First draft prepared by
D.J. Clegg, Carp, Ontario, Canada
Chlorpyrifos-methyl was evaluated by previous Joint Meetings in
1975 and 1991 (Annex 1, references 24 and 62). An ADI of 0.01 mg/kg
bw allocated in 1975 was reduced to 0.001 mg/kg bw in 1991. The
present Meeting re-evaluated the recently-conducted long-term
dietary study in rats on which the ADI of 0.001 mg/kg bw was based
in 1991. The major interest was the vacuolation of the zona
fasciculata of the adrenal gland.
EVALUATION FOR ACCEPTABLE DAILY INTAKE
Long-term toxicity/carcinogenicity studies
Five groups of 50 Fischer 344 rats (5-6 weeks old)/sex/dose
were fed chlorpyrifos-methyl (average purity 95.4 ± 0.8%) at dietary
concentrations of 0, 1, 2, 20 or 1000 ppm for 2 years, equivalent to
0.05, 0.1, 1 or 50 mg/kg bw/day. Ten additional rats/sex/dose were
administered the same diets for 53 weeks prior to sacrifice.
Analyses of the diet at 9 time intervals during the study showed
dietary concentrations to be acceptable. A single set of analyses
confirmed adequate homogeneity and stability in the diet for up to
24 days. Rats housed singly with food and water available ad
libitum, were observed daily, with a detailed examination weekly,
throughout the study. Animal weights were recorded weekly for 13
weeks and then once every 4 weeks. Feed intake, using 20
rats/sex/dose followed the same time pattern as body-weight
measurement. Blood samples (from fasted animals) for clinical
chemistry and haematology and urine for urinalyses were collected at
26, 52-53, 78, and 104 weeks. Brain cholinesterase was measured in
half brain samples in all interim sacrifice rates, and in 10
rats/sex/dose at terminal sacrifice. Organ weights (recorded only in
scheduled sacrifices) were determined in fasted rats at necropsy.
Histopathology was performed on 53 tissues in all rats receiving 0
and 50 mg/kg bw/day for 2 years and on all rats dying or terminated
in moribund condition. At 0.05, 0.1 and 1 mg/kg bw/day,
histopathological examination was limited to adrenal glands, brain,
epididimides, kidneys, liver, lungs, pituitary gland, spinal cord,
sciatic nerve, testes, tibial nerve and gross lesions in rats
sacrificed at term.
Mortality was not affected. Body-weight reduction
(statistically significant but less than 10%) was detected at 50
mg/kg bw/day in both sexes. Food intake was slightly reduced in
males at 50 mg/kg bw/day. Clinical signs were comparable in all
groups. Haematological parameters (PCV, Hb, RBC counts, WBC total
and differential counts and platelet counts) showed no differences
attributable to chlorpyrifos-methyl; neither did urinalyses
parameters (pH, protein, glucose, ketones, bilirubin, urobilinogen
or sediment macroscopic examination). Clinical chemistry values
(alanine transaminase, albumin, aspartate transaminase, alkaline
phosphatase, glucose, phosphorus, total protein, triglycerides,
blood urea nitrogen, chloride, sodium and potassium) were
sporadically significantly different, but results were inconsistent,
except for depressed levels of ALT and AST in males at 6, 12 and 18
months (but not at 24 months) and in females at 6 and 12 months
only. Cholinesterase inhibition was noted in plasma at 1 and 50
mg/kg bw/day in both sexes. Erythrocyte and brain cholinesterase
activities were depressed at 50 mg/kg bw/day in both sexes.
Incidence of gross lesions were comparable in all groups. Organ
absolute and relative weights were comparable in all groups except
for the adrenal gland. After deletion of adrenal glands bearing
tumours (i.e. where the organ weight was likely to be abnormal) both
absolute and relative adrenal weights were increased in both sexes
at 50 mg/kg bw/day at interim (1 year) and terminal (2 year)
sacrifice. At interim sacrifice, both sexes in the 50 mg/kg bw/day
group showed 10/10 rats with moderate vacuolation, consistent with
lipid accumulation in the zona fasciculata of the adrenal. At
terminal sacrifice, in males, the incidence of moderate vacuolation
was 49/50, 1/50, 0/50, and 0/50 at 50, 1, 0.1 and 0.05 mg/kg bw/day,
respectively. Slight vacuolation was noted in 1/50, 10/50, 9/50,
5/50 and 3/50 at 50, 1, 0.1, 0.05 and 0 mg/kg bw/day. In the
females, adrenal zona fasciculata vacuolation was noted in 30/50,
0/50, 0/50, 0/50 and 0/50 as being moderate, and in 19/50, 0/50,
1/50, 2/50 and 2/50 as being slight at 50, 1, 0.1, 0.05 and 0 mg/kg
An increased incidence of adrenal vacuolation was observed in
both sexes at 50 mg/kg bw/day. This increase was probably compound-
related. At lower doses, percentage incidence of adrenal zona
fasciculata vacuolation, consistent with lipid accumulation were,
in males, 22%, 18%, 10% and 6% and in females, 0%, 2%, 4% and 4% at
1, 0.1, 0.05 and 0 mg/kg bw/day. Historical control data from 5
contemporary studies from the same laboratory and in the same strain
of rat showed 40%, 10%, 42%, 0% and 20% incidence of vacuolation of
the adrenal zona fasciculata in males, and 26%, 0%, 10%, 4% and 0%
in females. Thus incidences observed in the current study at 1 mg/kg
bw/day and below are well within historical control data ranges, and
are unlikely to be compound-related. The NOAEL for the study was 1
mg/kg bw/day (Barna Lloyd et al., 1991).
Observations in humans
Fourteen male volunteers were divided into two treatment groups
of five men each and a control group of four men. Chlorpyrifos-
methyl was administered by gelatin capsule in a single daily dose of
0, 0.03 or 0.1 mg/kg bw/day for four weeks. Plasma and erythrocyte
cholinesterase activities were not depressed at levels tested.
Haematology, blood chemistry, urinalysis, blood pressure, pulse rate
and ophthalmology were not affected by treatment. The NOAEL was the
highest dose tested, 0.1 mg/kg bw/day (Coulston et al., 1975).
The two-year dietary study in rats, which utilized dietary
doses of 0, 0.05, 0.1, 1 or 50 mg chlorpyrifos-methyl/kg bw/day, did
not show any carcinogenic potential of the compound.
On histopathological examination, no correlation was found
between the incidence of vacuolation of the zona fasciculata of
the adrenal gland and of other organs. The incidence of vacuolation
of the zona fasciculata at all dose levels except the high dose
was within the range of occurrence noted in contemporary rat studies
performed in the same laboratory. The NOAEL for the study was
therefore interpreted as being 1 mg/kg bw/day.
The 1975 Joint Meeting reviewed a human study in which five
males/test group were given single doses of 0, 0.03, or 0.1 mg
chlorpyrifos-methyl/kg bw/day for four weeks. Test groups were
comparable to a control group of four males with respect to plasma
and erythrocyte cholinesterase activity, haematology, blood
chemistry, blood pressure, pulse rate and ophthalmology. The NOAEL
was the highest dose tested, 0.1 mg/kg bw/day.
The ADI allocated by the 1991 Joint Meeting was based on the
changes in rat adrenal pathology which were interpreted as showing a
NOAEL of 0.1 mg/kg bw/day, to which a 100-fold safety factor was
applied. With the revision of the NOAEL with respect to the rat
adrenal, the present Meeting allocated an ADI based on the human
data (NOAEL 0.1 mg/kg bw/day) using a 10-fold safety factor. This
ADI is supported by the NOAEL in studies in rats (1 mg/kg bw/day)
using a 100-fold safety factor.
Level causing no toxicological effects
Mouse: 50 ppm, equal to 3.9 mg/kg bw/day (78-week study)
Rat: 1 mg/kg bw/day (two-year feeding study)
Human: 0.1 mg/kg bw/day (four-week study).
Estimate of acceptable daily intake for humans
0-0.01 mg/kg bw.
Studies which will provide information valuable in the contimued
evaluation of the compound
Further observations in humans.
Barna-Lloyd, T., Szabo, J.R. & Davis, N.L. (1991) Chlorpyrifos-
methyl (Reldon R) rat chronic dietary toxicity/oncogenicity study.
Unpublished report TXT: K-046193-020 from Dow Chemical, Texas, USA.
Submitted to WHO by Dow Elanco, Indianapolis, USA.
Coulston, F., Rosenblum, I. & Griffin, T.B. (1975) Study of
chlorpyrifos-methyl in human volunteers. Unpublished report from
Institute of Comparative and Human Toxicollogy, Albany Medical
College and International Centre of Environmental Safety, Holloman
AFB, New Mexico. Submitted to WHO by Dow Chemical Company, Midland,