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    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 M [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 g/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)