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    PHORATE (addendum)

    First draft prepared by
    D. Grant & S. Geertsen
    Pesticide Evaluation Division, Health Evaluation Division,
    Health Canada, Tunney's Pasture, Ottawa, Ontario, Canada

    Explanation
    Evaluation for acceptable daily intake
       Absorption, distribution, and excretion
       Biotransformation
    Comments
    Toxicological evaluation
    References

    Explanation

         Phorate, an organophosphate insecticide that inhibits
    cholinesterase, was first reviewed for toxicological effects by the
    Joint Meeting in 1977 (Annex 1, reference 28). A temporary ADI of
    0-0.0002 mg/kg bw was established in 1982 (Annex 1, reference 38)
    In 1994, the Meeting re-evaluated phorate and allocated an ADI of
    0-0.0005 mg/kg bw. Because in a limited study in rats it was reported
    that less than 40% of the administered 32P label was excreted within
    144 h, adequate studies on absorption, distribution, excretion,
    and metabolism in rats were requested for review in 1996 (Annex 1,
    reference 71). Data available to address this issue are summarized in
    this monograph addendum.

    Evaluation for acceptable daily intake

    1.  Absorption, distribution, and excretion

         The 1994 review of phorate (Annex 1, reference 73) indicated
    that 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, largely unextractable
    residues (Bowman & Casida, 1958).

         Male rats were given a single dose of 0.8 mg/kg bw (8 µCi/mg) of
    14C-labelled phorate (purity, > 98%; specific activity, 40 µCi/mg;
    see Figure 1 for position of 14C) in corn oil by gavage. Most of the
    administered dose (77%) was excreted in the urine within the first
    24 h after dosing. After eight days, 83% of the administered dose had
    been eliminated in the urine and 13% in the faeces. Peak tissue
    residue levels were found 6 h after treatment, with 0.37 ppm in blood,
    0.29 ppm in kidney, 0.24 ppm in liver, 0.20 ppm in skin, 0.14 ppm in
    muscle, and 0.08 ppm in fat. The levels declined throughout the study,
    and by 48 h the concentrations of residue in muscle, fat, and skin
    were < 0.01 ppm. By 192 h, the residues in liver were only 0.02 ppm
    and those in kidney < 0.01 ppm. By the end of the study, 97% of the
    administered dose had been recovered from the urine, faeces, body
    tissues, and cage rinses. Oxidated, S-methylated, dephosphorylated
    metabolites represented 81% of the urinary radiolabel and the parent
    compound only 0.5%. Most of the radiolabel extracted from tissue was
    in dephosphorylated metabolites (Hussain, 1987).

    Figure 1.  Position of radiolabel in 14C-phorate

                               S
                               "
                      (C2H5O)2-P-S-14CH2-S-CH2CH3

         14C-Phorate (purity, > 98%; see Figure 1 for location of
    radiolabel) was rapidly absorbed and excreted by female rats given
    a single dose of 0.44 mg/kg bw (40 µCi/mg) in corn oil by gavage.
    The urine was the primary route of elimination, with 78% of the
    administered dose eliminated within 24 h. Faecal elimination accounted
    for only 8% of the administered dose. Peak tissue residue levels
    occurred after 6 h, with 0.168 ppm in blood, 0.163 ppm in kidney,
    0.142 ppm in liver, 0.109 ppm in skin, 0.100 ppm in muscle, and
    0.031 ppm in fat. By 192 h, the liver and kidney residues had dropped
    to only 0.008 and 0.010 ppm, respectively, while those in muscle, fat,
    skin, and blood residue were below the limit of detection by 48 h.
    At the end of the study, > 94% of the administered dose had been

    converted to non-phosphorylated metabolites arising from the cleavage
    of the phosphorus-sulfur bond, methylation of the liberated thiol
    group and oxidation of the resulting divalent sulfur moiety to the
    sulfoxide and sulfone (Miller & Wu, 1990).

    2.  Biotransformation

         The 1994 review of phorate (Annex 1, reference 73) indicated that
    the urine of male rats given daily doses of 1 mg/kg bw contained 17%
    diethyl phosphoric acid, 80%  O,O-diethylphosphorothioic acid, and 3%
     O,O-diethyl phosphorodithioic acid. When 32P-labelled phorate was
    incubated with rat liver slice preparations, < 1% of the radiolabelled
    compound was converted to hydrolysis products or unextractable
    residues. Phorate sulfoxide, phorate sulfone, phoratoxon sufoxide,
    and phoratoxon sulfone were formed (Bowman & Casida, 1958).

         In male rats given a single dose of 0.8 mg/kg bw 14C-phorate,
    the main urinary metabolites were the non-phosphorylated CL 180,298
    (43%), CL 180,296 (28%), and CL 180,297 (9.6%) (see Figure 2 and Table
    1 for identity of metabolites). Phosphorylated metabolites (CL 18,061,
    CL 18,161, CL 18,162, CL 18,177, and CL 4,259) accounted for < 15% of
    the recovered urinary metabolites, and unmetabolized parent compound
    accounted for < 1%. The main residues in liver, kidney, and muscle
    were also the non-phosphorylated metabolites, accounting for > 68,
    79, and 83% of the tissue metabolites, respectively (Hussain, 1987).

         In female rats given a single dose of 0.44 mg/kg bw 14C-phorate,
    the main urinary metabolites were non-phosphorylated CL 180,298 (43%),
    CL 180,296 (24%), CL 325,959 (15%), and CL 180,297 (4.6%). The main
    tissue metabolites were also non-phosphorylated, accounting for
    > 35, 24, and 74% of the metabolites in liver, kidney, and muscle,
    respectively. No phosphorylated metabolites were identified in
    the urine or tissue samples. Faecal samples contained primarily
    unmetabolized parent compound (33%) and the phosphorylated metabolites
    CL 18,177 (24%) CL 18,161 (8.8%), CL 18,162 (5.5%), and CL 4,259
    (4.3%). The proposed metabolic pathway is presented in Figure 2, and
    the chemical names of the metabolites are given in Table 1 (Miller &
    Wu, 1990).

    FIGURE 7

        Table 1.  Identity of metabolites of phorate

                                                                                                

    Metabolite no.      Chemical name
                                                                                                

    CL 35,024           Phosphorodithioic acid, O,O-diethyl S-(ethylthio)methylester
                        (parent compound)
    CL 18,162           Phosphorothioic acid, O,O-diethyl S-(ethylsulfinyl)methylester
    CL 18,177           Phosphorodithioic acid, O,O-diethyl S-(ethylsulfinyl)methylester
    CL 18,061           Phosphorothioic acid, O,O-diethyl S-(ethylsulfonyl)methylester
    CL 4,259            Phosphorothioic acid, O,O-diethyl S-(ethylthio)methylester
    CL 18,161           Phosphorodithioic acid, O,O-diethyl S-(ethylsulfonyl)methylester
    CL 180,298          Sulfoxide, (ethylsulfonyl)methyl
    CL 180,297          Sulfoxide, ethyl(methylsulfonyl)methyl
    CL 180,296          Methane, (ethylsulfonyl)(methylsulfonyl)-
    CL 325,959          Methane, (ethylsulfinyl)(methylsulfinyl)-
                                                                                                

    From Miller & Wu (1990)
    
    Comments

         14C-Labelled phorate was rapidly absorbed and excreted by rats
    after a single dose in corn oil given by gavage. The urine was
    the primary route of elimination, with approximately 80% of the
    administered radiolabel excreted within 24 h; faecal elimination
    accounted for about 10% of the label.

         The current studies showed essentially total excretion of 14C
    label after 192 h. The Meeting concluded that phorate and its
    metabolites are rapidly excreted and that accumulation of a toxic
    metabolite is not a concern. Thus, the new data did not indicate
    that the ADI allocated in 1994 should be reassessed. The ADI of
    0-0.0005 mg/kg bw allocated on the basis of an NOAEL of 0.05 mg/kg bw
    per day in a one-year study of toxicity in dogs and a two-year study of
    toxicity and carcinogenicity in rats, with a 100-fold safety factor,
    was confirmed.

    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 developmental toxicity)

         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

         Further observations in humans

    References

    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.

    Hussain, M. (1987) Thimet(R) insecticide, prorate (CL 35,024):
    Disposition and metabolic fate of carbon-14 labelled CL 35,024 in the
    rat. Unpublished report from American Cyanamid Co. Report No. PD-M
    Volume 27-59. Submitted to WHO by American Cyanamid Co., Princeton,
    NJ, USA.

    Miller, P. & Wu, D. (1990) Phorate (CL 35,024): Absorption,
    disposition, elimination and metabolic fate of carbon-14 CL 35,024 in
    the female rat. Unpublished report from XenoBiotic Laboratories Inc.,
    American Cyanamid Co. XBL Report No. RPT0043, PD-M Volume 27-59.
    Submitted to WHO by American Cyanamid Co., Princeton, NJ, USA.
    


    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: 1994 evaluations Part II Toxicology)