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    Pesticide residues in food -- 1999



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    with the support of the International Programme
    on Chemical Safety (IPCS)



    Toxicological evaluations




    Joint meeting of the
    FAO Panel of Experts on Pesticide Residues
    in Food and the Environment
    and the
    WHO Core Assessment Group

    Rome, 20-29 September 1999

    ETHOPROPHOS

    P.H. van Hoeven-Arentzen and J.G.M. van Engelen
    Centre For Substances And Risk Assessment 
    National Institute of Public Health and the Environment, 
    Bilthoven, The Netherlands

            Explanation
            Evaluation for acceptable daily intake
                Biochemical aspects
                    Absorption, distribution and excretion
                    Biotransformation
                    Effects on enzymes and other biochemical parameters
                Toxicological studies
                    Acute toxicity  
                    Short-term studies of toxicity 
                    Long-term studies of toxicity and carcinogenicity
                    Genotoxicity 
                    Reproductive toxicity 
                        Multigeneration reproductive toxicity
                        Developmental toxicity 
                    Special studies: neurotoxicity
                    Studies on metabolites
                        Acute toxicity 
                        Cholinesterase inhibition
                Observations in humans
            Comments 
            Toxicological evaluation 
            References


    Explanation

         Ethoprophos was evaluated for toxicological effects by the Joint
    Meeting in 1983 and 1987 (Annex 1, references  40 and  50). An ADI
    of 0-0.0003 mg/kg bw was established in 1987 on the basis of a NOAEL
    of 0.025 mg/kg bw per day in a 1-year study in dogs. Since that time,
    several studies have been conducted on the metabolism, acute,
    short-term and long-term toxicity, reproductive and developmental
    toxicity, neurotoxicity and mutagenicity of ethoprophos. The compound
    was evaluated at the present meeting within the periodic review
    programme of the Codex Committee on Pesticide Residues. 

    Evaluation for Acceptable Daily Intake

    1.  Biochemical aspects

    (a)  Absorption, distribution and excretion

         In a study conducted according to GLP with a statement of QA,
    groups of five male and five female Crl:CD(SD)BR rats were given
    [ethyl-1-14C]ethoprophos (radiochemical purity, 97%) by gavage in a
    0.9% solution of sodium chloride or intravenously in a solution of 35%

    ethanol in 0.9% sodium chloride. The animals received a single oral or
    intravenous dose of 4 mg/kg bw, an oral dose of 12.5 mg/kg bw, or an
    oral (pulse) dose of 4 mg/kg bw after 14 daily doses of 4 mg/kg bw of
    unlabelled ethoprophos (purity, 99.4%). Samples of urine and faeces,
    cage debris, and cage washings were taken for analysis at 6, 12, 24,
    48, 72, 96, 120, 144, and 168 h, at which time the animals were killed
    for analyses of radiolabel in various tissues. Expired air was
    captured in a trapping system at 6, 12, 18, 24, 36, 48, 60, 72, 96,
    120, 144, and 168 h. An additional group of five animals of each sex
    received a single oral dose of 4 mg/kg bw, a group of five males
    received a single oral dose of 25 mg/kg bw, and a group of five
    females received a dose of 12.5 mg/kg bw. (The original dose was 25
    mg/kg bw but was lowered to 12.5 mg/kg bw because of severe toxic
    effects.) Blood samples were taken from the lateral tail vein of these
    additional animals, before treatment, at 0.5, 1, 2, 3, 4, 6, 12, and
    24 h, and at subsequent 24-h intervals until 168 h for determination
    of radiolabel and pharmacokinetic parameters. 

         No abnormal clinical findings were noted after intravenous
    injection or after single or multiple oral administration of the low
    dose. Clinical signs were observed during the first few hours after
    oral dosing with the high dose, including teeth grinding, dyspnoea,
    poor posture, and hypoactivity in males and teeth grinding, severe
    tremors, and excessive salivation in females. One female was found
    dead and was replaced. 

         The patterns of excretion after the various dosing regimens are
    shown in Table 1. After the single intravenous injection, 94% of the
    administered dose was recovered in males and 90% in females. The
    primary route of excretion was the urine, which accounted for about
    57% of the administered dose, the the majority being excreted within
    12 h. Less was found in faeces (7-9%) and expired air (13-17%),
    although these were significant routes of elimination. Within 24 h,
    5.6% of the radiolabel was detected in faeces in males and 7.7% in
    females. The cage washings contained 11% of the administered dose for
    males and 8.6% for females. In animals of each sex, 2.0% of the
    administered dose was found in the carcass and 0.5% in liver; the
    total recovery in tissues, including carcass, was 2.7% of the
    administered dose. Residual radiolabel was found in all tissues, with
    the highest concentrations (expressed as equivalents) in liver, lung,
    kidney, abdominal fat, testis, and blood (0.3-0.5 g/g),
    concentrations of 0.1-0.2 g/g in heart, spleen, ovaries, and uterus,
    and < 0.1 g/g in other tissues including bone marrow.

         Similar profiles and rates of excretion were seen in animals
    treated orally with single or multiple doses of 4 mg/kg bw, although
    the amount of radiolabel in faeces was slightly higher (10-16%) than
    after intravenous injection (7-9%). As in animals treated
    intravenously, most of the radiolabel was recovered within 12 h in the
    urine and within 24 h in faeces. Less radiolabel was recovered in the
    tissues of animals given multiple doses (0.3% of the administered
    dose) than those given the single low dose (2%, with 1-1.5% in carcass
    and 0.6% in liver). The amounts of radiolabel were highest in liver,


        Table 1. Recovery of radiolabel, expressed as equivalents, after administration of 14C-ethoprophos to groups of five rats 
             of each sex

                                                                                                                              
    Treatment                           Sex    Recovery (% of administered dose)
                                                                                                                              
                                               Urine     Faeces    Expired air   Cage wash   Cage      Tissues        Total
                                                                                             debris    (+ carcass)
                                                                                                                              

    4 mg/kg bw intravenously            M      57        6.6       17            11          ND        2.8            94
                                        F      57        8.7       13            8.6         ND        2.7            90

    4 mg/kg bw orally                   M      52        16        19            3.0         ND        2.2            93
                                        F      50        12        12            13          ND        1.9            91

    4 mg/kg bw orally; repeated doses   M      54        12        14            10          ND        0.3            91
                                        F      59        10        13            10          ND        0.3            93

    12.5 mg/kg bw orally                M      58        12        13            4.1         0.2       1.5            88
                                        F      54        9.9       11            7.8         0.8       3.6            88
                                                                                                                              

    ND, not detected
    

    kidneys, and lungs, the organs associated with excretion, the
    concentrations being 0.2-0.8 g/g after the single low dose and
    0.1-0.2 g/g after mutliple doses. The concentrations were about 0.2
    and 0.1 g/g in abdominal fat for the respective dose groups and
    < 0.1 g/g in all other tissues including bone marrow.

         The pattern of excretion of radiolabel after the oral dose of
    12.5 mg/kg bw was similar to that with the intravenous and low single
    and multiple oral doses and was similar for males and females. Renal
    excretion predominated, and elimination in faeces and expired air
    accounted for a smaller but significant proportion of the administered
    dose. The highest concentrations of radiolabel, expressed as
    equivalents, were again found in liver, kidneys, and lungs and ranged
    from 0.4 to 0.9 g/g. Apart from abdominal fat, which contained about
    0.5 g/g, the concentrations in other tissues were 0.1-0.4 g/g. One
    female had a very high concentration in the uterus (190 g/g), which
    was considered to be due to contamination.

         Haematological pharmacokinetics also showed no apparent sex
    difference. A mean maximum concentration of about 1.5 g/g was
    achieved between 0.5 and 1 h after the single oral dose of 4 mg/kg bw.
    Female rats receiving 12.5 mg/kg bw achieved a mean maximal blood
    concentration of 4.6 g/g 0.5 h after dosing, while male rats
    receiving 25 mg/kg bw had a maximal blood concentration of 3.8 g/g
    around 0.6 h after dosing. The terminal elimination half-life of the
    radiolabelled material was 110-120 h at 4 mg/kg bw, 92 h at 12.5 mg/kg
    bw, and 140 h at 25 mg/kg bw. The systemic concentrations of
    radiolabel increased with dose but not in a directly proportional
    manner (the integrated area under the curve of
    concentration-time0-infinity being  60 g h/g at 4 mg/kg bw, 110 g
    h/g at 12.5 mg/kg bw, and 150 at 25 mg/kg bw g h/g (Yenne, 1990).

         Male and female rats given a single oral dose of
    [14C-ethyl]ethoprophos (1.3  106 counts/min) or
    [14C-propyl]ethoprophos (3  106 counts/min) excreted 55-65% of
    the administered radiolabel (actual doses not given) in the urine, and
    < 1% in the faeces. Most of the radiolabel in the urine was
    eliminated during the first 6 h after treatment and was in the form of
    hydrolytic products, including  O-ethyl- S-propyl phosphorothioic
    acid, which was the major urinary metabolite, accounting for
    approximately 40% of the administered dose. Other water-soluble
    metabolites identified in the urine included  O-ethylphosphoric acid,
     S-propyl-phosphorothiolic acid, and  S,S-dipropyl-phosphorodithioic
    acid. The urine of rats treated with the 14C-propyl-labelled
    compound also contained metabolites extractable in organic solvents,
    consisting of methyl propyl sulfide, methyl propyl sulfoxide, and
    methyl propyl sulfone, which accounted for about 2% of the
    administered radiolabel (Iqbal & Menzer, 1972).

    (b)  Biotransformation

         Samples of urine and faeces containing > 50 000 dpm/g from rats
    in the study of Yenne (1990) were pooled by sex, dose, and dose
    interval and examined for metabolites by both thin-layer and
    high-performance liquid chromatography. The biotransformation of
    [14C]ethroprophos did not appear to be dependent on sex, dose, or
    dose frequency. No parent compound was found in up to five
    chromatographically resolved fractions of urine and faeces. Analysis
    by gas chromatography and mass spectroscopy and 31P-nuclear magnetic
    resonance spectromtery confirmed the presence of ethyl phosphate and
     O-ethyl- S-propyl phosphorodithioate in the urine. One fraction was
    evaluated as a conjugate of ethylphosphate and two other fractions as
    conjugates of  O-ethyl- S-propyl phosphorothioate. The conjugates
    (not specified) of the two metabolites represent the majority (> 60%)
    of the total residue found in urine and faeces. Ethyl phosphate was
    the major metabolite in faeces. A proposed metabolic pathway for
    ethoprophos is shown in Figure 1.

    (c)  Effects on enzymes and other biochemical parameters

         The permeability of human, mouse, rat, and rabbit skin  in 
     vitro to undiluted or diluted [14C]ethoprophos (radiochemical
    purity, 95.5%) was compared over 24 h. An emulsified concentrate of
    [14C]ethoprophos was mixed with 10 g of unlabelled ethoprophos
    (purity, 70%), or one part of 70% unlabelled concentrate was mixed
    with 19 parts of water and [14C]ethoprophos was then added. Skin
    samples were obtained from laboratory animals and from the abdomens of
    human corpses; those from rabbits and humans were stripped of
    subcutaneous fat. The dermal sides of the samples were applied to
    penetrating wells containing 10 ml of normal saline buffer, and 2 ml
    were withdrawn for scintillation counting and replaced by 2 ml of
    normal saline at each interval. A 1.4-cm-wide plastic cylinder was
    fixed on the epidermal side of each specimen, and 0.02 ml of the test
    solutions was applied. The results are shown in Table 2. Less
    penetration was found through human skin than through that of the
    other species. The penetration rate was highest for mice and was equal
    for rats and rabbits. Undiluted ethoprophos penetrated more slowly
    than diluted ethoprophos, but a fairly consistent rate was
    established, especially in rabbits, mice, and rats, after 1 h
    (Stoughton, 1986). As radiolabel in the skin was not determined, this
    study provides only a qualitative indication of dermal penetration. 

         NADPH-dependent microsomal enzymes from the livers of rats and
    rabbits biotransformed [14C-ethyl]ethoprophos or
    [14C-propyl]ethoprophos to  O-ethyl- S-propylphosphorothioic acid,
    the major metabolite, and to  O-ethyl-phosphoric acid from
    [14C-ethyl]ethoprophos, or to  S-propyl phosphorothiolic acid from
    [14C-propyl]ethoprophos. Incubation of liver supernatant
    preparations from rats and rabbits with ethoprophos in the presence of
    reduced glutathione led to the formation of
     O-ethyl- S-propyl-phosphorothioic acid as the major metabolite.
     S-Ethyl glutathione and  S-S-dipropyl-phosphorodithioic acid were

    FIGURE 1

        Table 2. Penetration of undiluted or 1:19 diluted 14C-ethoprophos solutions through 
             the skin of various species (in % of applied dose)

                                                                                           
    Time       Undiluted ethoprophos                   Diluted ethoprophos
                                                                                           
               Human     Rabbit   Mouse      Rat       Human    Rabbit     Mouse     Rat
                                                                                           
    15 min     0         0.003    0.003      0.01      0.001    0.004      0.004     0.05
    30 min     0.0004    0.008    0.01       0.05      0.01     0.02       0.12      0.15
    1 h        0.0008    0.04     0.11       0.15      0.08     0.2        1.9       0.55
    2 h        0.002     0.2      0.91       0.4       0.31     1.1        4.2       1.7
    4 h        0.004     0.7      2.6        1.0       0.72     3.7        11        4.2
    6 h        0.08      1.5      5.0        1.7       1.1      7.7        29        7.8
    24 h       1.0       7.8      16         5.4       5.2      27         38        23
                                                                                           
    

    also formed when the 14C-ethyl- and the 14C-propyl-labelled
    compound, respectively, was the substrate (Iqbal & Menzer, 1972).

    2.  Toxicological studies

    (a)  Acute toxicity

         The acute toxicity of ethoprophos in animals is summarized in
    Table 3. In 1983, the Meeting reported that "in the studies evaluated
    the toxic symptoms in the treated species were characteristic of
    anticholinesterase poisoning and usually persisted for up to 48 h in
    mice and 72 h in rats surviving treatment. In general, deaths occurred
    within 4 h (mice) or 1 h to 3 days (rats) post-dosing. Information on
    duration of symptoms and time of death in rabbits was not available."

         Ethoprophos is considered to be toxic after single oral and
    dermal doses and very toxic after inhalation. The dermal and oral
    LD50 values for rabbits and mice are of the same order of magnitude
    and indicate efficient absorption of the compound after dermal
    exposure in these species, whereas less is absorbed dermally in rats. 

         A single application of 0.1 ml of undiluted technical-grade
    ethoprophos (equivalent to 44 mg/kg bw) into one eye of each of three
    New Zealand white rabbits immediately produced moderate erythema and
    vascularization of the sclera and nictating membrane. The substance
    was severely toxic, since all three animals died within 1 h of
    treatment (Weir, 1965).

         In a study of primary dermal irritation, all six male New Zealand
    white rabbits that received 0.5 ml of undiluted technical-grade
    ethoprophos (purity, 93%) at a dose equivalent to 240 mg/kg bw on
    clipped, abraded and unabraded skin and kept under an occlusive patch 
    died within the first 8 h of treatment (Becker & Parke, 1977).


        Table 3. Acute toxicity of ethoprophos

                                                                                                                                       
    Species     Strain            Sex      Purity    Vehicle           Route                LD50/LC50        Reference
                                           (%)                                              (mg/kg bw or 
                                                                                            mg/L)
                                                                                                                                       

    Mousea      OF1 (SPF)         M & F    NR        Water             Oral                 31               Pasquet & Mazuret (1982)

    Rat         SD                M        NR        Corn oil          Oral                 62b              Powers (1965) 
                                  F                                                         33b

    Ratc,d      Crl:CD (SD)       F        NR        Corn oil          Oral                 56               Weiler (1998)
                BR VAF/ Plus

    Rabbite     NZW               F        NR        NR                Oral                 33               Schwartz (1978)

    Mouse       CD-1              M        NR        Acetone           Dermal (24 h)        18b              Auletta & Rinehart (1979)

    Rata        CD (COBS)         M & F    NR        Water             Dermal (24 h)        226              Pasquet & Mazuret 
                                                                                                             (1982)

    Ratd,f      SD                M        NR        None              Dermal (24 h)        1280             Karcher et al. (1987); 
                                  F                                                         424              Blacker (1987)

    Rabbitg     NZW               M & F    NR        None              Dermal (24 h)        8.5              Karcher et al. (1986); 
                                                                                                             Blacker (1987)

    Pigh        Yorkshire white   M        94%       None              Dermal               327              Rucci (1979)

    Rabbith     Albino            M & F    NR        None or 40%       Dermal (intact       26b              Powers (1965)
                                                     solution in       and abraded 
                                                     corn oil          skin; 24 h) 

    Rat         Wistar            M & F    92%       None              Inhalation(4 h)      0.250j           Kopp et al. (1980)
                                                                                                                                       

    Table 3 (continued)

    NR, not reported
    a   Ethoprophos lot No. DA 232. Clinical signs: trembling, convulsions, hypomotility, and dyspnoea in both mice and rats; no dermal
        irritation in rats. Only 3 male and female mice and 2 male and female rats per dose.
    b   Doses reported as l/kg bw. They were converted to mg/kg bw by multiplying them by the specific gravity of ethoprophos at 
        20C, 1.094.
    c   Ethoprophos batch No. 51EAR122. Deaths occurred within 4 days. Clinical signs: thinness, staggered gait, salivation, 
        hypoactivity, hunched posture, and tremors.
    d   Statements of compliance with good laboratory practice and quality assurance included.
    e   Clinical signs: ataxia, diarrhoea, and decreased activity. Only 4 females per group. Information on duration of symptoms and 
        time of death not available.
    f   Technical-grade ethoprophos lot No. 302106001. Deaths occurred within 3 days. Clinical signs: urine staining, tremors, excess
        salivation, decreased activity, ataxia, soft stools, diarrhoea, laboured breathing, lachrymation, exophthalmus, and prostration, 
        in general increased with increasing dose. No signs of irritation. Considerable body-weight decreases in animals that died. 
    g   Ethoprophos lot No. 302106001. Deaths occurred within 4 days except for one on day 9. Clinical signs: prostration, laboured 
        breathing, salivation, loss of locomotor ability, tremor, ataxia, soft stools, diarrhoea, and lachrymation. No signs of 
        irritation. Considerable body-weight decreases in animals that died. Only statement of quality assurance included.
    h   Deaths occurred within 4 days. Clinical signs: laboured breathing, salivation, wobbly gait, and decreased activity. Erythema 
        was reported but no indication of severity was given. Only 2-4 males per group.
    i   Clinical signs: depression, laboured breathing, unsteadiness, tremors, salivation. Deaths occurred within 24 h. Very slight, 
        subsiding erythema observed in surviving animals 24-48 h after dosing on abraded (> 10 l/kg bw) and unabraded skin 
        (31.6 g/kg bw). Only 4 animals per group.
    j   4-h LC50 expressed as actual chamber concentration and particle size < 10 m; animals were exposed to the liquid 
        technical-grade material as an aerosol (nominal concentration, 1500 mg/m3) in a nose-only exposure chamber. Clinical signs: 
        apathy, dyspnoea, salivation (from JMPR, 1983, and slightly modified by reference to original report).
    


    (b)  Short-term studies of toxicity

         (i)  Oral administration

          Rats 

         Groups of 25 male and 25 female Charles River caesarean-derived
    rats were fed diets containing technical-grade ethoprophos (purity not
    stated) in acetone at concentrations of 0, 0.3, 1, or 100 ppm
    (equivalent to 0, 0.015, 0.05, and 5 mg/kg bw per day) for three
    months. The control group was given basal diet containing 'a volume of
    acetone equivalent to the volume used for the test diets'. No deaths
    or treatment-induced toxic signs were observed. Growth was depressed
    by < 10% in males at doses > 1 ppm and in females at 100 ppm
    during the last 6 weeks of the study. Food consumption was unaffected.
    Haematological, clinical chemical (only blood urea nitrogen, aspartate
    aminotransferase, and glucose determined), and urinary examinations in
    five males and five females per group after 1 and 3 months indicated
    no significant treatment-related findings. Cholinesterase activity in
    plasma, erythrocytes, and brain was measured in five rats of each sex
    per group at days 4, 8, 16, and 33 and terminally in the remaining
    rats. Marked inhibition (25-100%) of the enzyme activity was found at
    100 ppm at all sampling intervals, the depression being greatest for
    erythrocyte and least for brain acetylcholinesterase activity. Maximum
    inhibition of the enzyme occurred on day 8 or 16, in all tissues. Male
    rats at 0.3 or 1 ppm showed a 24-28% reduction in erythrocyte and
    plasma cholinesterase activity on day 8 and of brain cholinesterase
    activity at termination. In females, brain cholinesterase activity was
    inhibited by 20-25% at 0.3 and 1 ppm on day 8 and at 1 ppm on day 16,
    and plasma and erythrocyte cholinesterase activity was inhibited by
    23-26% at 1 ppm on day 16. At the end of the study, the absolute and
    relative weights of the adrenals were decreased, but not in relation
    to dose, in females in all treated groups. Histopathological
    evaluation of about 20 selected tissues, including the adrenals, from
    five males and five females in the control and highest-dose groups
    showed no significant changes attributable to treatment. No NOAEL
    could be identified, as brain acetylcholinesterase activity was
    inhibited at all doses (Weir, 1967a). As only five animals of each sex
    were examined for haematological, biochemical, and urinary parameters,
    instead of the 20 rats of each sex required by the OECD guideline, and
    few parameters were tested, the study was considered unreliable and
    was not used for further evaluation of the toxicology of ethoprophos.

          Dogs 

         In a study conducted according to GLP and with a statement of QA,
    ethoprophos (purity, 95.6%) was administered orally to groups of six
    male and six female beagle dogs aged 6-7 months at doses of 0, 0.01,
    0.025, or 1 mg/kg bw per day in corn oil in gelatin capsules for 20
    weeks.Two dogs of each sex at each dose were then allowed a 4-week
    recovery. The animals were observed for deaths and clinical signs.
    Body weights and food consumption were recorded, and ophthalmological
    examinations were performed before dosing and at sacrifice after the

    exposure (week 20) and recovery (week 24) periods. Haematological,
    biochemical, and urinary analyses were carried out before treatment
    and during weeks 17, 20, and 24. Cholinesterase activity in plasma and
    erythrocytes was measured before dosing and about 3 h after dosing
    during weeks 2, 4, 8, 12, 20, and 24. Brain cholinesterase activity
    was measured at sacrifice; at that time, the weights of about 10
    organs were determined and about 40 tissues and all gross lesions from
    all animals were examined macroscopically and microscopically.

         There were no deaths, and there were no treatment-related
    clinical signs or effects on body weight, body-weight gain, food
    consumption, ophthalmologic, haematological, biochemical, or urinary
    end-points, macroscopic or microscopic findings, or organ weights. No
    histopathological changes were found in the heart. The only finding
    was a statistically significant increase in platelet count in bitches
    at the high dose at weeks 17 and 20. This was considered to be of no
    toxicological relevance since the value was increased before
    treatment, and an elevated value was observed after recovery.
    Significant inhibition of plasma cholinesterase activity was observed
    in bitches at the intermediate dose and males and females at the high
    dose. In animals at the high dose, erythrocyte acetylcholinesterase
    activity was inhibited by more than 20% in males at all times during
    exposure and in females only at weeks 8 and 12, when the values
    reached statistical significance in males. Brain acetylcholinesterase
    activity was not inhibited. After the recovery period, plasma
    cholinesterase activity was found to have returned to normal, but the
    activity in erythrocytes showed some variation and it could not be
    concluded that recovery had occurred. The NOAEL was 1 mg/kg bw per
    day, the highest dose tested, in the absence of inhibition of brain
    acetylcholinesterase activity (Hamada, 1990). 

         Groups of three male and three female young adult pure-bred
    beagles weighing 6-12 kg were fed diets containing technical-grade
    ethoprophos (purity unknown) at concentrations of 0, 1, 3, or 100 ppm
    for 13 weeks, equal to 0, 0.034, 0.098, or 3.4 mg/kg bw per day for
    males and 0, 0.035, 0.11, or 4.0 mg/kg bw per day for females. There
    were no deaths. Emesis occurred once in two animals at 100 ppm. Body
    weights, food consumption, haematological, biochemical (glucose, blood
    urea nitrogen, and aspartate aminotransferase and alkaline phosphatase
    activity), and urinary parameters measured at 1 and 3 months were not
    significantly affected by treatment. At termination, however, males
    and females at the high dose had slightly decreased erythrocyte counts
    and erythrocyte volume fractions. Plasma cholinesterase activity was
    measured before dosing and on days 2, 4, 8, 16, 35, 65, and 87 of
    administration. The activity was inhibited by 27-88% at doses > 3
    ppm in animals of each sex at virtually all of the sampling intervals
    and by 20-27% at 1 ppm in males at the two last sampling times. A
    > 20% depression of erythrocyte cholinesterase activity was seen in
    animals at the highest dose at all but the first two or three sampling
    intervals and in females at 3 ppm on one occasion (day 65) only.
    Cholinesterase activity in brain was not measured. At termination, no
    compound-related effects on organ weights or gross pathological
    changes were noted. The only significant finding on histopathological

    evaluation of about 20 tissues from each animal in the control and
    high-dose groups was foci of perivascular myocardial swelling with
    loss of striations in one of three female controls and two of three
    males and one of three females at 100 ppm; all of these animals also
    showed swelling and vacuolation of Purkinje fibres. These changes were
    considered by the pathologist to represent 'an artefact induced in the
    processing of the tissues'. The NOAEL was 3 ppm, equal to 0.098 mg/kg
    bw per day, on the basis of depression of erythrocyte cholinesterase
    activity at 100 ppm (Weir, 1967b). 

         Groups of four male and four female pure-bred beagle dogs aged
    4-6 months were given capsules containing ethoprophos (purity, 96%)
    dissolved in peanut oil at doses of 0, 0.025, 1.0, or 10 mg/kg bw per
    day orally for 52 weeks. The doses were based on the results of a
    4-week range-finding study. The animals were offered 400 g/day of food
    and water  ad libitum and were observed daily for signs of toxicity;
    body weights were recorded weekly and food consumption daily. Blood
    and urine were collected before treatment and after 6, 13, 26, and
    52 weeks of treatment. The standard haematological, serum chemical,
    and urinary examinations were performed, in addition to measurements
    of plasma and erythrocyte cholinesterase activity. At the end of the
    study, the animals were killed by an overdose of thiopentone sodium
    followed by exsanguination. All tissues were examined grossly
     in situ, and the major organs were removed and weighed, and brain
    cholinesterase activity was measured. The standard set of tissues was
    collected for histopathological examination. A statement of QA was
    provided.

         No effect of treatment on the incidence of clinical signs was
    observed. Treatment did not affect the weight gain of treated males,
    but a dose-related trend to decreased body weight was seen in treated
    bitches throughout the study, and, at termination, bitches at the high
    dose weighed about 10% less than controls. Males and females at the
    high dose tended to consume about 10% less food than controls.
    Erythrocyte count, haemoglobin concentration, and erythrocyte volume
    fraction were statistically significantly lower in males at the high
    dose than in control males at all intervals. The only change in serum
    chemistry was an increase in mean aspartate aminotransferase activity
    associated with decreased total cholesterol and serum albumin in males
    at the high dose from 6 weeks of treatment throughout the study. This
    effect was due largely to an apparent hepatotoxic response in two of
    four males at this dose; in one of these dogs, aspartate
    aminotransferase activity was increased by 10-fold over that of
    controls at study termination, and serum alkaline phosphatase,
    gamma-glutamyl transferase, and alanine aminotrans-ferase activities
    were also greatly increased. The serum albumin concentration was
    decreased in all males at the high dose. Plasma and erythrocyte
    cholinesterase activities were depressed in a dose-related manner in
    dogs at the intermediate and high doses at all intervals. At the end
    of the study, plasma cholinesterase activity was decreased to 33% and
    17% of the control value in males and to 58%, 19%, and 17% in females
    at the low, intermediate, and high doses, respectively. The
    erythrocyte cholinesterase activity in males at the end of the study

    was 127%, 88%, and 39% of the control value at the low, intermediate,
    and high doses, respectively, whereas the values for females were
    103%, 62%, and 38% of control, respectively. Brain cholinesterase
    activity was inhibited only in males at the high dose (to 56% of the
    control value; significant) and females (72% of control) at the high
    dose. Animals at the intermediate dose showed inhibition to 91% and
    94% of the control values, respectively, which was considered not to
    be toxicologically relevant. Ophthalmoscopic examination conducted
    before and at the end of the study revealed no treatment-related
    effects. At necropsy, no effect of treatment on absolute or relative
    organ weights was seen, nor was there any effect on the incidence of
    gross findings. Treatment-related histopathological changes were
    restricted to the liver. Selected findings are tabulated in Table 4.
    The NOAEL was 0.025 mg/kg bw per day on the basis of findings in the
    liver at the low dose (Brown, 1986).

        Table 4. Pathological findings in the liver of dogs treated with ethoprophos for 52 weeks

                                                                                                 
    Lesion                   Dose (mg/kg bw per day) 
                                                                                                 
                             Males                               Females
                                                                                                 
                             Control  0.02     1        10       Control  0.025    1        10
                                                                                                 

    Vacuolation              0        0        3        4        0        0        3        4
    Focal necrosis           0        0        0        2        1        0        0        1
    Pigment                  0        0        1        4        0        0        0        3
    Kupffer-cell pigment     0        0        1        4        0        0        1        4
    Fibrosis                 0        0        0        4        0        0        0        4
    Biliary proliferation    0        0        0        4        0        0        0        4
                                                                                                 
    

         (ii) Dermal administration

          Rats 

         In a range-finding study for dermal toxicity conducted according
    to GLP and with a QA statement, groups of three Crl:CD(R)BR rats of
    each sex received dermal applications of ethoprophos (purity, 95.6%)
    in mineral oil at doses of 0, 0.3, 1, 10, 30, or 100 mg/kg bw per day
    for 6 h per day on 5 days per week for 3 weeks. The animals were
    observed for clinical signs and dermal irritation. Body weight and
    food consumption were recorded, and cholinesterase activity was
    measured in plasma and erythrocytes before dosing, at week 2, and at
    the end of study (week 4). At necropsy, brain acetylcholinesterase
    activity was measured. Macroscopic examinations were performed and
    organ weights recorded.

         Deaths occurred in the groups given 30 (2/6 rats on days 7 and 8)
    and 100 mg/kg bw per day (6/6 rats, days 2-7). The clinical signs in
    these animals were characteristic of cholinesterase poisoning. Small
    faeces, hunched posture, and low body temperature were also observed
    at 10 mg/kg bw per day, and the latter finding was also observed in
    one male and one female at 0.3 mg/kg bw per day and two males and one
    female at 1 mg/kg bw per day. Food consumption was lower (reaching
    statistical significance in females) at 10 mg/kg bw per day in females
    and 30 mg/kg bw per day in males and females during the first week of
    exposure. There were no signs of dermal irritation. Statistically
    significant inhibition of plasma cholinesterase activity was observed
    at weeks 2 and 4 in females at 0.3 and 1 mg/kg bw per day and in
    animals of each sex at 10 and 30 mg/kg bw per day. Inhibition of
    erythrocyte acetylcholinesterase activity by > 20% was observed in
    females at all doses at week 2 and at 10 and 30 mg/kg bw per day at
    week 4 and in males at 10 and 30 mg/kg bw per day at both times. Brain
    acetylcholinesterase activity was inhibited by > 50% in animals of
    each sex at 10 and 30 mg/kg bw per day; the effect was dose-related
    and significant at the high dose. No treatment-related changes were
    observed in organ weights or in gross appearance (Henwood, 1990a).

         In the main study, conducted according to GLP and with a QA
    statement, groups of 10 Crl:CD(R)BR rats of each sex aged about 2.5
    months received 1.4 ml/kg bw of ethoprophos (purity, 95.6%) dermally
    in mineral oil at doses of 0, 0.3, 1, or 10 mg/kg bw per day for 6
    h/day on 5 days per week for 3 weeks. The area of exposure on the
    dorsal trunk constituted about 10% of the total body surface. The
    animals were observed for clinical signs and dermal irritation. Body
    weights and food consumption were recorded, and cholinesterase
    activity was measured in plasma and erthrocytes before treatment, in
    week 2, and at the end of study (week 4). Haematological and clinical
    chemical parameters were evaluated on the day of necropsy, when the
    brain was collected for analysis of acetylcholinesterase activity.
    Macroscopic examinations were performed, and the weights of the brain,
    kidneys, and liver were recorded. Kidneys, liver, skin, and tissues
    with lesions from all rats in the control and high-dose groups were
    examined microscopically.

         The deaths of one control female and one at the high dose were
    considered to be unrelated to treatment. Clinical signs observed in
    some females at the high dose included soft and/or small faeces (three
    animals) and hunched posture (one animal). During the first week of
    exposure, a slightly lower (not statistically significant) body-weight
    gain was observed in animals at the high dose, but food consumption
    was not affected. There were no signs of dermal irritation that could
    be related to treatment. In all groups, including controls, slight
    erythema and desquamation associated with pustules or papules were
    observed. Moderate desquamation was observed in one male and one
    female at 0.3 mg/kg bw per day and in one male at 1 mg/kg bw per day.
    Dose-related, statistically significant inhibition of plasma
    cholinesterase activity was observed in weeks 2 and 4 in animals given
    1 or 10 mg/kg bw per day. Erythrocyte acetylcholinesterase activity
    was inhibited by > 20% in rats at 1 mg/kg bw per day in week 4 and at

    10 mg/kg bw per day at both times, with 30% inhibition in week 2 and
    37% (males) and 54% (females) in week 4. Brain acetylcholinesterase
    activity was statistically significantly inhibited by 50% in males and
    70% in females at 10 mg/kg bw per day. At 1 mg/kg bw per day, the
    inhibition was 12% in males and 16% in females (not significant). At
    0.3 mg/kg bw per day, brain acetylcholinesterase activity was not
    inhibited in males and was nonsignificantly inhibited by 11% in
    females. In female controls and in those at 0.3 mg/kg bw per day, but
    not those at 1 mg/kg bw per day, the standard deviation of the mean
    acetylcholinesterase activity was relatively high. As a result, the
    inhibition in brain at 0.3 mg/kg bw per day is considered not
    toxicologically relevant, whereas that at 1 mg/kg bw per day is
    relevant. Other clinical findings in females were a dose-related
    decrease in platelet count and a decrease unrelated to dose in the
    numbers of neutrophils and lymphocytes in animals at 1 and 10 mg/kg bw
    per day. These findings were considered to be due to the excessive
    formation of fibrin strands in the blood of these females. The
    statistically significant findings of increases in aspartate
    aminotransferase activity in males at 1 mg/kg bw per day and slight
    decreases in urea nitrogen in males at 0.3 and 10 mg/kg bw per day
    were considered not to be related to treatment. Increased plasma
    concentrations of sodium and chloride were observed in females at 0.3
    mg/kg bw per day and in animals of each sex at 1 and 10 mg/kg bw per
    day, but these changes were small and not related to dose. An
    increased calcium concentration was seen in males at 1 mg/kg bw per
    day and in males and females at 10 mg/kg bw per day. No
    compound-related changes were observed in terminal body weights or
    organ weights. At macroscopic examination, two of 10 males at the high
    dose were found to have crusted areas on untreated skin in the
    cervical and/or thoracic region, confirmed microscopically to be
    multifocal erosion or ulceration of the epidermis and chronic
    inflammation. Three of nine females at the high dose showed dark areas
    in the glandular stomach, which was confirmed microscopically in two
    rats as oedema or focal erosion or ulceration of the glandular mucosa;
    and one of these females and another at the high dose showed
    dilatation of the pelvis. The NOAEL was 0.3 mg/kg bw per day on the
    basis of inhibition of brain acetylcholinesterase activity in females
    at 1 mg/kg bw per day. Since no sign of treatment-related dermal
    irritation was seen, the NOAEL for this end-point was 10 mg/kg bw per
    day, the highest dose tested (Henwood, 1990b).

          Rabbits 

         In a study conducted according to GLP and with a QA statement,
    groups of 10 Hra:(NZW)SPF rabbits of each sex, aged about 3 months,
    received dermal applications of 1.4 ml/kg bw of ethoprophos (purity,
    95.6%) in 4% carboxymethylcellulose at doses of 0, 0.03, 0.1, or 1
    mg/kg bw per day for 6 h/day on 5 days per week for 3 weeks. The area
    of exposure on the dorsal trunk constituted about 10% of the total
    body surface. The animals were observed for clinical signs and dermal
    irritation. Body weight and food consumption were recorded and
    cholinesterase activity was measured in plasma and erythrocytes before
    treatment and at the end of study (week 4). Haematological and

    clinical chemical parameters were evaluated on the day of necropsy,
    when the brain was collected for measurement of acetylcholinesterase
    activity. Macroscopic examinations were performed, and the weights of
    the brain, kidneys, and liver were recorded. Kidneys, liver, skin, and
    tissues with lesions from all rabbits in the control and high-dose
    groups were examined microscopically.

         Deaths occurred in one male and three females at 0.03 mg/kg bw
    per day and two males at 1 mg/kg bw per day. These animals and a few
    others in each group except female controls showed clinical signs that
    were associated with an increased incidence of mucoid enteritis
    (diarrhoea, mucoid diarrhoea, and reduced food consumption), but this
    finding was considered to be unrelated to treatment. Females at the
    high dose had significantly lower body weights in weeks 2 and 4. The
    groups in which the deaths occurred had lower food consumption, but
    this was considered to be of no toxicological importance. An increased
    incidence of slight-to-moderate irritation (erythema and sometimes
    desquamation) was seen in all treated groups when compared with
    controls, and the frequency increased with dose. The only
    treatment-related change in haematological or clinical chemical
    parameters was statistically significant inhibition of cholinesterase
    activity in plasma, erythrocytes, and brain at the high dose. The
    inhibition in males was 37% in plasma, 42% in erythrocytes, and 49% in
    brain, and that in females was 35% in plasma, 42% in erythrocytes, and
    49% in brain. No inhibition of brain acetylcholinesterase activity was
    found at lower doses; erythrocyte acetylcholinesterase activity was
    inhibited by 12% in females at the intermediate dose, but this is
    considered to be of no toxicological relevance. The terminal body
    weights were significantly lower for females given 0.03 or 1 mg/kg bw
    per day, which might be due to their lower food consumption. The
    absolute kidney weights were significantly lower in females and
    slightly lower in males at the high dose, and the relative kidney
    weights were slightly (and not significantly) lower in females at this
    dose. There were no treatment-related macroscopic or microscopic
    changes. The NOAEL for dermal toxicity was 0.1 mg/kg bw per day on the
    basis of inhibition of cholinesterase activity in brain and
    erythrocytes and decreased kidney weights at 1 mg/kg bw per day. No
    NOAEL could be identified for dermal irritation, as slight irritation
    of the skin was observed at all doses (Henwood, 1989).

    (c)  Long-term studies of toxicity and carcinogenicity

          Mice 

         Randomly assigned groups of 80 male and 80 female B6C3F1 (SPF)
    mice were fed diets containing 0, 0.2, 2, or 30 ppm of technical-grade
    ethoprophos (purity, 94.6%) for 2 years. The test material and diets
    were analysed periodically to ensure that the concentrations were
    within acceptable limits of the nominal values. The diets and water
    were provided  ad libitum. Animals were examined daily for signs of
    toxicity, and moribund animals were killed. Body weights were recorded
    weekly for the first 26 weeks of treatment and twice weekly
    thereafter. Food consumption was determined weekly. Haematological,

    clinical chemical, and urinary parameters and cholinesterase activity
    were measured after 26, 52, 78, and 104 weeks of treatment. Ten mice
    of each sex at each dose were killed after 26, 52, and 78 weeks of
    treatment, and all surviving animals were killed after 104 weeks of
    treatment. Complete post-mortem examinations were conducted on all
    animals killed at scheduled sacrifice and on those that died or were
    killed in a moribund condition during the study.

         No effect of treatment was seen on survival or the incidence of
    clinical signs. Mean body-weight gain was decreased by 5-10% in males
    and females at the high dose during the first 80 weeks of treatment,
    but by the end of the study there was no decrease in males and only a
    slight decrease (6%) in females. Treatment had no effect on food
    consumption, the mean intake levels over the course of the study being
    0, 0.026, 0.25, and 4.0 mg/kg bw per day for males and 0, 0.032, 0.32,
    and 4.9 mg/kg bw per day for females. The only change in
    haematological parameters was a decrease in the mean total leukocyte
    count in all treated males: at the end of the study, the mean total
    counts were: 4.1  1.7, 2.3  0.9, 2.1  1.1, and 1.6  0.5 
    103/mm3 for controls and males at the low, intermediate, and high
    doses, respectively, which were statistically significantly different
    from the controls in all groups. The significance of this finding is
    unclear as it was not associated with any other toxic effect that
    might result from an impaired immune function. Historical control data
    would have been useful for evaluatng this finding, but they were not
    submitted.

         Plasma and erythrocyte cholinesterase activity was inhibited in a
    dose-related manner in animals at the intermediate and high doses
    during the first 78 weeks of treatment, and in most cases statistical
    significance was reached. The cholinesterase activity remaining in
    plasma represented 65-90% of control values at the intermediate dose
    and 23-34% at the high dose, whereas in erythrocytes it was 83-89% of
    control values at the intermediate dose and 19-26% at the high dose.
    By the end of the study, the plasma and erythrocyte cholinesterase
    activities in the group at the intermediate dose were similar to those
    of controls, except that the activity in plasma of females at the
    intermediate dose was significantly decreased by about 20%. Brain
    cholinesterase activity was inhibited only in males and females at the
    high dose, with statistical significance reached in week 26 (64% of
    control value for males and 71% for females), week 52 (82% of control
    value in males), and week 104 (81% of control value in males and 83%
    in females).

         Necropsy of animals that died on test or were killed in a
    moribund condition did not reveal any treatment-related lesions or an
    effect on absolute or relative organ weights. No treatment-related
    lesions were found at gross and microscopic examinations at interim
    sacrifices. At final sacrifice, an increased incidence of
    calcification of the kidney was noted in males at the high dose
    (13/42, 2/45 controls) with 'basophilic changes' in the kidneys of
    males only (0/45 control, 9/31 at the low dose, 13/38 at the
    intermediate dose, and 24/42 at the high dose). The historical control

    data that were supplied showed a high spontaneous incidence of these
    lesions, with incidences of calcium deposits in males of 2.4-72% and
    basophilic changes in males of 0-87%. The study authors reported that
    these lesions are of spontaneous origin and do not represent
    treatment-related pathological lesions. Treatment had no effect on the
    incidences of tumours in specific tissues or on the total tumour
    burden. 

         The NOAEL for toxicity was 2 ppm, equal to 0.25 mg/kg bw per day,
    on the basis of inhibition of brain acetylcholinesterase activity at
    30 ppm (Yamagata et al., 1984a,b). 

          Rats 

         Groups of 10 male and 20 female Fischer 344 rats were fed diets
    containing technical-grade ethoprophos (purity, 95.3%) at
    concentrations of 0, 60.5, 131, or 262 ppm for 8 weeks before mating
    (one male to two females). (The section on methods indicates that the
    F0 generation comprised 10 males and 20 females per group, but later
    in the report it is stated that approximately 16 males and 32 females
    at each dose were mated. The complete absence of data on the
    reproduction phase of the study precluded verification of the
    information. The report stated that analytical data for dietary
    analyses were not available.) Ten days after the detection of a
    positive vaginal smear, the females were separated from the males and
    were maintained on the test diets until weaning of their pups. This
    part of the study constituted the reproductive phase. Weanlings from
    the reproductive phase (60 males and females per group) were selected
    randomly and fed diets containing ethoprophos at 0, 4.5, 9, or 18 ppm
    during weeks 0-12 and at 0, 49, 98, or 196 ppm during weeks 13-109 in
    a study of carcinogenicity. The dietary levels during weeks 13-109
    were equivalent to doses of 0, 2.5, 4.9, and 9.8 mg/kg bw per day.
    (The mean intake over the whole exposure period was not reported.) In
    this 109-week study, all animals that died or were killed in a
    moribund condition, all survivors at the end of the study, and the 10
    males and 10 females per group killed after 52 weeks of treatment were
    subjected to gross necropsy and histopathological examination of a
    wide range of tissues and all gross lesions. 

         The mortality rate was increased in males of the highest dose
    during the first 7 months, although the rates at the end of the study
    (53-63%) were comparable in all groups, including the controls. Other
    than emaciation in animals at the high dose, there were no
    treatment-related clinical signs. Growth was depressed at the highest
    dose throughout the study and at the intermediate dose during most of
    the study. Food consumption was reduced in animals at the intermediate
    and high doses during the first 52 weeks. Weekly water consumption,
    monitored between weeks 102 and 109, was unaffected. Haematological,
    blood chemical (glucose, blood urea nitrogen, gamma-glutamyl
    transferase, alanine and aspartate aminotransferase and alkaline
    phosphatase activities, total protein, and cholesterol concentrations)
    and urinary (pH, glucose, ketones, and protein) parameters measured at
    the end of weeks 52 and 109 revealed decreased erythrocyte count,

    erythrocyte volume fraction, and haemoglobin values in males at the
    two higher doses and in females of the highest dose after 52 weeks.
    Assay of cholinesterase in plasma, erythrocytes, and brain, conducted
    at termination only, indicated a dose-related, statistically
    significant 81-93% depression of plasma cholinesterase activity and a
    30-68% decrease in brain cholinesterase activity in all treated
    groups. Erythrocyte cholinesterase activity was not inhibited. The
    weights of the spleen, liver, kidney, and testis deviated from those
    of controls at the 52-week and terminal sacrifices, but essentially
    only at the highest dose, with no accompanying microscopic lesions.
    The gross pathologic changes seen were not significantly different
    from those in controls, but histopathological examination of males at
    the highest dose showed an increased incidence of 'scleral
    mineralization' of the eye. No other microscopic lesions that might be
    attributable to treatment were found.

         The only notable oncogenic finding was a statistically
    significant increase in the incidence of thyroid C-cell adenoma in
    males at the highest dose at terminal sacrifice, with incidences of
    2/34 in controls, 3/31 at the low dose, 1/29 at the intermediate dose,
    and 9/33 at the high dose, expressed as the ratio of the number of
    survivors bearing the tumour to the number examined
    histopathologically. C-cell adenomas were also observed at the interim
    sacrifice in one male in the control group and one at the low and one
    at the high dose. The background incidence of the tumour was not
    reported. The incidences of animals with benign and/or malignant
    tumours, malignant tumours, or multiple primary tumours were not
    significantly different in control and treated groups.
    Interstitial-cell adenoma of the testis in males and pituitary adenoma
    in females were the most frequently observed spontaneous tumours,
    occurring in nearly 90% of males and over 50% of females in the
    concurrent control group (Barnett et al., 1983). The authors
    considered that the increased incidence of C-cell adenomas in males at
    the high dose was a direct treatment-related effect as there was no
    trend with dose. No NOAEL could be identified since inhibition of
    brain acetylcholinesterase activity was seen at all doses. The LOAEL
    was 49 ppm, equivalent to 2.5 mg/kg bw per day.

         Groups of 70 male and 70 female Fischer 344 rats were assigned
    randomly to receive diets containing technical-grade ethoprophos
    (purity, 95.9%) at concentrations of 0, 1, 10, or 100 ppm (equivalent
    to 0, 0.05, 0.5, or 5 mg/kg bw per day; mean intake over entire
    exposure not reported) for 105 consecutive weeks. The test material
    and diets were analysed periodically to ensure the stability and
    homogeneity of diets. Food and water were provided  ad libitum. The
    animals were examined daily for mortality and morbidity and underwent
    detailed physical examinations every week. The body weights were
    recorded weekly for the first 26 weeks and twice weekly thereafter
    until the end of the study, as were measurements of food and water
    consumption. Ophthalmoscopic examinations were performed on all rats
    at the beginning of the study and after 6, 12, 18, and 24 months of
    treatment, and routine haematological, serological, and urinary
    studies were conducted at the same intervals after the beginning of

    treatment. Plasma and erythrocyte cholinesterase activity was measured
    at the same intervals as other parameters, and brain
    acetylcholinesterase activity was measured at interim sacrifice of 10
    rats of each sex per dose after 12 and 18 months and at final
    sacrifice at the end of 24 months of treatment. All animals were
    killed on schedule, and those that died on test or were killed in a
    moribund condition were subjected to a complete post-mortem
    examination. A statement of QA was provided.

         Treatment had no effect on survival, body-weight gain, or food or
    water consumption. The only potential treatment-related finding at
    physical examination was an increased incidence of anogenital staining
    in females at the high dose during weeks 14-78 of treatment.
    Haematological examination revealed decreased erythrocyte counts,
    haemoglobin, and erythrocyte volume fraction, with increased mean
    corpuscular volume in males and females at the high dose at all
    intervals except 18 months. This change was statistically significant
    only after 6 and 12 months of treatment, but not at final sacrifice. A
    slight (10-20% over controls) increase in blood urea nitrogen was also
    seen in these animals, which was statistically significant only at 6
    months in females and at 18 months in males. A statistically
    significant decrease in serum globulin content was noted after 6 and
    12 months of treatment but not at final sacrifice. No toxicologically
    significant alterations were found in urinary or ophthalmic
    end-points.

         Plasma and erythrocyte cholinesterase activity was significantly
    inhibited in a dose-related manner at most intervals in males and
    females at the intermediate and high doses. Erythrocyte activity was
    inhibited by 28-44% in animals at the high dose, by < 20% in males at
    the intermediate dose, and by 19-27% in females at 6, 12, and 18
    months and 4% at the end of the study. Brain cholinesterase activity
    was significantly inhibited in animals at the high dose at all
    measured intervals, with inhibition of 27-35% in males and 36-48% in
    females.

         At the 12-month sacrifice, a statistically significant increase
    of about 10% was noted in the relative weight of the spleen of males
    and females at the high dose, with decreases of a similar magnitude in
    the absolute and relative weights of the kidney in males at the high
    dose. No significant treatment-related changes were seen on gross or
    microscopic examination. At the 18-month sacrifice, no significant
    alterations in organs weights were found, nor were any significant
    changes noted on gross or microscopic examination. At final sacrifice,
    statistically significant, 16-20% increases in the absolute and
    relative weights of the thyroid/parathyroid were seen in males at the
    high dose, with non-significant, 14-16% increases in males at the
    intermediate dose. Gross examination revealed an increased incidence
    of enlarged thyroids in males at these doses: 5/35 and 5/39,
    respectively, compared with 1/36 controls. The only potentially
    treatment-related lesion found on microscopic examination was an
    increased incidence of parafollicular C-cell neoplasms in males at the
    high dose (Table 5). The authors concluded that these changes were

    statistically significant and the incidence of thyroid neoplasia was
    'random and unrelated to the test article'.

        Table 5. Incidence of C-cell neoplasms in male rats fed diets containing 
             ethoprophos

                                                                                      
    Neoplasm                                     Dose (ppm)
                                                                                      
                                                 0         1         10        100
                                                                                      

    C-cell adenoma
        Deaths and unscheduled sacrifices        2/13      0/7       0/13      1/9
        Final sacrifice                          6/36      5/39      5/35      11/39
        Total                                    8/49      5/46      5/48      12/48

    C-cell carcinoma
        Deaths and unscheduled sacrifices        0/13      0/7       0/13      1/9
        Final sacrifice                          0/36      0/39      1/35      2/39
        Total                                    0/49      0/46      1/48      3/48

    All C-cell neoplasms                         8/49      5/46      6/48      15/48
                                                                                      
    

         The NOAEL for toxicity was 10 ppm, equivalent to 0.5 mg/kg bw per
    day, on the basis of inhibition of brain acetylcholinesterase
    activity, effects on erythrocyte parameters, and effects on the
    thyroid at 100 ppm (Spicer, 1985).

         In a study conducted according to GLP and with a QA statement,
    ethoprophos (purity, 95.6%) was mixed into the diet of groups of 70
    randomly assigned Crl:CD(R)(SD)BR VAF/Plus(R) rats of each sex at
    concentrations of 0, 1, 60, and 600 ppm for 105 weeks. During week 3
    of the study, the highest dose was lowered to 400 ppm because of the
    occurrence of tremors, ataxia, and death in females. The average doses
    throughout treatment were 0, 0.04, 2.7, and 20 mg/kg bw per day for
    males and 0, 0.06, 3.4, and 26 mg/kg bw per day for females in the
    four groups, respectively. Ten additional animals of each sex per dose
    were killed after 54 weeks, while another 10 animals of each sex were
    allocated to the control and high-dose groups to study recovery after
    dosing for 52 weeks followed by 4 weeks of control diet. The test
    material and diets were analysed regularly to ensure that the
    concentrations in the diet were within an acceptable range. Food and
    water were provided  ad libitum, and animals were monitored twice
    daily for their clinical condition. Body weights and food consumption
    were determined weekly up to week 13 and at least every other week
    thereafter. The clinical pathological examinations comprised
    haematology, clinical chemistry, including erythrocyte and plasma
    cholinesterase activity, and urinalysis at weeks 12, 26, 52, and 78 on

    10 animals per group and on all surviving animals at termination of
    the study, when brain cholinesterase activity was determined in all
    animals. All animals were necropsied, and 41 organs from animals in
    the control and high-dose groups and all those that died or were
    killed before the end of the study were examined histologically.
    Additionally, all macroscopic lesions and lung, liver, and kidneys
    from animals at the low and intermediate doses were studied. The
    thyroids of all animals were examined, and ophthalmic examinations
    were done on all animals at weeks 0, 52. and 104. 

         Animals at the high dose showed depressed food consumption (up to
    week 70 in males and up to week 20 in females) and cumulative
    body-weight gain, and in males food efficiency was depressed up to
    week 20. The survival rate was essentially the same in all groups up
    to week 78, but by week 104 the survival rate of animals at the high
    dose was higher than that of other groups. The major causes of death
    were renal disease and pituitary tumours. Females at the high dose had
    significantly reduced erythrocyte count, haemoglobin, and erythrocyte
    volume fraction after 12, 26, 52, and 78 weeks of treatment, although
    the decreases were not statistically significant at 53 or 104 weeks.
    In males at this dose, these parameters were significantly reduced
    only at week 26; at weeks 12, 52, 53, and 78, nonsignificant decreases
    were seen, and at week 104 there was no difference from controls.
    These effects were reversible in the animals allowed to recover. Males
    and females at the high dose had significant reductions in total
    plasma protein and globulin at weeks 12 and 26 and males also at week
    52 and 53. In females, the decrease was significant only for total
    protein at week 52 and was nonsignificant for both total plasma and
    globulin at week 53. At week 78, only a significant decrease for total
    protein was observed, while in females both parameters showed a
    nonsignificant decrease. At week 104, the decreases in total plasma
    protein and globulin were no longer significant in animals of either
    sex. The effects on total plasma protein and globulin were reversible
    after 4 weeks of control diet. Other significant changes in
    haematological and blood biochemical parameters were incidental and
    considered not to be related to treatment. No changes in
    haematological or general blood biochemical parameters were observed
    in rats at the other doses. The results of urinary analysis were
    summarized in the report and indicated that males at the high dose had 
    significantly decreased urine volume and urea nitrogen and creatinine
    concentrations at the end of the study. These findings were attributed
    to a lower incidence of chronic renal disease in these animals.

         Plasma cholinesterase activity was significantly inhibited
    throughout the experiment, by 48-64% in males at the intermediate
    dose, 62-77% in males at the high dose, 61-77% in females at the
    intermediate dose, and 75-82% in females at the high dose. Erythrocyte
    cholinesterase activity was also significantly inhibited throughout
    the study, by 34-44% in males at the intermediate dose, 35-51% in
    males at the high dose, 36-48% (36% inhibition not significant) in
    females at the intermediate dose, and 41-51% in females at the high
    dose. At the low dose, inhibition of plasma and erythrocyte
    cholinesterase activity was highly variable but never exceeded 18% in

    plasma or 6% in erythrocytes in either males or females and was not
    statistically significant. Inhibition of brain cholinesterase activity
    was significant at weeks 52 and 104 and amounted to 35-33% of control
    values in males at the intermediate dose, 53-64% in males at the high
    dose, 28-32% in females at the intermediate dose, and 64-66% in
    females at the high dose. At the low dose, inhibition of brain
    cholinesterase activity was maximal in females at week 104 (8%), but
    the differences (whether increased or decreased) were not
    statistically significant in either males or females. Both plasma and
    erythrocyte cholinesterase activity recovered to about 80% of the
    control activity after 4 weeks of control diet from week 52, and brain
    acetylcholinesterase activity was nearly fully restored.

         Both males and females at the high dose tended to have lower
    terminal body weights, but the differences were not statistically
    significant. At week 52, the weight of the left thyroid glands of
    females at this dose was reduced both absolutely and relative to the
    weight of the brain. Males that were allowed to recover showed reduced
    absolute left and right adrenal weights, left thyroid plus parathyroid
    weights, and heart weight; the organ:brain weight and the kidney:brain
    weight ratios were also reduced. The weight of the left testis
    relative to that of the body was increased. At 104 weeks, females at
    the high dose had increased brain weights and decreased kidney:brain
    weights. The males in this group had reduced absolute and relative (to
    body) weights of the right and left kidney, heart, and right adrenal
    gland.

         Males at the high dose had a decreased incidence and reduced
    severity of chronic nephropathy as compared with control animals,
    whereas females at this dose had a reduced incidence of mineral
    calcareous deposits in the renal pelvis. Males and females at the high
    dose showed increased incidences of inflammation of tissues of the
    tail (joints and skin) and of alveolar macrophage infiltration.
    Probably as a result of this inflammation, higher incidences of
    lymphoreticular cell hyperplasia, sinal ectasia, and congestion were
    observed in lymph nodes at various sites in the body. The inflammatory
    changes were considered to be age-related, and their increased
    incidence in animals at the high dose was assumed to reflect the poor
    physical condition of these animals. In females at the high dose,
    increased incidences of gastric erosion and submucosal oedema were
    found at terminal sacrifice. No treatment-related ophthalmic changes
    were observed.

         Tumours of various cell types occurred in many tissues. The only
    changes that showed a clear relationship to treatment were increased
    proliferative cell lesions in thyroid C-cells, the adrenal medulla,
    and the uterine endometrium (Table 6). A statistical evaluation of the
    tumour incidence in all animals was not provided, but according to a
    statistical analysis of data on tumours found at final sacrifice,
    females at the low and high doses had a significantly reduced
    incidence of C-cell hyperplasia at terminal sacrifice, while males at
    the low and intermediate doses had significantly increased incidences
    of C-cell adenomas. The increase in C-cell carcinomas at termination


        Table 6. Occurrence (%) of proliferative lesions in organs of rats given ethoprophos in the diet

                                                                                                                    
    Groups of animals and tumour type           Dose (ppm)
                                                                                                                    
                                                Males                               Females 
                                                                                                                    
                                                 0        1       60      400        0        1       60      400
                                                                                                                    

    No. of animals
    No. of unscheduled deaths                   50       42       42       30       42       47       37       27
    No. at terminal sacrifice                   20       28       28       41       29       23       33       44
    Total                                       70       70       70       71       71       70       70       71

    Thyroid
        C-cell hyperplasia                      31       29       41       39       61       39       63       46
        C-cell adenomas                         11        9       13       17       14       11       16       17
        C-cell carcinomas                        0        0        1        4        1        1        1        3
        All tumours                             11        9       14       21       15       12       17       20

    Adrenal
        Benign phaeochromocytomas               20       10       10        7        4        3        1        3
        Malignant phaeochromocytomas             0        3        3        7        0        0        0        0
        All tumours                             20       13       13       14        4        3        1        3

    Uterus
        Endometrial stromal polyps                                                   1        1        4        8
                                                                                                                    

    Data are incidences in percentages of the total number of animals (terminal sacrifice plus intercurrent deaths)
    

    was not significant. Males at the high dose showed a significant
    decrease in the incidence of benign phaeochromocytomas but a
    significant increase in that of malignant phaeochromocytomas at study
    termination. The increased incidence of uterine endometrial stromal
    polyps in females at the high dose was also significant. The study
    authors indicated that C-cell and uterine proliferative lesions are
    more likely to occur in old animals and their increased incidences are
    attributable to the greater longevity of the animals at the high dose.

         The NOAEL was 1 ppm, equal to 0.04 mg/kg bw per day, on the basis
    of inhibition of brain acetylcholinesterase activity at the next
    highest dose (Williams, 1992).

    (d)  Genotoxicity

         The results of studies on the genotoxicity of ethoprophos are
    summarized in Table 7.

    (e)  Reproductive toxicity

         (i)  Multigeneration reproductive toxicity

         In a two-generation study of reproductive toxicity conducted
    according to GLP and with a QA statement, groups of 28
    Crl:CD(R)(SD)BR rats of each sex, 6 weeks of age, were given diets
    containing ethoprophos (purity, 95.3%) in acetone at concentrations of
    0, 1, 30, or 300 ppm, equal to 0, 0.04, 1.3, or 23 mg/kg bw per day
    for males and 0, 0.09, 2.6, or 27 mg/kg bw per day for females. After
    10 weeks of exposure, the F0 parents were mated 1:1 to produce the
    F1a generation. Treatment was continued through mating, gestation,
    parturition, and lactation. On day 4 after parturition, each litter
    was culled to four pups of each sex. After 3 weeks of lactation, the
    pups were weaned. Owing to significant mortality among weanlings at
    the high dose, pups in the control, low-, and intermediate-dose groups
    were not used as parents for the next generation but instead 10 pups
    of each sex were selected for necropsy. All weanlings at the high dose
    and control F1a weanlings selected as parents were maintained on
    their respective diets until day 49  post partum; the weanlings at
    the high dose and 10 controls of each sex were then necropsied. From
    about 1 week after weaning of the last F1a pups, F0 parents at 300
    ppm were fed a diet containing 150 ppm of ethoprophos (equal to 7.1
    mg/kg bw per day for males and 14 mg/kg bw per day for females), and
    all F0 animals at the other two doses were maintained on their
    original diets. After 3 weeks, the F0 parents were mated again to
    produce the F1b generation, mating, gestation, parturition,
    lactation, and weaning being conducted as described above. One week
    after weaning, 28 F1b pups of each sex at each dose were selected as
    parents of the F2 generation. The remaining F1b pups and all F2
    pups were necropsied. Parental F0 animals aged 35-36 weeks and
    parental F1 animals aged 20-21 weeks were killed after weaning of
    the F1b and F2 progeny, respectively, and neccropsied grossly.
    Blood was taken just before sacrifice, and brain tissue was collected,
    for analyses of cholinesterase in plasma, erythrocytes, and brain. The


        Table 7. Results of studies of the genotoxicity of ethoprophos

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

     In vitro 
    Reverse mutationa,b   S. typhimurium           10-1000 g/plate in                  NR       Negative  S9   Barfknecht et al. (1985a)
                          TA98, TA100, TA1535,     DMSO
                          TA1537, TA1538

    Gene mutationa        Mouse lymphoma           0.24-0.032 g/ml                     95       Negative        Thomson et al. (1981)
                          L5178Y cells, Tk locus   (total growth between 18
                                                   and 126%, respectively), 
                                                   - S9 
                                                   0.024-0.0032 g/ml 
                                                   (total growth between 10 
                                                   and 106%), + S9

    Gene mutationc,d      Chinese hamster ovary    0-500 g/ml - S9                     NR       Negative        Stankowski et al. (1985)
                          cells (CHO-K1-BH4);      0-150 g/ml + S9
                          Hprt locus               in DMSO

    Chromosomal           Chinese hamster ovary    0-300 g/ml - S9                     NR       Negative        SanSebastian et al. (1985)
    aberrationsc,e        cells (CHO-K1-BH4)       0-60 g/ml + S9 in DMSO                       Positivef

    Unscheduled DNA       Rat hepatocytes          2.5-100 nl/ml in DMSO;               NR       Negative        Myhr & Brusick (1981)
    synthesisg,h                                   toxic from 50 nl/ml

    Unscheduled DNA       Rat hepatocytes          0-333 g/well in DMSO                NR       Negative        Barfknecht et al. (1985b)
    synthesisg,i          (male Fischer 344)

    Unscheduled DNA       Rat hepatocytes          0-333 g/well in DMSO                NR       Negative        Barfknecht et al. (1985b)
    synthesisg,i          (male Fischer 344)

    Sister chromatid      Chinese hamster ovary    0-350 g/ml - S9                     NR       Negative        SanSebastian et al. (1986)
    exchangec,j           cells (CHO-K1-BH4)       0-60 g/ml + S9 in DMSO                       Positivek
                                                                                                                                           

    Table 7. (continued)

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

     In vivo 

    Chromosomal           Rat (SD) bone            0-20 mg/kg bw per day for 5 days     95.7     Negative        Skinner et al. (1981)
    aberrationl           marrow                   by gavage in Methocel K4M 
                                                   premium; sacrifice 6 h after dosing

    Chromosomal           Rat (SD) bone            0-25 mg/kg bw once by gavage;        95.5     Negative        Ivett (1989)
    aberrationm,n         marrow                   sacrifice 6, 18, and 30 h after 
                                                   dosing 0-25 mg/kg bw per day for 
                                                   5 days by gavage; sacrifice 6 h 
                                                   after dosing
                                                   Vehicle, corn oil.

    Dominant lethal       Rat (SD)                 0-20 mg/kg bw per day for 5 days     NR       Equivocal       Putman & Schechtman
    mutationo                                      by gavage in corn oil                                         (1981)

    Dominant lethal       Rat (Crl:COBS            0-20 mg/kg bw per day for 5 days     95       Negative         Dearlove (1987)
    mutationm,p           CD(SD)BR)                by gavage in carboxymethyl 
                                                   cellulose
                                                                                                                                           

    NR, not reported; S9, 9000  g supernatant of rat liver; DMSO, dimethylsulfoxide; SD, Sprague-Dawley
    a   Test in triplicate; positive controls included; S9 fraction of Aroclor 1254-induced rat liver; GLP and QA statements included
    b   Cytotoxicity  (small colonies) seen in preliminary test at 1666 g/plate and no growth at 5000 g/plate in TA1538 and TA100 
    c   Test in duplicate; positive controls included; S9 fraction of Aroclor 1254-induced rat liver; GLP and QA statements included
    d   Cytotoxicity observed at doses > 167 g/ml; relative initial survival, 18% at 350 g/ml without S9 and 10% at 150 g/ml with S9 
    e   Only 100 cells/dose scored instead of 200/dose required by OECD; cytotoxicity at doses > 500 g/ml without S9 and at 80 g/ml 
        with S9, and average proliferation time increased at 400 and 60 g/ml, respectively
    f   Statistically significant increase in aberrations seen with metabolic activation at 60 g/ml. In a second test at concentrations 
        of 50-70 g/ml, statistically significant but not dose-related increase at all concentrations (14-22 breaks compared with 2 breaks 
        in control). The authors considered this a weakly positive result. Although the results of the second test were not dose-related, 
        they confirm the positive result of the first test.

    Table 7 (continued)

    g   Test in triplicate; positive controls included; GLP and QA statements included
    h   Purity, 95% (personal communication from Dr Rao, Rhone Poulenc). Toxicity observed as 73% survival at 50 nl/ml and 0% at 100 nl/ml.
    i   Cytotoxicity (abnormally low nuclear and cytoplasmic grains) at doses > 333 g/well
    j   Cytotoxicity at doses > 500 g/ml without S9 and > 80 g/ml with S9, and average proliferation time was increased at 400 and 
        60 g/ml, respectively.
    k   Statistically significant, dose-dependent increase with metabolic activation at 50-60 g/ml. In a second test at concentrations of 
        50-75 g/ml, statistically significant, dose-related increase at all concentrations (22-35 aberrations/cell compared with 16 per 
        cell in controls). Since there was no twofold increase at any dose, the authors considered the compound a weak inducer.
    l   Aberrations analysed in 5 males/group; positive control included; absorption of compound tested in 1 animal/group and confirmed 
        by a minimum of a 10-fold depression in cholinesterase activity in plasma at 20 mg/kg bw per day for 5 consecutive days. Clinical 
        signs: decreased motor activity at high dose (day 2); mitotic index not measured. Not clear that the bone-marrow cells were 
        exposed to the test substance. Only 50 cells scored instead of 100 cells/animal required by OECD. 
    m   GLP and QA statements included 
    n   Each group of dose and harvest time consisted of 5 rats of each sex. Positive control group included in single-dose study only. 
        Deaths observed only in females at high dose. Clinical signs at high single dose: dyspnoea, languid appearance, and diarrhoea.
        Additional signs at high multiple doses: tremor, rough coat, hunched posture, and dark crusts around eyes. No decrease in mitotic 
        index after single dose, but slightly decreased (54-75%) in males in short-term study. It is not clear whether the bone-marrow 
        cells were exposed to the test substance. Aberrations were studied in only 50 cells/animal instead of 100 cells and mitotic 
        index studied in only 500 cells instead of 1000 as required by OECD. 
    o   10 male rats/group. Three days after the last dose, males were mated with 2 untreated virgin females. Mating process repeated 
        another 6 times once a week with new virgin females. Females were killed about 2 weeks after mating; scoring for number of corpora 
        lutea number of dead and live implants per pregnant female. Absence of body-weight gain in males at high dose. No effect on 
        fertility index, number of corpora lutea, or number of implantations. Pre-implantation loss statistically significantly increased 
        in week 3 at high and low doses. Number of dead implants increased in week 1 at low dose, weeks 2-3 at intermediate dose, and weeks 
        1-6 at high dose, with a positive dose-response relationship. The proportion of females with one or more dead implants was 
        significantly increased at the high and intermediate doses (week 2 and weeks 2-3, respectively) and at the high dose also of females
        with two or more dead implants. The number of dead implants per total number of implants was significantly increased at the high 
        dose (weeks 2-5) and intermediate dose (week 5) (0.08-0.12 in test groups compared with 0.02-0.05 in control group). The number of 
        live implants per pregnant female, however, was not affected (change in live implant ratio is the criterion of dominant lethality). 
        In the positive control group, more marked increases in dead implants per total implants observed in weeks 1-4 (0.6-1.0), and the 
        number of live implants per pregnant female was significantly decreased (0.0-8.3 compared with 9.9-11.9 in controls). The authors of 
        the report concluded that the compound was mutagenic; however, the shortcomings of the test and the fact that the number of live
        implants was not affected indicate that the result is equivocal. The shortcomings of the study were that only 12-18 pregnant females
        were included in each treated group and 15-17 in the control group, whereas according to OECD 478 30-50 pregnant females per mating
        interval are required; mating was not confirmed. Only QA statement included.

    Table 7 (continued)

    p   24 males/group. Doses based on a range-finding study. Two days after the last dose, males were mated with 2 untreated virgin 
        females. Mating process was repeated another 7 times once a week with new virgin females. Females were killed 14 days after 
        presumed gestation; scoring for number of corpora lutea and for number of dead and live implants per pregnant female. Other
        observations: viability, clinical signs, body weight, food consumption, gross necropsy of males and females, especially reproductive
        organs (including histopathological examination). Three males at high dose died showing clear signs of intoxication. One male was
        replaced. Clinical signs at high dose were characteristic of cholinesterase poisoning. Body-weight gain was inhibited (dose-related)
        during treatment at 5 and 20 mg/kg bw per day, and food consumption was lower at the high dose. No compound-related findings at 
        gross necropsy except a single finding of an epididymal mass in one male at 5 mg/kg bw per day; no effect on testis weight, mating
        index, fertility index, or pregnancy rate. Average numbers of corpora lutea, implantations, litter sizes, resorptions, percentage 
        of dead conceptuses, and number of females with resorptions similar in treated and control groups, except a single finding of dams 
        with increased resorptions in week 4 of mating at the intermediate dose. 
    

    thyroid glands were weighed, and selected tissues (especially from the
    reproductive organs) from control and high-dose animals were examined
    histopathologically. 

         The dietary concentrations of 1, 30, and 300/150 ppm during the
    first premating and mating periods were equal to 0.07, 2.2, and 23/12
    mg/kg bw per day for F0 and F1 males and 0.09, 2.7, and 27/14
    mg/kg bw per day for F0 and F1 females. During the second mating
    period, the doses of 1, 30, and 150 ppm given to F0 males were equal
    to 0.04, 1.3,and 7.1 mg/kg bw per day; the intake of females,
    calculated only for weeks 20-24, was 0.07, 2.0, and 11 mg/kg bw per
    day. 

         The clinical signs in F0 parents at the high dose were soft
    faeces during the first 2 weeks of exposure and tremors in females
    during gestation and lactation. No clinical signs were observed in
    F1 parents. The body weights of F0 males at 300 ppm and F1 males
    at 150 ppm were significantly decreased throughout the exposure
    period. F0 males at 300 ppm had significantly lower body-weight
    gains during several weeks, whereas an increase in body-weight gain
    was observed occasionally when the dose was changed to 150 ppm. The
    weight gain of F1 males at the high dose was reduced during the
    premating period and occasionally thereafter. Significantly decreased
    body-weight gains were observed occasionally in males at 30 ppm but
    these did not result in significantly lower body weights. F0 females
    at the high dose had significantly reduced body-weight gains in weeks
    1 and 9, but these did not result in lower body weights during the
    premating period for the F1a litter. The total body-weight gain
    during the 0-20 days of gestation with the F1a litter was
    significantly reduced, and the body weights were lower during
    lactation. These lower (but not statistically significant) body
    weights of F0 females at the high dose persisted during the
    premating and mating periods for F1b progeny and reached statistical
    significance on days 4-21 of lactation, although there was no effect
    on body-weight gain. The body weights of F1 females at the high dose
    were significantly reduced throughout premating, gestation, and
    lactation, but with no effect on weight gain. Food intake was affected
    only at the high dose, with lower consumption observed for F0 males
    in weeks 1, 2, and 20 and for F1 males in weeks 9, 11, and 16-20.
    The food consumption of females of the F0 generation at the high
    dose was lower during the first week of exposure, but food consumption
    was affected only on days 4-14 of gestation of the F1a generation.
    Length of gestation and indices of mating, fertility, and gestation
    were not affected in either generation. The final body weights of F0
    and F1 males at the high dose were significantly reduced, and only
    in F1 males at this dose the absolute, but not the relative, weight
    of the thyroid was significantly lower. No treatment-related lesions
    were observed on gross or histopathological examination.
    Cholinesterase activity was significantly inhibited in plasma and
    brain of F0 and F1 animals of each sex at the high dose, in F1
    animals at 30 ppm, and in F0 females at 30 ppm; plasma
    cholinesterase activity was also inhibited in F0 males at 30 ppm.
    There was no effect on erythrocyte acetylcholinesterase activity.

         Analyses of data for the litters revealed lower body-weight gains
    in F1a pups at 300 ppm throughout lactation, resulting in lower body
    weights from day 7 onwards in males and from day 4 in female pups. The
    survival of F1a pups was not affected up to weaning, but the
    survival index was only 51% 1 week after weaning on day 28. Necropsy
    of dead F1a pups showed blood in the stomach and/or intestines. The
    body weights of surviving F1a pups at the high dose remained reduced
    up to sacrifice on day 49, and their body-weight gain was reduced up
    to day 35. F1b pups at the high dose of 150 ppm showed lower
    body-weight gain, from the second week  post partum onwards for
    females and from the third week for males, resulting in lower body
    weights from day 14 and day 21, respectively. Survival was not
    affected. Significant reductions in average litter size at 1 ppm,
    beginning on day 4, were considered spurious owing to the lack of a
    dose-response relationship. The litter sizes and sex ratios of F2
    litters were unaffected by treatment. The decreased litter sizes, and
    as a consequence higher pup body weights, at 1 and 30 ppm and the
    reduced sex ratio at 30 ppm were considered not to be related to
    treatment in the absence of a dose-response relationship. The
    body-weight gain of pups at the high dose was reduced from day 4
    onwards, resulting in lower weights from lactation day 7. Reduced
    weight gain was found on days 4-7 of lactation in the group receiving
    30 ppm, with a (probably) compensatory increase on days 14-21, but
    this was considered irrelevant toxicologically. Deaths among F2 pups
    at the high dose on days 7-14 resulted in a significantly reduced
    14-day survival index and lactation index. No treatment-related
    lesions were observed in dead F2 pups or in F1a, F1b, and F2
    pups subjected to scheduled necropsy 1 week after weaning.

         The NOAEL was 1 ppm, equal to 0.04 mg/kg bw per day, on the basis
    of lower body-weight gain in F0 males and inhibition of brain
    acetylcholinesterase activity in parents of each sex in each
    generation. No effects were observed on reproductive parameters. The
    NOAEL for toxicity to pups was 30 ppm, equal to 1.3 mg/kg bw per day,
    on the basis of effects on body weight and body-weight gain and deaths
    in F2 litters at 150 ppm (Neeper-Bradley, 1991).

         Groups of 10 male and 20 female Fischer 344 rats, 44 days old,
    were fed diets containing technical-grade ethoprophos (purity, 95.3%)
    at concentrations of 0, 60.5, 131, or 262 ppm (equivalent to 0, 3.0,
    6.6, and 13 mg/kg bw per day) for 56 days before mating. Weanlings
    from the second litters were selected to be the parents of the next
    generation. All parental animals and weanlings of each generation were
    subjected to gross pathological examination. Additionally, all F1b
    and F2b parents and five male and five female weanlings from each of
    the F1a, F2a, F3a, and F3b litters were evaluated
    histopathologically. A statement of QA was provided.

         The mortality rate was unaffected but growth was slightly
    depressed at the two higher doses in all parental generations. Data on
    food consumption were not available. A dose-related increase in the
    number of dams that cannibalized their young was noted in all treated
    groups of the F0 generation, mainly with regard to the second

    litters. The percentage of animals becoming pregnant was reduced in
    all treated F1a litters (75, 60, 55, and 55%, respectively, at 0,
    60.5, 131, and 262 ppm) and for F3a and F3b litters at 262 ppm.
    The lactation index (pup survival on days 4-21) was decreased at both
    131 and 262 ppm in the F1b (56% and 40%, respectively, compared with
    89% in controls) and the F2a generation (86% and 67%, respectively,
    compared with 100% in controls) and at the high dose in the F2b
    generation (67% compared with 88% in controls). Weanling weight (not
    determined for F1a and F1b litters) was depressed in the F2a and
    F2b litters at 131 and 262 ppm and in the F3a litters at 262 ppm.
    Other effects that were observed only at the highest dose included a
    tendency for decreased litter size at birth in almost all generations
    of progeny, prolongation of the mean gestation period of F1b litters
    (this parameter was not evaluated in all generations), the occurrence
    of bilateral lenticular opacity (cataracts; in 29%) and anophthalmia
    (3%) in F2a weanlings (information on the litter distribution of
    these abnormalities was not available), and an increased incidence of
    multifocal granuloma of the mesenteric lymph nodes in F2b adults.
    The increased incidence of cannibalization among F0 dams and the
    slight decrease in pregnancy rate in F1a litters at 60.5 ppm did not
    recur in all generations and were probably related to maternal
    toxicity. This concentration was considered to be the minimal
    no-effect level for reproductive toxicity. As enzootic pneumonia was
    observed in all parental animals and in some pups, the results of this
    test are unreliable (Fletcher et al., 1980).

         (ii) Developmental toxicity

          Rats 

         A preliminary dose-range finding study was conducted according to
    GLP and with a statement of QA, in which five female Sprague-Dawley
    COB SC rats were given ethoprophos (purity, 95.6%) in corn oil at
    doses of 0, 0.2, 1, 4, 8, or 16 mg/kg bw per day by gavage on days
    7-15 of gestation. Fetuses were removed surgically on day 20 and
    examined for external anomalies. The only finding was body-weight loss
    in dams at the highest dose during days 6-9 of gestation and reduced
    body-weight gains (24%) throughout treatment. There were no signs of
    developmental toxicity (Rodwell, 1989a).

         In the main study, conducted according to GLP and with a
    statement of QA, groups of 25 pregnant female
    Crl:COBS(R)CD(R)BR(R)VAF rats aged 3 months were given
    ethoprophos (purity, 95.6%) by gavage in corn oil at doses of 0, 2, 9,
    or 18 mg/kg bw per day on days 6-15 of gestation. The dams were
    observed for clinical signs, and body weight and food consumption were
    measured. Fetuses were removed surgically on day 20, weighed, and
    examined for external, visceral, and skeletal anomalies. 

         Dose-dependent maternal toxicity was observed at 9 and 18 mg/kg
    bw per day, with treatment-related findings including soft stools at
    both doses and faecal staining at the high dose. Significantly lower
    body-weight gain was observed at 9 mg/kg bw per day on days 6-9 of

    gestation, and during this period females at the high dose showed
    body-weight loss which resulted in lower body weights by the end of
    the study. The food consumption of animals at the high dose was
    reduced on days 6-16 of gestation. No effect was found on survival, no
    morphological changes were observed at necropsy, and no changes in
    gravid uterine weight or fetal parameters (viability, resorptions,
    body weight, and sex ratio) were found. The incidences of external,
    visceral, and skeletal variations and of irreversible structural
    changes were unchanged. The NOAEL for maternal toxicity was 2 mg/kg bw
    per day, and that for developmental toxicity was 18 mg/kg bw per day
    (Rodwell, 1989b).

         Groups of 25-35 mated female TAC:N(SD)FBR rats were intubated
    with technical-grade ethoprophos (purity, 94%) in corn oil at doses of
    0, 0.16, 1.6, or 16 mg/kg bw per day on days 6-15 of gestation, the
    day a vaginal sperm plug was observed being considered day 0. The dams
    were killed on day 20 and the fetuses removed for gross, skeletal, and
    visceral examination. A positive control group was included.

         The pregnancy rate was comparable in all groups. During
    gestation, 1/24 pregnant dams at 1.6 mg/kg bw and 18/30 at 16 mg/kg bw
    died or aborted; in addition, three non-pregnant dams at the high dose
    were found dead. Growth was depressed on days 15 and 20 of gestation
    in dams at 16 mg/kg bw. No significant difference was found between
    control and treated groups in the mean numbers of corpora lutea,
    implantation sites, live fetuses, dead fetuses or resorptions, mean
    fetal weight, or sex ratio. According to a summary of the skeletal
    findings and soft-tissue abnormalities in fetuses, the only notable
    observation was a significant increase in the incidence of litters
    containing fetuses with extra lumbar ribs in animals at 1.6 mg/kg bw.
    Since no dose-response relationship was seen, the authors could not
    attribute this finding to exposure to ethoprophos. Incomplete
    ossification of vertebrae was observed in all groups, including the
    controls, with incidences of 18, 10, 7, and 17% in control, low-,
    intermediate-, and high-dose groups, respectively). The NOAEL for
    maternal toxicity was 1.6 mg/kg bw per day on the basis of deaths and
    depressed body-weight gain, and the NOAEL for developmental toxicity
    was 16 mg/kg bw per day (Knickerbocker & Re, 1979, and amendment in
    1985). 

          Rabbits 

         A preliminary dose-range-finding study was conducted according to
    GLP and with a statement of QA in which groups of eight female New
    Zealand white rabbits received ethoprophos (purity, 95.6%) in corn oil
    at doses of 0, 0.1, 0.5, 2, 5, or 10 mg/kg bw per day by gavage on
    days 7-18 of gestation. The fetuses were removed surgically on day 29
    and observed for external anomalies. Dose-dependent maternal toxicity
    was observed at 5 and 10 mg/kg bw per day, the clinical signs
    including emaciation, an increased incidence of soft stools, and
    faecal and urine staining. Consistent body-weight loss and a
    dose-dependent mortality rate were also seen in these two groups.
    There were no signs of developmental toxicity (Rodwell, 1989c).

         In the main study, conducted according to GLP and with a
    statement of QA, groups of 20 artificially inseminated New Zealand
    white rabbits aged 6 months received ethoprophos (purity, 95.6%) by
    gavage in corn oil at doses of 0, 0.625, 1.25, or 2.5 mg/kg bw per day
    on days 6-18 of gestation. The does were observed for clinical signs,
    and body weight and food consumption were measured. The fetuses were
    removed surgically on day 29, weighed, and examined for external,
    visceral, and skeletal anomalies.

         There were no clinical signs of maternal toxicity, no adverse
    effects on survival, body weight, or food consumption, and no
    morphological changes. Gravid uterine weight and fetal parameters
    (viability, resorptions, body weight, and sex ratio) were also
    unchanged. The finding of a significantly reduced mean number of
    viable fetuses per doe at 1.25 mg/kg bw per day was due to the absence
    of this effect in the group at the high dose and considered not to be
    related to treatment. The incidences of external, visceral, and
    skeletal variations were not altered, as the finding of a decreased
    incidence of 27 presacral vertebrae in fetuses at the intermediate and
    high doses was considered not to be toxicologically relevant.
    Increased incidences of cleft palate, flexed paw, gastroschisis, and
    cephalocoele seen on external examination and of atelectasis and
    hydrocephaly seen on visceral examination of the group at the
    intermediate dose when compared with controls and with historical
    control values were observed in fetuses of only a single doe and were
    considered not to be related to treatment. The incidences of fetuses
    with malformations were 2.4, 4.1, 4.0, and 6.6% and those of litters
    with malformed fetuses were 12, 11, 11, and 35% in the control, low-,
    intermediate-, and high dose groups, respectively. The malformations
    seen at the high dose were dissimilar and occurred once or at very low
    frequency; furthermore, they were types that occur spontaneously in
    this strain of rabbits and the incidences were within the historical
    control values. In the absence of effects on fetal weight, the
    increased incidences of irreversible structural changes at the high
    dose were considered to be unrelated to treatment. The NOAEL for both
    maternal and developmental toxicity was 2.5 mg/kg bw per day, the
    highest dose tested (Rodwell, 1989d).

         Groups of 17 artificially inseminated female New Zealand white
    rabbits were intubated with technical-grade ethoprophos (purity,
    95.7%) in corn oil at doses of 0, 0.125, 0.5, or 2 mg/kg bw per day on
    days 6-18 of gestation, the day of insemination being considered day
    0. The surviving does were killed on day 29 of gestation, and their
    fetuses were removed for external, visceral, and skeletal
    examinations. A statement of QA was provided.

         No treatment-related deaths occurred, but does at all doses
    showed an increased (but not dose-related) incidence of anorexia
    during and after treatment. A dose-related but not statistically
    significant decrease in mean body-weight gain was observed between
    days 6 and 18 in the does at the two higher doses. Control and treated
    groups did not differ significantly with respect to pregnancy rate,
    mean numbers of corpora lutea, implantations, resorptions, dead

    fetuses, or live fetuses, mean fetal weight, mean fetal crown-rump
    length, sex ratio, uterine weight with or without fetuses, or the
    frequency of gross and visceral abnormalities. An increased total
    incidence of skeletal variants was observed in the litters of all
    treated does, with 14% at the low dose, 13% at the intermediate dose,
    and 17% at the high dose, and 6% in controls. The increase was not
    dose-related, and the frequency of any particular type of skeletal
    variant, when considered alone, did not show a dose-response
    relationship. The NOAEL for maternal toxicity was 0.12 mg/kg bw per
    day on the basis of reduced body-weight gain. The NOAEL for
    developmental toxicity was 2 mg/kg bw per day (Wolfe et al., 1981).

    (f)  Special studies: Neurotoxicity

          Rats 

         In a study conducted according to GLP and with a statement of QA,
    groups of 24 Crl:CD(SD)BR VAF/Plus rats of each sex, about 6 weeks
    old, were given a single dose of ethoprophos (purity, 95.7%) by gavage
    in corn oil. Male rats received doss of 0, 30, or 60 mg/kg bw and
    females received 0, 20, or 40 mg/kg bw. The doses were based on a
    range-finding study. The animals were observed daily for deaths and
    clinical signs, and body weights were recorded weekly. Blood samples
    were taken from six animals of each sex per group 2 h and 3, 8, and
    15 days after dosing to determine cholinesterase inhibition in plasma
    and erythrocytes. Brain acetylcholinesterase activity was determined
    in the frontal cortex, caudate/putamen, hippocampus, and cerebellum of
    the same animals at the same times. 

         One male rat given 60 mg/kg bw was found moribund. The clinical
    signs observed in females at 40 mg/kg bw and males at 60 mg/kg bw were
    tremors and excessive salivation. Males also showed hunched posture,
    laboured breathing, rough and stained coats, pale bodies, ocular
    discharge, incoordination, hypoactivity, and were cold to touch. Among
    animals at the lower dose, the only clinical sign was cold to touch in
    one female at 20 mg/kg bw. There were no effects on body weight.
    Cholinesterase activity was inhibited in all tissues examined by 2 h
    after treatment, and the effects were dose-, time-, and
    tissue-dependent (Table 8). Recovery of cholinesterase activity was
    apparent by day 3 or 8 after dosing and was complete by day 15 for
    plasma and cerebellar cholinesterase activity. Statistically
    significant inhibition of acetylcholinesterase activity persisted in
    the hippocampus through day 15 at the high dose and in erythrocytes
    and the frontal cortex of males at both low and high doses. Persistent
    inhibition was observed in the caudate/putamen at both doses, but the
    inhibition was not statistically significant (Weiler, 1994a).

         In a study conducted according to GLP and with a statement of QA,
    groups of 17 Crl:CD(R) BR VAF/Plus rats of each sex, about 6 weeks
    old, were given a single oral dose of ethoprophos (purity, 96.2%) by
    gavage in corn oil. The doses were 0, 5, 50, or 75 mg/kg bw for males
    and 0, 5, 25, or 50 mg/kg bw for females, which were based on
    range-finding studies. The animals were observed daily for deaths and


        Table 8. Cholinesterase activity in various tissues of rats after a single oral dose of ethoprophos

                                                                                                                                    

    Tissue                             Dose           Inhibition from control mean (%)a
                                       (mg/kg bw)                                                                                   
                                                      Males                                   Females
                                                                                                                                    
                                                      Day 1b    Day 3     Day 8     Day 15    Day 1b    Day 3     Day 8     Day 15
                                                                                                                                    

    Plasma cholinesterase              30/20          83*       43*       6         12        90*       31*       27        6
                                       60/40          93*       68*       19*       14        94*       52*       23        0
    Erythrocyte cholinesterase         30/20          43*       46*       31*       23*       44*       48*       29*       17
                                       60/40          53*       46*       33*       22*       49*       50*       35*       17
    Brain cholinesterase in            30/20          45*       38        30        9         72*       25        54        25
      caudate/putamen                  60/40          93*       75*       51        32        92*       63*       39        40
    Brain cholinesterase in            30/20          45*       30*       19*       0         50*       14        8         4
      hippocampus                      60/40          72*       47*       27*       18*       75*       29*       21*       17*
    Brain cholinesterase in            30/20          48*       38*       34        19*       55*       10        0         32
      frontal cortex                   60/40          76*       50*       49*       27*       77*       40*       24*       29
    Brain cholinesterase in            30/20          46*       11        0         0         60*       11        0         3
      cerebellum                       60/40          81*       35*       7         6         80*       29*       9         3a
                                                                                                                                    

    a  100% - mean treated/mean control  100%
    b  2 h after dosing
    *  Significantly different from control at  p < 0.05
    

    clinical signs, and body weights were recorded before treatment and on
    days 1, 8, and 15. Twelve animals of each sex per group were subjected
    to a functional observational battery of tests and tests for motor
    activity before treatment and at 2 h and 8 and 15 days after dosing.
    Cholinesterase activity was determined in plasma and erythrocytes from
    the remaining five animals of each sex per group before treatment and
    on days 2, 8, and 15 after dosing. After blood collection on day 15,
    the animals were killed and their brains collected for determination
    of acetylcholinesterase activity. Neuropathological examinations were
    conducted at the end of the study on all appropriate tissues from six
    rats of each sex per group that had not been used to determine
    cholinesterase activity. All animals were examined macroscopically and
    only the control and high-dose groups microscopically.

         Treatment-related deaths were seen in two males given 75 mg/kg bw
    and six females given 50 mg/kg bw; four of the females were replaced.
    The deaths of one male at 50 mg/kg bw and one female at 5 mg/kg bw
    were considered to be unrelated to treatment. Clinical signs in
    animals at the high doses included thin or hunched appearance,
    tremors, laboured respiration, protruding eyes, incoordination,
    hypoactivity, excessive salivation, and cold to touch. A few males at
    50 mg/kg bw also showed some of these signs. The results of the
    functional battery showed mild to severe functional or behavioural
    changes only on the day of dosing, and the changes were characteristic
    of cholinesterase poisoning. No effects were seen at 5 mg/kg bw dose.
    Mild effects were seen in two females at 25 mg/kg bw and in several
    males at 50 mg/kg bw, which included salivation, lip smacking, ataxia,
    negative pupillary response, and tremors. Moderate and severe effects
    were observed in females at 50 mg/kg bw and males at 75 mg/kg bw,
    which included, in addition to the effects described above, low
    carriage and prostrate position, lethargy, changes in the ease of
    handling, lachrymation, laboured or gasping respiration, increased
    latency until first step, paralytic gait, negative corneal response,
    and negative air drop and startle reflexes. The analgesic reflex to a
    mild heat stimulus was delayed significantly on day 1 in males and
    females at 50 mg/kg bw and in males at 75 mg/kg bw. Body temperature
    was statistically significant decreased by 3-4C in females at 50
    mg/kg bw and in males at 75 mg/kg bw; a slight decrease in body
    temperature was seen in males at 50 mg/kg bw. Mean forelimb grip
    strength was decreased only on day 1 in males at 75 mg/kg bw and
    females at 50 mg/kg bw and was statistically significant only in
    males. The NOAEL for behavioural and functional effects was 5 mg/kg
    bw. Motor activity was markedly and statistically significantly
    decreased in males at 50 and 75 mg/kg bw (dose-related) and in females
    at 50 mg/kg bw on day 1 (Table 9). Differences were no longer seen on
    day 8 or 15. The NOAEL for motor activity was 5 mg/kg bw.

         The effects on cholinesterase activity are summarized in Table
    10. On day 2, plasma cholinesterase activity was dose-dependently
    inhibited for males (45-94%) and females (49-94%) at all doses but had
    recovered within 8 days of dosing. Erythrocyte acetylcholinesterase
    activity was inhibited in females at all doses (not dose-related) and
    in males at 50 and 75 mg/kg bw. Generally nonsignificant inhibition of


        Table 9. Motor activity (mean total counts) measured shortly after dosing (day 1) and during 10-min 
             intervals for 60 min in rats given single doses of ethoprophos

                                                                                                                        

    Dose         Motor activity (mean (standard deviation)) 
    (mg/kg bw)                                                                                                          
                 Males                                               Females
                                                                                                                        
                 0-10 min     10-20 min    2-30 min     Total        0-10 min     10-20 min    20-30 min    Total 
                                                        (0-60 min)                                          (0-60 min)
                                                                                                                        

    0            720 (130)    280 (140)    100 (120)    1300 (510)   580 (86)     280 (140)    98 (100)     1200 (330)
    5            630 (110)    300 (160)    51 (64)      1000 (270)   650 (160)    200 (180)    69 (110)     1200 (460)
    25                                                               480 (270)    170 (210)    78 (140)     1100 (960)
    50           290* (240)   150* (110)   20 (25)      670* (430)   38* (34)     28* (24)     44 (80)      240* (190)
    75           33* (28)     25* (22)     17 (19)      180* (100)
                                                                                                                        

    *  Significantly different at  p < 0.05 

    Table 10. Cholinesterase activity in plasma, erythrocytes, and brain of rats given a single 
              dose of ethoprophos

                                                                                                          
    Dose           Day of       Inhibition of cholinesterase from mean control valuea
    (mg/kg bw)     sampling                                                                               
                                Males                                Females
                                                                                                          
                                Plasma     Erythrocyte    Brain      Plasma       Erythrocyte     Brain
                                                                                                          

    5               2            45*           8                       49*           33*
                    8            -4           27                       11            20 
                   15            -8           -7           -3          -3             3             7
    25              2                                                  73*           49*
                    8                                                 -12            21 
                   15                                                 -14            33*           11
    50              2            87*          45*                      94*           35*
                    8            -2           26                        2            30 
                   15             0           18           17          -3            20            10
    75              2            94*          55*
                    8             6           25 
                   15            -4           31*          18
                                                                                                          

    a  100% - mean treated/mean control  100%
    *  Significantly different at  p < 0.05
    

    > 20% was still present on days 8 and 15 in females at 25 and 50
    mg/kg bw and in males at 50 and 75 mg/kg bw. In these groups, brain
    acetylcholinesterase activity was inhibited by about 10% in females
    and 18% in males at day 15, but this effect was not statistically
    significant. Macro- and microscopic neuropathological examinations
    showed no treatment-related changes. As brain acetylcholinesterase
    activity was not measured directly after exposure, the NOAEL could not
    be based on this end-point. The inhibition in females was not
    dose-related, and there was a large standard deviation at 5 mg/kg bw.
    The NOAEL for neurotoxicity was 5 mg/kg bw on the basis of behavioural
    changes and inhibition of erythrocyte acetylcholinesterase activity
    (Weiler, 1994b).

         Groups of 27 Crl:CD BR VAF/Plus rats of each sex aged 6 weeks
    were fed diets containing ethoprophos (purity, 95.7%) at
    concentrations of 0, 4, 40, or 400 ppm, equal to 0, 0.26, 2.6, and 27
    mg/kg bw per day for males and 0, 0.31, 3.0, and 31 mg/kg bw per day
    for females, for 13 weeks. Twelve rats of each sex at each dose were
    selected for functional battery and motor activity testing before
    dosing and in weeks 4, 8, and 13. Of these, six of each sex per dose
    were selected for neuropathological investigations, and the remaining
    rats were observed only macroscopically. Cholinesterase activity was
    measured in the plasma and erythrocytes of 10 rats of each sex per
    group and acetylcholinesterase activity in four regions of the brain
    (frontal cortex, caudate/putamen, hippocampus, and cerebellum) at
    sacrifice in five rats of each sex per dose in weeks 4, 8 and 14. All
    animals were observed for deaths and clinical signs, and body weight
    and food consumption were recorded.

         The only clinical sign considered to be related to treatment was
    brown staining of the anal region in males at 400 ppm. Significantly
    lower body-weight gain was observed in males at 400 ppm throughout
    treatment and in females up to week 10, which resulted in
    significantly lower body weights in animals at this dose. The mean
    food consumption of animals of each sex at the high dose was
    significantly lower during the first week and remained low in males
    throughout treatment. In males at 40 ppm, the food consumption was
    higher than that of controls. Changes in the results of the functional
    battery tests considered to be related to treatment were seen only at
    the high dose and included mild tremor (one female), vocalization
    during handling (two females), slight salivation (one male), unkempt
    fur (two males, one female), latency to first step (one male, one
    female), and jumping to touch (one male) in week 4; in week 8, the
    observations included intermittent vocalization in cage (three
    females), slight lachrymation (one female, and one female at 40 ppm),
    slight salivation (one male, two females, and one female at 40 ppm),
    latency to first step (two females), aggressive behaviour (one
    female), jump to touch response (one female), negative air drop reflex
    (one male), and negative pupillary response (one female); in week 13,
    the observations included aggressive behaviour (one female), latency
    to first step (one female, and two males at 40 ppm), and no movement
    in open arena (one male). Because the clinical signs observed at 40
    ppm also occurred occasionally as single findings in controls, they

    were considered to be unrelated to treatment. Other clearly
    treatment-related observations in the functional battery test at the
    high dose were decreased analgesic reflex in males in weeks 4, 8, and
    13, and lower grip strength in fore- and hindlimbs of males and in
    forelimbs of females. The motor activity of males at 400 ppm was
    generally significantly lower than that of controls at all times;
    females showed a slight decrease in activity only in week 4. 

         Cholinesterase activity in plasma, erythrocytes, and brain is
    shown in Table 11. A > 20% inhibition of erythrocyte
    acetylcholinesterase activity was found in males and females at doses
    > 40 ppm. Inhibition of acetylcholinesterase activity in one or two
    regions of the brain by > 20% was observed in males at 40 ppm at
    weeks 4 and 8 and in all regions at 400 ppm. In females, inhibition of
    acetylcholinesterase activity by > 20% was seen in three brain
    regions at weeks 8 and 14 at 4 ppm, which was significant in the
    frontal cortex in week 14, and in all regions at all times at higher
    doses. No macroscopic or microscopic changes were observed. 

         An NOAEL could not be identified because brain
    acetylcholinesterase was inhibited at all doses. The NOAEL for
    neurotoxic effects was 40 ppm, equal to 2.6 mg/kg bw per day, on the
    basis of the results of the functional observational battery and motor
    activity tests (Weiler, 1994c).

          Chickens 

         Two groups of 10 deep-litter hens weighing 1.4-2.1 kg (age not
    specified) were intubated with technical-grade ethoprophos (purity
    unknown) at a single dose of 5.6 l/kg bw, equivalent to 6.2 mg/kg bw
    and stated to represent the oral LD50 value in a previous study
    performed at the testing laboratory, or tri- ortho-cresyl phosphate
    (positive control) at 500 mg/kg bw. Four untreated hens were retained
    as untreated controls. The hens given ethoprophos showed no gross
    signs of ataxia or paralysis, although symptoms of transient
    inactivity and 'depression' were noted between 1 and 48 h. Four hens
    died within 48 h of dosing. The positive controls showed clinical
    signs characteristic of delayed neurotoxicity. Histopathological
    examination of sections of the spinal cord and sciatic nerve, stained
    by the Weil-Weigert method (not referenced), from birds in all groups
    at the end of a 21-day observation period revealed no evidence of
    demyelination (Weir & Murphy, 1967). 

         In an initial study conducted to determine the LD50 of
    ethoprophos in hens, six groups of 10 domestic hens ('a hybrid brown
    laying strain') were given ethoprophos (purity, 94.5%) at doses of
    0-16 mg/kg bw in a volume of 2 ml/kg bw by gavage. An LD50 of 6.4
    mg/kg bw was calculated, with a 95% confidence interval of 4.8-8.8
    mg/kg bw. On this basis, a dose of 6.5 mg/kg bw was selected for the
    main study. The hens were divided randomly into six groups of 10. The
    untreated controls received only corn oil, whereas the positive
    controls received 500 mg/kg bw of tri- ortho-cresyl phosphate by
    gavage. The treated groups first received 10 mg/kg bw of atropine 

        Table 11. Cholinesterase activity in plasma, erythrocytes, and brain regions at various 
              times during 13-week dietary exposure of rats to ethoprophos

                                                                                                
    Week    Tissue                 Inhibition of cholinesterase activity from mean in controlsa
                                                                                                
                                   Males                          Females
                                                                                                
                                   4 ppm    40 ppm     400 ppm    4 ppm      40 ppm    400 ppm
                                                                                                
    4       Plasma                   3         64*       90*        20         80*       97*
            Erythrocyte              7         30*       26*       -14         19        14
            Brain
              Caudate/putamen       17         23*       82*         8         32        83*
              Hippocampus           -7          4        48*         9         18        50*
              Frontal cortex        19         -2        71*       -28         30        74*
              Cerebellum            -7          0        52*        -3         13        53*
    8       Plasma                  -5         58*       85*        34*        87*       98*
            Erythrocyte             10         23        35         19         32        40
            Brain
              Caudate/putamen        3         11        82*       -11         -2        53*
              Hippocampus           11         26*       59*        23         33*       67*
              Frontal cortex        10         23        61*        33         49        80*
              Cerebellum             0          0        46*        21         38*       62*
    14      Plasma                  -2         54*       87*        18         86*       97*
            Erythrocyte             19         33*       40*        -6         22        11
            Brain
              Caudate/putamen       -3          0        73*        34         49*       88*
              Hippocampus            0         -9        54*        23         36*       66*
              Frontal cortex        10          7        53*        52*        56*       80*
              Cerebellum             9        -11        49*         6         27*       59*
                                                                                                

    a  100% -  mean treated/mean control  100%
    *  Significantly different from controls at  p < 0.05
    

    sulfate by intramuscular injection and then 6.5 mg/kg bw of
    ethoprophos by gavage in corn oil at a constant volume of 2.5 ml/kg
    bw. The atropine was found to have had only a minimal protective
    effect, as 31/40 of the treated birds died within 4 days, most within
    the first 24 h. Because of excessive mortality, two additional groups
    were added, consisting of 12 and 11 birds, respectively, but it was
    not specified whether they were treated concurrently with the
    untreated controls. These birds were given pyridine 2-aldoxime
    methiodide at 50 mg/kg bw by intramuscular injection in addition to
    atropine before treatment with 6.5 mg/kg bw of ethoprophos. The
    surviving birds were given further injections of pyridine 2-aldoxime
    methiodide and atropine 24 and 48 h after treatment, and two birds in
    the last group were injected 72 h after treatment. This procedure was
    only marginally successful, as 14/23 birds died within 48 h of

    treatment. On the basis of the deaths after the first dose of
    ethoprophos, the LD50 was recalculated as 5.2 mg/kg bw, and this
    dose was administered on day 22 to all 18 surviving birds, which were
    treated with pyridine 2-aldoxime methiodide and atropine immediately
    before administration of ethoprophos and again 5 h after the second
    (day 22) treatment with ethoprophos; birds in the initial groups were
    given additional injections 24 h after the second treatment. Two of
    the 18 treated birds died within 72 h of the second treatment.

         No clinical signs of neurotoxicity were noted in untreated
    control or ethoprophostreated birds, whereas 9/10 birds treated with
    tri- ortho-cresyl phosphate showed slight to marked signs of
    neurotoxicity, usually by 11 days after treatment. These birds were
    killed on day 21 and examined for histopathological changes. All other
    surviving birds were killed on day 43. No remarkable macroscopic
    changes were observed at necropsy. Portions of the forebrain, mid- and
    hind-brain, cervical, thoracic, and lumbar spine, proximal and distal
    sciatic nerve, and tibial nerve were evaluated for microscopic
    evidence of neurological change. Lesions were noted in the spinal cord
    and peripheral nerves of all positive control birds that reflected
    'significant axonal degeneration'. Minimal lesions (grade 2 out of a
    maximum of 5) were also found in the spinal cord of 9/10 control
    birds, and one control had a minimal lesion in the proximal sciatic
    nerve; no lesions were found in the brain or distal sciatic or tibial
    nerves. A similar distribution of lesions of the spinal cord was seen
    in treated birds, except that minimal lesions of the mid- or
    hind-brain and lesions of the proximal sciatic nerve were each found
    in 2/16 birds. One of the latter lesions was graded as moderate (grade
    3) and occurred in a bird that also had a lesion of the mid- and
    hind-brain. Three of 16 birds, including this one, also had minimal
    lesions of the distal sciatic nerve. The authors concluded that
    treatment with ethoprophos produced no clinical signs of neurotoxicity
    and that the result was confirmed by the histological examination,
    which showed no treatment-related changes in the nerve tissue. The
    changes noted in the proximal sciatic nerve of the bird with multiple
    lesions were considered to be unrelated to treatment and to represent
    the extreme upper limit of background change (Roberts et al., 1986).
    The Committee concluded that the study does not provide clear evidence
    of neurotoxicity, but because the high rate of mortality reduced the
    sensitivity of the study, the equivocal findings in some birds cannot
    be dismissed. Historical control data from the testing facility
    suggest that the lesions in proximal sciatic nerve are not
    spontaneous.

    (g)  Studies on metabolites

          O-Ethyl- S-propylphosphorothioate, a metabolite in rats, and
     O-ethyl- O-methyl- S-propylphosphoro-thioate and
     O-ethyl- S-methyl- S-propylphosphorodithioate, two metabolites
    identified only in corn and potatoes (Rao, 1999), were tested for
    toxicity and for their ability to inhibit cholinesterase activity in
    female rats given single oral doses.

         (i)  Acute toxicity

         The LD50 values of the three metabolites of ethoprophos in
    female Crl:CD (SD)BR VAF/ Plus rats treated orally with the substances
    in corn oil were 50 mg/kg bw for
     O-ethyl- S-methyl- S-propylphosphorodithioate, 22 mg/kg bw for
     O-ethyl- O-methyl- S-propylphosphorothioate, and 1600 mg/kg bw for
     O-ethyl- S-propylphosphorothioate. The clinical signs were similar
    for each of the metabolites and differed primarily in time of onset
    and duration; the signs were also similar to those caused by the
    parent compound. GLP and QA statements were included (Welier, 1998).

         (ii) Cholinesterase inhibition

         Three groups of 10 female Crl:CD(R)(SD)BR VAF/Plus rats aged
    about 6 weeks were given a single dose of ethoprophos at 19 mg/kg bw,
     O-ethyl- S-methyl- Spropylphosphorodithioate at 17 mg/kg bw, or
     O-ethyl- O-methyl- S-propylphosphorothioate at 8 mg/kg bw by
    gavage; a control group received the vehicle, corn oil. The animals
    were observed for clinical signs before dosing, 0.5 and 2 h after
    dosing, and the next day before sacrifice. Cholinesterase activity was
    evaluated in plasma, erythrocytes, and brain 24 h after dosing.

         Body-weight gain over the 2-day period was significantly lower in
    all treated groups than in controls but resulted in a statistically
    significant lower terminal body weight only with
     O-ethyl- S-methyl- S-propylphosphorodithioate. In the other two
    groups, the only clinical finding was a slightly increased incidence
    of soft stools, whereas the animals given
     O-ethyl- S-methyl- S-propylphosphorodithioate showed signs of
    cholinesterase inhibition consisting of staggered gait and tremors.
    All three compounds reduced cholinesterase activity in all three
    tissues, the strongest inhibition being due to
     O-ethyl- S-methyl- S-propylphosphorodithioate (Table 12; Weiler,
    1998).

        Table 12. Cholinesterase activity in plasma, erythrocytes, and brain of female rats 24 h 
              after a single oral dose of ethoprophos and two metabolites

                                                                                                 

    Compound                                       Dose        Inhibition from control mean (%)a
                                                   (mg/kg bw)                                    
                                                               Plasma    Erythrocytes   Brain
                                                                                                 
    Ethoprophos                                    19          73*       37*            32*
    O-Ethyl-S-methyl-S-propylphosphorodithioate    17          78*       30             71*
    O-Ethyl-O-methyl-S-propylphosphorothioate       8          40*       47*            48*
                                                                                                 

    a   100% - mean treated/mean control  100%
    *   Significantly different from control at  p < 0.05
    
    4.  Observations in humans

         In a study of occupational exposure to ethoprophos in Salinas
    Valley, Monterey County, California, USA, four male volunteers were
    recruited among regular employees of an experienced pesticide
    application firm who were thoroughly familiar with both the practices
    and equipment involved in pesticide application and had not worked
    with cholinesterase inhibitors for at least 90 days. The occupation
    under consideration was that of mixer-loader-applicator, who applied
    an emulsifiable concentrate containing 70% ethoprophos in xylene for 3
    days (two men), 4 days (one man), or 5 days (one man) to field plots
    of 2-3 ha; the only operator working on day 5 treated a field plot of
    about 8.6 ha. During the mixing and loading phase, the concentrate,
    supplied in 5-gallon (~ 20-L) containers, was diluted to 5% in two
    160-gallon (~ 40-L) tanks. Transfer of the pesticide into the tank,
    rinsing the container, and filling the spray tanks were done in a
    closed system to minimize exposure. The operators wore long-sleeved,
    twill work shirts, denim jeans, and rubber boots. They used rubber
    gloves and disposable respirators which were changed each day. Face
    shields and hard hats were available, especially for the mixing and
    loading operations. The application rate of active ingredient was
    about 12 kg/ha (range, 11-15 kg/ha). Blood was collected for
    determination of cholinesterase activity in plasma and erythrocytes
    three times before exposure and as soon as possible after each man's
    work session. During application of a commercial preparation
    containing 69.6% ethorprop (as an emulsifiable concentrate in xylene),
    dermal exposure was monitored on the hands (three hand washings with
    2-propanol in water) and face (passive facial patch collector
    consisting of 12-layer surgical gauze), corresponding to a surface of
    24 cm2. Inhalation was not monitored. Exposure to ethoprophos was
    monitored by a standardized gas chromatographic method.

         No clinical signs or adverse effects were noted during exposure,
    and the changes in plasma and erythrocyte cholinesterase activity were
    not statistically significantly different from the mean baseline
    values. The maximal variation around the mean was - 8.9% to 7.9%,
    which is considered to be normal day-to-day variation. Exposure on the
    head and neck was 3.1-78 g/h, with an average of 34 g/h
    (calculations based on a default head area of 910 cm2). The total
    exposure of the head to ethoprophos was 7.6-153 g, with means of 96,
    53, 67, and 36 g for the four men. The hands were exposed to 0.2-18
    g/h, with an average value of 6.3 g/h and a value of 0.52-42 g for
    both hands; the mean values were 29, 2.0, 12, and 6.3 g for the four
    men. The overall exposure during the entire period of observation of
    the man who worked for 5 days was 620 g, and the quantity of active
    ingredient handled per day was 31-92.4 kg. The overall exposure of the
    other operators was 220 g for the man who worked for 4 days and
    handled 26-44 kg daily; 240 g for the man who worked for 3 days and
    handled 19-27 kg daily; and 130 g for the man who worked for 3 days
    and handled 17-44 kg daily (Leffingwell, 1986).

    Comments

         After oral administration to rats, 14C-ethoprophos was rapidly
    and virtually completely absorbed, metabolized and excreted. The main
    route of excretion was the urine (51-56%), but significant proportions
    were excreted in expired air (about 15%) and faeces (10-14%). Little
    radiolabel was found in tissues at 168 h, representing less than 2.5%
    of the dose, and the highest concentrations were found in excretory
    organs (liver, kidneys, and lungs). There was no evidence that
    bioaccumulation would occur after repeated doses. The kinetics of the
    radiolabel and the biotransformation appeared to be independent of the
    route of administration (oral or intravenous), magnitude and frequency
    of dose and sex. Ethoprophos was metabolized by dealkylation of one or
    both  S-propyl groups, followed by conjugation. The dermal absorption
    of ethoprophos has not been studied  in vivo, but radiolabelled
    ethoprophos penetrated the skin  in vitro, the greatest penetration
    being seen through mouse skin, lesser penetration through rat and
    rabbit skin (at equal rates), and the least penetration through human
    skin. In all of the species tested, the rate of penetration was
    greater with ethoprophos (emulsified concentrate) diluted 1:19 in
    water than with undiluted material.

         Single oral doses of ethoprophos were toxic to rats, mice and
    rabbits (LD50 values, 31-62 mg/kg bw), and it was highly toxic to
    rats exposed once by inhalation (LC50, 0.25 mg/L). Mice and rabbits
    were more sensitive to dermal exposure than rats, and female rats were
    more sensitive than males, with LD50 values of 8.5 mg/kg bw for
    rabbits, 18 mg/kg bw for mice, 420 mg/kg bw for female rats, and 1300
    mg/kg bw for male rats. The toxic signs observed after exposure by any
    route were characteristic of cholinesterase inhibition. WHO (1999) has
    classified ethoprophos as 'extremely hazardous'.

         Ethoprophos not only irritates the eyes of rabbits but is very
    toxic after administration into the eye, causing the death of tested
    animals within 1 h. In a test for dermal irritation, all treated
    animals died within 8 h of exposure to the undiluted compound. In a
    three-week study in rabbits, the compound caused slight dermal
    irritation at all doses tested (0.03, 0.1, and 1 mg/kg bw per day),
    whereas no dermal irritation was observed in a 3-week study in rats
    given doses of up to 10 mg/kg bw per day. 

         In studies in dogs (13, 20 and 52 weeks) and mice and rats (2
    years) given ethoprophos orally, inhibition of cholinesterase was the
    most sensitive parameter. In dogs, the NOAEL for inhibition of brain
    acetylcholinesterase activity was 1 mg/kg bw per day, the highest dose
    tested. In mice, brain acetylcholinesterase activity was inhibited at
    30 ppm, resulting in a NOAEL of 2 ppm, equal to 0.25 mg/kg bw per day;
    in rats, the overall LOAEL was 30 ppm (equal to 1.3 mg/kg bw per day)
    and no effects were seen at 10 or 1 ppm (equal to 0.5 and 0.04 mg/kg
    bw per day, respectively). In a two-generation study of reproductive
    toxicity in rats, inhibition of brain acetylcholinesterase activity
    was seen in animals of each sex of both parent generations at 30 ppm,
    resulting in a NOAEL of 1 ppm, equal to 0.04 mg/kg bw per day. In a

    13-week study of neurotoxicity in rats, the activity of brain
    acetylcholinesterase was inhibited in females at all doses tested, the
    lowest dose being 4 ppm, equal to 0.26 mg/kg bw per day. Clinical
    signs characteristic of cholinesterase inhibition were observed only
    in the 2-year study in rats, in which the highest dose was reduced
    from 600 to 400 ppm because of clinical signs.

         Inhibition of brain acetylcholinesterase activity was also
    demonstrated after dermal exposure of rabbits and rats to ethoprophos
    for three weeks, with NOAELs of 0.1 mg/kg bw per day (LOAEL, 1 mg/kg
    bw per day) in rabbits and 0.3 mg/kg bw per day (LOAEL, 1 mg/kg bw per
    day) in rats. 

         Ethoprophos was not carcinogenic in a long-term study in mice
    treated in the diet. The highest dose tested was 30 ppm, equal to 4.9
    mg/kg bw per day. In three long-term studies of toxicity and
    carcinogenicity in rats, some evidence was obtained that ethoprophos
    may stimulate progression of C-cell tumours in the thyroid and adrenal
    neoplasms (phaeochromocytomas), but this effect was considered to have
    little relevance for human risk. The highest dose tested was 400 ppm,
    equal to 26 mg/kg bw per day. The Meeting concluded that ethoprophos
    is unlikely to pose a carcinogenic risk to humans.

         Ethoprophos was tested in an adequate set of assays for
    genotoxicity  in vitro and  in vivo. Positive results in studies for
    chromosomal aberrations and sister chromatid exchanges  in vitro 
    after metabolic activation indicate that the compound has intrinsic
    genotoxic activity. Chromosomal aberrations were not induced in rats
    in two assays and dominant lethal mutations were not induced in rats
    in another study, although a slightly positive or equivocal result was
    obtained in a further study for dominant lethality. The Meeting
    concluded that ethoprophos is unlikely to be genotoxic  in vivo.

         In a two-generation study of reproductive toxicity in rats, no
    effect was observed on reproductive parameters. Clinical signs were
    observed only in the F0 parents at the highest dose (300 ppm). The
    NOAEL was 1 ppm, equal to 0.04 mg/kg bw per day, on the basis of
    reduced body-weight gain in F0 males and inhibition of brain
    acetylcholinesterase activity in animals of each sex in both parental
    generations. The NOAEL for developmental toxicity was 30 ppm, equal to
    1.3 mg/kg bw per day, on the basis of effects on body weight and
    body-weight gain in pups of both generations and deaths of pups in the
    F2 litters at 150 ppm. 

         In studies of developmental toxicity, maternal toxicity was
    characterized by growth depression in rats in one study (NOAEL,
    2 mg/kg bw per day) and in rabbits in one study (NOAEL, 0.12 mg/kg bw
    per day), whereas no maternal toxicity was observed in rabbits in
    another study (NOAEL, 2.5 mg/kg bw per day, highest dose tested).
    Since in the first study in rabbits the effect was only marginal (not
    statistically significant), the overall NOAEL for maternal effects in
    rabbits was 2.5 mg/kg bw per day. No effects on the fetuses of either
    species were observed. Cholinesterase activity was not measured in
    these studies.

         The relationship between neurotoxicity and cholinesterase
    inhibition was examined in rats in two studies. In rats exposed by
    gavage to single doses of 0, 20, or 40 mg/kg bw (females) or 0, 30, or
    60 mg/kg bw (males), cholinesterase activity in plasma, erythrocytes,
    and brain was inhibited, with a maximum effect 2 h after dosing. While
    cholinesterase inhibition was found in tissues at the lowest doses
    tested, clinical signs characteristic of cholinesterase inhibition
    were seen only at the highest doses (60 mg/kg bw for males and
    40 mg/kg bw for females). In the second study, in which male rats were
    exposed to a single dose of ethoprophos at 5, 50, or 75 mg/kg bw and
    females at 5, 25, or 50 mg/kg bw, effects on motor activity were
    observed in animals of each sex at doses of 50 mg/kg bw and higher,
    and functional and behavioural effects were observed at 25 mg/kg bw
    and higher. These neurotoxic signs were seen only on the day of
    dosing. Significant inhibition of acetylcholinesterase activity in
    erythrocytes, measured 1 day after exposure, was observed in females
    at doses of 5 mg/kg bw and higher and in males at 50 mg/kg bw and
    higher. The inhibition in females was not dose-related, and there was
    a large standard deviation at 5 mg/kg bw . The Meeting concluded that
    the NOAEL in this study was 5 mg/kg bw. 

         In a 13-week study in rats, neurotoxicity was observed in tests
    for motor activity and for functional and behavioural abnormalities
    only at the highest dose (400 ppm). The NOAEL for neurotoxic effects
    was 40 ppm, equal to 2.6 mg/kg bw per day. The activity of brain
    acetylcholinesterase was inhibited in females at all doses tested; the
    lowest dose was 4 ppm, equal to 0.26 mg/kg bw per day. 

         A study of delayed neurotoxicity in hens was performed with doses
    of ethoprophos that caused a high mortality rate despite antidotal
    treatment. Equivocal findings were reported in some of the treated
    birds; data on the effect of this compound on neuropathy target
    esterase activity in hens would be useful in order to allow full
    assessment of the neuropathological potential of ethoprophos.

         A study of occupational exposure to ethoprophos did not reveal
    significant effects on human plasma or erythrocyte cholinesterase
    activity. The calculated rates of exposure of head-and-neck areas were
    3.1-78 g/h (average, 34 g/h), and the rate of exposure of the hands
    was calculated to be 0.2-18 g/h (average, 6.3 g/h). 

          O-Ethyl  S-propyl phosphorothioate, a metabolite in rats, and
     O-ethyl  O-methyl  S-propyl phosphorothioate and  O-ethyl
     S-methyl  S-propyl phosphorodithioate, two metabolites that have
    been identified only in corn and potatoes, were tested for toxicity
    and for their ability to inhibit cholinesterase activity in female
    rats given single oral doses. The two plant metabolites were of
    approximately the same toxicity as the parent compound, while the
    animal metabolite was less toxic (LD50, 1600 mg/kg bw) than the
    parent. 

         The 1987 Meeting derived the ADI on the basis of a slight effect
    on the liver in a 52-week study in dogs at a dose of 1 mg/kg bw per
    day. The NOAEL for this effect was 0.025 mg/kg bw per day. Since this
    marginal effect was not observed in other studies in dogs or in other
    species and there is a 40-fold difference between the LOAEL and the
    NOAEL, this NOAEL was not used as the overall NOAEL to derive the ADI
    in the present evaluation.

         The present Meeting established an ADI of 0-0.0004 mg/kg bw on
    the basis of the NOAEL of 1 ppm, equal to 0.04 mg/kg bw per day, for
    inhibition of brain acetylcholinesterase activity in the 2-year study
    of toxicity and carcinogenicity in rats and in the study of
    reproductive toxicity in rats, and a 100-fold safety factor. 

         An acute reference dose of 0.05 mg/kg bw was established on the
    basis of the NOAEL of 5 mg/kg bw in the study of acute neurotoxicity
    in rats, in which functional and/or behavioural effects and inhibition
    of erythrocyte acetylcholinesterase were observed at the next highest
    dose, and a 100-fold safety factor. 

    Toxicological evaluation

     Levels that cause no toxic effect 


    Mouse     2 ppm, equal to 0.25 mg/kg bw per day (2-year study of
              carcinogenicity and toxicity)

    Rat       1 ppm, equal to 0.04 mg/kg bw per day (2-year study of
              carcinogenicity and toxicity)

              1 ppm, equal to 0.04 mg/kg bw per day (parental toxicity in
              a study of reproductive toxicity)

              5 mg/kg bw (single dose, study of neurotoxicity by gavage)

              2 mg/kg bw (maternal toxicity, study of developmental
              toxicity; acetylcholinesterase activity not determined);

              18 mg/kg bw (fetotoxicity, highest dose tested, study of
              developmental toxicity; acetylcholinesterase activity not
              determined)

    Rabbit    2.5 mg/kg bw (maternal toxicity and fetotoxicity, study of
              developmental toxicity; acetylcholinesterase activity not
              determined);

    Dog       0.025 mg/kg bw per day (1-year study of toxicity) 

     Estimate of acceptable daily intake for humans 

         0-0.0004 mg/kg bw 

     Estimate of acute reference dose 

         0.05 mg/kg bw

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

    1.   Information on effects on neuropathy target esterase activity in
         hens

    2.   Further observations in humans 


        Toxicological end-points relevant for setting guidance values for dietary and non-dietary exposure to ethoprophos

     Absorption, distribution, excretion and metabolism in mammals 

    Rate and extent of oral absorption           > 70%, mainly within 12 h (rats)
    Dermal absorption                            Tested  in vitro, no quantitative data
    Penetration rate                             Mouse skin > rat, rabbit skin > human skin
    Distribution                                 Widely distributed, highest levels in organs of elimination (rats)
    Potential for accumulation                   No
    Rate and extent of excretion                 Excreted in urine (55%), expired air (13-17%), and faeces (7-9%) (rats)
    Metabolism in animals                        In rats, metabolized by dealkylation of one or both  S-propyl groups, 
                                                 followed by conjugation
    Toxicologically significant compounds        Parent compound and plant metabolites ( O-ethyl  O-methyl  S-propyl 
                                                 phosphorothioate and  O-ethyl  S-methyl S-propyl phosphorodithioate) 

     Acute toxicity 

    Rat, LD50, oral                              33-62 mg/kg bw (vehicle, corn oil)
    Rat, LD50, dermal                            420 mg/kg bw, females
    Rat, LD50, inhalation                        0.25 mg/L
    Dermal irritation                            Not reported, but death occurred within 8 h of dermal exposure 
    Ocular irritation                            Yes, but also death within 1 h of ocular exposure 
    Sensitization                                Not tested

     Short-term toxicity 

    Target/critical effect                       Acetylcholinesterase inhibition
    Lowest relevant oral NOAEL                   < 0.26 mg/kg bw per day (LOAEL, 13-week study of neurotoxicity 
                                                 in rats)
    Lowest relevant dermal NOAEL                 0.1 mg/kg bw per day (3-week study of dermal  toxicity in rabbits)
    Lowest relevant inhalation NOAEL             Not tested

     Long-term toxicity and carcinogenicity 

    Target/critical effect                       Acetylcholinesterase inhibition
    Lowest relevant NOAEL                        0.04 mg/kg bw per day (105-week study in rats treated in the diet)
    Carcinogenicity                              Not carcinogenic (mice, rats)

    Genotoxicity                                 Not genotoxic  in vivo 

     Reproductive toxicity 

    Reproductive target/critical effect          Reduced body weight and survival at maternally toxic doses
    Lowest relevant reproductive NOAEL           Maternal toxicity: 1 ppm, equal to 0.04 mg/kg bw per day; 
                                                 fetotoxicity: 30 ppm, equal to 1.3  mg/kg bw per day 
    Developmental target/critical effect         Only maternal toxicity (highest dose tested), no fetotoxicity 
    Lowest relevant developmental NOAEL          Rabbit: maternal and fetotoxicity: 2.5 mg/kg bw per day (highest 
                                                 dose tested)
                                                 Rat: maternal toxicity: 2 mg/kg bw per day; fetotoxicity: 
                                                 18 mg/kg bw per day 

    Neurotoxicity/ Delayed neurotoxicity         5 mg/kg bw per day (acute toxicity in rats)
                                                 < 4 ppm, equal to 0.26 mg/kg bw per day (13-week study in rats)
                                                 No evidence for delayed neurotoxicity in hens, but some equivocal findings 

    Other toxicological studies                  No significant effects on erythrocte acetylcholinesterase activity in 
                                                 exposed workers

    Medical data                                 None

                                                                                                                  
    Summary                  Value                  Study                                     Safety factor
                                                                                                                  

    ADI                      0-0.0004 mg/kg bw      2-year study, rat; two-generation        100
                                                    study of reproductive toxicity, rats

    Acute reference dose     0.05 mg/kg bw          Acute neurotoxicity, rats                100
                                                                                                                  
    

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         mammalian cell forward gene mutation assay. Unpublished report
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    See Also:
       Toxicological Abbreviations
       Ethoprophos (ICSC)
       Ethoprophos (Pesticide residues in food: 1983 evaluations)
       Ethoprophos (Pesticide residues in food: 1984 evaluations)
       Ethoprophos (Pesticide residues in food: 1987 evaluations Part II Toxicology)