PESTICIDE RESIDUES IN FOOD - 1982
Sponsored jointly by FAO and WHO
EVALUATIONS 1982
Data and recommendations of the joint meeting
of the FAO Panel of Experts on Pesticide Residues
in Food and the Environment and the
WHO Expert Group on Pesticide Residues
Rome, 23 November - 2 December 1982
Food and Agriculture Organization of the United Nations
Rome 1983
ACEPHATE
CH3S O O
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P - NH - C - CH3
/
CH3O
Explanation
Acephate was evaluated by the Joint Meeting in 1976 (FAO/WHO
1977)1/ and a full ADI was allocated, based on no-effect levels taken
exclusively from Industrial Bio-Test Laboratories (IBT) data.
Additional data have become available and are summarized in this
Monograph Addendum.
EVALUATION FOR ACCEPTABLE DAILY INTAKE
BIOCHEMICAL ASPECTS
Effects on Enzymes and Other Biochemical Parameters
The anticholinesterase activity of acephate (99.3%), technical
acephate (93.6%), methamidophos (99.6%), a metabolite of acephate and
mixtures of acephate and methamidophos in brain and erythrocytes of
rats and monkeys were determined in vitro. The I50 values were:
Based on the data, brain cholinesterase appeared to be
considerably more sensitive than erythrocyte cholinesterase to
anticholinesterase activity of acephate (99.3%) in rats. Additionally,
technical acephate (93.6%) was more potent than acephate (99.3%) as a
cholinesterase inhibitor, probably owing to contamination of the
technical material with methamidophos as an impurity. The latter was
substantially more potent as an anticholinesterase agent than acephate
(99.3%or 93.6%) (Wong et al 1979).
Rat
Four control and four treated groups of rats (Sprague-Dawley
derived) comprising 5 males per group were fed technical acephate in
their diet at 0 or 75 ppm for a maximum of 20 days. One control and
one treated group were sacrificed on days 6, 11, 15 and 20. Inhibition
(31-41%) of brain cholinesterase was evident in the treated groups at
all sampling intervals. Neither plasma cholinesterase nor erythrocyte
cholinesterase was depressed (<20%) at any time during the
1/ See Annex 2 for WHO and FAO documentation.
I50
Brain cholinesterase Erythrocyte Cholinesterase
Rat Monkey Rat Monkey
Acephate (99.3%) >1 × 10-3 1 × 10-3 9 × 10-3
Technical acephate (93.6%) 4.5 × 10-4 9 × 10-5 5 × 10-4 1 × 10-4
Methamidophos 5 × 10-6 3.5 × 10-6 9 × 10-7 9 × 10-6
Mixture I 4.5 × 10-4 1.0 × 10-4
Mixture II 8.0 × 10-5 1.5 × 10-4
Mixture III 8.0 × 10-5 9.0 × 10-5
Mixture I: "Acephate concentration adjusted to 93.6% with
1% methamidophos added"
Mixture II: "Technical acephate (93.6%) with 2% methamidophos added".
Mixture III: "Acephate concentration adjusted to 93.6% with
3% methamidophos added".
experiment. In another simultaneously conducted study, 5 control
groups and 5 treated groups of 10 male rats were given dietary levels
of technical acephate at 0 or 75 ppm for up to 7 days and then
sacrificed at different time intervals. Activity of cholinesterase in
brain was reduced by 34% while that in plasma or erythrocytes was
reduced by 21-25% on test day 7. No significant depression (<20%) of
the enzyme in plasma, erythrocytes or brain was observed at any time
during the recovery period, i.e. 1, 2, 4 and 6 weeks after treatment
withdrawal (Chevron 1980).
TOXICOLOGICAL STUDIES
Special Studies on Teratogenicity
Rabbit
A pilot study with groups of 5 artificially inseminated Dutch
Belted rabbits was undertaken to determine dosage levels of technical
acephate to be used in a teratology study. Results showed that oral
doses up to 10 mg/kg body weight/day given on day 6 through day 27,
inclusive of gestation, induced no mortality or adverse effects on
body weight. Mortality and toxic signs occurred at 30 mg/kg body
weight/day, and all rabbits at 100 mg/kg body weight/day died. The 3
surviving does at 30 mg/kg body weight/day had only viable foetuses
with no resportions at sacrifice on gestation day 28. However, 2/3
pregnant does at 10 mg/kg body weight/day and 1/4 pregnant does at
3 mg/kg body weight/day had only resportions with no viable foetuses
(Rodwell and Jessup 1980).
Groups of 16 artificially inseminated Dutch Belted rabbits were
intubated with an aqueous solution of technical acephate at 0, 1.0,
3.0 or 10 mg/kg body weight/day on gestation days 6 through 27
(Day 0 = day of insemination). All surviving does were sacrificed on
day 28 of gestation and foetuses were removed by caesarean section for
external, visceral and skeletal examination. No mortality was
observed. The pregnancy rate was comparable in all groups. Maternal
body weight during gestation was not adversely affected. Does at both
3 and 10 mg/kg body weight/day showed a slightly increased incidence
of nasal discharge. Two of the pregnant does at 10 mg/kg body
weight/day aborted. Sex ratio (M/F) of foetuses was increased at
10 mg/kg body weight/day. A slight decrease (not dose-related) in the
mean number of viable foetuses and total implantations occurred in all
treated groups. There were no significant differences between control
and treated groups with respect to early and late resorptions, post-
implantation loss, mean number of corpora lutea and mean foetal
weight. Anasarca was detected in 3/66 foetuses (from 1/12 litters) and
dome-shaped head in an additional foetus from another litter at
10 mg/kg body weight/day. Neither of these abnormalities was observed
in concurrent controls, lower dosage groups or in a total of 741
foetuses from 118 litters that served as historical controls. No
significant increase in frequency of any other type of malformation
was observed, although a non-dose-related increase in incidence of
total (soft tissue and skeletal) malformations was noted in all
treated groups, as compared to concurrent controls. Such an increase
was not evident when compared to historical controls. Overall, 3 mg/kg
body weight appears to be a teratogenic no-effect level (Rodwell
et al 1980).
Special Studies on Neurotoxicity
Twenty-four white Leghorn hens (about 7 months old) were
intubated with a single dose of 375 mg/kg body weight of acephate
(98.9% pure) followed immediately by 5 mg/kg body weight of atropine
intramuscularly. (The oral LD50 of acephate in hens was determined to
be 360 mg/kg body weight prior to initiation of the neurotoxicity
study.) Twelve hens, given distilled water only, were used as
controls. In the treated group, 3 hens died and 2 were sacrificed in
moribund condition within 5 days of dosing. During the 21-day post-
exposure period, symptoms of acute poisoning, such as depressed
spontaneous activity, were seen up to 11 days but no delayed
neurotoxic symptoms were noted at any time. Histopathological
evaluation of the sciatic nerve and several sections of the spinal
cord revealed no morphological changes suggestive of delayed
neurotoxic activity of acephate. Positive controls, treated orally
with 500 mg/kg body weight of TOCP, exhibited signs and histological
changes in the nervous tissue typical of delayed neurotoxicity
(Hayashizaki et al undated).
Special Studies on Eye and Skin Irritation
One-tenth milliliter (=31.8 mg) of Orthene Specialty Concentrate
(a formulation containing 97.8% acephate) produced slight conjunctival
redness, chemosis and discharge at one hour when placed in the eyes of
New Zealand White rabbits. At this time, slight chemosis was also
noted in treated eyes that had been rinsed with distilled water. All
eyes were normal at 24 hours. No corneal opacity or iritis was
observed at any time during the study (Levy et al 1979a).
In a patch test with 6 New Zealand rabbits, 0.5G of Orthene
Specialty Concentrate induced well-defined erythema in the abraded
skin of 2 rabbits at 24 hours. Slight erythema was noted in the intact
skin of an additional rabbit at 48 hours. Treated sites of all 6
animals were normal by 72 hours (Levy et al 1979b).
Acute Toxicity
Dermal
No mortality occurred in groups of 6 male rabbits (New Zealand
White) with clipped trunk skin (3 with intact skin and 3 with abraded
skin per group) exposed for 24 hours to 5 000 or 10 000 mg/kg body
weight of technical acephate (97.8%) in physiological saline. The
LD50 was over 10 000 mg/kg body weight. Toxic signs observed were
tremors after both dosage levels and diarrhoea after 10 000 mg/kg body
weight (Rittenhouse and MacGregory 1977).
Inhalation
Five male and 5 female rats (Sprague-Dawley derived) were exposed
in an inhalation chamber for 4 hours to an aerosol of Orthene
Specialty Concentrate dissolved in distilled water at a calculated
nominal concentration of 61.7 mg/l air (particle size of aerosol not
given). There was no mortality. Tremors, ataxia and decreased
spontaneous activity were observed in all the animals after exposure.
No toxic signs were evident the following day or during the subsequent
14-day observation period (Rittenhouse et al 1979).
Short-Term Studies
Rat
Groups of Sprague-Dawley rats (70 males and 70 females per
control or treated group) were fed diets containing technical acephate
at 10 or 50 ppm for 190 days and 138 days, respectively. A high dosage
group was given 250 ppm for 119 days and then 350 ppm for 71
subsequent days. The study, originally designed to evaluate chronic
toxicity and carcinogenic potential of the compound, was terminated
early after the discovery of an impurity (not identified) in the test
material. No compound-related findings were noted with respect to
mortality, physical condition, food consumption or ophthalmoscopic
observations at 3 months. Growth was depressed in males of the top
dosage group at week 18 and thereafter. Except for a slight decrease
in total serum protein and serum albumin in females of all treated
groups and in males of the top dosage group at 3 months, there were no
significant differences between control and treated groups in
haematological and urinalysis parameters. Assay of tissue
cholinesterase several times over the course of the experiment
indicated significant inhibition (>20%) of plasma, erythrocyte and
brain cholinesterase in the top dosage group at most intervals. At
50 ppm, males exhibited depression (>20%) of brain cholinesterase at
weeks 7 and 17 and females displayed inhibition (>20%) of plasma
cholinesterase at weeks 6 and 15 and of brain cholinesterase at week
17. (Brain cholinesterase at week 17 was reduced in both sexes at
10 ppm by only about 12%.) Organ weight analysed at terminal sacrifice
(190 days) revealed a significant increase in absolute weight and
organ/body weight ratio of thyroid in females of the 10 ppm and top
dosage groups. Thyroid/body weight ratio was, however, significantly
elevated in males in the top dosage group. No histopathology data were
available (Chevron 1979).
Long-Term Studies
Rat
Groups of 75 male and 75 female rats (Sprague-Dawley CD strain;
45 days old) were fed technical acephate (92.4%) in their diet at 0,
5, 50 or 700 ppm for 28 months to assess the chronic toxicity and
carcinogenic potential of the compound. All animals dying during the
study, those subjected to interim sacrifice (generally 10 males and
10 females/group at 4 and 12 months, 5 males and 5 females/group at
22 months) and all terminal survivors were examined grossly.
Histopathological examination was conducted on a set of over 30
tissues plus unusual lesions and tissue masses from all control and
top dosage animals that died or were sacrificed during the study or
terminally and on gross lesions, tissue masses, eyes and adrenals from
presumably all animals of 5 and 50 ppm groups.
Survival was generally good and not adversely affected by
treatment. Growth was depressed in males at 700 ppm throughout the
study. Food consumption was consistently increased in both sexes at
700 ppm throughout the study and at 5.0 ppm and 50 ppm during the
first 5 weeks and 17 weeks, respectively. A slight, transient increase
in the frequency of aggressive behaviour and/or increased activity at
700 ppm in both sexes, especially in the males, was noted during the
first 6 months of the study. Alopecia was also evident in animals of
all groups, including the control, but the incidence (not consistently
dose-dependent) was slightly increased in both sexes at 700 ppm during
the entire duration of the study and in males of all treated groups
occasionally. Ophthalmoscopic examination performed 5 times during the
experiment revealed that a) "focal retinopathy appeared more
frequently in the high dose rats than in other groups", b) "some
animals in the mid- and high-dose groups developed a more diffuse
retinal degeneration, at least one of which was preceded by focal
retinopathy, and some rats developed posterior subcapsular or complete
cataracts", and c) "there was a greater incidence of various types of
cataracts (i.e. complete, focal posterior polar and posterior
subcapsular cataracts) in treated animals as compared to controls. No
consistent dose-related differences were apparent. About half of the
cataracts were unilateral." (Ophthalmological findings in individual
animals were not included in the report.) Periodic haematological and
biochemical studies showed occasional variations from controls in
values of a number of parameters such as haemoglobin, reticulocytes,
haematocrit, serum potassium and inorganic phosphorous, but these were
essentially confined only to the top dosage group. Urinalysis
conducted periodically during the study showed no abnormalities
associated with the compound. Activity of cholinesterase in
erythrocyte and plasma, monitored nine times over course of the study,
was reduced (>20%) at all of the intervals in the top dosage group.
At 50 ppm there was depression (>20%) of erythrocyte cholinesterase
in males after 28 months and of both plasma and erythrocyte
cholinesterase in females after 6 and 7 weeks and after 28 months.
Erythrocyte cholinesterase was inhibited by 29% and plasma
cholinesterase was marginally depressed (19.3%) in females even at
5 ppm after 7 weeks. Brain cholinesterase was inhibited (>20%) at
both 50 and 700 ppm after 7 and 19 weeks and 12, 22 and 28 months.
Inhibition of the tissue (brain, plasma and erythrocyte)
cholinesterase was dose-dependent and of a greater magnitude with
brain cholinesterase than with plasma or erythrocyte cholinesterase.
An increase in organ/body weight ratio of several organs, e.g.
thyroid, lung, liver, kidney, testis, was detected sporadically in
males of the top dosage group at interim or terminal sacrifice.
Liver/body weight ratio was elevated with no concomitant
histopathological changes of the tissue at both 50 and 700 ppm in
females sacrificed at the conclusion of the study. Based on summary
histopathology data unsubstantiated with histopathological findings on
individual animals, there appeared to be an increase in incidence, as
compared to concurrent controls, of adrenal medullary tumours in males
of all treated groups. A variety of gross pathological changes and
other neoplastic and non-neoplastic findings also occurred, which
probably were not related to treatment.
The unavailability of data for individual animals (supposedly
contained in appendices not included in the report) particularly on
ophthalmologic observations and gross and histopathological findings
rendered the present evaluation only preliminary in nature. No
conclusion, therefore, could be made as to the no-effect level or the
carcinogenic potential of acephate in the rat (Chevron 1981).
COMMENTS
Both the in vitro and in vivo cholinesterase studies pointed
to a higher sensitivity in brain cholinesterase than plasma or
erythrocyte cholinesterase to the anticholinesterase activity of
acephate. In a rabbit teratology study, 3 mg/kg body weight was
considered as a teratogenic no-effect level. An acute delayed
neurotoxicity in hens, while negative, could only be considered as a
screen. A long-term toxicity/carcinogenic study in rats was available,
but, due to the absence of individual animal data in the study,
particularly on gross and histopathological findings and
ophthalmoscopic observations, a full evaluation of the study could not
be made.
Except for a 2-year oral study in dogs and a teratogenicity study
in rats, all the IBT studies (including a 2-year study in rats and a
cholinesterase study in volunteers), which were essential to the
allocation of an ADI, were found to be invalid. Consequently, the
Meeting considered it necessary to change the ADI to a temporary
status using an increased safety factor.
TOXICOLOGICAL EVALUATION
Level Causing no Toxicological Effect
Rat: 5 ppm in the diet, equivalent to 0.25 mg/kg bw
Dog: 30 ppm in the diet, equivalent to 0.75 mg/kg bw
Estimate of a Temporary Acceptable Daily Intake for Man
0 - 0.003 mg/kg bw
FURTHER WORK OR INFORMATION
Required (by 1984)
1. A multigeneration reproduction study.
2. An appropriate delayed neurotoxicity study.
3. Data for individual animals in the 28-month toxicity/
carcinogenicity rat study on ophthalmoscopic observations and
gross and histopathological findings.
Desirable
Further studies to elucidate the metabolic fate of acephate,
preferably in a non-rodent species.
REFERENCES
Chevron. Oral toxicity/carcinogenicity study of technical RE-12420
1979 in rats. Intended duration: 2 years, study terminated at 190
days. Report from Bio/dynamics Inc., U.S., submitted to the
World Health Organization by Chevron Chemical Co.
(Unpublished)
1980 Recovery from cholinesterase inhibition following dietary
exposure to Orthene in the rat. Report from Bio/dynamics
Inc., U.S., submitted to the World Health Organization by
Chevron Chemical Co. (Unpublished)
1981 A life-time oral toxicity/carcinogenicity study with
technical RE-12420 in rats. Final report, from Bio/dynamics
Inc. U.S., submitted to the World Health Organization by
Chevron Chemical Co. (Unpublished)
Hayashizaki, A., Kawai, M. and Sunomata, K. Studies on acute delayed
undated neurotoxicity of Orthene. Report from Bozo Research Center,
Inc., Japan, submitted to the World Health Organization by
Chevron Chemical Co. (Unpublished)
Levy, J.E., Wong, Z.A. and MacGregor, J.A. The eye irritation
1979a potential of Orthene Speciality Concentrate. Report from
Chevron Environmental Center, U.S., submitted to the World
Health Organization by Chevron Chemical Co. (Unpublished)
Levy, J.E., Wong, Z.A. and MacGregor, J.A. The skin irritation
1979b potential of Orthene Specialty Concentrate. Report from
Chevron Environmental Center, U.S., submitted to the World
Health Organization by Chevron Chemical Co. (Unpublished)
Rittenhouse, J.R. and MacGregor, J.A. The acute dermal toxicity of
1977 Orthene technical. Report from Safety and Health Division,
Standard Oil Company of California, U.S., submitted to the
World Health Organization by Chevron Chemical Co.
(Unpublished)
Rittenhouse, J.R., Wong, Z.A. and MacGregor, J.A. The acute
1979 inhalation toxicity of Orthene Specialty Concentrate. Report
from Chevron Environmental Health Center, U.S., submitted to
the World Health Organization by Chevron Chemical Co.
(Unpublished)
Rodwell, D.E. and Jessup, D.C. Technical RE-12420, pilot teratology
1980 study in rabbits. Report from International Research and
Development Corporation, U.S., submitted to the World Health
Organization by Chevron Chemical Co. (Unpublished)
Rodwell, D.E. Jones, J.M. and Jessup, D.C. Technical RE 12420
1980 teratology study in 1980 rabbits. Report from International
Research and Development Corporation, U.S., submitted to the
World Health Organization by Chevron Chemical Co.
(Unpublished)
Wong, Z.A., Kodama, J.K. and MacGregor, J.A. Characterization of the
1979 in vitro inhibition of rat and monkey red blood cell and
brain acryl cholinesterase by acephate, technical acephate
and methamidophos and their mixtures. Report from Chevron
Environmental Health Centre, U.S., submitted to the World
Health Organization by Chevron Chemical Co. (Unpublished)