ACEPHATE EXPLANATION First draft prepared by Dr. E.M. den Tonkelaar, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands Acephate toxicity has been reviewed by several Joint Meetings between 1976 and 1988 (Annex 1, FAO/WHO 1977ab, 1983ab, 1985bc, 1987b, 1988b, 1988c, and 1989a). Data that have been reviewed include pharmacokinetic studies, short-term tests in mice, rats and dogs, long-term studies in mice and rats, mutagenicity data, reproduction and teratogenicity studies and data on humans. Since the last review in vitro and in vivo studies on cholinesterase inhibition, a teratogenicity study in rats and additional mutagenicity studies have been submitted, which were reviewed at the present Meeting. EVALUATION FOR ACCEPTABLE DAILY INTAKE BIOLOGICAL DATA Biochemical aspects Special study on in vitro metabolism Post-mitochondrial liver supernatant fractions (S9) were prepared from Sprague-Dawley rats, beagle dogs, rhesus monkeys and humans. The S9 fractions were incubated with 20 to 30 µM [O-methyl-14C]-acephate for 0, 1 and 4 hours. Metabolites were separated by HPLC and detected by a flow-through radioactivity monitor. Only a small fraction of acephate was metabolized by the liver S9 fractions from all 4 species. For dogs and monkeys this was 13-14%, for rats about 9% and for humans about 4%. The main metabolites were methamidophos and an unidentified metabolite, which is possibly 0,S- dimethyl phosphorothioate (DMPT), a known metabolite of methamidophos which is also found in rat metabolism studies with acephate (Annex 1, FAO/WHO, 1977b). In addition 3 other (unidentified) metabolites were observed in minor quantities, of which only one was found in monkey and only one was only found in humans. The relative proportions of methamidophos and presumed "DMPT" differed. In humans and dogs the percentages for "DMPT" were higher than for methamidophos, for rats it was about the same and for monkeys methamidophos was higher than "DMPT". Because of the low quantities of the metabolites found it is difficult to draw a conclusion about a difference in metabolism between the four species (Green, 1989). The low percentage which was metabolised by liver fractions is also reflected in the in vivo study in rats, in which 73-77% was excreted as unchanged acephate (Annex 1, FAO/WHO, 1977b). Effects on cholinesterase activity Groups of Sprague-Dawley rats (10/sex/group) were fed diets containing 0, 2, 5, 10 or 150 ppm acephate technical (purity 98.2%) for 4, 9 or 13 weeks. There were no effects of treatment on mortality, clinical signs, body weight, food consumption or macroscopy. The only effect observed was a dose-related depression of cholinesterase activity in plasma, erythrocytes (RBC) and brain, which was already maximal after 4 weeks. At doses of 2, 5 and 10 ppm brain cholinesterase was only slightly inhibited (8, 10 and 15%, respectively). At 150 ppm marked inhibition was found in brain, erythrocyte and plasma cholinesterase (Brorby et al., 1987). In an in vitro experiment the cholinesterase inhibition of methamidophos, acephate and paraoxon (a known strong anticholinesterase) were determined in human erythrocytes and on brain samples of rats, mice and rainbow trout. In all cases, except trout brain cholinesterase, acephate and methamidophos were found to be six and three orders of magnitude weaker than paraoxon, respectively (Hussain et al., 1985). Results of this study, are tabulated at Table 1 in the monograph on methamidophos. Toxicological Studies Special study on embryo/fetoxicity Rats Groups of 25 pregnant Charles River Crl:Cd rats received 0, 5, 20 or 75 mg acephate (purity 98.4%)/kg b.w./day orally by gavage from days 6 through 15 of gestation. At day 20 of gestation all animals were sacrificed and the fetuses were examined. No mortality occurred. Tremors and decreased motor activity were observed in rats at 75 mg/kg bw/day. Food consumption and growth of dams was significantly decreased at 20 and 75 mg/kg bw/day. There was no effect of acephate administration on the number of implantations, early and late resorptions and live and dead fetuses. Pup weight at 75 mg/kg bw/day was decreased (significantly in female fetuses). After examination for gross external, soft tissue and skeletal alterations slight decreases in the average numbers of ossified caudal vertebrae, sternal centers, metacarpals and fore- and hindpaw phalanges were observed in fetuses at 75 mg/kg bw. The NOAEL for fetotoxicity in this study was 20 mg/kg bw/day (Lochry,1989). Special studies on genotoxicity Three new genotoxicity reports were received. They are summarized in Table 1. The studies by Carver et al., (1985), were reviewed at the 1984 JMPR (Annex 1, FAO/WHO 1985c), but were listed under different authors. In the study of Behera and Bhunya (1989), i.p. dosing was used instead of oral dosing and the purity of the compound is not known. The doses were higher than in earlier (oral) in vivo studies. COMMENTS In an in vitro metabolism study of acephate, only minor amounts were metabolized. No substantial differences were observed between rats, dogs, monkeys and humans. A comparative in vitro study showed the same rate of inhibition for both human erythrocyte and rat brain cholinesterases. Acephate is a less potent inhibitor of cholinesterase than its metabolite, methamidophos. Data from a special 90-day dietary study with acephate in rats on cholinesterase inhibition demonstrated that inhibition of brain cholinesterase is the most sensitive indicator. A NOAEL of 10 ppm, equivalent to 0.5 mg/kg bw/day was demonstrated. In a rat teratogenicity study, maternal toxicity was observed at doses of 20 mg/kg bw/day and above, but no teratogenic effects were seen. The NOAEL for embryotoxicity/fetotoxicity was 20 mg/kg bw/day. For maternal toxicity, the NOAEL was 5 mg/kg bw/day. Additional data on genotoxicity included positive responses in in vivo chromosomal aberration studies. The purity of the compound used in these studies is not known. A review of data that had already been evaluated by the JMPR in 1894 showed some positive results in in vitro tests, but in vivo tests were negative. A new study showed no effect in an in vivo somatic cell mutation assay (spot test). The Meeting concluded that acephate has genotoxic properties in in vitro studies, but in vivo studies were negative for gene mutations and showed conflicting results for chromosomal aberrations. The data reviewed by the present Meeting did not warrant any change in the value of the ADI established in 1988. The present ADI is based on the rabbit NOAEL for teratogenicity and the human volunteer study. The latter was determined to be the definitive study. Table 1. Results of genotoxicity assays on acephate Test system Test object Concentration Results Reference In vitro Ames test 1 S. typhimurium TA98, TA1537 data not given negative Carver et al., 19855 S. typhimurium TA100 0 - 93.5 mg/pl positive Mouse lymphoma assay 2 Mouse L5718Y TK+/- cells 0-5000 µg/pl positive In vivo CD-1 mice bone marrow cells 0-96 mg/kg bw negative Sister chromatid exchange assay Macaca monkey lymphocytes 2.5 mg/kg bw negative Cytogenetic study CD-1 mice bone marrow cells 0-112 mg/kg bw negative (chromosomal aberration) Macaca monkey lymphocytes 2.5 mg/kw bw negative Swiss albino mice bone i.p. 150, 200, 250 positive Behera and Bhunya, 1989 marrow cells or 5x50 mg/kg bw Micronucleus test Swiss Webster male mice bone 2x75, 2x150 negative Carver et al., 19855 marrow cells or 2x300 mg/kg bw In vivo Micronucleus test Swiss albino mice 2x150, 2x200 positive3 Behera and Bhunya, 1989 or 2x250 mg/kg bw i.p. CD-1 male mice 0-1000 ppm for 5 days negative Carver et al., 19855 Dominant lethal test Swiss albino mice 5x40 or 5x50 mg/kg bw i.p. negative Behera and Bhunya, 1989 Table 1 (contd) Test system Test object Concentration Results Reference Sperm-shape abnormality assay Swiss albino mice 5x30, 5x40 or 5x50 mg/kg i.p. positive Behera and Bhunya, 1989 (mice killed after 35 days) Somatic cell mutation assay T-strain male C57B1/6 50, 200, 600 or 800 ppm negative4 Zimmerman and (spot test) female mice orally from day 8-13 of Glickman, 1986 gestation 1 Without metabolic activation 2 With and without metabolic activation (S-9) 3 Positive at the highest dose (2x250 mg/kg) only 4 Ethylnitrosourea was used as a positive control 5 Reviewed at 1984 JMPR TOXICOLOGICAL EVALUATION Level causing no toxicological effect Rat: 10 ppm in the diet, equivalent to 0.5 mg/kg bw/day Rabbit: 3 mg/kg bw/day Dog: 30 ppm in the diet, equivalent to 0.75 mg/kg bw/day Human: 0.3 mg/kg bw/day Estimate of acceptable daily intake for humans 0-0.03 mg/kg bw Studies which will provide information valuable in the continued evaluation of the compound Further observations in humans. REFERENCES Behera, B.C. and Bhunya, S.P. (1989). Studies on the genotoxicity of asataf (acephate) an organophosphate insecticide, in a mammalian in vivo system. Mutation Res. 223, 187-193. Brorby, G.P., Rosenberg, D.W. and Wong Z.A. (1987). The cholinesterase inhibition potential of acephate technical (SX-1102) following 4-, 9-, or 13-week dietary administration in male and female rats. Unpublished report no. CEHC 2821, December 30, 1987 from Chevron Environmental Health Center. Submitted to WHO by Chevron Chemical Company, Richmond, CA, USA. Carver, J.H., Bootman, J., Cimino, M.C., Esber, H.J., Kirby, P., Kirkhart, B., Wong, Z.A. and MacGregor, J.A. (1985). Genotoxic potential of acephate technical: in vitro and in vivo effects. Toxicology, 35, 125-142. Green, C.E. (1989). Comparative metabolism of [14C] acephate by in vitro preparations from rat, dog, monkey and human liver tissue. Unpublished report from Stanford Research Institute project no. LSC-6402. Submitted to WHO by Chevron Chemical Company, Richmond, CA, USA. Hussain, M.A., Mohamad, R.B. and Oloffs, P.C. (1985). Studies of the toxicity, metabolism and anticholinesterase properties of acephate and methamidophos. J. Environ. Sci. Health B20(1), 129-147. Lochry, E.A. (1989). Oral teratogenicity and developmental toxicity study in rats with Chevron acephate technical. Unpublished report nr.303-008 from Argus Research Laboratories, Inc. Perkasie, PA 18944. Submitted to WHO by Chevron Chemical Company, Richmond, CA, USA. Zimmerman, R.A. and Glickman, A.H. (1986). Evaluation of chevron acephate technical in the mouse somatic cell mutation assay. Unpublished report (project nr.: 2107-141) from Hazleton Laboratories America, Inc. Rockville, Maryland USA.
See Also: Toxicological Abbreviations Acephate (ICSC) Acephate (Pesticide residues in food: 1976 evaluations) Acephate (Pesticide residues in food: 1979 evaluations) Acephate (Pesticide residues in food: 1981 evaluations) Acephate (Pesticide residues in food: 1982 evaluations) Acephate (Pesticide residues in food: 1984 evaluations) Acephate (Pesticide residues in food: 1984 evaluations) Acephate (Pesticide residues in food: 1987 evaluations Part II Toxicology) Acephate (Pesticide residues in food: 1988 evaluations Part II Toxicology) Acephate (JMPR Evaluations 2002 Part II Toxicological) Acephate (JMPR Evaluations 2005 Part II Toxicological)