CHLORPYRIFOS-METHYL First draft prepared by Dr. S. Caroldi, University of Padua, Padua, Italy EXPLANATION Chlorpyrifos-methyl was previously evaluated by the Joint Meeting in 1975 and an ADI of 0.01 mg/kg was allocated (Annex I, 24). The Meeting stated that further desirable work included appropriate mutagenicity study and neurotoxicity study with histological examination of nervous tissue. Six acute toxicity studies, short-term studies in mice, rats and dogs, a carcinogenicity study and a long-term study in mice, two special studies on delayed neurotoxicity, and five teratogenicity and mutagenicity studies were submitted for evaluation. EVALUATION FOR ACCEPTABLE INTAKE Toxicological studies Acute toxicity studies Eye irritation in the rabbit Chlorpyrifos-methyl (100 mg ground into fine powder) was placed into the left eye of three female New Zealand White rabbits (Ranch Rabbits, Crawley 11-20 weeks old). The right eye served as a control. Both eyes were examined for signs of irritation 1, 24, 48 and 72 hours after instillation. Slight transient conjunctivitis was observed 1 hour after treatment but this effect disappeared within 24 hours. The test article was non-irritant for the eyes (Jones 1985c). Dermal irritation in the rabbit Chlorpyrifos-methyl (500 mg moistened with distilled water) was applied under occlusive dressing topically to the skin (approximately 6 cm2) of three New Zealand White rabbits (Ranch Rabbits, Crawley, at least 12 weeks old) for a contact period of 4 hours. Skin was evaluated at 1, 24, 48 and 72 hours after removal of patches. Very slight redness was observed 1 hour after removal of patches in two animals but skin was normal from 24 hours. Chlorpyrifos-methyl was not irritating for the skin (Jones, 1984). Dermal sensitization in the guinea pig Twenty-eight young albino Dunkin-Hartley guinea pigs (14 males, 14 females) were dosed with technical chlorpyrifos-methyl. Induction: a lint pad (2.5 cm x 2.5 cm) wetted with test article (0.3 ml) at a concentration of 100% w/v in polyethylene glycol was applied to the left flank of each guinea pig for a contact period of 6 hours on days 1, 7, 14. Challenge: On day 29 chlorpyrifos-methyl (100% w/v) was applied on the right flank of animals induced as described above and of controls. No adverse skin reactions were noted after exposure to chlorpyrifos-methyl either test or control animals. Chlorpyrifos-methyl does not cause delayed contact hypersensitivity in the guinea pig using the Buehler Test (Jones, 1985d). Table 1. Acute toxicity of Chlorpyrifos-methyl* Species Sex Route LD50 LC50 Reference (mg/kg bw) (mg/m3) Rats M oral 2680 (1) Lackenby (1985) F 3069 (1) M&F oral > 5000 (2) Jones (1985a) M&F dermal > 2000 (2) Jones (1985b) (24 h exp.) M&F inhalation > 670 (3) Hardy & Jackson (4 h exp.) (1984) * Analysis of formulations not reported 1) Signs of toxicity were detected from 2000 mg/kg bw which disappeared in survivors within 4 days. Pathology of animals that died before scheduled sacrifice showed abnormalities of the gastrointestinal tract and of the liver. 2) No spontaneous deaths, minor toxicity signs disappeared within 24 hr of intoxication. 3) No spontaneous deaths. Signs of respiratory tract and eye irritation during exposure. Short-term studies Mice Twelve male and 12 female ICR mice (4 weeks old at the beginning of the study) were orally dosed with chlorpyrifos-methyl (91.8% purity) at concentrations of 0, 1, 5, 10, 1000 or 10 000 ppm, equal to 0, 0.125, 0.651, 1.3, 122, 523 mg/kg bw/day for males and 0, 0.14, 0.75, 1.45, 139, 318 mg/kg bw/day for females (calculated as average daily intake throughout the duration of the study) for 28 days. Chlorpyrifos-methyl was incorporated into the basal diet (fresh preparation performed weekly) up to nominal concentrations. The actual content of the test substance in the formulations was 89% of nominal (mean of 2 determinations at each dose level). Clinical signs, mortality, body weight, food consumption, and water consumption in each group were recorded during treatment. Gross pathology was performed on all animals; full histopathology on all mice in the 0, 1000, and 10 000 ppm groups and histopathology of adrenal glands in the 1, 5 and 10 ppm groups. All animals of the high dose group died within 9 days of treatment. Marked reduction of body weight and of food consumption were observed. At 1000 ppm, mortality rate was not different from that of controls and clinical signs of toxicity were not observed. A slight decrease of body weight was observed in both sexes with respect to controls (less than 10% in both sexes at the end of the study) which corresponded to lower food intake (less than 10%) and lower water intake (7% and 18% in males and females, respectively). The results of urinalysis, haematological and biochemical tests were not different from controls apart from a slight increase of alanine aminotransferase and total cholesterol observed in both sexes (statistically significant in females only). Cholinesterase activity was reduced in plasma, erythrocytes and brain in both sexes. Organ weight data showed significant increases of absolute and relative liver weights in males and relative liver weight in females, though no gross nor microscopical abnormalities were detected. Significant increases in absolute and relative adrenal weights which corresponded to swelling of cortical cells on microscopic examination were observed in males. At 10 ppm, significant reductions of both plasma (53%) and erythrocyte (33%) cholinesterase activities were observed in male rats and of plasma (23%) cholinesterase activity only in females. Brain cholinesterase activities were within normal values in both sexes. No other parameters were different from control. At 5 ppm, trivial reduction of plasma cholinesterase activities was observed in both sexes. No other parameters were different from controls. At 1 ppm, no parameters were different from controls. The NOAEL in this study was 10 ppm for both sexes corresponding to 1.3 and 1.5 mg/kg bw/day for males and females, respectively (Yoshida et al., 1985). Rats Ten male and 10 female Fischer-344 rats (4 weeks old at the beginning of the study) were treated with chlorpyrifos-methyl (technical grade 94.2% purity) at concentrations of 0, 0.1, 1, 10, 250 mg/kg bw/day for 13 weeks (primary groups). Ten additional rats/sex/ fed with 0 or 250 mg/kg bw/day after the 13 weeks of treatment were allowed to recover for 4 weeks (recovery groups). The concentrations of the test substance in the diets were adjusted weekly. Homogeneity, stability and correspondence to targeted concentrations of the diets were checked and found acceptable. No animal died during the study. At 250 mg/kg bw/day, body weight and body weight gain were significantly reduced throughout the duration of the study and this effect was still detectable during the 4 week recovery period. The mean body weight gain over the 13 week period was decreased 16% in males and 23% in females. Food consumption was not reduced. At the end of the 13-week test period, packed cell volume, red blood cells and haemoglobin values were decreased and the platelet count was increased in both sexes. Alanine amino-transferase, alkaline phosphatase and aspartate aminotransferase were decreased, cholesterol values were increased and triglycerides were decreased in both sexes. Plasma, erythrocyte and brain cholinesterase activities were significantly reduced at the 6th and 13th week of the study. At the 17th week plasma cholinesterase activity almost recovered to control values but erythrocyte and brain cholinesterase activities were still reduced. Urine staining, worse condition of the hair coat and increased lacrimation were observed. Adrenals and liver weights were increased at the end of the study. Histology showed alterations in adrenal glands and liver. In adrenals changes consisted of varying degrees of hypertrophy and vacuolation of the cells of the zona fasciculata and scattered necrotic foci. The incidence of these lesions was reduced after stopping treatment. Slight hypertrophy of centrilobular hepatocytes was observed in males only which disappeared after recovery period. At 10 mg/kg bw/day, no differences of body weight or body weight gain were detected. Haematological and biochemical parameters were not different from controls. Plasma cholinesterases were reduced at 6 and 13 weeks of the study while erythrocyte cholinesterase was reduced at week 6 but had recovered at the end of the study. Brain cholinesterases were reduced at the end of the study by 17% and 15% in males and females, respectively. Histology showed alterations in the adrenals similar to those found in animals at the higher dose level. At 1 mg/kg bw/day, only plasma cholinesterase activity was slightly reduced in both sexes while erythrocyte and brain cholinesterase activities were not different from controls. Other parameters and results of pathology were not different from controls. At 0.1 mg/kg bw/day, all parameters were unaffected. On the basis of slight inhibition of brain cholinesterase and histological alterations detected in adrenals at 10 mg/kg bw/day, the NOAEL in this study was 1 mg/kg bw/day for both sexes (Barna-Lloyd et al., 1990). Dogs Four male and 4 female beagle dogs (16 weeks old) were treated with chlorpyrifos-methyl (purity 94.2%) at concentrations of 0, 0.1, 10 or 50 mg/kg bw/day for 13 weeks. Homogeneity, stability and correspondence of the diets to the target concentrations of the test substance were checked throughout the study and found acceptable. At 50 mg/kg bw/day, marked reduction of food consumption, body weight and body weight gain were observed in both sexes. Female dogs showed weakness and generalized muscle wasting at the end of the study. Significant differences of some haematological and biochemical parameters were detected at the end of the study. Plasma and erythrocyte cholinesterase activities were reduced during and at the end of the study. Brain cholinesterase activity at the end of the study was 34% of the control value. Increased absolute and relative liver weights were observed in both sexes. Histology showed slight hypertrophy of centrilobular hepatocytes. At 10 mg/kg bw/day, reductions of plasma and, to a lesser extent, erythrocyte cholinesterase activities, but not of brain cholinesterase activity, were measured. At 0.1 mg/kg bw/day, slight reduction of plasma cholinesterase was found. Based on brain cholinesterase inhibition and on clinical effects at 50 mg/kg bw/day, the NOAEL level in this study was 10 mg/kg bw/day (Szabo & Davis 1990). Long-term carcinogenicity studies Mice Fifty-two male and 52 female ICR mice (Charles River Japan, Inc.) were treated with chlorpyrifos-methyl (97.4% pure) at concentrations of 0, 1, 5, 50, or 500 ppm, equal to 0, 0.08, 0.42, 4.4 or 44.0 mg/kg bw/day for males and 0, 0.08, 0.4, 3.9 or 41.5 mg/kg bw/day for females (calculated as average daily intake throughout the duration of the study) for 78 weeks. Forty-four additional mice/sex/dose level were treated for 26 and 52 weeks, then sacrificed for intermediate investigations. Mice were 5 weeks old at the beginning of the study. Standard diet and tap water were available ad libitum. The test compound was incorporated into basal diet (diet freshly prepared 2-3 times a week) up to the nominal concentrations. The actual content of chlorpyrifos-methyl in the formulations was checked monthly and results showed 94%, 91%, 95%, and 94% of nominal (mean of the 19 determinations) for 1, 5, 50, and 500 ppm, respectively. Urinalysis, haematological examinations, biochemical tests in blood, cholinesterase activity determinations in plasma, erythrocyte and brain were performed in 10 mice/sex/group at 26, 52, and 78 weeks. Pathology was performed on all animals, organ weights were recorded at 26, 52, and 78 weeks in 10 mice/sex/group. After 52 weeks full histopathology was performed only on mice in the 0 and 500 ppm groups (on lung, liver, kidneys, adrenals, and gross lesions in mice of the other groups). Histopathology was also performed on all survivors at the end of treatment and on mice found dead during the treatment. Mortality rate at the end of treatment was 38%, 33%, 33%, 38% and 27% in males and 29%, 37%, 27%, 29% and 25% in females at 0, 1, 5, 50, and 500 ppm, respectively. Small reductions of body weight (less than 10%) were detectable at the highest dose level in both sexes (in females during the first year of treatment only) which corresponded to a small statistically significant reduction in food and water intake. No other treatment-related signs of toxicity were detectable in either sex at any dose level. At 26, 52 and 78 weeks total cholesterol was increased in females in the group at the highest dose but not in males; no biologically significant alterations of other parameters were observed for either sex at any dose level. Plasma and erythrocyte cholinesterases were significantly reduced in both sexes at 50 and 500 ppm throughout the duration of the study. At 500 ppm cholinesterase activity in brain was consistently reduced throughout the study (the average percent of control activity in 3 determinations performed after 26, 52 and 78 weeks was 43 and 52 in males and females, respectively). Cholinesterase activity in brain was not affected at lower dose levels. The incidence of gross lesions and neoplastic histological lesions was not different among groups. Some non-neoplastic histological alterations as centrilobular hepatocellular fatty change and cortical cell swelling in the adrenals were increased at 500 ppm in both sexes. The incidence of renal tubular atrophy and cortical cyst(s) was increased in male only. The NOAEL level in this study is 50 ppm equal to 4.4 mg/kg bw/day for males and 3.94 mg/kg bw/day for females (Yoshida et al., 1988). Fifty-six male and 56 female CD-1 mice were treated with chlorpyrifos-methyl (96% purity) at concentration of 0, 15, 30, 60 ppm for two years. Dietary concentrations of the test substances were prepared at approximately 3 to 4 week intervals. The actual content of chlorpyrifos methyl in the formulations was checked 15 times throughout the duration of the study and showed 196% (70-580), 128% (80-420), 138% (56-579) of nominal (mean and range of 15 determinations) for 15, 30, and 60 ppm, respectively. Food consumption and body weight were not influenced by administration of the test substance up to the highest dose. No relevant clinical observations were recorded. Mortality rate at the end of the study was 70%, 71%, 61% and 61% in male and 55%, 66%, 48% and 59% in females at 0, 15, 30, 60 ppm, respectively. There were no statistically significant treatment-related differences in organ weights. A variety of non-neoplastic and neoplastic lesions was observed in treated and control groups of both sexes. The lesions were of types commonly found in mice and the incidence of neoplasms in treated mice was not different from that in controls. Under the conditions of this study, chlorpyrifos-methyl was not carcinogenic to CD-1 mice. The determination of a NOAEL is hampered in this study by poor consistency of the concentrations of the test substance in the diet (Molello et al., 1980). Rats Sixty male and 60 female Fischer 344 rats/dose level were administered chlorpyrifos-methyl (94-95% pure) at dietary concentrations of 0, 1, 2, 20, and 1000 ppm for two years. Ten additional rats/sex/dose level were treated for 12 months and then sacrificed for intermediate investigations. Rats were 5-6 weeks old at the beginning of the study. Rats were housed one/cage and feed and tap water were available ad libitum. The actual content of chlorpyrifos-methyl in the diet was checked on days 1, 97, 181, 272, 363, 454, 545, 636, and 727, and concentrations were found to be within 2% of nominal. Animals were observed daily and weighed on a weekly basis for the first 13 weeks and then every 4 weeks. Urinalysis, haematological examinations, clinical chemistry, cholinesterase activity determinations in plasma, erythrocyte and brain were run at 26, 52, 78 and 104 weeks. Gross pathology was performed on all animals, and organ weights were recorded at terminal sacrifice. Full histopathology was performed only on rats in the control and high-dose groups and those animals sacrificed in moribund condition or found dead. Sections of adrenal glands, brain, epididymides, kidneys, liver, lungs, pituitary, spinal cord, sciatic nerve, testes, tibial nerve and gross lesions were histologically examined in animals from other dose levels at terminal sacrifice. Small but statistically significant reductions of body weight (less than 10%) were detectable at the highest dose level in both sexes after the first year on test. There was no compound-related effect on mortality in either sex. Plasma cholinesterase was significantly reduced in both sexes at 20 and 1000 ppm throughout the duration of the study. At 1000 ppm acetylcholinesterase activity in erythrocyte and brain was consistently reduced throughout the study. Slightly lower erythrocyte cholinesterase activity was observed at the 6 month evaluation but was not considered to be biologically significant. Cholinesterase activity in brain was not affected at lower dose levels (< 1000 ppm). The incidences of gross lesions and neoplasms were similar among groups. Biologically significant changes in organ weights were not found with the exception of the adrenal, which was increased (absolute and relative) in both high-dose males and females after one year and at final sacrifice. Urinalysis and clinical chemistry measurements were unremarkable. Although RBC, Hb and PCV were decreased and platelets increased at the high-dose level at intervals prior to final sacrifice in both males and females, no change was found at the final measurement. An increased incidence of diffuse, moderate vacuolation consistent with lipid accumulation was found in the adrenal glands of both males and females at the high-dose level at the interim sacrifice and in the 20 ppm groups at the final sacrifice. Based on the increased incidence of adrenal, vacuolation observed at the 20 and 1000 ppm dietary concentrations, the NOAEL was 2 ppm, equivalent to 0.1 mg/kg bw/day (Barna-Lloyd et al., 1991). Special studies on delayed neurotoxicity Four groups of 10 adult White Leghorn hens were treated orally with 30 mg/kg of atropine sulfate. One group received no additional treatment (negative controls), the second group received 250 mg/kg of tri-o-tolyl phosphate orally (positive controls). The remaining groups received either 2500 or 5000 mg/kg of chlorpyrifos-methyl orally (95.9% purity). In a previous study 6 hens had survived a single oral dose of chlorpyrifos-methyl of 6400 mg/kg. Hens were observed daily for abnormal gait, they were weighed weekly and killed 21 days after dosing and histology performed on sciatic nerves and cervical, thoracic and lumbar sections of the spinal cord. Cholinesterase activity was not measured but 4 out of 10 hens developed acute symptoms (likely cholinergic) during the first 5 days after receiving 5000 mg/kg chlorpyrifos-methyl. None of the negative controls and all of the positive controls showed clinical (starting day 11-15 after dosing) and histological evidence (in spinal cord and sciatic nerves) of delayed neurotoxicity. None of the chlorpyrifos-methyl dosed birds showed clinical evidence of delayed neurotoxicity. At 2500 mg/kg one hen showed histological alterations in sciatic nerve while spinal cord was unaffected. Six out of 10 birds receiving 5000 mg/kg of chlorpyrifos-methyl showed positive and/or equivocal histological evidence of neurotoxicity of the spinal cord and/or sciatic nerve(s) (Clark et al., 1979). Groups of 10 adult White Leghorn hens were orally dosed with chlorpyrifos-methyl (97% purity) at concentrations of 0, 5, 50 or 500 mg/kg in corn oil for 5 days a week for 13 weeks. Two additional groups received tri-ortho-cresyl phosphate (TOCP) p.o. at concentration of 10 or 30 mg/kg/day for 5 days a week. Dosing was discontinued after 7 weeks of treatment at 30 mg/kg TOCP when 6 hens were killed because of pronounced signs of neurotoxicity. Actual concentrations of the test substance in the diets, homogeneity and stability were acceptable. Hens were observed daily and body weight recorded weekly. Histopathology of brain, of spinal cord and of sciatic nerve was performed on all hens at the end of the study. Apart from a consistent reduction of mean body weight in hens dosed with chlorpyrifos-methyl at the highest dose level, no other signs of toxicity were observed. The body weight reduction was apparent from day 3 of the study up to termination and early onset was considered indicative of acute toxicity at this dose level. Cholinesterase activity was not measured. Clinical signs of delayed neuropathy developed only in hens dosed with TOCP at the highest dose level starting in the 4th week of treatment. Histological signs of delayed neuropathy were observed in spinal cord and sciatic nerve of each animal dosed with TOCP. Hens dosed with chlorpyrifos-methyl did not show clinical nor histological signs of delayed neuropathy (Barna-Lloyd et al., 1984). Special study on embryotoxicity and teratogencity Ten female rabbits (Japan white, six to seven months old) were dosed with chlorpyrifos-methyl orally by stomach catheter at concentrations of 0, 4, 8, 16 mg/kg bw/day on days 6-18 of gestation. Selection of dose levels was based on approximately 20% inhibition of plasma cholinesterase observed in a preliminary study at 16 mg/kg bw/day of chlorpyrifos-methyl for 2 weeks. All rabbits were checked daily for toxicity. Maternal body weights, food and water consumption were recorded daily. All of the pregnant rabbits were sacrificed on day 29 of gestation. Body weight was not affected by chlorpyrifos-methyl treatment even though a slight reduction of food intake was observed in rabbits at the 8 and 16 mg/kg/day dose levels from 7 to 20 days of gestation. Number of corpora lutea and implantations was not different between dosed rabbits and controls. No significant differences were detected in the number of dead embryos and fetuses pre- and post-implantation, number of live fetuses, nor individual average pup weights. No abnormalities related to treatment were found in external, visceral or skeletal examinations. Chlorpyrifos-methyl produced no effects on teratology, mortality or weight loss. However, a higher dose level could have been utilized based on the lack of maternal toxicity at any tested dose level (Asai et al., 1976). Table 2. Results of genotoxicity assays on chlorpyrifos-methyl Test system Test object Concentration of test Purity Results Reference substance Ames test (both S. typhimurium 1, 10, 100, 500, 1000, 99.9% Negative (1) De Graff (1983) with and without TA98, TA100, TA1535, 2500, 5000, 10 000 µg/plate activation) TA1537, TA1538 dissolved in DMSO CHO/HGPRT assay Chinese hamster ovary 5, 20, 40, 60, 80, 100 95.2% Negative (2) Mendrala (1985) (both with and cells (CHO-K1-BH4) µM/plate dissolved in DMSO without metabolic activation) Chromosome aberration Chinese hamster ovary Nonactivated: 95.2% Negative Gollapudi (1985) assay (both with and cells (CHO-K1-CCL61) 4, 12, 40 µg/ml without metabolic Activated: Positive (3) activation) 5, 15, 50 µg/ml dissolved in DMSO Unscheduled DNA Male CDF Fischer rat 1 x 10-4 M 95.2% Negative (4) Mendrala & Dryzga (1985b) synthesis liver primary cell 3.16 x 10-5 M cultures 1 x 10-5 M 3.16 x 10-6 M 1 x 10-6 M Table 2 (contd). Test system Test object Concentration of test Purity Results Reference substance Mouse micronucleus Male/female CD-1 146 mg/kg bw 95.2% Negative (5) Bruce et al. (1985) test BR mice 460 mg/kg bw 1460 mg/kg bw (1) Positive control (without activation: TA-100/TA-1535,sodium azide at 10 µg/plate; TA-98/TA-1538,2-nitrofluorene at 10 µg/plate; TA-1537, 9-aminoacridine at 50 µg/plate. With activation: 2-anthramine at 2.5 µg/plate for all strains) gave expected positive response. (2) Positive control (3 mM EMS; 100 µM 3-MC) yielded expected positive response. The non-activation assay gave positive response at 80 µM concentration but not at 100 µM concentration. A second run with addition of 30 µM concentration gave negative response at all concentration levels. (3) Positive control (1242 µg/ml EMS; 21 µg/ml CP) yielded expected positive response. A dose-related increase of the frequency of aberrations was observed only in the assay with activation. (4) Positive control (2-AAF 1x10-5,1x10-6,1x10-7 M) yielded expected positive response. (5) Positive control (120 mg/kg bw CP) yielded expected positive response. COMMENTS Chlorpyrifos-methyl was evaluated by the 1975 JMPR which allocated an ADI of 0-0.01 mg/kg bw/day. Chlorpyrifos-methyl is moderately acutely toxic by the oral route. No significant differences between sexes were observed. In a 28-day study in mice at dietary concentrations of 0, 1, 5, 10, 1000 or 10 000 ppm, the NOAEL was 10 ppm, equal to 1.4 and 1.5 mg/kg bw/day for males and females respectively based on brain cholinesterase inhibition and alterations in the adrenal glands at 1000 ppm. In a 13-week study in rats at dietary concentrations yielding doses of 0, 0.1, 1, 10, or 250 mg/kg bw/day, the NOAEL was 1 mg/kg bw/day based on histological alterations in the adrenals at 10 mg/kg bw/day. In a 13-week study in dogs at dietary concentrations yielding doses of 0, 0.1, 10, or 50 mg/kg bw/day, the NOAEL was 10 mg/kg bw/day based on brain acetyl-cholinesterase inhibition, increased liver weight and reduction of body weight gain at 50 mg/kg bw/day. In a 78-week study in mice at dietary concentrations of 0, 1, 5, 50 or 500 ppm the NOAEL was 50 ppm, equal to 4.4 and 3.9 mg/kg bw/day in males and females, respectively. At 500 ppm, 50% inhibition of brain acetylcholinesterase occurred; other effects noted at this dietary concentration were centrilobular hepatocellular fatty change and cortical cellular swelling of the adrenals. The incidence of neoplastic lesions was similar in all groups. In a 2-year dietary study in rats the NOAEL was 0.1 mg/kg bw/day based on dose-related alterations in adrenal glands detected at 1 and 50 mg/kg bw/day. There was no evidence of carcinogenicity in rodents. Chlorpyrifos-methyl did not cause delayed neurotoxicity in hens. A teratology study in rabbits was negative at all doses tested. The NOAEL was 16 mg/kg bw/day, the highest dose tested. After consideration of all available in vitro and in vivo genotoxicity data, the Meeting concluded that chlorpyrifos-methyl was not genotoxic, despite a significant clastogenic response in an in vitro study. The ADI was based on the results of the 2-year study in rats using a 100-fold safety factor. The Meeting was not able to use the human acetylcholinesterase inhibition data reviewed in 1975 as the basis for the ADI because adverse effects on adrenals were observed in rats in the absence of cholinesterase inhibition. TOXICOLOGICAL EVALUATION Level causing no toxicological effect Mouse: 50 ppm, equal to 3.9 mg/kg bw/day Rat: 0.1 mg/kg bw/day Human: 0.1 mg/kg bw/day Estimate of acceptable daily intake for humans 0-0.001 mg/kg bw Studies which will provide information valuable in the continued evaluation of the compound Further observations in humans REFERENCES Asai, M., Hirose, K., Maita, K., Masuda, H., Matsunuma, N., Nagata, K., Okada, T., Okino, Y. & Yamashita, K. (1976) Study of the effects of Chlorpyrifos-methyl on rabbits embryonal and fetal development. Unpublished Report GHF-R 4 from Laboratory of Toxicology and Safety, Sankyo, Japan. Submitted to WHO by DowElanco, Indianapolis, USA. Barna-Lloyd, T., Jersey, G.C., McDermott, M., Hinze, C.A., Davis, N.L. & Rachunek, B.L. (1984) Chlorpyrifos-methyl insecticide: subchronic (3-month) delayed neurotoxicity study in laying chicken hens. Unpublished Report TXT: K-046193-(18) from Dow Chemical, Texas, USA. Submitted to WHO by Dow Elanco, Indianapolis, USA. Barna-Lloyd, T., Szabo, J.R. & Davis, N.L. (1990) Chlorpyrifos-methyl (Reldan R) rat subchronic dietary toxicity and recovery study. Unpublished Report TXT: K-046193-026 from Dow Chemical, Texas, USA. Submitted to WHO by Dow Elanco, Indianapolis, USA. Barna-Lloyd, T., Szabo, J.R. & Davis, N.L. (1991) Chlorpyrifos-methyl (Reldan R) rat chronic dietary toxicity/oncogenicity study. Unpublished Report TXT: K-046193-031 from Dow Chemical, Texas, USA. Submitted to WHO by Dow Elanco, Indianapolis, USA. Bruce, R.J., Gollapudi, B.B. & Hinze, C. (1985) Evaluation of chlorpyrifos-methyl in the mouse bone marrow micronucleus test. Unpublished Report TXT: K- 046193-020 from Dow Chemical, Texas, USA. Submitted to WHO by DowElanco, Indianapolis, USA. Clark, W.E., Warner, S.D. & Johnston, R.V. (1979) Acute delayed neuro-toxicologic evaluation of chlorpyrifos-methyl in white leghorn hens. Unpublished Report TXT:3280.0 from Dow Chemical, Texas, USA. Submitted to WHO by DowElanco, Indianapolis, USA. DeGraff, W.G. (1983) Evaluation of Dowco 214 in the Ames Salmonella/ mammalian microsomal bacterial mutagenicity assay. Unpublished Report HET K-46193-(16) from Litton Bionetics, Inc., Kensington, MD, USA. Submitted to WHO by DowElanco, Indianapolis, USA. Gollapudi, B.B., Linscombe, V.A. & Sinha, A.K. (1985) Evaluation of chlorpyrifos-methyl in an in vitro chromosomal aberration assay utilizing Chinese hamster ovary (CHO) cells. Unpublished Report TXT:K-046193-023 from Dow Chemical, Texas, USA. Submitted to WHO by DowElanco, Indianapolis, USA. Hardy, C.J. & Jackson, G.C. (1984) Reldan R technical: Acute inhalation toxicity in rats. Unpublished Report No. DET 366 from Huntington Research Centre, UK. Submitted to WHO by DowElanco, Indianapolis, USA. Jones, J.R. (1984) Reldan R F: Primary skin irritation and corrosivity study in the rabbit. Unpublished Report DET 376 from Hazleton Laboratories, Harrogate, UK. Submitted to WHO by DowElanco, Indianapolis, USA. Jones, J.R. (1985a) Reldan R F: Acute oral median lethal dose in the rat. Unpublished Report DET 418 from Hazleton Laboratories, Harrogate, UK. Submitted to WHO by DowElanco, Indianapolis, USA. Jones, J.R. (1985b) Reldan R F: Acute dermal median lethal dose in the rat. Unpublished Report DET 419 from Hazleton Laboratories, Harrogate, UK. Submitted to WHO by DowElanco, Indianapolis, USA. Jones, J.R. (1985c) Reldan R F: Eye irritation study in the rabbit. Unpublished Report DET 423 from Hazleton Laboratories, Harrogate, UK. Submitted to WHO by DowElanco, Indianapolis, USA. Jones, J.R. (1985d) Reldan R F: Delayed contact hypersensitivity study in the Guinea Pig (Buehler test). Unpublished Report DET 474 from Hazleton Laboratories, Harrogate, UK. Submitted to WHO by DowElanco, Indianapolis, USA. Lackenby, F. (1985) Acute oral toxicity study in the rat. Unpublished Report DET 685 from Hazleton Laboratories, Harrogate, UK. Submitted to WHO by DowElanco, Indianapolis, USA. Mendrala, A.L. (1985) Evaluation of chlorpyrifos-methyl in the Chinese hamster ovary cell-hypoxanthine (guanine) phosphoribosyl transferase (CHO/HGPRT) forward mutation assay. Unpublished Report HET K-046193-022 from Dow Chemical, Michigan, USA. Submitted to WHO by DowElanco, Indianapolis, USA. Mendrala, A.L. & Dryzga, M.D. (1985) Evaluation of chlorpyrifos-methyl in the rat hepatocyte unscheduled DNA synthesis assay. Unpublished Report HET K-046193-021 from Dow Chemical, Michigan, USA. Submitted to WHO by DowElanco, Indianapolis, USA. Molello, J.A., Gerbig, C.G., Starret, M.G., Ehalt, W.L., Solomon, J.L. & Cheng, W. (1980) Bioassay of chlorpyrifos-methyl for possible carcinogenicity to mice. Unpublished Report NBX-403 from Dow Chemical, USA. Submitted to WHO by DowElanco, Indianapolis, USA. Szabo, J.R. & Davis, N.L. (1990) Chlorpyrifos-methyl (Reldan R): 13-week dietary toxicity study in beagle dogs. Unpublished Report TXT:K-046193-027 from Dow Chemical, Texas, USA. Submitted to WHO by DowElanco, Indianapolis, USA. Yoshida, A., Kosaka, T., Miyaoka, T., Maita, K., Goto, S. & Shirasu, Y. (1985) Chlorpyrifos-methyl: 28-day oral toxicity study in mice. Unpublished Report No. GHF-R 80 from the Institute of Environmental Toxicology, Tokyo, Japan. Submitted to WHO by DowElanco, Indianapolis, USA. Yoshida, A., Kosaka, T., Miyaoka, T., Maita, K., Goto, S. & Shirasu, Y. (1988) Chlorpyrifos-methyl: 18-month oral chronic toxicity and oncogenicity study in mice. Unpublished Report GHF-R 166 from the Institute of Environmental Toxicology, Tokyo, Japan. Submitted to WHO by DowElanco, Indianapolis, USA.
See Also: Toxicological Abbreviations Chlorpyrifos-methyl (WHO Pesticide Residues Series 5) Chlorpyrifos-methyl (Pesticide residues in food: 1979 evaluations) Chlorpyrifos-methyl (Pesticide residues in food: 1992 evaluations Part II Toxicology)