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.