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.