METHOMYL

    EXPLANATION

          Methomyl was evaluated for acceptable daily intake by the Joint
    Meeting in 1978 and in 1986 (Annex 1, FAO/WHO, 1979a, 1986d).  A
    temporary ADI was estimated in 1986 with a request for additional
     in vitro studies in human blood to demonstrate the time-course of
    plasma and red blood cell cholinesterase inhibition and
    reactivation.  Data on the kinetics of human and rat
    acetylcholinesterase inhibition and spontaneous reactivation have
    been received and are summarized in this monograph addendum.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOLOGICAL DATA

    Special study on cholinesterase activity kinetics

          Acetylcholinesterase from human erythrocytes or from hemolyzed
    whole blood from male rats was incubated with technical grade
    methomyl (purity >98%) at 30°C for a fixed time period (apparently
    10 minutes) and the residual activity was assayed.  Three
    concentrations of methomyl were assayed with human enzyme and two
    with rat enzyme (at least duplicate assays with two concentrations
    with each enzyme).  Control assays were run to determine the
    activity of the uninhibited enzymes.  Enzyme activity was determined
    at 30°C with 18 ml saline solution (0.08 M MgCl2 and 0.2 M NaCl), 
    1 ml enzyme solution and 1 ml acetylcholine bromide solution
    (0.055 M) adjusted immediately to pH 7.6 with 0.01 M NaOH.  During
    the reaction the pH was kept constant at 7.6 (± 0.01 pH units) by
    addition of the sodium hydroxide solution.

          The logarithm of the ratio of the activity of inhibited enzyme
    over the activity of uninhibited enzyme was plotted against time and
    the slope determined.  The rate constant (k1) and the I50 value
    (the molar concentration of inhibitor resulting in a 50% inhibition
    of the enzymatic activity at a fixed time of incubation, in this
    study (10 minutes) were calculated.

          Spontaneous reaction of enzymatic activity was also determined
    for both enzymes.  Enzyme solution was incubated for 10-15 minutes
    at 30°C with enough methomyl to give 74-81% inhibition of human
    enzyme or 32-47% inhibition of rat enzyme and then passed through a
    Sephadex G-25 column to separate the enzyme and the methomyl. 
    Fractions 5 and 6 containing the enzyme were combined and incubated. 
    Periodically, aliquots were removed for cholinesterase activity
    measurement.  From the slope of the least squares line of log %
    enzyme inhibited versus time, the regeneration constant (k3) was
    determined and the time for one-half of the enzyme activity to be
    regenerated (t1/2) was calculated.

          The human enzyme was about six times more sensitive to methomyl
    inhibition than the rat enzyme.  I50 for human enzyme was
    2.65 x 10-6M while that for rat enzyme was 1.56 x 10-5M.  Rat
    enzyme regenerated about 1.4 times faster than the human enzyme;
    t1/2 = 26.6 min for rat vs. 38.0 minutes for humans (Carakostas,
    1987).

    Actual Cholinesterase Kinetics

    Inhibition

                           K1 (M-1 min-1)                 I50

          Human             2.96 x 104                2.65 x 10-6
          Rat               4.73 x 103                1.56 x 10-5

    Regeneration

                           K3 (min-1)                 t1/2 (min-1)

          Human            1.85 x 10-2                   38.0
          Rat              2.62 x 10-2                   26.6

    COMMENTS

          Rate constants of inhibition of human and rat AChE by methomyl
    and its spontaneous reactivation have been reported.

          The human enzyme is about five times more sensitive to methomyl
    inhibition than the rat, but the rates of spontaneous reactivation
    do not differ in the two species.

    TOXICOLOGICAL EVALUATION

    Level causing no toxicological effect

          Mouse:      50 ppm in the diet, equal to 8.7 mg/kg bw/day
          Rat:        50 ppm in the diet, equivalent to 2.5 mg/kg bw/day
          Dog:       100 ppm in the diet, equal to 3.1 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

          Observations in humans.

    REFERENCE

    Carakostas, M.C. (1987)  Inhibition and regeneration kinetics for
    human and rat acetyl-cholinesterase exposed to methomyl  in vitro. 
    Unpublished Report No.  HLR-379-88 from Haskell Laboratory. 
    Submitted to WHO by E.I. du Pont de Nemours and Co., Inc.,
    Washington, Delaware, USA.

    See Also:
       Toxicological Abbreviations
       Methomyl (EHC 178, 1996)
       Methomyl (HSG 97, 1995)
       Methomyl (ICSC)
       Methomyl (WHO Pesticide Residues Series 5)
       Methomyl (Pesticide residues in food: 1976 evaluations)
       Methomyl (Pesticide residues in food: 1977 evaluations)
       Methomyl (Pesticide residues in food: 1978 evaluations)
       Methomyl (Pesticide residues in food: 1986 evaluations Part II Toxicology)
       Methomyl (JMPR Evaluations 2001 Part II Toxicological)