ETHOPROPHOS
EXPLANATION
Toxicological data for Ethoprophos were previously examined for
acceptable intake by the 1983 JMPR (Annex 1, WHO/FAO, 1984a). An
Acceptable Daily Intake could not be estimated at the meeting "because
the data were inadequate". However, a specific data set was not
required by that meeting as a prerequisite to reconsideration of an
ADI for this chemical.
The following studies were submitted for consideration by the
1987 JMPR: (1) an acute delayed neurotoxicity study in the hen; (2) a
52-week feeding study in the dog; (3) a 2-year feeding study in the
rat; and (4) a 2-year feeding study, in mice.
EVALUATION FOR ACCEPTABLE INTAKE
BIOLOGICAL DATA
Toxicological studies
Special Study on Neurotoxicity
An initial study was conducted to determine the LD50 of
ethoprophos in the hen. After a preliminary range-finding study, 6
groups of 10 female domestic hens ("a hybrid brown laying strain")
were administered by savage dosages of the test material (94.5%
purity) that ranged from 0 to 16 mg/kg in a volume of 2 ml/kg. An LD50
of 6.44 mg/kg was calculated, with 95% confidence limits of
4.78-8.83 mg/kg. Based on these results, a dosage of 6.5 mg/kg was
selected for the main neurotoxicity study.
For an assessment of neurotoxicity, test animals were randomly
divided into 6 groups of 10 hens each. Control hens (group 1) received
only corn oil. Positive control hens were administered 500 mg/kg of
triorthocresyl phosphate (TOCP) by savage. Groups 3-6 were first
administered 10 mg/kg of atropine sulfate by intramuscular injection,
followed by administration of 6.5 mg/kg of ethoprophos by gavage. All
oral doses were administered in corn oil at a constant volume of
2.5 ml/kg.
After treatment of the test groups it was observed that atropine
had only a minimal protective effect, as 31/40 treated birds died
within 4 days, with the majority of deaths occurring within the first
24 hours. Because of excessive mortality in test groups 3-6, two
additional groups (#7 and #8) were placed on test. These two groups
contained 12 and 11 birds, respectively. The submitted study report
did not specify that these additional test groups were treated
concurrently with the negative control groups. Birds in groups 7 and 8
were administered 2-PAM (50 mg/kg by i.m. injection) in addition to
atropine prior to treatment with 6.5 mg/kg of the test chemical.
Surviving birds were given repeat injections of 2-PAM and atropine at
24 and 48 hours after treatment, and two birds from group 8 were
injected 72 hours after treatment. This procedure had only marginal
success as 14/23 birds died within 48 hours of treatment.
On the basis of the mortality observed in test birds after the
first dose of ethoprophos, the LD50 was recalculated as 5.2 mg/kg.
This dose was administered on day 22 to all 18 surviving test birds,
who were pretreated with 2-PAM and atropine immediately before
administration of ethoprophos. All test birds were given repeat
injections of 2-PAM and atropine 5 hours after the second (day 22)
treatment with ethoprophos, and birds from groups 3-6 were given
additional injections 24 hours after the second treatment. Two of the
18 treated birds died within 72 hours of the second treatment.
No clinical signs of neurotoxicity were noted in any negative
control or ethoprophos-treated birds, whereas 9/10 birds treated with
TOCP exhibited signs of neurotoxicity that ranged from slight to
marked. The majority of these birds began exhibiting these signs by
11 days after treatment with TOCP. As these birds displayed signs of
neurotoxicity after a single dose of TOCP, they were sacrificed on day
21 and examined for histopathological changes. All other surviving
birds were sacrificed on day 43. No remarkable macroscopic changes
were observed at necropsy. Portions of forebrain, mid- and hind-brain,
cervical, thoracic and lumbar spine, proximal and distal sciatic
nerve, and tibial nerve were evaluated for microscopic evidence of
neuropathologic change. Lesions were noted in the spinal cord and
peripheral nerve of all positive control birds that demonstrated
"significant axonal degeneration...related to TOCP treatment". Lesions
were noted in the spinal cord of 9/10 control birds that were graded
as minimal (grade 2 out of a maximum of 5), and 1/10 control birds was
observed to have a minimal lesion of the proximal sciatic nerve. No
lesions were noted in the brain, distal sciatic or tibial nerves of
control birds. A similar distribution of lesions of the spinal cord
was noted in treated birds, however, lesions of the mid/hindbrain that
were graded minimal were also noted in 2/16 treated birds, and lesions
of the proximal sciatic nerve were noted in 2/16 birds. One of these
lesions was graded as moderate (grade 3), and occurred in a bird that
was also noted to have a lesion of the mid/hindbrain (bird #211).
Three of 16 birds also had minimal lesions of the distal sciatic
nerve, and included bird #211.
The study authors concluded that treatment with ethoprophos "did
not produce any clinical signs of neurotoxicity", and that "this
result was confirmed by the histological examination, which showed no
treatment-related changes in the nerve tissue. The changes noted in
proximal sciatic nerve of bird #211 were considered to be unrelated to
treatment and to represent the extreme upper limit of background
change in this instance" (Roberts et al., 1986).
The present reviewer concludes that the study does not provide
any clear evidence of neurotoxicity. However, because a large degree
of mortality has reduced the sensitivity of this study, the equivocal
findings in some of the birds cannot be dismissed. Historical control
data from the testing facility suggest that the lesions in proximal
sciatic nerve are not spontaneous. Data on the effect of ethoprophos
on neuropathy target esterage (NTE) activity would aid in the
evaluation of this compound.
Special Study on Carcinogenicity
Mouse
Groups of male and female B6C3F1 (SPF) mice were randomly
assigned to test groups (80/sex/group) and fed diets containing
0, 0.2, 2 or 30 ppm of technical grade ethoprophos (94.6% purity) for
two years. Treatment commenced on May 21, 1981, and was terminated on
May 19, 1983. The test material and test diets were analysed
periodically to insure that test diet concentrations were within
acceptable limits of nominal values. Diets and water were provided
ad libitum. Animals were examined daily for signs of toxicity, and
moribund animals were sacrificed. Body weights were recorded weekly
for the first 26 weeks of treatment and biweekly thereafter. Food
consumption was determined weekly. Clinical pathology determinations
(hematology, clinical chemistry, cholinesterase and urinalysis) were
conducted after 26, 52, 78 and 104 weeks of treatment. Interim
sacrifices of 10 mice/sex/dose were conducted after 26, 52 and 78
weeks of treatment, and all surviving animals were sacrificed after
104 weeks of treatment. Complete post-mortem examinations were
conducted on all animals after scheduled sacrifice as well as on those
mice that died spontaneously or were sacrificed in a moribund
condition.
No effect of treatment on survival or incidence of clinical signs
were apparent. Mean body weight gain was decreased by about 5-10% in
high dose males and females over the first 80 weeks of treatment.
However, by study termination a decrease in mean body weight between
any of the male test groups, whereas for high dose females a
statistically significant decrease in mean body weight gain of about
6% was noted at study termination. Although occasional statistically
significant changes in food consumption were noted in all of the
treatment groups, no relation to treatment was apparent. Mean compound
intake over the course of the study was reported to be 0, 0.032,
0.306, and 4.66 mg/kg/day for males and 0, 0.038, 0.381, and
5.90 mg/kg/day for females.
Hematology values were unremarkable with the exception of the
mean leukocyte count for males, which appeared to be decreased in a
dose related fashion at each of the intervals measured. At study
termination, mean leukocyte counts for males were: 4.1 +1.7, 2.3 ±0.9,
2.1 ±1.1, and 1.6 ±0.5 (1000/mm3) for controls, low, mid and high
dose mice, respectively. The study authors noted that all treated male
groups were statistically different from control at termination,
however they stated that the values from the low and mid dose groups
were within the historical control range for that laboratory. The
significance of this finding is unclear as it was not associated with
any other toxic effects such as an increase in mortality or incidence
of intercurrent disease, however the pattern of response does suggest
a treatment-related effect. Historical control data would aid in the
evaluation of this potential finding.
Plasma and erythrocyte cholinesterase activities were inhibited
in a dose-related manner in the mid and high dose groups (male and
female) over the first 78 weeks of treatment. However, by study
termination plasma and erythrocyte cholinesterase activities in the
mid dose group were similar to control with the exception of plasma
values for mid dose females which were significantly decreased by
about 20%. Brain cholinesterase was inhibited only in high dose males
and females, and ranged from a value of 65% of control at week 26 to
about 80% of the control values at termination. The study authors
concluded that the NOEL for cholinesterase inhibition was the low
dose, 0.2 ppm.
Other serum chemistry and urinalysis values did not appear to be
affected by treatment with the test compound.
Necropsy of animals that died on test or were sacrificed in a
moribund condition did not reveal any treatment-related lesions.
At the interim and final sacrifices, occasional alterations in
organ weights were noted in high dose mice that were likely related to
the decreased weight gain in this group. A consistent effect on both
absolute and relative organ weights was not apparent. No
treatment-related lesions were noted after gross and microscopic
examinations for the interim sacrifices. At final sacrifice, an
increased incidence of calcification of the kidney was noted in high
dose males (13/42 vs. 2/45 control) along with "regenerating
epithelium" of the kidney (0/45 control, 9/31 low, 13/38 mid and 23/42
high dose males, respectively). A similar response was not noted in
the female. The study authors indicated that this lesion is
spontaneous, and concluded that "pathological lesions caused by
Ethoprop were not found".
No effect of treatment on the incidence of tumors in specific
tissues, nor on the total tumor burden of treated animals, was
apparent. The study authors concluded that the NOEL for chronic
toxicity in this study was 0.2 ppm, based on inhibition of plasma and
erythrocyte cholinesterase activity at 2 and 30 ppm
(Yamagata et al., 1984).
The present reviewer concludes that additional clarification of
the apparent decrease in leukocyte count, and of the lesion described
as "regenerating epithelium" in the kidney, would aid in the
evaluation of this study.
Long Term Studies
Rats
Groups of male and female Fischer 344 rats were randomly assigned
to test groups (70/sex/dose) and fed diets containing 0, 1, 10 or
100 ppm of technical grade ethoprophos (95.9% purity) for 105
consecutive weeks. The technical test material and test diets were
analysed periodically to insure stability and homogeneity of test
diets. Diets and water were provided ad libitum. Animals were
examined daily for mortality and morbidity, and were given detailed
physical examinations on a weekly basis. Body weights were recorded
weekly for the first 26 weeks, and biweekly thereafter until study
termination, as were measurements of food and water consumption.
Ophthalmoscopic examinations were performed on all rats at study
initiation and after 6, 12, 18 and 24 months of treatment. Routine
hematological, serological and urinalysis studies were also conducted
after 6, 12, 18 and 24 months of treatment. Plasma and erythrocyte
cholinesterase measurements were performed at the same intervals as
other clinical chemistry studies, and brain cholinesterase was
measured at the interim sacrifices and at final sacrifice. Ten
rats/sex/dose were sacrificed after 12 and 18 months of treatment for
interim evaluation, and all surviving animals were sacrificed at the
end of 24 months of treatment. All animals were killed on schedule,
and those that died on test or were sacrificed in a moribund
condition, were subjected to a complete post-mortem examination.
No effect of treatment on survival, body weight gain, or food and
water consumption was apparent. The only potential treatment-related
finding during physical examinations was an increased incidence of
anogenital staining in high dose female rats over weeks 14-78 of
treatment. Hematological examinations revealed evidence of decreases
in red cell count, hemoglobin and hematocrit with increases in mean
corpuscular volume in high dose males and females at all intervals.
This change was statistically significant only after 6 and 12 months
of treatment, but not after 18 months of treatment nor at final
sacrifice. A slight increase in BUN (10-20% compared to controls) was
also noted in high dose males and females. This finding was
statistically significant only at 6 months in females and at 18 months
in males. In addition, a statistically significant decrease in serum
globulins was noted after 6 and 12 months of treatment, but was not
apparent at final sacrifice.
Plasma and erythrocyte cholinesterase activities were
significantly inhibited in a dose-related manner at most time
intervals in mid and high dose males and females, whereas brain
cholinesterase activity was inhibited only in high dose male and
female rats, also at all measured intervals.
No toxicologically-significant alterations were revealed by
urinalysis studies, nor by ophthalmoscopic examinations.
At the 12 month interim sacrifice, statistically significant
increases of about 10% were noted in the relative spleen weights of
high dose males and females, along with similar decreases in absolute
and relative kidney weights in high dose males only. No significant
treatment-related changes were noted after gross or microscopic
examinations.
At the 18 month interim sacrifice, no significant alterations in
organs weights were noted, nor were any significant changes noted
after gross and microscopic examinations of tissues.
At final sacrifice, statistically significant increases (16-20%)
in absolute and relative organ weights of thyroid/parathyroid were
noted in high dose males, along with non-significant increases
(14-16%) in mid dose males. Gross examination revealed an increased
incidence of enlarged thyroid in mid and high dose males: 5/35 and
5/39, respectively, compared to 1/36 control. The only potentially
treatment-related lesion revealed by microscopic examination was an
increased incidence of parafollicular ("C") cell neoplasms in high
dose males (animals dying between 18 months and final sacrifice are
included):
MALES
0 ppm 1 ppm 10 ppm 100 ppm
C-cell adenoma DOT 2/13 0/7 0/13 1/9
FS 6/36 5/39 5/35 11/39
TOTAL 8/49 5/46 5/48 12/48
C-cell carcinoma DOT 0/13 0/7 0/13 1/9
FS 0/36 0/39 1/35 2/39
TOTAL 0/49 0/46 1/48 3/48
Total C-cell neoplasms 8/49 5/46 7/48 15/48
(16.3%) (10.9%) (12.5%) (31.3%)
FEMALES
0 ppm 1 ppm 10 ppm 100 ppm
C-cell adenoma DOT 0/9 0/9 0/4 1/8
FS 2/40 4/39 0/41 4/41
TOTAL 2/49 4/48 0/45 5/49
C-cell carcinoma DOT 0/9 0/9 0/4 0/8
FS 0/40 1/39 0/41 1/41
TOTAL 0/49 1/48 0/45 1/49
Total C-cell neoplasms 2/49 5/46 0/48 6/49
(4.1%) (10.4%) (0%) (12.2)
The study authors did not find these changes to be statistically
significant, and concluded that the observed incidence of thyroid
neoplasia was "random and unrelated to the test article". No other
potential treatment-related lesions were apparent.
The present reviewer is unable to conclude that the apparent
increase in C-cell tumors noted at the high dose is spontaneous. In a
previous 2-year rat feeding study conducted in the same strain of rats
(Barnett et al., 1983, reviewed at the 1983 JMPR), a remarkably
similar response was demonstrated in the thyroid. The 1983 JMPR
Evaluation of that study reported that the incidence of C-cell adenoma
in high dose (196 ppm) males was about 27%, compared to an incidence
of about 6% in control males. The response was also similar in that
tumors were noted with increased frequency only in high dose males at
terminal sacrifice, and other treatment groups were apparently
unaffected.
The study author concluded that NOEL for chronic toxicity was
1 ppm, based on inhibition of cholinesterase activity at 10 and
100 ppm (Spicer, 1985)
Dogs
Male and female purebred beagle dogs (4/sex/dose) were
administered 0, 0.025, 1.0 or 10.0 mg/kg/day by oral capsule. The test
material was dissolved in peanut oil. Doses were based on the results
of a 4-week range finding study. Dogs were offered 400 grams/day of
food, and were provided water ad libitum. Animals were observed
daily for signs of toxicity, and body weights were recorded weekly.
Food consumption was determined daily. Blood and urine were collected
prior to study initiation and after 6, 13, 26 and 52 weeks of
treatment. The standard hematology, serum chemistry and urinalysis
procedures were performed in addition to measurements of plasma and
erythrocyte cholinesterase activities. At study termination, dogs were
sacrificed by overdose of thiopentone sodium followed by
exsanguination. A gross examination of all tissues was performed
in situ, and major organs were removed and weighed. The standard set
of tissues was collected for histopathological examination.
No effect of treatment on the incidence of clinical signs was
reported. No effect of treatment on weight gain in treated males was
apparent, however a dose-related trend toward decreased body weights
in treated females was noted throughout the study. At termination,
high dose females weighed about 10% less than control. High dose males
and females tended to consume about 10% less food than control dogs,
low however and mild dose animals were not affected.
Red cell count, hemoglobin concentration and hematocrit were
statistically significantly lower in high dose males than in control
males at all measured intervals, however the pretest values for this
group were also lower than the control and other treatment groups.
Serum chemistry values were unremarkable with the exception of an
increase in mean SGPT activity associated with decreased total
cholesterol and serum albumin in high dose males that was noted after
6 weeks of treatment and persisted through study termination. This
effect was largely due to an apparent hepatoxic response in 2/4 high
dose males; for one of these dogs (#345) SGPT was elevated by 10-fold
over control at study termination, and serum alkaline phosphatase,
gamma-glutamyl transferase, and SGOT activities were also grossly
elevated in this animal. Serum albumin was decreased in all high dose
males equally.
Plasma and erythrocyte cholinesterase activities were depressed
in a dose related manner in mid and high dose dogs at all measured
intervals. At study termination, plasma cholinesterase activity was
decreased to 33% and 17% of control in mid and high dose males,
whereas this activity was reduced to 58%, 19% and 17% in low, mid and
high dose females, respectively. Erythrocyte cholinesterase activities
for males at study termination were 127%, 88% and 39% of control for
low, mid and high dose respectively, whereas for females red cell
cholinesterase activities were 103%, 62% and 38% of control,
respectively. Brain cholinesterase activities at study termination
were inhibited only in high dose males and females, to 56% and 62% of
control, respectively. The effects on plasma cholinesterase activity
in the low dose group were considered by the study authors to be
"biologically not significant", and the NOAEL for cholinesterase
activity was considered to be the low dose, 0.025 mg/kg/day.
Ophthalmoscopic examinations, conducted prior to study initiation
and termination, did not reveal any treatment-related effects.
At necropsy, no effect of treatment on absolute or relative organ
weights was apparent, nor was any effect on the incidence of gross
findings noted. Upon microscopic examination, treatment-related
pathological findings were restricted to the livers of males and
females. Selected findings are tabulated below (4 dogs examined from
each group; 1 = control, 2 = 0.025 mg/kg/day, 3 = 1 mg/kg/day,
4 = 10 mg/kg/day):
Males Females
1 2 3 4 1 2 3 4
Vacuolation 0 0 3 4 0 0 3 4
Focal necrosis 0 0 0 2 1 0 0 1
Pigment 0 0 1 4 0 0 0 3
Kuppfer Cell pigment 0 0 1 4 0 0 1 4
Fibrosis 0 0 0 4 0 0 0 4
Biliary proliferation 0 0 0 4 0 0 0 4
The study authors felt that the hepatotoxic response was
restricted to high dose animals, and that the NOEL was at the mid
dose. The present reviewer concludes that a more conservative
interpretation of the data would place the NOEL for this effect at the
low dose.
The study authors concluded that the overall NOEL for toxicity in
this study was the low dose, 0.025 mg/kg/day, based on inhibition of
plasma and erythrocyte cholinesterase activities at higher doses
(Brown, 1986).
COMMENTS
Toxicology data for ethoprophos were evaluated by the 1983 JMPR.
An ADI was not allocated by that meeting because the available data
were inadequate. Supplementary data were submitted to the present
meeting.
A acute delayed neurotoxicity study in the hen did not reveal any
clear evidence of a neurotoxic response. However, because of the high
mortality in this study, and equivocal findings in some of the treated
birds, data on the effect of this compound on neuropathy target
esterage (NTE) activity in the hen would be useful.
A two-year feeding study in the mouse did not reveal any evidence
of a carcinogenic effect of ethoprophos in this species. A NOAEL of
0.2 ppm (equal to 0.035 mg/kg/day) was established for erythrocyte
cholinesterase inhibition.
A two-year feeding study in rats produced equivocal evidence of a
carcinogenic response in the thyroid of high-dose males, but this
effect was restricted to the high-dose group. No other treatment-
related lesions were identified. The overall NOAEL for this
study was determined to be 1 ppm (equal to 0.05 mg/kg bw/day), based
on the inhibition of erythrocyte cholinesterase activity at higher
doses.
A 52-week oral dose study in dogs provided evidence of a
hepatotoxic response in mid- and high-dose males and females as
indicated by alterations in liver histology accompanied by
disturbances in related serum chemistry values. The NOAEL for this
study was determined to be 0.025 mg/kg bw/day, based on inhibition of
erythrocyte cholinesterase activity and evidence of hepatotoxicity at
higher doses.
TOXICOLOGICAL EVALUATION
LEVEL CAUSING NO TOXICOLOGICAL EFFECT
Mouse: 0.2 ppm in the diet, equal to 0.035 mg/kg bw/day
Rat: 1 ppm in the diet, equal to 0.05 mg/kg bw/day
Dog: 0.025 mg/kg bw/day
ESTIMATE OF ACCEPTABLE DAILY INTAKE FOR MAN
0 - 0.0003 mg/kg bw.
STUDIES WHICH WILL PROVIDE INFORMATION VALUABLE IN THE CONTINUED
EVALUATION OF THE COMPOUND
1. Observations in man.
2. Data on the effect of ethoprophos on neuropathy target
esterase (NTE) activity in the hen.
REFERENCES
Brown, D., 1986. Ethoprophos: 52 week oral (capsule administration)
toxicity study in the beagle. Unpublished report No. 4923-198/16 from
Hazleton Laboratories Europe, Ltd., North Yorkshire, England.
Submitted to WHO by Rhône-Poulenc Agrochimie, Lyon, France.
Roberts, N.L., Gopinth, C., Anderson, A., & Dawe, I.S., 1986. Acute
delayed neuro-toxicity study with ethoprophos in the domestic hen.
Unpublished report No. RNP 244/86189 from Huntingdon Research Centre
Ltd., Huntingdon, Cambridgeshire, England. Submitted to WHO by
Rhône-Poulenc Agrochimie, Lyon, France.
Spicer, E.J.F., 1985. Lifetime dietary toxicity and oncogenicity study
in rats. Unpublished report No. 347-029 from International Research
and Development Corporation, Mattawan, Michigan, USA. Submitted to WHO
by Rhône-Poulenc Agrochimie, Lyon, France.
Yamagata, S., Inoue, H. & Enomoto, M., 1984. Chronic feeding and
oncogenicity studies in mice with Ethoprop. Unpublished report No. 497
from Biosafety Research Center (AN-PYO Center), Shizuoka-ken, Japan.
Submitted to WHO by Rhône-Poulenc Agrochimie, Lyon, France.