actual content of the test substance in diets was 65% (0-101), 100%
    (55-241) and 85% (42-100) of nominal (mean of 12 determination,
    range in brakets) at 1, 5 and 25 ppm, respectively. Daily
    observations for overt toxicity signs, abnormalities and masses and
    weekly observations for feed consumption and body-weights were
    recorded. Haematology determinations and brain cholinesterase
    activity on 10 mice/sex/ dose level were performed at 12 months and
    at termination of the study. Serum and erythrocyte cholinesterase
    activities were measured on 10 mice/sex/dose at 0, 3, 6, 12, 18
    months and at termination of the study. Pathology was performed on
    all animals found dead or sacrificed at the end of the study.

         In both sexes, food intake and body-weight gain were unaffected
    by pyrazophos administration. The incidence of clinical signs did
    not differ between groups. The low survival rate noted at 1 ppm
    (week 92) for the females and at week 96 for the males, prompted the
    early termination of the study. The mortality rate was 67%, 75%,
    65%, 70% (males) and 72%, 80%, 63% and 75% (females) at 0, 1, 5 and
    25 ppm pyrazophos, respectively. Pyrazophos did not effects
    haematological parameters tested. Dose-related inhibition of serum
    and erythrocyte cholinesterase activities was observed in mice at 5
    and 25 ppm (marginal inhibition of erythrocyte cholinesterase at 5
    ppm in both sexes). There were no significant changes of brain
    cholinesterase activity up to the highest dose level.

         Trivial differences in organ weights were observed between
    controls and dosed mice. Both neoplastic and non-neoplastic
    histopathological observations were spontaneous or naturally
    occurring lesions of aging mice. Renal amyloidosis was considered to
    be the most frequent "cause of death" among control and treated
    animals, the incidence being comparable in all groups. A higher
    incidence of testicular atrophy and degeneration was observed in
    treated mice but a dose-effect relationship was lacking. No
    differences in the incidence of neoplastic lesions were found when
    treated mice were compared to controls.

         Pyrazophos did not cause adverse effects up to the highest
    nominal dose of 25 ppm, equal to 3.5 and 4.1 mg/kg bw/day in males
    and females, respectively. However, the poor correspondence of
    actual and nominal concentrations of pyrazophos in the diets and the
    lack of toxic effects up to the highest dose level hampered a
    thorough evaluation of this study (Griggs  et al., 1982).

    Rats

         Five groups of 30 Wistar albino rats/sex were treated with
    technical grade pyrazophos (purity 90%) at dietary concentrations of
    0, 5, 8, 10 and 50 ppm for 104 weeks (except the 8 ppm group which
    was discarded at week 20). General condition, behaviour, body-
    weight, food and water intake were recorded throughout the duration
    of the study. Haematology, blood biochemistry, cholinesterase
    activity in plasma, erythrocytes and brain and urinalysis were
    measured. Pathology (organ weights) and histopathology (incomplete)
    were performed at the end of the study.

         Pyrazophos administration did not affect general condition,
    behaviour, body-weight gain, food and water intake of animals at any

    dose level. At the end of the study the mortality rate was 40%, 70%,
    47% and 60% in males and 27%, 47%, 30% and 33% in females at 0, 5,
    10 and 50 ppm, respectively. In the 50 ppm dose group, white blood
    cell counts significantly decreased in males at week 102 only
    (differential count normal). Glicemia was increased in the 10 and 50
    ppm dose groups in males at week 52. Significant inhibition of
    plasma and erythrocyte cholinesterase activity was measured in both
    sexes at the 10 and 50 ppm dietary levels. Brain cholinesterase
    activity was unaffected at the end of the study. A decrease in the
    relative spleen weight was observed in females only at all dietary
    levels. It was not dose-related and was not accompanied by
    treatment-related histological changes. Both neoplastic and non-
    neoplastic histopathological observations were those of spontaneous
    or naturally occurring lesions of aging albino rats. No differences
    in the incidence of neoplastic or non-neoplastic lesions were found
    when treated rats were compared to controls. In conclusion scattered
    differences in blood parameters were not suggestive of a clear toxic
    effect of pyrazophos. The NOAEL in this study was 50 ppm, equivalent
    to 2.5 mg/ kg bw/day. (Til  et al., 1979a)

         Four groups of fifty Wistar rats/sex, 4 weeks of age, were
    treated via pelleted feed with technical pyrazophos (95.7% purity)
    at dietary concentrations of 0, 2, 80 or 320 ppm equal to 0, 0.1,
    4.0 or 15.9 mg/ kg bw/ day for males and 0, 0.1, 4.8 or 19.3 mg/kg
    bw/ day for females for 117 weeks. Ten and 20 additional
    rats/sex/dose were treated with pyrazophos for 52 and 105 weeks,
    respectively. Pyrazophos was dissolved in acetone, mixed with
    microgranulated feed and pelleted. This preparation was performed
    every two weeks. Pyrazophos was stable at room temperature for at
    least 21 days. Homogeneity (range of variation between -15% and 22%
    of the mean concentration) and content of the test article in the
    feed (mean concentrations 98.5%, 95.6% and 94.5% of the nominal
    concentrations at 2, 80 and 320 ppm, respectively) were determined
    before the beginning of the study and every two months throughout
    the duration of the study. Parameters monitored were
    viability/mortality, clinical signs, food and water consumption,
    body-weight, ophthalmoscopic examination, hearing test, haematology,
    clinical biochemistry including cholinesterase activity
    determination, urinalysis and pathology.

         A slight but statistically significant increase of body-weight
    was detected in both sexes at 320 ppm during the second year of the
    study which corresponded to a slight increase of food intake.

    Absolute water intake was not affected in any group resulting in a
    slight decrease of relative water intake at the highest dose level.
    The incidence of clinical signs and palpable masses during life and
    the results of ophthalmoscopic examinations and hearing tests were
    comparable between groups. At the end of the 117 weeks treatment,
    mortality rate was 60%, 40%, 54% and 52% (male) and 60%, 56%, 38%
    and 48% (female) at 0, 2, 80 and 320 ppm, respectively.

         Slight decrease of total lipid content in blood which
    corresponded to lower cholesterol and triglyceride concentrations
    was measured in both sexes at 80 and 320 ppm but was not seen
    consistently throughout the duration of the study. Other haemato-
    logical and biochemical parameters and urinalysis measured at 26,
    52, 78 and 105 weeks were unaffected by pyrazophos administration.
    Dose-related inhibition of plasma and erythrocyte cholinesterase was
    detected at 80 and 320 ppm throughout the duration of the study.
    Absent or marginal (between 13% and 23%) inhibition of brain
    acetylcholinesterase was measured at 320 ppm in both sexes. No
    biologically significant differences in organ weights were detected
    at 52, 105 or 117 weeks. Histopathology showed a slight increase of
    gastric lesions (ulcers and erosions) in males at 320 ppm; several
    other non-neoplastic lesions were considered unrelated to test
    substance administration. An increased incidence of hemangiomas in
    mesenteric lymph nodes was observed at 105 and 117 week
    necroscopies. Hemangiomas occurred at incidences of 12%, 13%, 30%
    and 33% (males) and 6%, 4%, 26% and 16% (females) at 0, 2, 80 and
    320 ppm, respectively. This type of neoplasm was pratically absent
    at 52 weeks necroscopies. A statistically significant positive trend
    was present for males only. Hemangiosarcomas were not reported in
    mesenteric lymph nodes. The incidence of this type of neoplasm in
    historical controls is 8% (0-24) and 2% (0-8) in males and females,
    respectively (mean, range in brackets). Increased incidence of skin
    fibroma (still within the range of historical controls) was detected
    in males at the highest dose level (10%) in comparison with controls
    (0%). The NOAEL was 2 ppm, equal to 0.1 mg/kg bw/day in both sexes
    based on a higher incidence of hemangiomas in mesenteric lymph nodes
    detected in males at the higher doses (Tennekes  et al., 1991).

         In a supplementary study, two groups of 20 Wistar rats/sex were
    treated via pelleted feed with technical pyrazophos (95.7% purity)
    at dietary concentrations of 0 or 1 ppm equal to 0 or 0.05 mg/kg
    bw/day for males and 0 or 0.06 mg/kg bw/day for females for 104
    weeks. Monitored parameters were observation for

    viability/mortality, clinical signs, food and water consumptions,
    body-weight, ophthalmoscopic examination, hearing test,
    cholinesterase determination in plasma, erythrocytes and brain and
    pathology (organ weights and macroscopic examinations). Mortality
    rate at 104 weeks was 25% in both groups for males and 25% and 15%
    at 0 and 1 ppm, respectively for females. All tested parameters were
    unaffected by pyrazophos treatment. The NOAEL was 1 ppm of
    pyrazophos, equal to 0.05 mg/kg bw/day and 0.06 mg/kg bw/day for
    male and female rats, respectively (Tennekes  et al., 1991)

    Reproduction studies

    Rats

         In a 3-generation, 2-litter/generation study, four groups of 10
    male and 20 female rats (CIVO-colony) approximately 3-4 weeks old at
    the beginning of the study were treated with pyrazophos (90% purity
    grade) at dietary concentrations of 0, 5, 10 or 50 ppm. The diets
    were freshly prepared every two weeks and stored at room temperature
    (homogeneity and stability of test substance in diets not reported).
    Rats of the F0 generation were maintained on their respective
    diets for 13 weeks prior to mating and then up to weaning of the
    F1b generation. Litters were culled to 8 pups on day 1. Rats from
    F1b litters (10 males and 20 females selected at weaning) were
    mated at weeks 14 and 23 post-weaning. Ten male and 20 females of
    F2b litters were selected at weaning and mated at weeks 13 and 22
    after weaning. F1a, F2a and F3a litters were killed post-
    weaning. Rats from the F3b generation (10 males and 10 females)
    were selected and continued on the same diet for a 90-day feeding
    study.

         The following parameters were evaluated in the present study:

         -     Reproduction phase: litter size, pup survival and litter
               weight at day 1, 10 and 20.

         -     90-day study: signs of toxicity, food intake, body-weight,
               haematology, blood chemistry (included cholinesterase
               activity, brain cholinesterase activity, urinalysis and
               pathology.

         No relevant changes in reproductive parameters were observed.
    An increased percent mortality of pups both at day 10 and 20 was

    detected at the highest dose level after the second mating of the
    F0 generation only. Signs of toxicity were not detected during the
    90-day study. Dose-related inhibition of whole blood cholinesterase
    activity was measured at 10 and 50 ppm but brain cholinesterase
    activity was normal up to the highest dose level. Blood chemistry
    and urinalysis did not show changes related to the administration of
    the test substance. Total leucocyte counts were increased at 50 ppm
    in both sexes (likely because of lymphocyte increase). The absolute
    and relative weights of the thymus were increased at the two highest
    dose groups in both sexes. This change did not correspond to any
    histological abnormality. Pathology did not show changes
    attributable to pyrazophos administration. The overall NOAEL in this
    combined study (reproduction study and 90-day study) was 5 ppm equal
    to 0.45 and 0.42 mg/kg bw/day for males and females, respectively,
    based on increased weight of thymus observed at 10 and 50 ppm and
    increased lymphocyte count observed at 50 ppm in both sexes (Til  et
     al., 1979b)

         In a two-generation one-litter study/generation, groups of 25
    male and 25 female rats (Wistar/HAN) approximately 6 weeks old at
    the beginning of the study were treated with pyrazophos technical
    (95.7% purity) at dietary concentrations of 0, 2, 20 or 200 ppm.
    Homogeneity and stability of pyrazophos in the diet were checked and
    found acceptable. Parental rats (F1-generation) were maintained on
    their respective diets for 70 days prior to mating. The F0
    generation received the test compound in the diet for 126 days prior
    to mating. Both F0 and F1 generations received the test
    substance during the pairing, gestation and lactation periods.

         Trivial differences in food intake and body-weight gain were
    observed throughout the duration of the study in F0 and F1
    generations. Fertility index, gestation index and viability index
    were unaffected by pyrazophos administration in both generations.
    Lactation index was not affected in litters of the F0 generation
    but it was significantly reduced in litters of the F1 generation
    at 200 ppm. In both the F1 and F2 pups, no treatment-related
    abnormal findings were observed at external examination and the sex
    ratio of the pups was unaffected by treatment. At 200 ppm, reduced
    body-weight gain of the pups during lactation was evident in both
    the F1 and F2 pups; additionally, in the F2 pups, the initial
    body-weight was significantly reduced. Trivial differences in body-
    weight gain were also observed at 20 ppm. Dose-related inhibition of
    both plasma and erythrocyte cholinesterase activity was measured in

    both sexes at 20 and 200 ppm. The inhibition was evident in F0 and
    F1 generations and also in pups (plasma cholinesterase only was
    measured in pups). The extent of cholinesterase activity inhibition
    was higher in erythrocyte than in plasma. Slight inhibition of brain
    cholinesterase activity was measured only in females, at the highest
    dose level in both generations. Statistically significant inhibition
    of brain cholinesterase of no biological relevance was measured in
    F2 pups of both sexes at 200 ppm. Several statistical differences
    of organ weights between groups were judged of no biological
    relevance. Pathology did not show treatment-related abnormal
    findings either in parent animals or in pups. The NOAEL in this
    study was 20 ppm, equivalent to 1 mg/kg bw/day, based on retardation
    of body-weight gain of pups and brain cholinesterase inhibition
    measured in F0 and F1 generations (females) and in pups of the
    F2 generation. However, neither thymus weight nor lymphocyte
    counts (affected parameters in the previously performed reproduction
    study) were checked in this study (Suter  et al., 1991).

    Special studies on delayed neuropathy

    Hens

         Six white Leghorn hens were treated orally with 150 mg/kg of
    pyrazophos diluted (10%) in sesame oil on two separate occasions 21
    days apart. Another six hens received a single dose of
    atropine/toxogonin i.v. before being similarly dosed with
    pyrazophos. In preliminary studies of acute toxicity in hens, the
    LD50 was set between 150 and 400 mg pyrazophos/kg bw . A third
    group of 6 hens received a single dose of 500 mg tri-o-
    cresylphosphate/kg bw (positive controls) and a fourth group of 6
    animals received sesame oil (negative controls). All hens were
    sacrificed on day 42 after first dosing and histological examination
    of cerebrum, cerebellum, spinal cord and the nerves of the brachial
    and lumbosacral plexus was performed.

         Pyrazophos-treated hens (both groups) showed symptoms of the
    type expected for cholinergic toxicity. Positive controls showed
    symptoms suggestive of delayed neuropathy. Negative controls did not
    develop toxic symptoms. Histopathology did not reveal significant
    changes in pyrazophos treated hens and in negative controls. In
    positive controls demyelinization and glial cell proliferation were
    observed in the optic tract, in the medulla and in the cervical,
    thoracic and lumbar segments of the spinal cord. Peripheral nerves

    were unaffected in all animals. According to the results of this
    study, pyrazophos did not cause delayed neuropathy (Hollander &
    Weigand, 1979a)

    Special studies on embryotoxicity and teratogenicity

    Rats

         Four groups of twenty mated female Wistar rats were dosed with
    pyrazophos at concentrations of 0, 0.5, 1.6 or 5.0 mg/kg bw/day
    orally by gavage (vehicle, starch mucilage) on days 7, 10 and 16 of
    gestation. On day 21 of gestation, animals were killed and fetuses
    were examined for developmental disorders. Neither toxic signs nor
    mortality were observed in pyrazophos-treated or control pregnant
    rats. There were no significant differences in body-weight or feed
    consumption between the groups. Pathology did not reveal induced
    changes. There were no significant differences in the number of
    implantations per litter, incidence of live, dead or resorbed
    fetuses per litter or in the average weight of live fetuses between
    groups. The fetuses developed normally and showed no pyrazophos
    related external abnormalities, anomalies of the internal organs, or
    skeleton. The NOAEL in the present study was 5.0 mg pyrazophos/kg
    bw/day for both maternal toxicity and teratogenicity. Cholinesterase
    activity was not measured (Baeder  et al., 1978).

    Rabbits

         Groups of fifteen pregnant Himalayan rabbits were treated by
    gavage with pyrazophos (purity 95.6%) during gestation days 6-18 at
    doses of 0, 10, 32 or 100 mg/kg bw/day (vehicle, starch mucilage).
    On day 29 of gestation, the dams were killed. Internal, external and
    skeletal examination were performed on the fetuses. A slight
    reduction of food intake was observed in the pregnant rabbits at the
    highest dose level which corresponded to some reduction of body-
    weight gain (not statistically significant). Intra-uterine
    developement of the embryos and the viability of fetuses were not
    affected at any dose level of pyrazophos. The morphological
    examination of the fetuses for development, external anomalies,
    alterations of internal organs and skeleton yielded no evidence of a
    teratogenic effect of pyrazophos. The NOAEL was 100 mg/kg bw/ day
    for both maternal toxicity and terato-genicity. Cholinesterase
    activity was not measured (Baeder & Kramer, 1985; Baeder  et al.,
    1986).


    
    Special studies on genotoxicity

    Table 2. Results of genotoxicity assays on pyrazophos

                                                                                                                               
    Test system            Test object           Concentration of pyrazophos      Purity  Results        Reference
                                                                                                                               

    Ames test (1)          S. typhimurium        0.2-5000 g/plate dissolved in   ?       Negative (2)   Gericke (1977)
                           TA98, TA100, TA1535,  DMSO
                           TA1537

    Reversion assay (1)    E. coli WP2 hcr       10-5000 g/plate dissolved in    92.8%   Negative (2)   Shirasu  et al. (1979)
                           S. typhimurium        DMSO
                           TA98, TA100, TA1535,
                           TA1537, TA1538

    Rec-assay              B. subtilis           20-2000 g/disc dissolved in     92.8%   Negative (3)   Shirasu  et al. (1979)
                           H17 Rec + M45 Rec-    DMSO

    Mitiotic gene-         S. cerevisiae D4 (1)  800-1600 g/ml                   Afugan  Negative (4)   Bertoldi  et al. (1980)
    conversion             A. nidulans (5)       200-690 g/ml                    Afugan  Negative

    CHO/HGPRT mutation     Chinese hamster lung  6.25-75 g/ml (-act.)            97.3%   Negative (6)   Muller (1988)
    assay (1)              fibroblasts V79       25-150 g/ml (+ act.)

    Chromosome             Human lymphocytes     1-50 g/ml x 47 h (- act.)       95.6%   Negative (7)   Taalman  et al. (1985)
    aberrations                                  10-175 g/ml x 1 h (+ act.)
                                                 dissolved in DMSO

    Micronucleus test      Mouse (NMRI strain)   50, 75, 100 mg/kg p.o. treated   95.6%   Negative (8)   Mayer  et al. (1985)
                                                 twice 24 h apart

                                                                                                                               

    Table 2 (continued)

    (1)  Both with and without metabolic activation.
    (2)  Positive controls yielded expected positive responses.
    (3)  Kanamycin (10 g/disc) and Mitomycin C (0.1 g/disc) gave expected negative and positive response, respectively.
    (4)  Positive control (methylmethanesulphonate) gave expected positive response at 500 g/ml.
    (5)  Also using germinating conidia.
    (6)  Positive control (EMS 1 mg/ml (- act.); DMBA 7.7 g/ml ( + act.) gave expected positive responses.
    (7)  Positive controls (EMS 0.2 l/ml (- act.); cyclophosphamide 18.7 g/ml (+ act.) gave expected positive responses.
    (8)  Positive control (cyclophosphamide) gave expected positive response at 50 mg/kg bw (single dose).


    
    Special study on antidote effect

    Rats

         Female SPF-Wistar rats were dosed orally with a single
    pyrazophos (94% pure) dose of 800 mg/kg bw (4 x LD50). A single
    dose (10 minutes after pyrazophos) of either atropine methyl
    nitrate, atropine sulphate or obidoxime or combinations of these
    antidotes given i.p. delayed the time of death without modifying
    survival rate. The best treatment was a combination of atropine
    sulphate (3.75 mg/kg bw) and obidoxime (4 mg/kg bw) repeated several
    times starting 10 min after intoxication up to 57 h later, which
    increased survival rate from 0% to 90% (Sholz & Weigand, 1971).

         Male and female Wistar rats were dosed orally with a single
    pyrazophos dose (93.6% pure), approximately 2 x LD50. Where no
    therapeutic measures were taken, all animals with the exception of
    one female, died within three days. Prolonged treatment with
    atropine sulphate only did not increase survival rate. The
    combinations of both atropine sulphate + 2-PAM methiodide or
    atropine sulphate plus obidoxime proved to be efficient antidotes by
    reducing mortality to zero (Leist & Weigand, 1983).

    Observations in humans

         Five healthy humans received 0.15 mg/kg bw of pyrazophos daily
    for 10 days. Other 6 subjects received 0.15 mg/kg bw of pyrazophos
    for 3 days, followed by 7 daily doses of 0.07 mg/kg bw. Another 11
    subjects received 0.07 mg/kg bw of pyrazophos for 10 days.
    Pyrazophos was always administered orally, in orange juice, at
    breakfeast. Males and females were similarly represented in the
    groups. Controls received orange juice only. The great majority of
    controls were the same subjects who had previously taken pyrazophos.

         All subjects were checked for appearance of clinical symptoms,
    alterations of cardiovascular parameters (blood pressure, pulse
    rate, ECG). Moreover, haematology, blood biochemistry, urinalysis
    and quantitative assay of cholinesterase in plasma, whole blood and
    erythrocytes were performed on several occasions before, during and
    up to 7 days after the last administration of pyrazophos.
    At the highest dose, only two subjects completed the study as two
    withdrew because of upper respiratory infection and dosing was
    discontinued in one subject because of low baseline cholinesterase
    level after 3 doses (sic). Three out of six subjects in the
    0.15/0.07 mg/kg bw group reported adverse reactions of the type:
    mild headache, "feeling faint" and slight abdominal disconfort. Two
    subjects in the lowest dose group referred: dizziness and
    lightheadedness. The other two in the same group referred symptoms
    which could be related to upper tract respiratory infection. No
    alterations of cardiovascular parameters were found. Blood and urine
    analysis were within the normal range except for a sharp increase of

    serum CPK measured in one subject of the lowest-dose level. CPK was
    increased from the first up to the last day of pyrazophos
    administration (a pre-dosing normal level was reported) showing a
    maximum on day 4 (CPK was not measured during the 7 day recovery
    period). This subject showed similar inhibitions of plasma and whole
    blood cholinesterase activities of about 25% during the last 3 days
    of dosing (average inhibition calculated by comparison of the mean
    of cholinesterase activity on days 9-10-11 of the study with the
    mean of the three pre-dosing values). Cholinesterase activities in
    plasma and erythrocytes were normal on the day when CPK was at its
    maximum. Other biochemical parameters were all in the normal range.
    The possible explanation for increased CPK offered by authors was
    running activity performed by the subject during the study.

         Cholinesterase values within each group showed that, at 0.15
    mg/kg bw inhibition of plasma cholinesterase was observed starting
    on day 6 (approximately 20-50%) and persisting up to 4 days after
    the last dose of pyrazophos. In this group marginal inhibition of
    whole blood cholinesterase was noted but cholinesterase activity was
    not affected in erythrocytes. Cholinesterase activity was not
    affected in the other groups.

         Evaluation of this study is hampered by the presence of several
    methodological problems and lack of details. Controls were mainly
    the same subjects who had previously taken pyrazophos. Upper
    respiratory disorders are not substantiated by alterations of
    haematological parameters like ESR or increased WBC and cholinergic
    toxicity was not ruled out. Cholinesterase activities show a great
    intra-individual variability (greater than 10-20% which is the
    normal intra-individual variation in day-by-day analysis). The NOAEL
    for erythrocyte cholinesterase inhibition in this study is 0.15 mg
    of pyrazophos/kg bw/day for 10 days, however 3/5, 3/6 and 5/11
    subjects in the three groups reported symptoms of the type expected
    for cholinergic toxicity (Dinsdale & Protheroe, 1983).

         An unknown quantity of Afugan 30 EC squirted out of a damaged
    metal container into the face of a 36-year-old male worker, mainly
    in the orbital region. The worker was soon hospitalized. Both eyes
    developed conjunctivitis. Mild systemic clinical effects of the
    cholinergic type developed within 2 hours, namely miosis, tremors,
    muscle twitchings, isolated spasms, psychic agitation, bronchial
    spasticity and bradycardia. The patient received a total of 4 mg
    atropinum sulfuricum i.v. and as soon as systemic symptoms
    disappeared on the same day of poisoning, he was transferred to the
    out-patients department. Corrosion of the conjunctivae of both eyes
    and corrosion of the cornea of the right eye were diagnosed.
    Cholinesterase activity measured both in the serum and in
    erythrocytes at approximately 3 and 7 h after intoxication, in the
    presence of clinical symptoms, resulted in the normal range (Stasik,
    1987).

    COMMENTS

         In rats after a single or 14 daily (only the last dose
    radiolabelled) oral administrations of 14C-pyrazophos, blood
    radioactivity peaked within six hours after administration. The
    half-life was approximately five hours. Radioactivity was eliminated
    mainly via urine (71-78%) and faeces (16-24%). The parent compound
    accounted for most of the radioactivity detected in faeces,
    indicating incomplete absorption from the gastrointestinal tract.
    Intact pyrazophos was not found in urine, where the radioactivity
    mostly corresponded to its hydrolysis products.

         Pyrazophos was moderately toxic after single oral doses to
    mice, rats and dogs. No significant differences between sexes or
    routes of administration were detected. WHO has classified
    pyrazophos as moderately hazardous (WHO, 1992).

         In two 28-day studies in mice at dietary concentrations of 0,
    1, 5, 25 or 125 ppm (the highest dose in only one study), the NOAEL
    was 25 ppm equal to 4.7 and 5.0 mg/kg bw/day for males and females,
    respectively. The NOAEL was based on 20% inhibition of brain
    cholinesterase observed at 125 ppm.

         In a 13-week study in rats at dietary concentrations of 0, 2.5,
    50 or 1000 ppm the NOAEL was 2.5 ppm, equal to 0.21 mg/kg bw/day, in
    both sexes, based on brain cholinesterase inhibition at the end of
    the study at 50 ppm.

         In a 52-week study in rats at dietary concentrations of 0, 2,
    20 or 200 ppm the NOAEL was 20 ppm, equal to 1.0 and 1.4 mg/kg
    bw/day in males and females, respectively. The NOAEL was based on
    30% inhibition of brain cholinesterase activity in females at 200
    ppm.

         In a 92-day (moist semi-solid diet) study in dogs at dietary
    concentrations of 0, 0.5, 2.0, 5.0 or 10/125/320 ppm the NOAEL was 5
    ppm, equivalent to 0.4 mg/kg bw/day, based on inhibition of
    erythrocyte cholinesterase activity at the next highest dose (brain
    acetylcholinesterase activity was not determined). Clinical signs of
    the cholinergic type were observed when the dietary concentration
    was raised to 320 ppm.

         In a 6-month study in dogs at dietary levels of 0, 1.2, 18 or
    320 ppm pyrazophos (moist semi-solid diet), the NOAEL was 1.2 ppm,
    equivalent to 0.09 mg/kg bw/day, based on marginal brain
    acetylcholinesterase inhibition observed at 18 ppm.

         In a two-year study in dogs at dietary concentrations of 0, 2,
    5 or 320 pm (moist semi-solid diet), the NOAEL was 5 ppm, equivalent
    to 0.4 mg/kg bw/day based on erythrocyte cholinesterase inhibition,

    reduced body-weight gain and histopathological abnormalities
    observed in the kidneys of dogs fed 320 ppm.

         In a 92/96 (female/male)-week study in mice at dietary
    concentrations of 0, 1, 5 or 25 ppm, pyrazophos did not cause
    adverse effects up to the highest nominal concentration of 25 ppm,
    equal to 3.5 and 4.1 mg/kg bw/day in males and females,
    respectively. Inhibition of serum and erythrocyte cholinesterase
    activities, but not of brain acetylcholinesterase activity, was
    observed at 5 ppm and above. The poor correspondence between actual
    and nominal concentrations of pyrazophos in diets hampered
    definitive evaluation of this study.

         In a two-year study in rats at dietary levels of 0, 2, 80 or
    320 ppm, the NOAEL was 2 ppm equal to 0.1 mg/kg bw/day, based on a
    higher incidence of hemangiomas in mesenteric lymph nodes detected
    in males at the higher doses. Marginal brain acetyl cholinesterase
    inhibition was noted at 320 ppm only.

         In a two-year study in rats at dietary concentrations of 0, 5,
    8, 10 or 50 ppm, the NOAEL was 50 ppm, equivalent to 2.5 mg/kg
    bw/day, based on the absence of adverse effects including brain
    acetyl cholinesterase inhibition at this dose level. No compound-
    related abnormalities were detected in mesenteric lymph nodes.

         In a two-litter, three-generation study in rats (a 90-day
    toxicity study was also conducted on the F3b generation) at
    dietary levels of 0, 5, 10 or 50 ppm, the NOAEL was 5 ppm, equal to
    0.45 and 0.42 mg/kg bw/day for males and females, respectively,
    based on increased thymus weight observed at 10 and 50 ppm and
    increased lymphocyte counts observed at 50 ppm in both sexes in the
    90-day toxicity study.

         In a two-generation reproduction study in rats at dietary
    concentrations of 0, 2, 20 or 200 ppm of pyrazophos, the NOAEL was
    20 ppm, equivalent to 1 mg/kg bw/day, based on retardation of body-
    weight gain of pups of both generations, reduced lactation index of
    the F1 generation and slight inhibition of brain acetyl
    cholinesterase activity in parental females and in pups of the F2
    generation.

         Pyrazophos did not cause delayed neuropathy in hens.

         Pyrazophos was not teratogenic in rats or rabbits. The NOAELs
    for both maternal and embryofetal toxicity in rats was 5 mg/kg
    bw/day and in rabbits 100 mg/kg bw/day, the highest doses tested.
    Maternotoxicity was not observed. However, cholinesterase activity
    was not measured.

         Male and female human volunteers received pyrazophos orally at
    0.07, 0.07/0.15 or 0.15 mg/kg bw/day for 10 days. At the highest

    dose level only plasma cholinesterase activity was inhibited (20-
    40%), with marginal inhibition of erythrocyte cholinesterase
    activity. Symptoms which could be attributed to cholinergic toxicity
    were observed in all groups. The study was considered inadequate
    because of deficiencies in its design and conduct.

         After reviewing the available genotoxicity data, it was
    concluded that pyrazophos was not genotoxic.

         The Meeting concluded, after consideration of the long-term
    studies and the genotoxicity data that pyrazophos was unlikely to
    pose a carcinogenic hazard for humans.

         An ADI was allocated on the basis of NOAELs in the two-year
    study in dogs and the three-generation study in rats, using a 100-
    fold safety factor.

    TOXICOLOGY EVALUATION

    Level causing no toxicological effect

         Mouse:      25 ppm, equal to 4.7 mg/kg bw/day (28-day study)

         Rat:        5 ppm, equal to 0.4 mg/kg bw/day (three-generation
                          reproduction study)

         Dog:        5 ppm, equivalent to 0.4 mg/kg bw/day (two-year
                          study)

    Estimate of acceptable daily intake for humans

         0-0.004 mg/kg bw

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

         Further observations in humans.

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    Baeder, C.H. & Kramer, M. (1985) Hoe 002873 - Active ingredient
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    Unpublished report No. GT85.1105 by Hoechst Pharma Fo.To., Germany.
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    Hollander, H. & Weigand, W. (1977a) Acute intraperitoneal toxicity
    of O,O-diethyl-O-(5-methyl-6-ethoxycarbonyl -pyrazolo -(1.5-a) -
    pyrimid-2-yl)thiono-phoaphate (Hoe 02873 OF AS201) to the male SPF-
    Wistar-rat (vehicle:sesame oil). Unpublished report No. 55/77 from
    Hoechst Pharma Fo. To. Germany. Submitted to WHO by Hoechst.

    Hollander, H. & Weigand, W. (1977b) Acute intraperitoneal toxicity
    of O,O-diethyl-O-(5-methyl-6-ethoxycarbonyl -pyrazolo- (1.5-a)-
    pyrimid-2-yl)thiono-phoaphate (Hoe 02873 OF AS201) to the female
    SPF-Wistar-rat (vehicle:sesame oil). Unpublished report No. 56/77
    from Hoechst Pharma Fo. To. Germany. Submitted to WHO by Hoechst.

    Hollander, H. & Weigand, W. (1977c) Hoe 02873 O F AS201. Irritance
    to the rabbit skin and eye mucose Unpublished report No. 252/77 from
    Hoechst Pharma Fo. To. Germany. Submitted to WHO by Hoechst.

    Hollander, H. & Weigand, W. (1977d) Hoe 02873 O F AS201. Acute oral
    toxicity to the male SPF-NMRI-mouse. Unpublished report No. 351/77
    from Hoechst Pharma Fo. To. Germany. Submitted to WHO by Hoechst.

    Hollander, H. & Weigand, W. (1977e) Hoe 02873 OF AS201. Acute oral
    toxicity to the female SPF-NMRI-mouse. Unpublished report No. 351/77
    from Hoechst Pharma Fo. To. Germany. Submitted to WHO by Hoechst.

    Hollander, H. & Weigand, W. (1977f) Acute oral toxicity of Hoe 02873
    OF AS201 to the male SPF-Wistar-rat (vehicle: sesame oil).
    Unpublished report No. 353/77 from Hoechst Pharma Fo. To. Germany.
    Submitted to WHO by Hoechst.

    Hollander, H. & Weigand, W. (1977g) Acute subcutaneous toxicity of
    Hoe 02873 O F AS201 to the male SPF-Wistar-rat (vehicle:sesame oil).
    Unpublished report No. 354/77 from Hoechst Pharma Fo. To. Germany.
    Submitted to WHO by Hoechst.

    Hollander, H. & Weigand, W. (1977h). Acute subcutaneous toxicity of
    Hoe 02873 O F AS201 to the female SPF-Wistar-rat (vehicle:sesame
    oil). Unpublished report No. 355/77 from Hoechst Pharma Fo. To.
    Germany. Submitted to WHO by Hoechst.

    Hollander, H. & Weigand, W. (1977i). Acute subcutaneous toxicity of
    Hoe 02873 O F AS201 to the male SPF-NMRI-mouse (vehicle: sesame

    oil). Unpublished report No. 356/77 from Hoechst Pharma Fo. To.
    Germany. Submitted to WHO by Hoechst.

    Hollander, H. & Weigand, W. (1977l) Acute subcutaneous toxicity of
    Hoe 02873 O F AS201 to the female SPF-NMRI-mouse (vehicle: sesame
    oil). Unpublished report No. 357/77 from Hoechst Pharma Fo. To.
    Germany. Submitted to WHO by Hoechst.

    Hollander, H. & Weigand, W. (1978a) Acute oral toxicity of Hoe 02873
    O F AS201 to the female SPF-Wistar-rat (vehicle: sesame oil).
    Unpublished report No. 1024/77 from Hoechst Pharma Fo. To. Germany.
    Submitted to WHO by Hoechst.

    Hollander, H. & Weigand, W. (1978b) Hoe 02873 OF AS 201:Acute oral
    toxicity to the male SPF-Wistar-rat. Unpublished report No. 293/78
    from Hoechst Pharma Fo. To. Germany. Submitted to WHO by Hoechst.

    Hollander, H. & Weigand, W. (1978c) Acute oral toxicity to the
    female SPF-Wistar-rat. Unpublished report No. 294/78 from Hoechst
    Pharma Fo. To. Germany. Submitted to WHO by Hoechst.

    Hollander, H. & Weigand, W. (1978d) Hoe 02873 OF AS201 Acute oral
    toxicity to the male and female Beagle dog (vehicle: starch
    suspension). Unpublished report No. 295/78 from Hoechst Pharma Fo.
    To. Germany. Submitted to WHO by Hoechst.

    Hollander, H. & Weigand, W. (1978e) Range finding test with Hoe
    02873 OF AS201 in a 28-day study with SPF-mice. Unpublished report
    No. 604/78 from Hoechst Pharma Fo. To. Germany. Submitted to WHO by
    Hoechst.

    Hollander, H. & Weigand, W. (1979a) Hoe 02873 OF AS201.
    Neurotoxicity study in hens. Unpublished report No. 16/79 from
    Hoechst Pharma Fo. To. Germany. Submitted to WHO by Hoechst.

    Hollander, H. & Weigand, W. (1979b) Hoe 02873 OF AS201 (active
    ingredient techn.). Acute oral toxicity to the male and female
    Beagle dog. Unpublished report No. 461/79 from Hoechst Pharma Fo.
    To. Germany. Submitted to WHO by Hoechst.

    Hollander, H. & Weigand, W. (1981) Acute percutaneous toxicity of
    Hoe 028731 - Active ingredient (Code: Hoe 02873 OF AS207) to the
    female rat. Unpublished report No. 573/81 from Hoechst Pharma Fo.
    To. Germany. Submitted to WHO by Hoechst.

    Hollander, H. & Weigand, W. (1982) Aerosol inhalation of Hoe 02873 -
    Active ingredient (Code: Hoe 02873 OF AS207) to the male and female
    SPF-Wistar-rat. 4 hr- LC 50. Unpublished report No. 152/82 from
    Hoechst Pharma Fo. To. Germany. Submitted to WHO by Hoechst.

    Lachmann G. (1986). Metabolism of 14C-pyrazophos in male and
    female rats after a single oral administration of 2 mg per animal.
    Unpublished report No. BIeV-V-66.588 from Battelle-Institute
    Toxikologie und pharmakologie Am Romerhof 35, 6000 Frankfurt am Main
    90, FRG. Submitted to WHO by Hoechst.

    Lachmann G. (1987). Metabolism of 14C-pyrazophos in male and
    female rats after a oral administration of 0.4 mg per animal (single
    and repeated dosing). Unpublished report No. BIeV-V-66.736 from
    Battelle-Institute Toxikologie und pharmakologie Am Romerhof 35,
    6000 Frankfurt am Main 90, FRG. Submitted to WHO by Hoechst.

    Leist, K. H. & Weigand, W. (1982) Test for sensitizing properties of
    Hoe 02873- Active ingredient (Code: Hoe 02873 OF AS207) in the
    guinea-pig according to Buehler. Unpublished report No. 9/82 from
    Hoechst Pharma Fo. To. Germany. Submitted to WHO by Hoechst.

    Leist, K. H. & Weigand, W. (1983) Pyrazophos-active ingredient
    technical. Effects of antidotes tested on male and female Wistar
    rats. Unpublished report No. 83.0241 from Hoechst Pharma Fo. To.
    Germany. Submitted to WHO by Hoechst.

    Mayer, D. & Leist, K.H. (1985) Hoe 002873 OF ZD94 0001. Micronucleus
    test in male and female NMRI mice after oral administration.
    Unpublished report No. GT85.0492 from Hoechst Pharma Fo. To.
    Gremany. Submitted to WHO by Hoechst.

    Muller, W. (1988). Pyrazophos substance technical (Code: Hoe 002873
    OF ZD96 0001) detection of gene mutations in somatic cells in
    culture. HGPRT-test with V79 cells. Unpublished report No. 88.0333
    from Hoechst Toxicology, Germany. Submitted to WHO by Hoechst.

    Otaka, T., Nakayoshi, H. & Abe, M. (1981a) Acute toxicity study on
    pyrazophos. Oral administration in male rats. Unpublished report No.
    NRI79-2843 726/81a. Nomura Res.Inst., JPN. Submitted to WHO by
    Hoechst.

    Otaka, T., Nakayoshi, H., & Abe, M. (1981b) Acute toxicity study on
    pyrazophos. Oral administration in female rats. Unpublished report
    No. NRI79-2843 727/81a. Nomura Res.Inst., JPN. Submitted to WHO by
    Hoechst.

    Otaka, T., Nakayoshi, H., & Abe, M. (1981c) Acute toxicity study on
    pyrazophos (Hoe 02873 O F AS201). Subcutaneous administration in
    male rats. Unpublished report No. NRI79-2843 728/81a. Nomura
    Res.Inst., JPN. Submitted to WHO by Hoechst.

    Otaka, T., Nakayoshi, H., & Abe, M. (1981d) Acute toxicity study on
    pyrazophos (Hoe 02873 OF AS201). Subcutaneous administration in
    female rats. Unpublished report No. NRI79-2843 729/81a. Nomura
    Res.Inst., JPN. Submitted to WHO by Hoechst.

    Otaka, T., Nakayoshi, H., & Abe, M. (1981e) Acute toxicity study on
    pyrazophos (Hoe 02873 O F AS201). Intraperitoneal administration in
    male rats. Unpublished report No.NRI79-2843 730/81a. Nomura
    Res.Inst., JPN. Submitted to WHO by Hoechst.

    Otaka, T., Nakayoshi, H., & Abe, M. (1981f) Acute toxicity study on
    pyrazophos (Hoe 02873 OF AS201). Intraperitoneal administration in
    female rats. Unpublished report No. NRI79-2843 731/81a. Nomura
    Res.Inst., JPN. Submitted to WHO by Hoechst.

    Otaka, T., Nakayoshi, H., & Abe, M. (1981g) Acute toxicity study on
    pyrazophos. Oral administration in male mice. Unpublished report No.
    NRI79-2843 732/81a. Nomura Res.Inst., JPN. Submitted to WHO by
    Hoechst.

    Otaka, T., Nakayoshi, H., & Abe, M. (1981h). Acute toxicity study on
    pyrazophos. Oral administration in female mice. Unpublished report
    No. NRI79-2843 733/81a. Nomura Res.Inst., JPN. Submitted to WHO by
    Hoechst.

    Otaka, T., Nakayoshi, H., & Abe, M. (1981i) Acute toxicity study on
    pyrazophos (Hoe 02873 O F AS201). Subcutaneous administration in
    male mice. Unpublished report No. NRI79-2843 734/81a. Nomura
    Res.Inst., JPN. Submitted to WHO by Hoechst.

    Otaka, T., Nakayoshi, H., & Abe, M. (1981l) Acute toxicity study on
    pyrazophos (Hoe 02873 O F AS201). Subcutaneous administration in
    female mice. Unpublished report No. NRI79-2843 735/81a. Nomura
    Res.Inst., JPN. Submitted to WHO by Hoechst.

    Otaka, T., Nakayoshi, H., & Abe, M. (1981m) Acute toxicity study on
    pyrazophos (Hoe 02873 O F AS201). Intraperitoneal administration in
    male mice. Unpublished report No. NRI79-2843 736/81a. Nomura
    Res.Inst., JPN. Submitted to WHO by Hoechst.

    Otaka, T., Nakayoshi, H., & Abe, M. (1981n) Acute toxicity study on
    pyrazophos (Hoe 02873 O F AS201). Intraperitoneal administration in
    female mice. Unpublished report No. NRI79-2843 737/81a. Nomura
    Res.Inst., JPN. Submitted to WHO by Hoechst.

    Otaka, T., Nakayoshi, H., & Abe, M. (1981o) Subacute toxicity study
    on pyrazophos (Hoe 02873 OF AS20). Unpublished report No. NRI79-2532
    738/81a. Nomura Res.Inst., JPN. Submitted to WHO by Hoechst.

    Otaka, T. (1983) Chronic (52 weeks) toxicity study of pyrazophos
    (Hoe 02873 OF AS 206) in rats. Unpublished report No. NRI82-7581,
    gt84/0336. Nomura Res.Inst., JPN. Submitted to WHO by Hoechst.

    Shirasu, Y., Moriya, M., & Koyashiki, R. (1979) Microbial
    mutagenicity testing on pyrazophos. From Inst. Environ. Toxicol.,
    JPN. Submitted to WHO by Hoechst.

    Sholz, J. & Brunk, R. (1973) W11099 = Hoe 2873. Toxicological
    examination. 92 day feeding trial in beagles.

    Sholz, J. & Brunk, R. (1973) Pyrazophos, active ingredient. 90 day
    oral toxicity study (feeding) in beagle-dogs. Supplement. Individual
    data. Analysis. Unpublished report No. 363/73 from Hoechst Pharma,
    Germany. Submitted to WHO by Hoechst.

    Scholz, J. & Weigand, W. (1971) Determination of the antidote effect
    in case of poisoning by Hoe 2873 = W11 099. By Hoechst Pharma Fo.
    To. Germany. Submitted to WHO by Hoechst.

    Scholz, J. & Weigand, W. (1972a) Acute oral toxicity determined
    with male and female rats. Hoechst Pharma Fo. To. Germany. Submitted
    to WHO by Hoechst.

    Scholz, J. & Weigand, W. (1972b) Acute intraperitoneal toxicity
    determined with male and female rats. Hoechst Pharma Fo. To.
    Germany. Submitted to WHO by Hoechst.

    Stasik, M.J. (1987) Acute human poisoning with the organophosphate
    pyrazophos (Afugan(R) 30EC). Doc. No. A44268 from Hoechst
    Werksaerztl. Abt., Germany. Submitted to WHO by Hoechst.

    Suter, P., Vogel, W., & Luetkemeier, H. (1991) Pyrazophos substance
    technical (Code: Hoe 002873 OF ZD96 0001) two-generation
    reproduction study in the rat. Unpublished report No. 071583 from
    RCC, Swiss. Submitted to WHO by Hoechst.

    Taalman, R. & Horn, A. (1985). Mutagenicity evaluation of
    pirazophos. Substance technical grade (Code: Hoe 002873 OF ZD94
    0001) in an  in vitro cytogenic assay measuring chromosome
    aberration frequencies in human lymphocytes. Final report.
    Unpublished report No. GT85.1217 from Litton Bionetics, NLD.
    Submitted to WHO by Hoechst.

    Tennekes, H., Janiak, T., Probst, D., Luetkemeier, H., Vogel, O.,
    Schlotke, B., Biedermann, K., & Heusner, W. (1991) Pyrazophos
    substance technical. Chronic toxicity/oncogenicity feeding study in
    rats. Unpublished report No. 071526 from RCC, Research and
    Consulting Company AG, Itingen/Switzerland. Submitted to WHO by
    Hoechst.

    Tennekes, H., Janiak, T., Probst, D., Luetkemeier, H., Vogel, O.,
    Schlotke, B., Biedermann, K., & Heusner, W. (1991) Pyrazophos
    substance technical. Chronic toxicity addendum to RCC project 071526
    satellite feeding study in rats with a supplementary test
    concentration. Unpublished report No. 209226 from RCC, Research and
    Consulting Company AG, Itingen/Switzerland. Submitted to WHO by
    Hoechst.

    Til, H.P., Leegwater, D.C., & Feron, V.J. (1978) Range-finding (28
    days) toxicity study in albino rats. Unpublished report No. R 5786.
    CIVO/TNO Netherlands. Submitted to WHO by Hoechst.

    Til, H.P., Dreef-van der Meulen, H.C., Leegwater, D.C., & Huismans,
    J.W. (1979a) Chronic (two-year) toxicity study with Hoe 2873 in
    rats. Unpublished report No. R6027. TNO, NDL. Submitted to WHO by
    Hoechst.

    Til, H.P., Spanjers, M. T., Dreef-van der Meulen, H.C., & Leegwater,
    D.C. (1979b) Multi-generation study with Hoe 2873 in rats.
    Unpublished report No. R5948 from TNO/NDL. Submitted to WHO by
    Hoechst.

    WHO (1992) The WHO recommended classification of pesticides by
    hazard and guidelines to classification 1992-1993 (WHO/PCS/92.14).
    Available from the International Programme on Chemical Safety, World
    Health Organization, Geneva, Switzerland.


    See Also:
       Toxicological Abbreviations