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.
See Also: Toxicological Abbreviations Ethoprophos (ICSC) Ethoprophos (Pesticide residues in food: 1983 evaluations) Ethoprophos (Pesticide residues in food: 1984 evaluations) Ethoprophos (JMPR Evaluations 1999 Part II Toxicological)