PHORATE First draft prepared by M. Caris, Bureau of Chemical Safety, Health Canada, Ottawa, Canada Explanation Evaluation for acceptable daily intake Biochemical aspects Absorption, distribution and excretion Biotransformation Toxicological studies Acute toxicity Short-term toxicity Long-term toxicity and carcinogenicity Reproductive toxicity Embryotoxicity and teratogenicity Genotoxicity Special studies Delayed neuropathy Studies of metabolites Enzymes and biochemical parameters Acute toxicity Short-term toxicity Observations in humans Comments Toxicological evaluation References Explanation Phorate, an organophosphorus insecticide which inhibits cholinesterase, was first reviewed by the Joint Meeting in 1977 (Annex I, reference 28). A temporary ADI of 0-0.0002 mg/kg bw was established in 1982 (Annex I, reference 38). In 1985, after review of a study of delayed neurotoxicity in chickens, an ADI of 0-0.0002 mg/kg bw was established (Annex I, reference 44). The purpose of the present evaluation was to review additional information on toxicity, submitted to complement and update the database. This included a 90-day study in mice treated in the diet, 14-day and one-year studies of toxicity in dogs treated orally, a two-generation study of reproductive toxicity in rats, studies of teratogenicity and preliminary range-finding studies in rats and rabbits, and studies to determine genotoxicity. In order to facilitate a comprehensive review of data on the toxicology of phorate, relevant summaries from previously published monographs and monograph addenda (Annex I, references 29, 39 and 46) are included in this monograph. Evaluation for acceptable daily intake 1. Biochemical aspects (a) Absorption, distribution and excretion Male rats given a single oral dose of 2 mg/kg bw of 32P-labelled phorate excreted 35% of the administered radiolabel in the urine and 3.5% in the faeces within 144 h. Male rats given six daily doses of 1 mg/kg bw excreted 12% of the administered radiolabel in the urine and 6% in the faeces within seven days. Brain, liver and kidney tissues from the latter animals contained unidentified and largely unextractable residues (Bowman & Casida, 1958; Annex I, reference 29). (b) Biotransformation The urine of male rats given daily oral doses of 1 mg/kg bw contained 17% diethyl phosphoric acid, 80% O,O-diethyl phosphorothioic acid and 3% O,O-diethyl phosphorodithioic acid. After 32P-labelled phorate was incubated with rat liver slice preparations, less than 1% of the radiolabelled compound was converted to hydrolysis products or unextractable residues. Phorate sulfoxide, phorate sulfone, phoratoxon sulfoxide and phoratoxon sulfone were formed (Figure 1) (Bowman & Casida, 1958; Annex I, reference 29). 2. Toxicological studies (a) Acute toxicity The acute toxicity of phorate is summarized in Table 1. Phorate was extremely toxic to rats when administered by the oral, dermal and intravenous routes, with acute LD50 values ranging from 1.1 to 10 mg/kg bw. Phorate was also severely toxic to rabbits after dermal administration and to mice when given either orally or intraperitoneally.Table 1. Acute toxicity of technical-grade phorate Species Sex Route LD50 (mg/kg bw per day) Reference or LC50 (mg/m3) Mouse NR Oral 11 Blinn, 1982 NR Intraperitoneal 3 Blinn, 1982 Rat NR Oral 1.9-10 Blinn, 1982 M&F Oral M: 2.3; F: 1.1 Gaines, 1969 M&F Oral M: 2.8; F: 1.6 Anon., 1976 M&F Oral M: 3.7; F: 1.4 Newell & Dilley, 1978 NR Dermal 3 Blinn, 1982 M&F Dermal M: 6.2; F: 2.5 Gaines, 1969 M Dermal 5.7 American Cyanamid Co., 1976 M&F Dermal M: 9.3; F: 3.9 Newell & Dilley, 1978 M&F Intravenous M: 2.2; F: 1.2 Newell & Dilley, 1978 M&F Inhalation (1 h) M: 60 Newell & Dilley, 1978 F: 11 Newell & Dilley, 1978 Rabbit M Dermal 5.2 Anon., 1976 From Annex I, references 29 and 39; strains not specified NR, not reported (b) Short-term toxicity Mice Phorate (purity, 92.1%) was administered to groups of 20 male and 20 female Crl:CD-1 (ICR)BR outbred Swiss mice at dietary levels of 0, 1, 3 or 6 ppm, equal to 0.18, 0.55 or 1.10 mg/kg bw per day for males and 0.23, 0.67 or 1.38 mg/kg bw per day for females, for 13 weeks. A single male mouse treated at the highest dietary level had convulsions on one occasion during the final week of treatment, but the relationship of this isolated finding to treatment could not be ascertained; no other remarkable clinical signs were reported. Treatment had no effect on survival, food consumption or body-weight gain. Histopathological examinations were not performed. Plasma, erythrocyte and brain cholinesterase activities were measured before terminal sacrifice. Plasma cholinesterase activity was significantly ( p < 0.05) inhibited in animals of each sex at 3 and 6 ppm and in females at 1 ppm. Erythrocyte cholinesterase levels were significantly decreased in males and females at the highest dose; the inhibition, in relation to values in concurrent controls, was 50% in males and 61% in females. A slight, non-significant decrease in erythrocyte cholinesterase activity (17%) was recorded in females at 3 ppm. Brain cholinesterase levels, assayed in both halves of the brain, were significantly ( p < 0.05) decreased in animals of each sex treated at 3 and 6 ppm, by slightly more than 10% at 3 ppm and to about 50% of control values at 6 ppm. Minimal inhibition of plasma cholinesterase levels (15%) in females at 1 ppm was not considered to represent an adverse effect of treatment, particularly in the absence of corresponding decreases in erythrocyte and brain cholinesterase levels. The NOAEL was 1 ppm, equal to 0.18 mg/kg bw per day, on the basis of significantly reduced brain cholinesterase activity at dietary levels of 3 and 6 ppm (Trutter, 1990). Rats Groups of 50 male and 50 female rats [strain not specified] were fed phorate (purity, 92%) in the diet at levels of 0, 0.22, 0.66, 2 or 6 ppm, equivalent to 0.01, 0.03, 0.1 or 0.3 mg/kg bw per day, and groups of 25 male and 25 female rats were fed 12 or 18 ppm, equivalent to 0.6 or 0.9 mg/kg bw per day. Treatment continued for 13 weeks, and cholinesterase activity was monitored each week. Occasional episodes of excitability and intermittent tremors were noted in females given 6 ppm, and animals of each sex receiving 12 and 18 ppm showed severe excitability, intermittent tremors and ataxia, culminating in death of 50% of animals at 12 ppm and no survivors at 18 ppm. Levels of 6 ppm and higher produced significant depression of plasma, erythrocyte and brain cholinesterase activity in animals of each sex and decreased erythrocyte cholinesterase activity in females at 2 ppm. In all groups receiving 6 ppm or less, growth, food consumption, survival and liver and kidney weights and ratios were within normal limits, and no abnormalities were seen at gross necropsy or histological examination. The NOAEL was 2 ppm, equivalent to 0.1 mg/kg bw per day, on the basis of inhibition of brain cholinesterase activity at 6 ppm and above (Tusing et al., 1956a; Annex I, reference 29). Dogs Groups of two male and one female mongrel dogs received capsules containing 0, 0.01, 0.05, 0.25 or 1.25 mg/kg bw per day of phorate (purity, 92%) in corn oil on six days per week for 15 weeks. Two males received a single dose of 2.5 mg/kg bw. Plasma and erythrocyte cholinesterase activities were monitored weekly; brain cholinesterase activity was not measured. At 0.05 mg/kg bw per day, plasma enzyme activity was significantly depressed. Erythrocyte cholinesterase activity was not affected during the first 12 weeks but was depressed slightly (not significantly) during the last three weeks of the study. Significant decreases in plasma and erythrocyte enzyme activity were observed at 0.25 mg/kg bw per day, and 1.25 mg/kg bw per day induced total inhibition of plasma cholinesterase activity and a significant reduction in erythrocyte cholinesterase activity. All of the dogs administered the single dose of 2.5 mg/kg bw per day died within 3-4 h; enzyme activity was not determined. No signs of systemic toxicity were seen, and haematological, clinical chemistry and urine analyses showed no effects in dogs receiving 0.01 or 0.05 mg/kg bw per day. Histopathological examination revealed no consistent findings related to the treatment (Tusing et al., 1956b; Annex I, reference 29). Groups of three male and three female beagle dogs were fed phorate in the diet at 0, 0.5 or 1.0 ppm, equivalent to 0.012 or 0.025 mg/kg bw per day, for six weeks. Cholinesterase activity in plasma and erythrocytes was determined before and every two weeks during the test. Analyses of variance revealed no significant difference between the treated and control animals with respect to plasma and erythrocyte enzyme activities (Kay & Calandra, 1961; Annex I, reference 29). In a preliminary range-finding study, groups of two male and two female beagle dogs were given capsules containing phorate (purity, 92.1%) in corn oil at doses of 0.01, 0.05, 0.10, 0.25 or 0.50 mg/kg bw per day for 14 days. A control group of three male and three female dogs was given capsules containing corn oil. Plasma and erythrocyte cholinesterase levels were measured twice before treatment, and after 3, 7, 10 and 14 days of treatment; brain cholinesterase levels were determined in the cerebellum and cerebrum of each dog at sacrifice. Excessive salivation and tremors were seen in one female dog given the highest dose, and body-weight gain was decreased in comparison with the controls in animals of each sex. Slight decreases in total serum protein were recorded in males and females at the high dose. Treatment did not affect survival, food consumption, haematological parameters or organ weights, and gross pathology showed no effects; histopathological examinations were not performed. Plasma cholinesterase levels were significantly decreased in animals of each sex given 0.10 mg/kg bw per day or more and in males given 0.05 mg/kg bw per day, in comparison with values before treatment and in control animals, and erythrocyte cholinesterase activity was significantly (> 20%) inhibited in both males and females at the highest dose of 0.50 mg/kg bw per day. Brain cholinesterase activity was inhibited (31-69%) in both males and females at 0.10 mg/kg bw per day or more. The NOAEL was 0.05 mg/kg bw per day (Piccirillo et al., 1987). Phorate (purity, 92.1%) in corn oil was administered orally by capsule to groups of six male and six female beagle dogs at doses of 0.005, 0.01, 0.05 or 0.25 mg/kg bw per day for one year. Eight male and eight female controls received capsules containing only corn oil. Plasma and erythrocyte cholinesterase activities were measured twice before treatment, at six weeks, at three and six months and at sacrifice; brain cholinesterase levels were measured in the cerebrum and cerebellum at termination of treatment. Mild tremors were observed occasionally in one male and two females during weeks 23-52 of treatment with the high dose. Mean body weights and overall weight gain (26% less than control) were decreased in males at 0.25 mg/kg bw per day. Ophthalmological examination of all dogs before treatment, at six months and at one year showed no treatment-related effects. There were no adverse effects on food consumption or haematological or urinary parameters. Decreased ( p < 0.05) total protein levels were recorded at week 6, at three and six months and at termination of treatment in males treated at 0.25 mg/kg bw per day. Although the values were reported to be within the range in historical controls, no supporting data were provided. As the individual levels of total protein in males at the high dose were consistently below concurrent values before treatment and in controls, the finding appeared to be related to treatment. The decreases ( p < 0.05) in total protein values in females and the decreased albumin values in males at the high dose were within the lower limits of control values. Gross and histopathological examination revealed no lesions that could be attributed to treatment with phorate. The highest dose induced inhibition of erythrocyte (> 20%) and brain cholinesterase activity (43-54%); plasma cholinesterase levels were significantly decreased in animals of each sex at doses of 0.05 mg/kg bw per day and above. The decrease in plasma cholinesterase activity at this dose was not considered to be adverse in the absence of correlative inhibition of brain and erythrocyte cholinesterase activity and evidence of clinical toxicity. The NOAEL was 0.05 mg/kg bw per day on the basis of decreased body weight, significant inhibition of erythrocyte and brain cholinesterase activity and clinical signs consistent with cholinergic toxicity at the high dose of 0.25 mg/kg bw per day (Shellenberger & Tegeris, 1987). (c) Long-term toxicity and carcinogenicity Mice Groups of 50 male and 50 female 41-day-old CD-1 outbred Swiss albino mice were fed technical-grade phorate (purity, 85.5%) in the diet at 0, 1, 3 or 6 ppm, equivalent to 0.15, 0.45 or 0.9 mg/kg bw per day for 18 months. All animals that died or were killed in moribund condition during the study or were sacrificed terminally were subjected to gross and histopathological examination. Survival was not adversely affected: 78-90% of males and 66-74% of females in the control and treated groups were still alive at the end of the experiment. The growth of females at 6 ppm was retarded almost throughout the experiment. All treated animals appeared to eat less food during the first three weeks and occasionally thereafter, but no consistent dose-response relationship was seen. Some clinical signs, such as tremors, hyperactivity and excessive salivation, occurred at higher incidence and more frequently in animals at the highest dose than in controls. Gross pathological examination showed no changes significantly different from those in the controls, and microscopic evaluation of a wide range of tissues from each animal revealed no alterations related to treatment. There was no significant dose-related increase in the incidence of any particular type of tumour, of animals with tumours, of animals with malignant tumours or of animals with multiple primary tumours. Although the incidence of alveolar and bronchiolar adenoma appeared to be increased in males at 6 ppm (8/50, compared with 3/50 in controls), this effect was not considered to be related to treatment since the increase was not statistically significant and this tumour is known to occur frequently in CD-1 mice. The NOAEL was 3 ppm, equivalent to 0.45 mg/kg bw per day, on the basis of decreased body weight and clinical signs of toxicity at 6 ppm (Litton Bionetics, 1981a; Annex I, reference 39). Rats Groups of 50 male and 50 female five-week-old Crl:COBS CD(SD) BR rats were fed dietary levels of technical-grade phorate (purity, 84.5%) at 0, 1, 3 or 6 ppm, equal to 0.05, 0.16 or 0.32 mg/kg bw per day in males and 0.07, 0.19 or 0.43 mg/kg bw per day in females, for 24 months. The mortality rate appeared to be increased in females at 6 ppm, as only 36% survived to the end of the experiment; however, more than 60% of animals in all groups, including the controls, lived at least 90 weeks. The only clinical sign related to treatment was tremors induced by over-dosing (327% of all the intended doses) during week 9. Growth was depressed in females at 6 ppm during the first 26 weeks and again between weeks 74 and 102. Food consumption showed no consistent dose-response pattern. On haematological examination, clinical chemistry and urinalysis performed at 6, 12 and 24 months, the only notable findings were decreased values for erythrocyte counts, haemoglobin and haematocrit in females at the highest dose at 12 months. Dose-related inhibition (> 20%) of plasma cholinesterase activity was noted in males at 6 ppm at 12 months, in all treated males at 24 months and in females at both 3 and 6 ppm at all sampling intervals (3, 6, 12 and 24 months). Erythrocyte cholinesterase activity was not significantly depressed (< 20%) at any time. The activity of brain cholinesterase was reduced (> 20%) in males at 6 ppm and in females at 3 ppm or more. At sacrifice, females given the highest dose had increased organ:body weight ratios with respect to the adrenals, brain, heart, liver and spleen. On gross pathological and histopathological examination of a variety of tissues, the only effect apparently related to treatment was a significant increase in the incidence of inflammation and epithelial hyperplasia of the forestomach in animals of each sex, but particularly in males, at 6 ppm. The most prevalent types of spontaneous tumours were pituitary adenomas in animals of each sex and mammary tumours in females. There were no significant differences between control and treated groups with regard to incidence, type or time of appearance of tumours. The marginal no-effect level was 1 ppm, equal to 0.05 mg/kg bw per day (Litton Bionetics, 1981b; Annex I, reference 39). (d) Reproductive toxicity Mice Groups of eight male and 16 female mice (strain not specified) were fed phorate (purity unknown) in the diet at levels of 0, 0.6, 1.5 or 3 ppm, equivalent to 0.09, 0.23 or 0.45 mg/kg bw per day, for three generations, with two litters per generation. There were no dose-related effects on indices of fertility, gestation, viability or lactation during the study, but the lactation index was slightly lowered in the group receiving 3 ppm, to below the control value in the first mating of the F0 generation, in both matings of the F1 generation and in the second mating of the F2 generation. Gross and microscopic examination of tissues revealed no consistent abnormalities related to treatment. The NOAEL was 1.5 ppm, equivalent to 0.23 mg/kg bw per day, on the basis of decreased lactation indices at 3 ppm (American Cyanamid Co., 1965; Annex I, reference 39). Rats A two-generation study of reproductive toxicity was conducted in groups of 25 male and 25 female COBS CD(SD) rats given phorate (purity, 92.1%) at dietary levels of 0, 1, 2, 4 or 6 ppm, equal to 0.09, 0.17, 0.35 or 0.52 mg/kg bw per day in males and 0.10, 0.20, 0.40 or 0.62 mg/kg bw per day in females, for a minimum of 60 days before mating, to produce two consecutive litters (F1a and F1b) of the first (F0) parental generation. Groups of 25 males and 25 females were then selected from the F1b litters to become the second-generation (F1) parental animals. Owing to a high incidence of mortality during the post-weaning period among F1b pups at 6 ppm, 30 males and 30 females at this dose were selected as F1 parents. The second-generation parental animals were treated for a minimum of 100 days before mating to produce two consecutive litters (F2a and F2b). Animals were exposed continuously to the test diets before mating and throughout weaning of the offspring in both generations. Cholinesterase levels were determined in plasma, erythrocytes and brain from 10 parental F1 rats of each sex in each group at terminal sacrifice. A treatment-related decrease in body weight and tremors were seen at > 4 ppm in parental animals; at 6 ppm, increased mortality and clinical signs were seen, manifested as convulsive movements and aggressive behaviour. Routine clinical observation of the animals revealed an increased incidence of ocular effects at 6 ppm, described as exophthalmus, 'protrusion of tissue off the cornea' and opacities. Subsequent ophthalmological evaluation revealed markedly higher incidence and severity of infectious sequelae affecting the cornea, anterior uveal tract and posterior segment of the eye in rats at this dose. The increased susceptibility of rats to ocular disease at the high dose was considered to be a secondary effect of treatment with phorate. Plasma cholinesterase levels were significantly decreased in females at 4 and 6 ppm and in males at 6 ppm. Erythrocyte activity was inhibited only slightly (10-11%) in animals of each sex at 6 ppm. Inhibition of brain cholinesterase activity was significant in males (40%) at 6 ppm and in females at 4 ppm (59%) and 6 ppm (83%). There was no significant inhibition of brain cholinesterase activity at the lower dietary levels. Pup survival during the lactation periods was significantly reduced in all litters from both parental generations at 6 ppm and in the F2a litter at 4 ppm. The size of both litters from the F1 generation was slightly decreased at 6 ppm. Mean pup weights were decreased in both F1a and F1b litters of animals receiving 4 or 6 ppm. The mean weights of pups of the F2a litter were decreased at 4 ppm (those of the F2b litter were not affected) and in pups of both F2a and F2b litters at 6 ppm. Anogenital staining was recorded in F1b and F2b pups at 6 ppm but not in control animals or in those receiving lower dietary levels. There were no adverse effects on mating or fertility indices, pregnancy rate, length of gestation, gestation index or sex distribution ratios, and no gross morphological alterations or pathological changes were seen in the reproductive organs at any of the dietary levels studied. The NOAEL was 2 ppm, equal to 0.17 mg/kg bw per day (Schroeder & Daly, 1991). (e) Embryotoxicity and teratogenicity Rats Groups of 25 mated female Crl:COBS CD (SD)BR rats were given technical-grade phorate (purity, 91.7%) by gastric intubaton at 0, 0.125, 0.25 or 0.5 mg/kg bw per day on gestation days 6-15 and were sacrificed on day 20 of gestation. The fetuses were removed for gross, skeletal and visceral examination. The pregnancy rate was comparable in all groups. During the gestation period, 7/23 pregnant dams at 0.5 mg/kg bw per day, none at 0.25 mg/kg bw per day and 1/24 at 0.125 mg/kg bw per day died. Fetuses at 0.5 mg/kg bw per day had an increased frequency of enlarged heart (not otherwise characterized). Clinical signs, body weight and food consumption of dams during gestation, the number of implantation sites, the number of resorptions, the number of dead fetuses, mean live litter size, average fetal weight, sex ratio, and gross, skeletal and visceral abnormalities of fetuses were not significantly different from those in the controls. The NOAEL for teratogenicity was 0.25 mg/kg bw per day (Litton Bionetics, 1978; Annex I, reference 39). In a preliminary range-finding study, groups of eight mated female Crl:CDBRVAF/Plus (SD) rats were administered phorate (purity, 92.1%) in corn oil by gavage at doses of 0, 0.25, 0.5, 0.7 or 0.9 mg/kg bw per day on days 6-15 of gestation. The presence of spermatozoa or a copulatory plug was considered evidence of mating, and the day on which it occurred was considered day 0 of gestation. Treatment with doses > 0.5 mg/kg bw per day was lethal to the dams, and there were no survivors at these doses after day 12 of gestation. Overt clinical signs of toxicity preceding death included twitches, tremors, excessive salivation, exophthalmos, ataxia, clonic convulsion, and decreased body weight and food intake. Gross examination of rats which died revealed enlarged and/or congested adrenal glands. The NOAEL was 0.25 mg/kg bw per day for both maternal and developmental toxicity (Lochry, 1990a). In a study of teratogenicity, groups of 24-25 mated female Crl:CDBRVAF/Plus(SD) rats were treated by gavage with phorate (purity, 92.1%) in corn oil at doses of 0, 0.1, 0.2, 0.3 or 0.4 mg/kg bw per day on days 6-15 of gestation. The presence of spermatozoa or a copulatory plug was considered evidence of mating, and the day on which this occurred was considered day 0 of gestation. Six of 25 females at 0.4 mg/kg bw per day that died during days 11-16 of gestation had enlarged adrenal glands. No deaths were reported at lower doses. Clinical signs of toxicity seen at 0.4 mg/kg bw per day were an increased number of rats with tremors, excessive salivation, chromodacryorrhoea and rhinorrhoea, urine-stained abdominal fur, decreased motor activity, hunched posture, impaired righting reflex and laboured breathing. Maternal body weights, corresponding weight gains and food consumption and fetal body weights were significantly decreased at 0.4 mg/kg bw per day when compared with control values. Increased incidences of skeletal variations were observed in fetuses and/or litters at this dose, which were related to delays in ossification of the sternebrae, incompletely and unossified pubes and incompletely ossified ischia. The NOAEL for maternal and developmental toxicity was 0.3 mg/kg bw per day on the basis of mortality, clinical signs of toxicity, and significantly decreased body weights and food consumption in the mothers and decreased fetal body weights and potentially reversible delays in skeletal ossification at 0.4 mg/kg bw per day (Lochry, 1990b). Groups of 10 pregnant Sprague-Dawley rats were exposed by nose only in an inhalation chamber to aerosols of technical-grade phorate (purity, 78-90%) with a count median diameter of 0.57 µm, generated from a 1% solution of phorate in xylene, for 1 h per day on days 7-14 of gestation at concentrations of 0.15 ± 0.04, 0.4 ± 0.15 or 1.94 ± 0.48 mg/m3. Three groups of 15 dams were exposed to xylene or air or to a restricted diet. (The reason for including the last group was not specified.) All dams were sacrificed on day 20 of gestation, and fetuses were removed by caesarean section for visceral and skeletal examination. Five dams at the highest dose died, and toxic signs (tremors, lachrymation and exophthalmus) were noted in animals at this level; one of the dead dams had resorbed the entire litter. Average percentage fetal mortality was markedly increased at this dose. The summary indicated no treatment-related effects on the body weight or food consumption of dams during gestation and no effects on pregnancy rate, average number of implants, average fetal weight, average number of sternal ossification centres or the incidence of supernumerary ribs. Specific information on the incidence of any gross or soft-tissue abnormalities was not provided (Newell & Dilley, 1978; Annex I, reference 39). Rabbits In a preliminary range-finding study, groups of five mated female New Zealand white rabbits were administered phorate (purity, 92.1%) in corn oil by gavage at doses of 0, 0.3, 0.6, 0.9, 1.2 or 1.5 mg/kg bw per day on days 6-18 of gestation. The day on which coitus with two successive males was observed was designated day 0 of gestation. The incidences of mortality in the six groups were reported as 0/5, 1/5, 1/5, 1/5, 2/5 and 4/5, respectively. Mean body-weight loss was seen at 1.2 mg/kg bw per day. Only one female at the highest dose survived to scheduled sacrifice. Food intake was generally decreased in the groups receiving 0.3, 0.6, 0.9 and 1.2 mg/kg bw per day, although no clear dose-response relationship was seen. Increased numbers of resorptions and post-implantation losses were reported at doses > 0.6 mg/kg bw per day. Decreased mean fetal body weights and shorter crown-rump lengths were noted at 1.2 mg/kg bw per day. External examination of the fetuses showed no treatment-related malformations or alterations. The only significant effects observed at the LOAEL of 0.3 mg/kg bw per day were a single maternal death and depressed food intake (Schroeder & Daly, 1986). Groups of 20 mated female New Zealand white rabbits were given phorate (purity, 92.1%) in corn oil by gavage on days 6-18 of gestation at doses of 0, 0.15, 0.5, 0.9 or 1.2 mg/kg bw per day. The day on which coitus with two successive males was observed was designated day 0 of gestation. At 1.2 mg/kg bw per day, 8/20 females died during days 14-19 of gestation, one female aborted and another female delivered prematurely on day 28 of gestation, so that only 10/20 dams survived to scheduled sacrifice. The deaths of two females at 0.9 and one at 0.5 mg/kg bw per day were also attributed to treatment with phorate. Anogenital staining was observed at a higher incidence in females treated with the highest dose. Mean body weights and corresponding weight gains were decreased at doses > 0.5 mg/kg bw per day. Food consumption was decreased in females treated at 1.2 mg/kg bw per day. Treatment had no effect on pre-implantation loss, number of resorptions, number of live fetuses, fetal body weight or fetal sex ratio. At 1.2 mg/kg bw per day, all three fetuses in a single litter (five of eight implantation sites showed early resorptions) had open eyelids, curved scapulae, an absent supraorbital process, an irregular margin of the frontals and a displaced anterior fontanel. Although these anomalies were not observed in the control group, the incidence of open eyes was within that of historical controls; curved scapulae had not previously been noted, however, in 1852 fetuses from 269 litters. Ocular and scapular defects were not seen in other fetuses from dams treated at this dose and were not recorded in any of the fetuses in the preliminary range-finding study. Although the defects seen in this single litter may have been a secondary response to the maternal toxicity of the high dose, the weight of evidence would suggest that the findings were spurious and perhaps genetically predicted. There was insufficient evidence to suggest that phorate is teratogenic to rabbits. The NOAEL for maternal toxicity was 0.15 mg/kg bw per day, as determined by a higher incidence of mortality and decreased body weights at > 0.5 mg/kg bw per day. In the absence of embryo- or fetotoxicity, the NOAEL for developmental toxicity was the highest dose, 1.2 mg/kg bw per day (Schroeder & Daly, 1987). (f) Genotoxicity A battery of assays were performed to test the genotoxicity of phorate, the results of which are presented in Table 2. Assays for gene mutation, chromosomal aberrations and other cytogenetics end-points in vitro and in vivo gave negative results. No unscheduled DNA synthesis was seen in human fibroblasts, and dominant lethal mutations were not induced in male mice. Table 2. Results of tests for the genotoxicity of phorate End-point Test system Concentration Purity Results Reference of phorate (%) In vitro Reverse S. typhimurium Up to 1000 mg/plate Technical Negativea Simmon et al., 1977b mutation TA100, 1535, 1537, grade 1538; E. coli WP2 Reverse E. coli p3478, 1 mg (on filter disc) Technical Negativec Simmon et al., 1977b mutation W3110; B. subtilis per plate grade Reverse Chinese hamster 30, 40, 50, 80, 100 92.1 Negativec Thilager & Kumarop, mutation ovary cells, hprt nl/ml 1985 locus 5, 10, 12, 14, 16, 18, Negatived 20 nl/ml Mitotic S. cerevisiae D3 5% w/v for 4-h Technical Negativea Simmon et al., 1977b recombination incubation before grade plating Unscheduled Human fibroblasts Up to 1 x 10-3 Technical Negativea Simmon et al., 1977b DNA synthesis WI-38 grade In vivo Chromosomal Male and female M: 0 (corn oil), 0.25, 92.1 Negative Ivett & Myhr, 1986 aberration Sprague-Dawley 1.25, 2.5 mg/kg bw rats, killed after per day; F: 0, 0.13, 6, 18, 30 h 0.63, 1.25 mg/kg bw per day Dominant Male mice 0, 5, 10, 20 mg/kg bw Technical Negative Simmon et al., 1977b lethal per day in diet for grade mutation 7 weeks, weekly matings for 8 weeks a In the presence and absence of metabolic activation b From Annex 1, reference 46 c In the absence of metabolic activation d In the presence of metabolic activation (g) Special studies Delayed neuropathy in chickens Groups of six adult hens were fed 0 or 40 ppm phorate (purity unspecified), equivalent to 5 mg/kg bw per day, in the diet for four weeks. A third group received 4000 ppm tri- ortho-tolyl phosphate as a positive control. Each hen was anaesthetized and immediately perfused with buffered formalin, and sections of brain, lower thoracic cord and each sciatic nerve were prepared for microscopic examination. Tri- ortho-tolyl phosphate induced myelin loss in each hen, but phorate had no adverse effects on nerve fibres or the myelin sheath (Levinskas et al., 1965; Annex I, reference 29). Phorate (purity, 89.5%) dissolved in corn oil was administered by gavage to fasted white Leghorn hens (22-23-months old). The LD50 by a single oral dose was 14.2 mg/kg bw. Fifty fasted hens were then given an intramuscular injection of 10 mg/kg bw of atropine sulfate and 1 h later given a single oral dose of 14.2 mg/kg bw phorate. An additional 15 fasted, atropinized hens were given corn oil only, and 15 hens that did not receive atropine were given tri- ortho-tolyl phosphate at 500 mg/kg bw as a positive control. All surviving hens in the test and vehicle control groups received the same doses 21 days later, except that atropine sulfate was administered at 30 mg/kg bw. All hens were observed daily for mortality, clinical signs and evidence of neurotoxic reactions. Body weights and food consumption were recorded every three days. All hens that died during the study and all hens that were killed at termination of the study at 42 days were subjected to gross necropsy. Those killed at the end of the study were perfused with 10% neutralized formalin, and brains, vertebral columns (with spinal cord in situ) and the entire right and left sciatic nerves were excised and fixed. Microscopic slides of neural tissue were prepared by taking a sagittal section of the entire brain (corpus striatum, cerebellum, pons), longitudinal and cross-sections of the cervical, thoracic and lumbrosacral levels of the spinal cord and both sagittal and longitudinal sections of the right and left sciatic nerves. Sections were stained with haematoxylin and eosin, and replicate sections were stained with luxol fast blue. Tissues from 10 hens in each group were examined histologically. None of the 15 hens fed corn oil died during the 42-day study, and all 15 hens given tri- ortho-tolyl phosphate were killed in extremis on day 16 of the study after clinical signs of neuropathy, first observed on day 11, became progressively more severe. These signs included generalized weakness, ataxia and paralysis of the legs and wings. Of the 50 hens treated with phorate, 27 died within 24 h of the first dose and 13 more within 24 h of the second dose. Ten hens survived to termination of the study. Vehicle control and phorate-treated hens had slight generalized weakness of the limbs, lasting about 2 h, shortly after each treatment with atropine sulfate; treated hens had slightly more severe reactions and, in addition, slight to moderate ataxia for up to 2 h after treatment with phorate. No clinical signs of delayed neuropathy were observed, however, in any vehicle-control or test birds. In comparison with vehicle controls, the mean body-weight gains of tested hens were greater at 0-21 days and smaller at 21-42 days, and the mean feed consumption of test hens was smaller at 0-21 days and greater at 21-42 days. No effects attributable to phorate were seen at gross necropsy. Histological examination of neural tissues from positive-control hens revealed treatment-related lesions involving the brain, spinal cord and/or sciatic nerves in all 10 birds. Generally mild to moderate axonal degeneration was observed in the brains of 4/10 hens, in the spinal cords of 10/10 hens and in the sciatic nerves of 10/10. In addition, demyelination was observed in the spinal cords and in the sciatic nerves of 7/10 hens; Schwann-cell hyperplasia was also observed in the sciatic nerves of 3/10 hens. These lesions were compatible with a delayed neurotoxic response induced by tri- ortho-tolyl phosphate. Minimal to mild focal axonal degeneration of the sciatic nerves was noted in 3/10 hens treated with phorate; no axonal degeneration was seen in vehicle-control hens. The axonal degeneration observed in the treated birds was associated with interstitial infiltration of lymphoid cells, which was also observed in other test and vehicle-control hens. This syndrome, which was distinctly different from that observed in the positive-control hens, was ascribed to lesions of a naturally occurring disease (Marek's disease) and was considered not to be related to treatment with phorate. Treatment did not induce clinical or histopathological signs indicative of acute delayed neuropathy (Fletcher, 1984; Annex I, reference 46). 3. Studies on metabolites (a) Enzymes and biochemical parameters The metabolites of phorate that inhibit cholinesterase are phorate sulfoxide and sulfone and the oxygen analogue phoratoxon and its sulfoxide and sulfone. The negative logarithms of the molar concentrations of these compounds that induce 50% inhibition of erythrocyte cholinesterase are as follows: phorate, 3.17; phorate sulfoxide, 3.35; phorate sulfone, 5.00; phoratoxon, 5.87; phoratoxon sulfoxide, 6.76; and phoratoxon sulfone, 7.02 (Annex I, reference 29). (b) Acute toxicity The acute toxicity of metabolites of phorate was studied in rats and mice; the results are shown in Table 3. Both the parent compound and the metabolites are extremely acutely toxic. Table 3. Acute toxicity of phorate and its metabolites Compound LD50 in rats (mg/kg bw per day) LD50 in mice (mg/kg bw per day) Oral Intraperitoneal Oral Intraperitoneal Phorate 1.9-10 3 11 3 Phorate sulfoxide 2-4 11 7 1 Phorate sulfone 1.8-2.0 27 9 2 Phoratoxon 0.6-0.8 - - - Phoratoxon sulfoxide 1.4-1.6 1 3 0.02 Phoratoxon sulfone 0.6-0.8 1.8 5 0.4 From Blinn, 1982; Annex I, reference 39 (c) Short-term toxicity Rats Groups of 35 male and 35 female rats (strain not specified) were fed phorate sulfoxide (purity, 92%) at dietary levels of 0.32, 0.8 or 2 ppm, equivalent to 0.02, 0.04 or 0.1 mg/kg bw per day, for 90 days. Fifty males and 50 females served as controls. Brain, erythrocyte and plasma cholinesterase activities were determined twice a week. A significant ( p < 0.05) depression of erythrocyte and brain cholinesterase activity was seen in females at 2 ppm; the decrease in plasma cholinesterase activity at this dose was less consistent and was considered to be borderline. Borderline depression of erythrocyte enzyme activity was seen at 0.8 ppm. At 0.32 ppm, all values were within acceptable statistical limits for animals of each sex. No effects were observed on other haematological parameters, organ weights or ratios, and no consistent histopathological effect was noted that could be attributed to feeding 2 ppm or less of phorate sulfoxide (Levinskas et al., 1968; Annex I, reference 29). Groups of 30 male and 30 female Charles River CD strain rats (50 male and 50 female controls), 51 days of age, were fed phorate sulfone (purity, 92%; containing 6% unchanged phorate and about 2% phorate sulfoxide) in the diet at 0, 0.32, 0.8 or 2 ppm, equivalent to 0.02, 0.04 or 0.1 mg/kg bw per day, for 90 days. None of the animals died before the end of the experiment, and there were no treatment-related changes in appearance or behaviour. Weight gain and increased food consumption were seen in males fed 0.8 or 2 ppm. Assay of tissue cholinesterase activity five times during the course of the experiment showed inhibition (> 20%) of erythrocyte cholinesterase at 2 ppm in animals of each sex at most time intervals. Plasma cholinesterase activity was reduced by 23-27% in males at 2 ppm after one, three and five weeks and by 25-72% in females at 2 ppm after all sampling intervals; plasma cholinesterase was also inhibited (39%) in females at 0.8 ppm after one and three weeks. The activity of brain cholinesterase was reduced (> 20%) only in females at 2 ppm after three, five and eight weeks. Control and treated groups did not differ significantly with regard to values for haematocrit, haemoglobin and total leukocyte count at the end of the experiment. No treatment-related effects were observed on the absolute weights of kidneys or liver, and no gross pathological changes were seen. Histopathological evaluation of a variety of tissues from five males and five females in the control and highest-dose groups revealed no morphological alterations attributable to treatment. The NOAEL was 0.32 ppm, equivalent to 0.02 mg/kg bw per day (Hutchison et al., 1978; Annex I, reference 39). 4. Observations in humans No relevant information was available. Comments The limited data available indicate that phorate given orally to rats is not readily eliminated, with less than 40% of the administered radioactivity recovered within six days after treatment. Urinary metabolites were identified as O,O-diethyl O-hydrogen phosphoro-thioate, diethyl hydrogen phosphorate and O,O-diethyl O-hydrogen phosphorodithioate. Phorate was severely acutely toxic in mice, rats and rabbits after administration by various routes. WHO (1992) has classified phorate as extremely hazardous. In a 13-week study of the toxicity study of dietary levels of 0, 1, 3 and 6 ppm phorate in mice, the NOAEL was 1 ppm, equal to 0.18 mg/kg bw per day, on the basis of inhibition of brain cholinesterase activity at dietary levels of 3 ppm and above. In rats that received phorate in the diet at 0, 0.22, 0.66, 2, 6, 12 or 18 ppm for 13 weeks, the NOAEL was 2 ppm, equivalent to 0.1 mg/kg bw per day, on the basis of inhibition of brain cholinesterase activity. At 6 ppm, cholinesterase inhibition was associated with tremors and hyperexcitability and at 12 and 18 ppm, with death. In a one-year study of toxicity in dogs, phorate was administered orally in capsules at doses of 0, 0.005, 0.01, 0.05 or 0.25 mg/kg bw per day. The NOAEL was 0.05 mg/kg bw per day, as determined by decreased body weights, significant inhibition of erythrocyte and brain cholinesterase activity and clinical signs consistent with cholinergic toxicity at 0.25 mg/kg bw per day. A preliminary 14-day range-finding study, a six-week study of dietary administration and a 15-week study in which phorate was given orally in capsules supported the findings reported in the one-year study. Mice and rats were treated with phorate at dietary levels of 0, 1, 3 or 6 ppm for 18 and 24 months, respectively. In mice, the NOAEL was 3 ppm, equivalent to 0.45 mg/kg bw per day, on the basis of decreased body weights and increased incidences of tremors, hyperactivity and excessive salivation at 6 ppm. Cholinesterase activity was not measured in this study. In the study of toxicity and carcinogenicity in rats, the NOAEL was 1 ppm, equal to 0.05 mg/kg bw per day, on the basis of significant inhibition of brain cholinesterase at 3 ppm and above. There was no apparent effect on erythrocyte cholinesterase activity. Phorate was not carcinogenic when fed to mice or rats at dietary levels of up to 6 ppm. A two-generation study of reproductive toxicity in rats given phorate at dietary levels of 0, 1, 2, 4 or 6 ppm showed no adverse effects on reproductive parameters. The NOAEL was 2 ppm, equal to 0.17 mg/kg bw per day, on the basis of decreased brain cholinesterase activity, tremors, decreased parental and pup body weights and decreased pup survival at 4 ppm and above. In a three-generation study of reproductive toxicity in mice fed dietary levels of 0, 0.6, 1.5 or 3 ppm, the NOAEL was 1.5 ppm, equivalent to 0.23 mg/kg bw per day, on the basis of slightly reduced lactation indices at 3 ppm. Two studies of teratogenicity were conducted with phorate in rats, at dose levels of 0, 0.125, 0.25 or 0.5 mg/kg bw per day and 0, 0.1, 0.2, 0.3 or 0.4 mg/kg bw per day. The NOAEL for maternal and developmental toxicity was 0.3 mg/kg bw per day on the basis of severe maternal toxicity culminating in death, decreased fetal body weights and delays in fetal skeletal ossification at 0.4 mg/kg bw per day. There was no evidence of teratogenic potential at doses as high as 0.4 mg/kg bw per day. At the maternally lethal dose of 0.5 mg/kg bw per day, there was an increased incidence of fetuses with enlarged hearts. In rabbits treated with doses of 0, 0.15, 0.5, 0.9 or 1.2 mg/kg bw per day, maternal mortality and decreased body weight were observed at doses of 0.5 mg/kg bw per day and above, resulting in an NOAEL of 0.15 mg/kg bw per day. In the absence of embryo- and fetotoxicity, the NOAEL for developmental toxicity was the highest dose, 1.2 mg/kg bw per day. Phorate was not teratogenic to rabbits. Phorate has been adequately tested for genotoxicity in vitro and in vivo in a battery of assays. The Meeting concluded that phorate is not genotoxic. No clinical or histopathological signs of delayed neuropathy were seen in chickens. Ninety-day studies of the toxicity of phorate administered in the diet were conducted in rats with the cholinesterase-inhibiting sulfoxide and sulfone metabolites of phorate. The metabolites of phorate were marginally more potent inhibitors of brain cholinesterase than phorate in female rats. Brain cholinesterase activity was inhibited in animals of each sex after administration of phorate at a dietary level of 6 ppm, but no significant inhibition was apparent at 2 ppm in either sex. The sulfoxide and sulfone metabolites of phorate inhibited brain cholinesterase only in females treated at the highest dietary level, 2 ppm. There were no significant differences in the erythrocyte cholinesterase inhibiting activity of the metabolites and the parent compound, phorate. An ADI was allocated on the basis of a NOAEL of 0.05 mg/kg bw per day in the one-year study of toxicity in dogs and the two-year feeding study in rats. The effect noted in both species was inhibition of brain cholinesterase activity, which was associated in dogs with clinical signs consistent with cholinergic toxicity. A safety factor of 100 was applied. Toxicological evaluation Levels that cause no toxic effect Mouse: 1 ppm, equal to 0.18 mg/kg bw per day (13-week study of toxicity) Rat: 1 ppm, equal to 0.05 mg/kg bw per day (two-year study of toxicity and carcinogenicity) Rabbit: 0.15 mg/kg bw per day (study of teratogenicity) Dog: 0.05 mg/kg bw per day (one-year study of toxicity) Estimate of acceptable daily intake for humans 0-0.0005 mg/kg bw Studies that would provide information useful for continued evaluation of the compound 1. Adequate studies on absorption, distribution, excretion and metabolism in rats. Studies known to exist may address this need, in whole or in part. In order to maintain the ADI, these data should be submitted in 1995, in time for review in 1996. 2. Observations in humans References American Cyanamid Co. (1965) Report on Thimet systemic insecticide: successive generation studies with mice. Unpublished report from Central Medical Department Cyanamid. Submitted to WHO by American Cynamid Co., Wayne, NJ, USA. American Cyanamid Co. (1976) Thimet soil and systemic insecticide. Unpublished technical information from Cyanamid International Corp. Submitted to WHO by American Cynamid Co., Wayne, NJ, USA. Blinn, R.C. (1982) Personal communication to WHO by American Cynamid Co., Wayne, NJ, USA. Bowman, J.S. & Casida, J.E. (1958) Further studies on the metabolism of Thimet by plants, insects and mammals. J. Econ. Entomol., 51, 838-843. Fletcher, D.W. (1984) 42-Day neurotoxicity study with phorate in mature white leghorn chickens. Unpublished report from Bio-life Associates Ltd, BLAL No. 83 DN 103, AC Protocol No. 981-84-114. 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See Also: Toxicological Abbreviations Phorate (ICSC) Phorate (Pesticide residues in food: 1977 evaluations) Phorate (Pesticide residues in food: 1982 evaluations) Phorate (Pesticide residues in food: 1984 evaluations) Phorate (Pesticide residues in food: 1985 evaluations Part II Toxicology) Phorate (Pesticide residues in food: 1996 evaluations Part II Toxicological)