Registration Dossier
Registration Dossier
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EC number: 240-815-0 | CAS number: 16752-77-5
Endpoint summary
Administrative data
Description of key information
| Study Type | Species | Findings | Guideline | Reliability |
| Oral 90-day Feed | Rat | LOAEL 540 ppm (body weight effects); 200/270 ppm (effects on hemoglobin) | no guideline |
2 |
| Oral 90-day Feed | Rat | LOAEL: 500/125 ppm (effects on body weight) | no guideline |
2 |
| Oral 90-day Feed | Mice | LOAEL: 150 ppm (effects on hemoglobin) | no guideline |
2 |
| Oral 2-year Feed | Dog | LOAEL: 400 ppm (pathology effects in kidney, spleen, liver, bone marrow) | no guideline |
2 |
| Oral 104-Week Feed | Mice | LOAEL 100/75 ppm effects on hematocrit, hemoglobin, and red blood cells. | no guideline |
2 |
| Oral 2-Year Feed | Rat | LOAEL: 400 ppm effects on hematocrit, hemoglobin, and red blood cells. | no guideline |
2 |
| Oral 22-Month Feed | Rat | LOAEL: 200 ppm body weight and food consumption effects, relative testes effects, pathology findings in spleen and kidney | no guideline |
2 |
| 21-Day Dermal | Rabbit | LOAEL: 50 mg/kg/d effects on plasma and brain cholinesterase activities | OECD 410, EPA OPP 82-2 | 1 |
| 21-Day Dermal | Rabbit | LOAEL: > 90 mg/kg/d (highest level tested) | EPA OPP 82-2 | 1 |
Key value for chemical safety assessment
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- Endpoint:
- chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The objective of the study was to evaluate the chronic toxicity and carcinogenic potential of the test substance in mice. The test substance was administered in the diet to mice at the levels of 0, 50, 100/75, and 800/200 ppm for 104 weeks. The animals were then observed for mortality, clinical signs, body weight, food consumption, hematology, gross pathology, organ weight and histopathology. Due to lower survival rates, the mid and high-dose group concentrations were lowered at Week 39.
- GLP compliance:
- no
- Specific details on test material used for the study:
- Test substance Name: Methomyl
H-11135
Purity: >99% - Species:
- mouse
- Strain:
- CD-1
- Sex:
- male/female
- Route of administration:
- oral: feed
- Vehicle:
- other: Feed
- Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 104 Weeks
- Frequency of treatment:
- Daily
- Dose / conc.:
- 50 ppm
- Dose / conc.:
- 100 ppm
- Remarks:
- Dose level was reduced from 100 to 75 ppm at Week 39
- Dose / conc.:
- 800 ppm
- Remarks:
- Because of unexpected high mortality, the high-dose level group was reduced to 400 ppm beginning Week 28; the dose level was further reduced to 200 ppm as of Week 39.
- No. of animals per sex per dose:
- 80
- Control animals:
- yes, plain diet
- Statistics:
- The growth rate, total food consumption, clinical pathology and organ weight data of the control groups were compared statistically to data of the treated groups of same sex using Bartlett’s test for homogeneity of variance and one-way ANOVA. If significant results were obtained from both and ANOVA, a multiple pairwise comparison procedure was used to compare the group mean values. If a significant result was not obtained from Bartlett’s test, but was obtained from ANOVA, Scheffe’s multiple pairwise comparison procedure was used to compare the group mean values.
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Clinical observations noted during this study included: hunched appearance; head tilt; circling behaviour; localized sores (legs, ears, base of tail, and back) sometimes accompanied by alopecia; swelling in the lower midline region; pale, red, small, or lacrimating eyes; and fur stains. The frequency of these findings increased in all groups as the study progressed; however, no treatment-related trends were noted.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Statistical analysis revealed that by Week 26, survival in the mid-dose females and both high-dose groups was significantly lower than that in control. Because of this unexpected high mortality beginning Week 28, the high-dose level group was reduced from 800 to 400 ppm. This was further reduced to 200 ppm as of Week 39. The mid-dose level was also reduced from 100 to 75 ppm at Week 39. Mortality noted in the high-dose females by Weeks 52, 80, and 104 continued to be significantly higher while the mid-dose females remained markedly higher at the same intervals. Mortality in the male groups was significantly higher in the high-dose males at Week 52, the mid- and high-dose males at Week 80, and all treated groups by Week 104. These differences in survival were considered related to the toxicity of the test substance.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Statistical analysis of weight gains through the first 26 weeks revealed that growth achieved by the treated groups of both sexes was equivalent to that of the control. Growth by the treated males was also equivalent to control through Week 46. Gains made by the high-dose females were significantly higher by Week 50. This finding was not considered biologically meaningful since the range of weights between control and high-dose females were one to two grams during the first 50 weeks. A comparison of the mean weights obtained at Weeks 60, 72, 84, 96, and 104 did not reveal any significant differences, except that the low-dose female weight was significantly increased at Week 72.
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Statistical analysis of the male data revealed significantly higher consumption levels in all treated groups during Weeks 20 through 26 and Weeks 20 through 80; significantly higher levels were noted from Week 20 through 52 in the low- and mid-dose groups. These changes were not considered biologically meaningful since concurrent body weight changes were not noted. Total food consumption by the treated males was statistically equivalent to control during Weeks 20 through 104. Statistical analysis of female data revealed a significantly higher intake in the low-dose group during Weeks 20 through 26. No other significant differences were noted.
The average daily theoretical compound consumption throughout the study was:
low dose: 8.720 mg/kg/day for males, 10.643 mg/kg/day for females
mid dose: 15.369 mg/kg/day for males, 19.082 mg/kg/day for females
high dose: 93.397 mg/kg/day for males, 118.542 mg/kg/day for females - Food efficiency:
- no effects observed
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Statistical analysis of the male data revealed the following differences: at Week 13, lower hematocrit in the mid- and high-dose group; at Week 26 lower hematocrit in the low-dose males, and lower hemoglobin and red blood cell count in the high-dose group. These changes are suggestive of compound-induced decrease in red cell mass. This trend was not apparent after Week 26; however, it should be noted that by Week 52, the high-dose level had been reduced from 800 to 200 ppm and the mid-dose level from 100 to 75 ppm.
In the females, decreases in hemoglobin levels occurred in the mid- and high-dose mice at Weeks 13 and 26 (a significantly lower level was noted in the mid-dose group at Week 13), and in the red cell count in mid- and high-dose animals at Week 26. These changes are suggestive of compound effect. Mean white blood cell count values were frequently influenced by moderate to marked elevation in a few animals in a group. No distinct treatment-related trends were detected in the white cell counts. - Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Organ weight data were unremarkable except for a significantly higher absolute and slightly elevated relative adrenal weight in the high-dose males. The biological significance of this finding is unknown.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Necropsy findings noted in the animals found dead/sacrificed moribund were generally considered incidental in nature.
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Test substance-related histomorphologic alterations were not observed in the tissues examined. Neoplastic and non-neoplastic spontaneous disease lesions were of the usual type and number observed in mice of this age and strain and were essentially comparable in incidence and severity between control and treated groups.
- Histopathological findings: neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Test substance-related histomorphologic alterations were not observed in the tissues examined. Neoplastic and non-neoplastic spontaneous disease lesions were of the usual type and number observed in mice of this age and strain and were essentially comparable in incidence and severity between control and treated groups.
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 75 ppm
- Basis for effect level:
- clinical biochemistry
- Remarks on result:
- other: The 75 ppm dose group started at 100 omm for the first 39 weeks.
- Conclusions:
- 104 Week study in mice: No histomorphologic alterations in tissues; no increase in neoplastic and non-neoplastic disease lesions
- Executive summary:
This study was designed to evaluate and characterize the chronic toxicity and carcinogenic potential of the test substance when administered in the diet to albino mice for 104 weeks. Three groups of animals (80/sex/group) received the test compound initially at levels of 50, 100, and 800 ppm. A fourth group of animals received the basal diet only. After 104 weeks of treatment, all surviving animals were sacrificed and necropsied. Criteria evaluated for compound-related effects were: mortality; body weights; food consumption; hematology profiles; tissue cultures; absolute and relative organ weights; and gross and microscopic pathology.
The survival in the high-dose males and females was significantly lower at Week 26. Survival in the mid-dose groups was also moderately lower. Due to these unexpected high mortality rates in the mid- and high-dose groups, the dose level of the high-dose group was decreased to 400 ppm at Week 28. For the same reason, the dose level was further reduced to 200 ppm beginning Week 39; at the same week, the mid-dose level was reduced from 100 to 75 ppm. Survival of the high-dose females was statistically significantly lower after 52, 80, and 104 weeks, while the mid-dose females remained markedly lower at the same intervals. Survival was statistically significantly lower after 52 weeks in the high-dose males, the mid- and high-dose males Week 80, and all treated males after 104 weeks. Lower survival rates were considered related to the toxicity of the test substance. Statistical analysis of the hemogram data revealed a significantly lower hematocrit in the mid- and high-dose males at Week 13. Significantly lower hemoglobin level, and red cell count were noted in the high-dose males at Week 26. Decreases in hemoglobin levels were noted in the mid- and high-dose females at Weeks 13 and 26 (a significantly lower level in the mid-dose females at Week 13). Decreases in red cell counts were also noted at Week 26 in the mid- and high-dose females. These changes are suggestive of a compound-related effect upon red cell mass. This trend was not noted after Week 26. No other treatment-related findings were seen with respect to body weight gains, food consumption and
utilization, or clinical observations. Test substance-related histomorphologic alterations were not observed in the tissues examined. Neoplastic and non-neoplastic spontaneous disease lesions were of the usual type and number observed in mice of this age and strain and were essentially comparable in incidence and severity between control and treated groups.
- Endpoint:
- chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The study was conducted to evaluate the chronic toxicity of test substance when administered orally in diet to rats for 24 months. The dietary levels were 50, 100, 200, and 400 ppm.
- GLP compliance:
- no
- Specific details on test material used for the study:
- Test substance Name: Lannate methomyl insecticide; S-Methyl N-[(methylcarbamoyl)oxy]thioacetimidate
Purity: S-Methyl N-[(methylcarbamoyl)oxy]thioacetimidate: 90% and Lannate methomyl insecticide: 100% active ingredient - Species:
- rat
- Strain:
- other: Albino (Caesarean-derived)
- Sex:
- male/female
- Route of administration:
- oral: feed
- Vehicle:
- other: Feed
- Duration of treatment / exposure:
- 22 Months
- Frequency of treatment:
- Daily
- Dose / conc.:
- 50 ppm
- Dose / conc.:
- 100 ppm
- Dose / conc.:
- 200 ppm
- Dose / conc.:
- 400 ppm
- No. of animals per sex per dose:
- 35
- Control animals:
- yes, plain diet
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test substance related effects were observed with regard to physical appearance and behavior was noted in rats received test substance at any dietary level tested.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Group observation of the deaths in each group indicated that the poor survival was due to spontaneous respiratory disease.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Growth for the high dose level (400 ppm) males (Weeks 0-52) was significantly lower compared with both male control groups. Growth during the first year for the 200 ppm males and the 400 ppm females was lower than, but not significantly different from, that for the corresponding control groups. Growth for the remaining male and female test groups was generally comparable with corresponding controls.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Food consumption for the 400 and 200 ppm males (Weeks 1-26) were significantly lower compared with both male control groups. Food consumption for all female test groups was comparable with that for female controls.
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- At 18 and 22 months, a dose-related trend toward decreased hemoglobin values was evident in the female test groups. Abnormal individual hematological values determined at 18 and 22 months were associated with incidental disease and were not considered meaningful.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The clinical biochemistry findings were comparable among control and test substance treated rats.
- Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The urine analysis results were comparable among control and test substance treated rats.
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- At terminal sacrifice, the testis/body weight ratio for 400 ppm group males was significantly higher than that of controls. This was probably due to weight suppression in the high level dose males.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No consistent gross pathological changes were observed in the organs and viscera of rats due to test susbtance at indicated levels.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Histopathologic evaluation of tissue sections from rats fed 400 ppm of test substance for one year did not reveal any test substance effect. After 22 months of dietary administration, microscopic examination revealed test substance related histopathologic alterations (cystic follicles, fibrosis, salpingitis and atrophy of ovary) in the kidneys of males and females at 400 ppm level and in the spleens of the females at 200 and 400 ppm levels of the test substance.
- Histopathological findings: neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The incidence of neoplastic lesions in the high-level test rats was generally comparable with that in the control animals.
- Key result
- Dose descriptor:
- LOEL
- Effect level:
- 200 ppm
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- histopathology: non-neoplastic
- organ weights and organ / body weight ratios
- Conclusions:
- - Histopathological evaluation did not reveal any test substance related effect when fed up to 400 ppm of test substance in diet at the one-year evaluation.
- After 22 months of dietary administration, microscopic examination revealed test substance related histopathologic alterations in the kidneys of males and females at 400 ppm level and in the spleens of the females at 200 and 400 ppm levels of the test substance. - Executive summary:
The study was conducted to evaluate the chronic toxicity of test substance when administered orally in diet to rats for 24 months. The dietary levels were 50, 100, 200, and 400 ppm.
No test substance related effects were observed with regard to physical appearance and behavior was noted in rats received test substance at any dietary level tested. Growth for the high level (400 ppm) males (Weeks 0-52) was significantly lower compared with both male control groups. Growth during the first year for the 200 ppm males and the 400 ppm females was lower than, but not significantly different from, that for the corresponding control groups. Growth for the remaining male and female test groups was generally comparable with corresponding controls. Food consumption for the 400 and 200 ppm males (Weeks 1-26) were significantly lower compared with both male control groups. Food consumption for all female test groups was comparable with that for female controls.
Survival in the test rats was not adversely affected. Survival at 22 months for the 100 ppm males and the 50 and 400 ppm females was significantly higher compared with the controls.
At 18 and 22 months, a dose-related trend toward decreased hemoglobin values was evident in the female test groups. Abnormal individual hematological values determined at 18 and 22 months were associated with incidental disease and were not considered meaningful. The biochemical values and the results of urine analysis were comparable among control and treated animals.
At 12 months, all organ weights and ratios, brain weights and organ/brain weight ratio values for the high-level test groups were comparable with control values. At terminal sacrifice, the testis/body weight ratio for 400 ppm group males was significantly higher than that of controls. This was probably due to weight suppression in the high-level dose males. Histopathologic evaluation of tissue sections from rats fed 400 ppm of test substance for one year did not reveal any test substance effect. After 22 months of dietary administration, microscopic examination revealed test substance related histopathologic alterations in the kidneys of males and females at 400 ppm level and in the spleens of the females at 200 and 400 ppm levels of the test substance
- Endpoint:
- chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The study was conducted to evaluate the chronic toxicity of test substance when administered orally in diet for 2 years. Test substance was fed to male and female rats, for two years at levels of 0, 50, 100, or 400 ppm in diet. During the period of administration the animals were observed for signs of toxicity. Animals that die or are euthanised during the test are necropsied and at the conclusion of the test surviving animals are euthanised and necropsied.
- GLP compliance:
- no
- Specific details on test material used for the study:
- Test substance Name: Technical methomyl (INX-1179-255)
Purity: >99 - <100% - Species:
- rat
- Strain:
- other: ChR-CD
- Sex:
- male/female
- Route of administration:
- oral: feed
- Vehicle:
- other: Feed
- Analytical verification of doses or concentrations:
- yes
- Duration of treatment / exposure:
- 2 years
- Frequency of treatment:
- Daily
- Dose / conc.:
- 50 ppm
- Dose / conc.:
- 100 ppm
- Dose / conc.:
- 400 ppm
- No. of animals per sex per dose:
- 80; 20 animals were added to all groups specifically for erythrocyte and brain cholinesterase determinations.
- Control animals:
- yes, plain diet
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no clinical signs of toxicity that could be attributed to the feeding of test substance for control and treated rats.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- No dose-mortality realtionship was demonstrated among various feeding levels of test substance.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Although slight decreases in body weight were observed periodically throughout the study, after two years of continuous feeding, the average final body weights and average body weight gains of male and female rats fed various dietary levels of test substance were not significantly different from those of the corresponding controls.
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no menaingful differences among the control and treated groups with respect to food consumption data.
- Food efficiency:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no menaingful differences among the control and treated groups with respect to food efficiency data.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Female rats fed 400 ppm test substance tended to have statistically significant lower erythrocyte counts, hemoglobin values and hematocrits than those of the corresponding controls. A similar trend occurred In the erythrocyte counts and hemoglobin values of male rats fed 400 ppm test substance but these changes were not statistically significant.
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- All clinical chemistry measurements were essentially within the normal range, as established by the controls.
- Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- The results of all urine analysis were essentially within the normal range, as established by the controls.
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no significant differences in absolute organ weights for either male or female rats and in relative organ weights for male rats fed various dietary levels of test substance, compared to those of controls. Female rats fed 100 or 400 ppm of test substance had significantly increased relative liver weights compared to those of controls. However, these differences did not appear to be related to any histopathological changes. Female rats fed 400 ppm test substance had increased relative spleen weights which may be correlated with rear footpad dermatitis, a common naturally occurring lesion in wire cage-reared aging rats.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test substance related findings were observed in either male or female rats that could be attributed to the dietary administration of the test substance.
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test substance related lesions were observed in either male or female rats that could be attributed to the dietary administration of the test substance.
- Histopathological findings: neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test substance related neoplastic lesions were observed in either male or female rats that could be attributed to the dietary administration of the test substance.
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 100 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- haematology
- Key result
- Dose descriptor:
- LOEL
- Effect level:
- 400 ppm
- Basis for effect level:
- haematology
- Conclusions:
- 2 year Rat NOEL (Male/Female): 100 ppm
- Executive summary:
The study was conducted to evaluate the chronic toxicity of test substance when administered orally in diet for 2 years. Test substance was fed to male and female rats for two years at levels of 0, 50, 100, or 400 ppm in diet.
Although slight decreases in body weight were observed periodically throughout the study, after two years of continuous feeding, the average final body weights and average body weight gains of male and female rats fed various dietary levels of test substance were not significantly different from those of the corresponding controls.
There were no clinical signs of toxicity that could be attributed to the feeding of test substance for control and treated rats and no meaningful differences among mortality rates for control and treated rats.
Female rats fed 400 ppm test substance tended to have statistically significant lower erythrocyte counts, hemoglobin values and hematocrits than those of the corresponding controls. All other hematological measurements and all clinical measurements and urine analysis were essentially within the normal range, as established by the controls.
There were no significant differences in absolute organ weights for either male or female rats and in relative organ weights for male rats fed various dietary levels of test substance, compared to those of controls. Female rats fed 100 or 400 ppm of test substance had significantly increased relative liver weights compared to those of controls. However, these differences did not appear to be related to any histopathological changes. Female rats fed 400 ppm test substance had increased relative spleen weights which may be correlated with rear footpad dermatitis, a common naturally occurring lesion in wire cage-reared aging rats.
No test substance related non-neoplastic or neoplastic lesions were observed in either male or female rats that could be attributed to the dietary administration of the test substance.
The No Observed Effect Level (NOEL) following dietary administration of test substance to male and female rats for two years was 100 ppm.
.
- Endpoint:
- chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The test substance was administered in the diet to four groups of four male and four female dogs each at levels of 0, 50, 100, 400, and 1000 ppm. 10 dogs, one of each sex and group, were sacrificed after one year on study. The remaining dogs were terminated after two years of test substance feeding.
- GLP compliance:
- no
- Limit test:
- no
- Specific details on test material used for the study:
- - Substance name: S-Methyl N-[(methylcarbamoyl)oxy]thioacetimidate
- Purity: 100% (1st sample) and 90% (2nd sample) - Species:
- dog
- Strain:
- Beagle
- Sex:
- male/female
- Route of administration:
- oral: feed
- Vehicle:
- other: diet
- Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- One year (10 dogs)
Two years (30 dogs) - Frequency of treatment:
- Daily
- Dose / conc.:
- 50 ppm
- Dose / conc.:
- 100 ppm
- Dose / conc.:
- 400 ppm
- Dose / conc.:
- 1 000 ppm
- No. of animals per sex per dose:
- 4
- Control animals:
- yes, plain diet
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Two male dogs at the 1000 ppm level, exhibited signs of test substance effect once during the 13th week of the study. These signs were noted in the afternoon and consisted of tremors, salivation, incoordination, and circling movements. The dogs were normal on the following morning. No further compound-related signs were evident in these animals. One male dog was placed on control diet from the 85th week through the 94th week because of anemia. From the 73rd week through the 81st week, eight scattered instances of diarrhea or emesis were observed in this dog; no other grossly observable signs were noted. One high dose level female dog maintained normal appearance and behavior during eight weeks of test substance administration. On the first morning of the 9th week the animal was found dead in its cage. Death had occurred during the night and was unobserved. Two weeks later, this dog with replaced with another female dog and was started on study at a level of 1000 ppm. After 17 days of the test substance administration, this replaced dog showed repeated convulsive seizures; it was treated intravenously with atropine. The animal went into coma and died in the afternoon of the next day. This dog was replaced with another female dog and was placed on study at a level of 1000 ppm. This dog did not show any grossly observable signs of test substance effect. No test substance related signs were seen in the four remaining high level dogs.
The test dogs at 50, 100, 400 ppm levels essentially were comparable to the control animals. - Mortality:
- mortality observed, treatment-related
- Description (incidence):
- The high dose level female dog maintained normal appearance and behavior during eight weeks of test substance administration. On the first morning of the 9th week the animal was found dead in its cage. Death had occurred during the night and was unobserved. Two weeks later, this dog with replaced with another female dog and was started on study at a level of 1000 ppm. After 17 days of the test substance administration, this replaced dog showed repeated convulsive seizures; it was treated intravenously with atropine. The animal went into coma and died in the afternoon of the next day. These two deaths were test substance related.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Three of the dogs which were sacrificed at one year, one at the 50 ppm level and two at the 100 ppm level, lost weight, the losses ranging from 0.6 to 12.0 kg. One male dog at the 100 ppm level gained 1.6 kg during the first 36 weeks of the study, and then gradually lost 4.4 kg during the remainder of the two-year study period.
Five dogs at 1000 ppm gained weight, the gains ranging from 0.7 to 1.6 kg. One male animal (sacrificed at 26 weeks) maintained weight (+0.2 kg). One female dog essentially maintained weight during 103 weeks on study but lost 0.9 kg during the last week. - Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- 1000 ppm: One male animal showed progressive anemia during the course of the study. At 18 months, the hemogram had markedly declined (HCT 16.0%; HGB 4.9 g%; RBC 2.04 million/cmm); the differential count revealed moderate hypochromia, severe polychromia, and 16 nucleated red blood cells per 100 WBC; the reticulocyte count was markedly elevated (30.5%), and the platelet count was low (172000/cmm). At the start of the 20th month the test substance was withdrawn and the dog was maintained on control diet for 10 consecutive weeks (85th through 94th weeks). Weekly hematological studies performed during this period showed gradual improvement. At the start of the 95th week the dog was placed back on test substance and continued to receive the compound during the last 10 weeks on study (95th through 104th weeks). This resulted in a renewed decline of the blood picture. Terminal studies prior to sacrifice showed severe anemia and moderate leukopenia. The remaining three male dogs in this group and two female dogs showed slight or moderate declines in their hemograms at three months. During the remainder of the study the blood picture for these dogs improved, and terminal values were comparable to corresponding initial values for most of the animals.
Control, 50, 100, 400 ppm: Clinical values for the control and the test substance-treated dogs at the three lower levels remained within the limits of normal variation. No test substance-related trends or alterations were evident. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- 1000 ppm: Biochemical values for the high dose level dogs generally remained within the limits of normal variation. At three months, one female dog showed slightly elevated values for serum glutamic-pyruvic transaminase and serum glutamic-oxaloacetic transaminase. At six and 12 months transaminase values similar to initial levels were obtained for this animal. Total protein for one male dog was slightly elevated at 18 months and to a Lesser degree at 24 months. At 13 weeks, plasma cholinesterase values for another male dog and female dog were slightly decreased in comparison with corresponding initial values.
Control, 50, 100, 400 ppm: Clinical values for the control and the test substance-treated dogs at the three lower levels remained within the limits of normal variation. No test substance-related trends or alterations were evident. - Urinalysis findings:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- No test substance related changes were noted at the one-year interval. Inspection of the data for the two-year sacrifice revealed an increased weight and ratio for the liver and kidneys of high dose male. Kidney/body weight ratio for one male dog at the 400 ppm level and for the remaining two high level males were also moderately increased in comparison with control measurements.
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- The female dog from high dose level that died after eight weeks of test substance administration showed following gross necropsy findings:
Moderate postmortem autolysis.
Lung: Trachea and bronchi contained moderate amount of froth, parenchyma dark purple in colour, with fluid exudate from cut surface
Liver: Enlarged; parenchyma showed scattered areas of white discolouration
Pancreas: Scattered diffuse subcapsular red areas suggesting hemorrhage
Kidneys: Cut surface blackish purple in colour
Another female dog, a replacement for the preceding dog, died after 18 days on study, showed the following gross necropsy findings:
GI tract: Mucosa of entire tract showed reddening and scattered petechiae
Heart: Scattered subendocardial hemorrhages in left ventricle
Lung: collapsed; dark red in colour
Kidneys: Cut surface dark red in colour
Spleen: Darker than usual
Ten dogs, one of each sex and group, were sacrificed after 12 months of test substance feeding. A possible test substance-related necropsy finding was a liver of an unusually pale yellow-brown colour in the male high Level dog. No other compound-related tissue alterations were seen.
The surviving dogs were sacrificed after 24 months of the test substance administration. Gross necropsy revealed an enormously enlarged spleen (weight: 179 g) with whitish deposits on the capsular surface and an enlarged liver in male at high dose level. This was compatible with a marked test substance-dependent anemia which was present in this animal at time of sacrifice. This dog, as well as one dog each at the 100 and 400 ppm levels had rather large prostate glands. One female at high dose level showed scattered ecchymoses in the mucosa of the jejunum. - Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test substance-related histopathological alterations were seen in the kidneys and spleens of the animals receiving 1000 and 400 ppm. Histopathological alterations were also seen in the livers and bone marrows of the animals receiving 1000 ppm.
In the kidneys of the males receiving 1000 and 400 ppm and the females receiving 1000 ppm, there an increase in the amount of pigment in the cytoplasm of the epithelial cells of the proximal convoluted tubules. The epithelial cells of the proximal convoluted tubules were also swollen slightly in both the males and the females receiving 1000 ppm.
Extramedullary hematopoiesis was present in the spleens of four of the five animals receiving 1000 ppm. A moderate increase in the amount of pigment in the spleen was present in the animals receiving 1000 and 400 ppm.
A minimal to slight bile duct proliferation wag present, in the livers of the animals receiving 1000 ppm. In the bone marrow, there appeared to be a slight increase in activity of both the erythroid and myeloid series in the animals receiving 1000 ppm. - Key result
- Dose descriptor:
- LOEL
- Effect level:
- 400 ppm
- Basis for effect level:
- clinical signs
- histopathology: non-neoplastic
- mortality
- organ weights and organ / body weight ratios
- Conclusions:
- 2-year dietary study in dogs: No signs of compound effect up to 400 ppm levels; two compound related deaths (females) at 1000 ppm; test substance related effects, anemia, enlarged spleen and liver at 1000 ppm, histopathological alterations in kidney and spleens at 400 and 1000 ppm, in livers and bone marrows at 1000 ppm, increase in bone marrow activity at 1000 ppm.
- Executive summary:
The test substance was administered in the diet to four groups of four male and four female dogs each at levels of 0, 50, 100, 400, and 1000 ppm. 10 dogs, one of each sex and group, were sacrificed after one year on study. The remaining dogs were terminated after two years of compound feeding.
Daily observations did not reveal any signs of test substance effect at the 50, 100, and 400 ppm levels. One female dog at 1000 ppm level died unexpectedly after 8 weeks on study without having shown any test substance-related signs. Another female dog, a replacement of the dog that died after 8 weeks, died after 18 days on study; death was preceded by convulsive seizures and coma. These two deaths were test substance related.
Two male dogs at the 1000 ppm level each showed signs of test substance effect once during the 13th week which included tremors, salivation, incoordination, and circling movements. The incidence of diarrhea was higher in male at high dose level when compared with the other dogs on study. The remaining four high level dogs appeared normal.
Hematological studies revealed slight to moderate anemia in five high level dogs at the three-month interval and persistent test substance dependent anemia in a high level male dog; temporary test substance withdrawal during 10 consecutive weeks resulted in improvement of the condition.
Gross necropsy findings in one dog included an enormously enlarged spleen and an enlarged liver. At two years, several of the males at the 1000 and 400 ppm levels had rather large prostate glands. Kidney/ body weight ratios for the three male dogs at the 1000 ppm level and one male dog at the 400 ppm level were slightly or moderately increased in comparison with control measurements.
Compound-related histopathological alterations were seen in the kidneys and spleens of the animals receiving 1000 and 400 ppm. Test substance-related histopathological alterations were also seen in the livers and bone marrows of the animals receiving 1000 ppm. The changes seen in the kidneys were characterized by an increase in pigmentation and slight swelling of the epithelial cells of the proximal convoluted tubules. Pigment deposition was seen in the males at both 1000 and 400 ppm but only at 1000 ppm in the females. Epithelial swelling was present only in the males and females receiving 1000 ppm. Four of the five animals receiving 1000 ppm had an increase in extramedullary hematopoiesis in the spleen. Pigment disposition was also slightly increased in the animals receiving both 1000 and 400 ppm. A minimal to slight increase in bile duct proliferation was seen in the livers of the animals receiving 1000 ppm. Bone marrow activity was increased slightly in the animals receiving 1000 ppm.
No characteristic signs of test substance effect were noted with respect to appetite, elimination, body weight changes, biochemical studies, and urine analysis.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The study was conducted to evaluate the sub-chronic toxicity of test substance in mice. Test substance was admixed in the diets of groups of male and female mice at levels of 75 to 960 ppm for 90 days. A similar group of mice received the basal diet only for 90 days. All animals were observed for clinical signs of toxicity and mortality twice daily. Clinical pathology was conducted in all animals. Hematologic evaluation, serum chemistry determination and urine analysis were perfomed. After 90 days of treatment, necropsy of animals was done and gross pathology and histopathological evaluations were done to evaluate the effect of test substance to organs and tissues of mice.
- GLP compliance:
- no
- Specific details on test material used for the study:
- Test substance name: Nudrin; (S-Nethyl-N-((methylcarbamoyl)oxy)thioacetimedate)
Purity: Not reported - Species:
- mouse
- Strain:
- B6C3F1
- Sex:
- male/female
- Route of administration:
- other: Diet
- Vehicle:
- other: Diet
- Duration of treatment / exposure:
- 90 Days
- Frequency of treatment:
- Daily
- Dose / conc.:
- 75 ppm
- Dose / conc.:
- 150 ppm
- Dose / conc.:
- 300 ppm
- Dose / conc.:
- 480 ppm
- Dose / conc.:
- 600 ppm
- Dose / conc.:
- 960 ppm
- No. of animals per sex per dose:
- 10 except 600 ppm where males were 11 and females were 9; Control animals: 20/sex
- Control animals:
- yes, plain diet
- Statistics:
- Differences between control and treated group values for weekly body weights and change in body weights, estimated food consumption, hematologic, serum chemistry, and organ weight data were analyzed for statistical significance by the method of Dunnett. This is a multiple comparison procedure for comparing several treatments simultaneously with a control.
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No remarkable clinical effects were noted during the twice daily observations of the mice.
- Mortality:
- mortality observed, non-treatment-related
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No consistent or apparent test substance related effects were noted during twice daily observations of the mice for 90 days in the weekly body weights.
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- No consistent or apparent test substance related effects were noted during twice daily observations of the mice for 90 days in estimated food consumption data.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Hematologic differences indicative of a systemic effect include decreased levels of hemoglobin in male mice receiving test substance at levels of 300 ppm or greater with apparent compensation in mice at levels of 600 and 960 ppm as indicated by increases in erythrocyte counts and hematocrit accompanied by decreased reticulocyte counts. Female mice at levels of 150 ppm or greater of test substance had decreased erythrocyte counts with increases in reticulocyte counts at and above levels of 400 ppm indicative of beginning compensation.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The only significant differences noted for serum chemistry variables determined at termination were elevated mean values of glucose for 75 and 480 ppm group males. These differences were considered to be spontaneous or incidental and not related to test substance administration since there are no dose-response relationship and differences were not observed in the female mice.
- Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No meaningful or consistent differences between treated and control groups were observed in 18 hour volume, pH, specific gravity, qualitative analysis or microscopic examination.
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- The absolute and relative weights of the liver of male mice at 480, 600 and 960 ppm were significantly greater than the corresponding control. In addition, the relative weights of the livers of 600 ppm group female mice were significantly greater than controls.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Necropsy of 5 male and 5 female mice randomly selected at pretest did not reveal any gross evidence of parasites, enteric pathogens or other disease conditions. There were no consistent or apparent changes indicative of a treatment related effect following test substance administration.
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The microscopic findings were considered spontaneous or incidental and not related to test substance administration.
- Key result
- Dose descriptor:
- other: Maximum Tolerable Dose (MTD)
- Effect level:
- > 960 mg/kg diet
- Sex:
- male/female
- Key result
- Dose descriptor:
- LOEL
- Effect level:
- 150 ppm
- Sex:
- female
- Basis for effect level:
- haematology
- Key result
- Dose descriptor:
- LOEL
- Effect level:
- 300 ppm
- Sex:
- male
- Basis for effect level:
- haematology
- Conclusions:
- 90 Day-Mice Maximum tolerable dose (Male/Female): >960 ppm in diet
- Executive summary:
The study was conducted to evaluate the sub-chronic toxicity of test substance in mice. Test substance was admixed in the diets of groups of male and female mice at levels of 75 to 960 ppm. A similar group of mice received the basal diet only for 90 days.
No consistent or apparent test substance related effects were noted during twice daily observations of the mice for 90 days neither in the weekly body weights nor in estimated food consumption data.
Hematologic differences indicative of a systemic effect includes decreased levels of hemoglobin in male mice receiving test substance at levels of 300 ppm or greater with apparent compensation in mice at levels of 600 and 960 ppm as indicated by increases in erythrocyte counts and hematocrit accompanied by decreased reticulocyte counts. Female mice at levels of 150 ppm or greater of test substance had decreased erythrocyte counts with increases in reticulocyte counts at and above levels of 400 ppm indicative of beginning compensation.
Male mice receiving 480 ppm or greater of test substance in the diet had increased absolute and relative weights of the livers. Slight increases (not statistically significant) in liver weights were also noted for female mice given test substance at levels of 480 ppm or greater. Other organ weight differences were due to minor differences in final body weights and not related to test substance administration.
These results conclude that the maximum tolerable dose of test substance in both male and female mice when administered for 90 days in diet was greater than 960 ppm.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The study was conducted to evaluate the 90-Day oral toxicity of test substance when administered through diet at nominal concentration of 10, 50, 125, 250 ppm to both male and female rats. The dietary level of 125 ppm was increased from 125 ppm to 500 ppm at the beginning of the 7th week and remained at this level for the remainder of the study. Control rats were fed plain diet. Weekly records were kept of body weight, food consumption, general appearance and behavior of each rat. Observations for signs of toxicity and pharmacological effects and for mortality were made daily. At initiation of the study and at 1, 2, and 3 months, hematological studies and urine analyses were performed on 5 male and 5 female rats from the control and each test group. On completion of 9th week 5 male and 5 female rats in 500 ppm and 5 stock rats of each sex of comparable age and weight which served as controls were sacrificed by exsanguination and gross necropsies, were performed. At termination of the study all remaining control and test rats were also sacrificed by this method. At both time intervals, the same necropsy procedures were applied.
- GLP compliance:
- no
- Specific details on test material used for the study:
- Test substance Name: Insecticide 1179
Purity: 100% - Species:
- rat
- Strain:
- other: Charles River Caesarean-derived
- Sex:
- male/female
- Route of administration:
- oral: feed
- Vehicle:
- other: Feed
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 3 Months
- Frequency of treatment:
- Daily
- Dose / conc.:
- 10 ppm
- Dose / conc.:
- 50 ppm
- Dose / conc.:
- 125 ppm
- Remarks:
- The dietarey level was increased from 125 ppm to 500 ppm at the beginning of seventh week and remained at this level for the remainder of the study.
- Dose / conc.:
- 250 ppm
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, plain diet
- Statistics:
- All criteria were examined by the analysis of variance, or F-test, at the 5% probability level. Growth rates were prepared for analysis using Rao's method. Before completing each F-test, the variances were tested for heterogeneity by the method of Bartlett. If the variances were homogeneous, the F-test could be completed in the normal fashion, and if a significant F-value was obtained, those groups significantly different from control could be determined by the method of Scheffe.
In those instances of heterogeneous variances, the samples were examined for extreme values by Sachs test for rejection of measurements. If no legitimate, unbiased adjustment to the variance could be made by rejection of outliers, comparison of test to control vas effected by the Fisher-Behrens modified t-test. - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The appearance and behavior of the test rats were generally comparable with those seen in the control animals. Intermittent tremors were seen in 1female rat in 250 ppm group at 13 weeks while the animal was handled and weighed. When handled again on the following day, the rat appeared excited but did not exhibit tremors.
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Body weight was suppressed in the 250 ppm males and, during the 7-13th weeks, in the 500 ppm males and females, but was significantly different from control values in only the 500 ppm males .
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Food consumption for the 500 ppm males was lower but not significantly different from that of the controls. Food consumption for the 250 ppm males and the 500 ppm females was slightly lower compared with the controls.
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- At three months hemoglobin values for the males in 50, 250 and 500 ppm groups were slightly below the normal range for male rats, but not significantly different from control values. At the three month interval the red blood counts for the 250 ppm females were significantly lower than those for controls but still within normal acceptable limits. Hematocrits for both sexes were within normal limits at all diet levels.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- At three months, the cholinesterase values for both sexes in the groups receiving 500 and 250 ppm were within normal limits and comparable with the corresponding control values.
- Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The results of the urinalysis were within normal limits and comparable among the control and test groups.
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- The significant reduction of liver weights and increased brain/body weight ratios was observed in males at 500 ppm and 250 ppm rats sacrificed after 3 months. These differences from control values were related to lower body weights of the animals in these groups in comparison with the controls.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Necropsy did not reveal consistent gross changes in the organs from test rats that could be attributed to ingestion of the test substance.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Moderate erythroid hyperplasia was found consistently in all male rats only in 250 ppm group. All other tissues examined were within normal variation.
- Key result
- Remarks on result:
- other: Growth was suppressed in 500 ppm males with no mortality and hematological changes. Decreased liver weight and increased brain/body weight ratio in 500 ppm and 250 ppm males was observed and erythroid hyperplasia in 250 ppm male rats.
- Key result
- Dose descriptor:
- LOEL
- Effect level:
- 500 other: 500/125 ppm group
- Basis for effect level:
- body weight and weight gain
- Conclusions:
- 90-Day Oral feed study in rats concluded that there was suppressed growth in 500 ppm males with no mortality and hematological changes. Decreased liver weight and increased brain/body weight ratio in 125-500 ppm and 250 ppm males was observed and histopathology revealed erythroid hyperplasia in 250 ppm male rats.
- Executive summary:
The study was conducted to evaluate the 90-Day oral toxicity of test substance when administered through diet at nominal concentration of 10, 50, 125, 250 ppm to both male and female rats. The dietary level of 125 ppm was increased from 125 ppm to 500 ppm at the beginning of the 7th week and remained at this level for the remainder of the study. Control rats were fed plain diet. Weekly records were kept of body weight, food consumption, general appearance and behavior of each rat. Observations for signs of toxicity and pharmacological effects and for mortality were made daily. At initiation of the study and at 1, 2, and 3 months, hematological studies and urine analyses were performed on 5 male and 5 female rats from the control and each test group. On completion of 9th week 5 male and 5 female rats in 500 ppm and 5 stock rats of each sex of comparable age and weight which served as controls were sacrificed by exsanguination and gross necropsies, were performed. At termination of the study, all remaining control and test rats were also sacrificed by this method. At both time intervals, the same necropsy procedures were applied.
No mortality of rats was observed in any dose. The appearance and behavior of the test rats were generally comparable with those seen in the control animals. Intermittent tremors were seen in one female rat in 250 ppm group at 13 weeks while the animal was handled and weighed. When handled again on the following day, the rat appeared excited but did not exhibit tremors.
Body weight was suppressed in the 250 ppm males and, during the seventh through the 13th weeks, in the 500 ppm males and females, but was significantly different from control values in only the 500 ppm males. Food consumption for the 500 ppm males was lower but not significantly different from that of the controls. Food consumption for the 250 ppm males and the 500 ppm females was slightly lower compared with the controls. The significant reduction of liver weights and increased brain/body weight ratios was observed in males at 125-500 ppm and 250 ppm rats sacrificed after three months. These differences from control values were related to lower body weights of the animals in these groups in comparison with the controls.
At three months, the cholinesterase values for both sexes in the groups receiving 125-500 and 250 ppm were within normal limits and comparable with the corresponding control values. The results of the urinalysis were within normal limits and comparable among the control and test groups. Necropsy did not reveal consistent gross changes in the organs from test rats that could be attributed to ingestion of the test substance.
Histopathological findings showed moderate erythroid hyperplasia was found consistently in all male rats only in 250 ppm group. All other tissues examined were within normal variation.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Groups of male and female, Fischer 344 rats were administered test substance admixed in the diet at levels of 100-600 ppm (males) and 135-810 ppm (females) for 13 weeks; 50 ppm (males) and 68 ppm (females for 4 weeks followed by 800 ppm (males) and 1080 ppm (females) for 9 weeks; or 20 ppm (males) and 27 ppm (females) for 9 weeks followed by 1000 ppm (males) and 1500 ppm (females) for 4 weeks. The animals were observed for clinical observations, clinical pathology, gross pathology and histopathology.
- GLP compliance:
- no
- Limit test:
- no
- Specific details on test material used for the study:
- - Substance name: S-Methyl N-[(methylcarbamoyl)oxy]thioacetimidate
- Substance ID: SD 14999-Tech
- Purity: Not reported - Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, Michigan
- Age at study initiation: males: 6 weeks, females: 7 weeks
- Housing: housed individually
- Diet: ad libitum except for 18 hour fast periods required prior to clinical pathology procedures and necropsy
- Water: ad libitum
- Acclimation period: 14 days
ENVIRONMENTAL CONDITIONS
- All animals were housed in an isolated room with filtered 100% fresh air supply and controlled temperature and humidity
- Photoperiod: 12 hrs dark / 12 hrs light - Route of administration:
- oral: feed
- Vehicle:
- other: acetone, corn oil, diet
- Details on oral exposure:
- - DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Purina Rodent Chow® #1500
- Storage temperature of food: Room temperature until use, but no longer than one week
- Preparation method: The appropriate amount of the test substance was dissolved in a small quantity of acetone and combined with an amount of corn oil such that when the mixture was added to the rodent chow, a 2% w/w level of corn oil in the ration resulted. Feed was weighed into the stainless steel bowl of a Hobart mixer and the test substance/acetone/corn oil mixture was slowly added while the mixer was running. A small acetone rinse was used to achieve a quantitative transfer. The diets were mixed for one hour to assure homogeneity of the mix and to allow the acetone to evaporate. The control diet was prepared in a similar manner but without addition of the test substance. - Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 13 Weeks
- Frequency of treatment:
- daily
- Dose / conc.:
- 20 ppm
- Remarks:
- Weeks 1-9 (males); Group DE2
- Dose / conc.:
- 27 ppm
- Remarks:
- Weeks 1-9 (females); Group DE2
- Dose / conc.:
- 1 000 ppm
- Remarks:
- Weeks 10-13 (males); Group DE2
- Dose / conc.:
- 1 500 ppm
- Remarks:
- Weeks 10-13 (females); Group DE2
- Dose / conc.:
- 50 ppm
- Remarks:
- Weeks 1-4 (males); Group DE3
- Dose / conc.:
- 68 ppm
- Remarks:
- Weeks 1-4 (females); Group DE3
- Dose / conc.:
- 800 ppm
- Remarks:
- Weeks 5-13 (males); Group DE3
- Dose / conc.:
- 1 080 ppm
- Remarks:
- Weeks 5-13 (females); Group DE3
- Dose / conc.:
- 100 ppm
- Remarks:
- males; Group DE4
- Dose / conc.:
- 200 ppm
- Remarks:
- males; Group DE5
- Dose / conc.:
- 400 ppm
- Remarks:
- males; Group DE6
- Dose / conc.:
- 600 ppm
- Remarks:
- males; Group DE7
- Dose / conc.:
- 135 ppm
- Remarks:
- females; Group DE4
- Dose / conc.:
- 270 ppm
- Remarks:
- females; Group DE5
- Dose / conc.:
- 540 ppm
- Remarks:
- females; Group DE6
- Dose / conc.:
- 810 ppm
- Remarks:
- females; Group DE7
- No. of animals per sex per dose:
- 20
- Control animals:
- yes, plain diet
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS:
- Time schedule: Twice daily
DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Twice daily
BODY WEIGHT:
- Time schedule for examinations: Once weekly
HAEMATOLOGY:
- Time schedule for collection of blood: At pretest and termination (from tip of the tail) and at Day 42 (from orbital sinus)
- Animals fasted: Yes
- How many animals: 10 animals/sex/dose
CLINICAL CHEMISTRY:
- Time schedule for collection of blood: At pretest and termination (from inferior vena cava) and at Day 42 (from orbital sinus)
- Animals fasted: Yes
- How many animals: 10 animals/sex/dose
URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine: An 18 hour urine sample was obtained from 10 animals/sex/group at 45 days and at end of the study
- Animals fasted: Yes
OTHER: Plasma and red blood cell cholinesterase activities were determined in blood samples taken shortly before the beginning of dosing and Weeks 1, 3, 6 and 13 . All blood samples were obtained from the orbital sinus and enzyme activity was determined by a modified Ellman method. Brain cholinesterase activity was determined at termination. - Sacrifice and pathology:
- GROSS PATHOLOGY: After 90 days of treatment, rats were fasted overnight and then sacrificed by exposure to chloroform vapor. Rats were incised to expose the contents of the abdominal, pelvic and thoracic cavities and a thorough visual examination was made of all organs and body tissues in situ. The following organs and tissues were examined for gross pathologic changes: brain, pituitary, eyes, nasal cavity, salivary gland (submaxillary), thyroid, trachea, esophagus, thymus, heart, lungs, liver, spleen, pancreas, stomach, duodenum, jejunum, ileum, colon, rectum, adrenals, kidneys, mesenteric lymph node, bladder, prostate, testes/epididymides, ovaries/with fallopian tubes, uterus, skeletal muscle, sternebrae (bone/marrow), spinal cord, unusual lesions.
HISTOPATHOLOGY: All organs, tissued and unusual lesions along with the carcass were placed in 10% formalin for preservation and possible subsequent histopathologic examination. The specimens from the organs and tissues were paraffin embedded, sectioned at 6 microns and stained with hematoxylin and eosin.
Groups DE1 and DE3 rats: brain, spinal cord (2 levels), eyes, pituitary, thyroid, adrenals, salivary gland, lymph node, esophagus, trachea, thymus, heart, lungs (2 sections), liver (2 sections), spleen, kidneys, urinary bladder, stomach, pancreas, small intestines (3 levels), large intestine (2 levels), testes/epididymides, prostate, ovaries/fallopian tubes, uterus/cervix, skeletal muscle, bone marrow (sternum or femur, after proper decalcification), nasal cavity (after proper decalcification), and unusual lesions.
Groups DE2, DE5, and DE7 rats: brain, heart, liver (2 sections), spleen, kidneys, testes/epididymides, ovaries/fallopian tubes, uterus/cervix, bone marrow (sternum or femur, after proper decalcification), and unusual lesions.
Groups DE4 and DE6 rats: heart, liver (2 sections), kidneys, bone marrow (sternum or femur, after proper decalcification), and unusual lesions. - Statistics:
- Differences between control and treated group values for weekly body weights and change in body weights, hematologic, serum chemistry, cholinesterase and organ weight data were analyzed for statistical significance by the method of Dunnett. This is a multiple comparison procedure for comparing several treatments simultaneously with a control.
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No consistent or apparent treatment related clinical effects were observed during the 90-day treatment period.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Animals at 135, 270, and 540 ppm were found dead during Week 2. The cause of the deaths were due to gastritis, enteritis and/or colitis, gastrointestinal distress. Within 2 days of water imbition for urinalysis, the female rats at 1500, 1080, 135, 270, 540, 810 ppm were found dead. These rats all had watery fluid in the pleural cavity and their lungs were darkened and congested. All rats apparently had esophageal ruptures due to attempts at regurgitation after water loading. Other rats found dead during Week 7 included the animals from 27, 800, 1080, 135, 540, 810 ppm groups. Based on the gross and/or microscopic findings, these deaths appear to be primarily related to systemic infections which are probably attributable to the water loading that occurred earlier in the Week. The animal at 810 ppm had diarrhea prior to death, dark fluid in the stomach and yellowish fluid and gas in the intestinal tract, indicative of enteritis and/or colitis. None of the deaths appear to be directly attributable to the test substance administration.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Significant differences that appear to be directly related to test substance administration include the following: less body weight gain during Week 3 and a significantly smaller value for the mean body weights during Weeks 3-6 for the 540 ppm; smaller mean body weight values during Week 7-12 for the 810 ppm females; decreased body weight gains for male and female rats and a smaller value for the mean body weight of the females from 1080 ppm group during Week 5, which coincides with the exposure level being increased from 50 and 65 ppm to 800 and 1080 ppm for the males and females, respectively; and decreased body weight gains for the males and females in 1500 ppm group during Week 10 and smaller mean values for body weights of the males during Weeks 10 and 11 (at 1000 ppm) and the females during Weeks 10-13. This latter finding in this group coincides with an increase in dosage at the beginning of Week 10 from 20 and 27 ppm to 1000 and 1500 ppm for the males and females, respectively.
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Statistical evaluation of the data revealed several significant (p ≤0.05) differences between treated and control animals in estimated food consumption at various weeks throughout the study. However, many of these differences, both increases and decreases were not consistent nor apparent treatment related changes.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- After 6 weeks of the test substance administration, there was an apparent treatment related decrease in hemoglobin in the male rats with significant differences noted between control and Groups 800/1080, 200/270, 400/540, and 600/810 ppm. In addition, the female rats at 810 ppm had a significantly lower mean value for erythrocytes and an increased mean MCV value. Groups at 200/270 and 600/810 ppm also had elevated mean values for MCV. The females at 1500 ppm had a significantly lower mean value for neutrophils with a concomitantly higher mean value for lymphocytes. This latter finding was considered to be incidental or spontaneous since it did not occur in other female groups or at the Week 13 evaluation. Platelet counts did not differ between treated and control groups.
After 13 weeks of treatment, the mean hemoglobin values at 800 and 400 ppm males, and 1500, 1080, 540, 810 ppm females were significantly less than the corresponding control values. However, the males at 600 ppm had a significantly elevated mean value for hemoglobin and MCV. In addition, the mean erythrocyte values for males and females and the mean hematocrit value for females at 600/810 ppm were significantly less than the corresponding control values. 1500 ppm females and 100 ppm males had significantly elevated platelet counts. 1000, 800, and 600 ppm males and 1500, 1080 and 270, 540, 810 ppm females had significantly greater values for reticulocyte counts than did the corresponding control groups. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- After 6 weeks of treatment, significant differences included smaller group mean glucose values for 800, 400, and 600 ppm males, smaller mean values of urea for the females of 1080 and 810 ppm, greater mean values of K+ for males at 100, 200, 400, 600 ppm and females at 540 ppm, and an elevated mean value for direct bilirubin for males at 400 ppm. The elevated K+ levels are probably of little importance since the K+ differences are slight (0.5 mEq/L) and the absolute values are well within the reference range (4.1-7.7). The elevated direct bilirubin value at 400 ppm appears to be an incidental or spurious finding since differences were not observed in male animals receiving higher doses of test substance or in any of the female groups.
Significant differences noted for serum chemistry variables determined at termination include elevated mean values of urea at 1080 ppm males and 1500 ppm females and smaller mean glucose values for the females at 1500, 1080, 540, and 810 ppm. The following significant differences between control and treated values were also noted but are considered to be spontaneous or incidental and not related to test substance administration: Elevated mean values of total protein and globulin for the females at 135, 270, and 810 ppm; smaller mean values of total bilirubin at 1080 and 540 ppm females; an increased mean value of direct bilirubin and a smaller mean value of cholesterol at 800 ppm males; an elevated mean K+ level at 135 ppm males; and an elevated activity level of SGPT at 270 ppm females. These differences were slight and/or inconsistent between dose groups or sex. - Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no consistent or distinct treatment related differences between treated and control values from the samples obtained during Week 7 or 13.
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Both the absolute and relative weights of the spleens from the male and female rats at 1000/1500, 800/1080, and 600/810 ppm were significantly greater than the corresponding control values. In addition, the relative weights of the spleens from the female rats at 540 ppm were significantly greater than controls. The absolute and relative weights of the ovaries (with fallopian tubes) were significantly smaller in the 1500 ppm females than control values. The absolute and relative liver weights at 1080 ppm females were significantly larger than control weights and the relative liver weights of the females at 1500, 540, and 810 ppm were significantly greater than relative control weights. In addition, the females at 1500, 1080, and 810 ppm had significantly greater values for relative kidney weights when compared to the relative kidney weights of control females.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no consistent or apparent changes indicative of a treatment related effect.
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No distinct or consistent compound related microscopic changes were found in the tissue sections evaluated.
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- Decreases in plasma cholinesterase activity was noted for females receiving 810 ppm or greater of the test substance but similar changes were not observed in the male rats. In contrast, there was an apparent treatment related decrease in cholinesterase activity for RBCs from the high dose (800 ppm) males at termination, but similar changes were not observed in the female rats. Statistical analysis of the brain cholinesterase activity data did not reveal any statistically significant differences between treated and control values.
- Key result
- Dose descriptor:
- other: MTD
- Effect level:
- > 1 000 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- clinical biochemistry
- haematology
- organ weights and organ / body weight ratios
- Key result
- Dose descriptor:
- LOEL
- Effect level:
- 540 ppm
- Basis for effect level:
- body weight and weight gain
- Key result
- Dose descriptor:
- LOEL
- Effect level:
- 270 other: 200/270 ppm
- Sex:
- male
- Basis for effect level:
- haematology
- Critical effects observed:
- no
- Conclusions:
- MTD (Rat): >1000 ppm
- Executive summary:
Groups of male and female rats were administered the test substance in the diet at levels of 100-600 ppm (males) and 135-810 ppm (females) for 13 weeks; 50 ppm (males) and 68 ppm (females for 4 weeks followed by 800 ppm (males) and 1080 ppm (females) for 9 weeks; or 20 ppm (males) and 27 ppm (females) for 9 weeks followed by 1000 ppm (males) and 1500 ppm (females) for 4 weeks.
No consistent or apparent test substance related clinical effects were noted during twice daily observations of the rats or 13 weeks. There were transient changes in weekly body weights observed for male and female rats receiving 1000 ppm or greater of test substance in the diet.
Clinical pathologic changes indicative of a systemic effect include decreases in hemoglobin and increases in reticulocytes at levels of 400 ppm (males) and 540 ppm (females) or greater and decreased RBC counts in males receiving 600 ppm and females receiving 810 ppm for 13 weeks. Decreased glucose levels were noted for male rats given 400 ppm or greater of test substance for 6 weeks but not after 13 weeks. Similar changes were noted in female rats after receiving 540 or 810 ppm for 13 weeks, or 68 ppm for 4 weeks followed by 1080 ppm for 9 weeks, or 27 ppm for 9 weeks followed by 1500 ppm for 4 weeks. No distinct or apparent treatment related differences were observed in the results of the urinalyses conducted during Week 7 or at termination. Decreases in plasma cholinesterase activity was noted for females receiving 810 ppm or greater of test substance but similar changes were not observed in the male rats. In contrast, there was an apparent treatment related decrease in cholinesterase activity for RBCs from the high dose (800 ppm) males at termination, but similar changes were not observed in the female rats.
Spleen weights, absolute and/or relative, were increased in males receiving 600 ppm or greater and in females receiving 540 ppm or greater of test substance. Relative weights of the livers and kidneys were increased for females receiving test substance at and above 540 and 810 ppm, respectively. No distinct or consistent test substance related microscopic changes were found in tissue sections evaluated.
Based on the data obtained in this study, the maximum tolerated dose (MTD) should be greater than 1000 ppm for both sexes and there were no apparent differences indicative of a real sex difference in response to the test substance administration.
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 3.6 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- Rat (diet): 90-day oral toxicity study, 50 ppm (males and females) equivalent to 3.6 mg/kg bw/day (males) and 4.1 mg/kg bw/day (females) based on decreased body weight, food consumption, and haematological effects at ≥250 ppm.
Rat (diet): 90-day oral toxicity study, 200 ppm (males) and 150 ppm (females) equivalent to 13.6 and 10.0 mg/kg bw/day, respectively based on hematology changes at ≥400 ppm in males and
≥270 ppm in females.
Mouse (diet): 13-week oral toxicity study, 150 ppm in males and 75 ppm in females equivalent to 26.6 and 15.6 mg/kg bw/day, respectively based on haematology changes at ≥300 ppm in males and
≥150 ppm in females.
Dog (diet): 13-week oral toxicity study, 400 ppm (highest dose tested) equivalent to 14.7 mg/kg bw/day for males and 12.5 mg/kg bw/day for females.
Rat (diet): 24 month oral toxicity study, The NOAEL is 100 ppm for males and females (4.8 mg/kg bw/day and 6.3 mg/kg bw/day respectively). This NOAEL was based on decreased weight gain and haematology changes at 400 ppm.
Rat (diet) 35-week: The NOAEL was 300 ppm (approximately 14.4 mg/kg bw/day) for males based on decreased body weight at 600 ppm and above and 100 ppm (approximately 6.4 mg/kg bw/day) for females based on haematology changes at 300 ppm and above.
Mouse (diet) 104-week: The NOAEL in the 104-week feeding study in mice was 50 ppm for both males
and females (8.7 mg/kg bw/day for males and 10.6 mg/kg bw/day for females). This NOAEL was based on increased mortality in 75 ppm and greater males and females.
Mouse (diet) 23-week study: The NOAEL in mice was 100 ppm for males and females (approximately 16.6 mg/kg bw/day and 23.2 mg/kg bw/day, respectively). This NOAEL was based on haematology changes in males and females at ≥300 ppm.
Dog (diet) 2-year: The NOAEL in the 2-year feeding study in dogs was 100 ppm for males and females (3.0 mg/kg bw/day). This NOAEL was based on histopathology changes in the kidney and spleen at 400 ppm and above. - System:
- haematopoietic
Repeated dose toxicity: inhalation - systemic effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: inhalation - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - systemic effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: dermal
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- Methomyl technical
Lot #: DPX-X1179-512
Purity: 98.6% - Species:
- rabbit
- Strain:
- New Zealand White
- Remarks:
- HM:(NZW)fBR
- Details on species / strain selection:
- Rabbits are considered an acceptable species suited for conducting dermal absorption studies. Their use is designated as appropriate according to the testing guidelines for toxicological evaluation of pesticides by the dermal route of exposure. In addition, rabbits were used as the test species in the previous study, and use of the same species for this new study facilitated comparison of results. The New Zealand White strain was chosen because extensive background information is available from the literature, the supplier, and previous studies conducted at the test facility.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Hare Marland
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Approximately 10 weeks old
- Weight at study initiation: 1603.6-1973.9 g
- Housing: The rabbits were housed singly in suspended, stainless steel, wire-mesh cages
- Diet: Purina certified high fiber rabbit chow # 5325, approximately 125 g per day
- Water: ad libitum
- Acclimation period: Quarantined for 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature: 20±1°C
- Humidity: 40-60%
- Photoperiod (hrs dark / hrs light): 12/12 - Type of coverage:
- semiocclusive
- Vehicle:
- other: deionized water
- Details on exposure:
- TEST SITE
- Area of exposure: Approximately 190 cm²
- % coverage: 10% of total body surface area
- Type of wrap if used: Wrapped with successive layers of porous dressing (stretch gauze and adhesive bandage)
REMOVAL OF TEST SUBSTANCE
- After an exposure period of approximately 6 hours, the bandages were removed and the test sites were washed with Ivory® soap and warm tap water, and the skin was patted dry.
TEST MATERIAL
- Amount applied: 1 mL
- Constant volume or concentration used: yes
- For solids, paste formed: yes
VEHICLE
- Amount applied: 1 mL
USE OF RESTRAINERS FOR PREVENTING INGESTION: yes - Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 6 hours
- Frequency of treatment:
- daily for 21 days
- Dose / conc.:
- 15 mg/kg bw/day
- Dose / conc.:
- 30 mg/kg bw/day
- Dose / conc.:
- 45 mg/kg bw/day
- Dose / conc.:
- 90 mg/kg bw/day
- No. of animals per sex per dose:
- 6
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: In a previous 21-day dermal study conducted with the LOEL was reported to be 50 mg/kg and the NOEL to be 5 mg/kg. Based on the results of the previous study and the pilot study, the dosages for this 21-day dermal study were selected.
- Pilot study: A dermal pilot study was conducted with the test substance to compare the sensitivity of the assay presently used to determine cholinesterase activity to the assay used in the previous study and to determine the washing procedures to be used in the main study. Additionally, the results of pilot study were used to determine the highest dosage tested in the main study. Before exposure, approximately 0.5 mL of blood was collected from the jugular vein of 4 male rabbits. Baseline cholinesterase activity in plasma and red blood cells was assessed.
Each rabbit was treated once dermally at a dosage of 100, 250, or 500 mg/kg. The test site was covered with a porous wrap. A control animal was similarly treated with deionized water only. After an exposure period of approximately 6 hours, the bandages were removed. The test site of each rabbit was washed twice with warm water. The test site was wiped with a dry swab after each washing; the swabs were analyzed for the presence of test substance to determine if the method of washing removed all of the test substance. After washing, the skin was patted dry. After the exposure period, the rabbits were examined for clinical signs of toxicity and dermal response.
Approximately 1 hour after the end of the exposure period, approximately 0.5 mL of blood was collected from the jugular vein of the rabbits. The rabbits were then euthanized by injection with a barbiturate into the auricular blood vessel and exsanguinated. Brains were collected and weighed, frozen at approximately -70°C, and analyzed for brain cholinesterase activity. Cholinesterase was evaluated in plasma and red blood cells.
No clinical signs of toxicity or dermal irritation were observed. The rabbit dosed at 500 mg/kg exhibited inhibition in plasma (56% of control) and brain (61% of control) cholinesterase activity.
Analyses of the swabs indicated there was residual test substance on the skin of the rabbits after washing with water. Therefore, the rabbits were washed with Ivory soap and warm water during the main study. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Rabbits were checked daily for mortality and for signs of illness, injury, or abnormal behavior.
OBSERVATIONS: Before treatment and after test substance removal each day, the rabbits were observed for the following clinical signs of toxicity indicative of cholinergic effects: salivation, excessive urination, convulsions, tremors, muscle fasciculations, diarrhea, hyperactivity, and hyperreactivity.
DERMAL IRRITATION: The animals were also observed for dermal irritation before treatment and after unwrapping each day. The Draize Scale was used to score skin responses. Adjacent areas of untreated skin were used for comparison.
BODY WEIGHT: The rabbits were weighed 2 times each week (at approximately 3-4 day intervals) during the dosing period.
FOOD CONSUMPTION and EFFICIENCY: The amount of food consumed by each rabbit was determined weekly. From these determinations and body weight data, individual daily food consumption and mean food efficiency were calculated.
CLINICAL CHEMISTRY: One hour following the last exposure (day 21), approximately 0.5-1 mL blood was collected from the jugular vein of all study rabbits and evaluated for cholinesterase activity in plasma and red blood cells. - Sacrifice and pathology:
- Following blood collection on day 21, all rabbits were euthanized by injection with a barbiturate into the auricular blood vessel and then exsanguinated. The rabbits were given a gross pathological examination. Brains were collected and weighed, frozen at approximately -70°C, and analyzed for brain cholinesterase activity. The remaining tissues were discarded without microscopic evaluation.
- Statistics:
- Body weights, body weight gains, and pathology data were analyzed by a one-way analysis of variance. Pairwise comparisons between test and control groups were made with Dunnett's test. Cholinesterase measurements were analyzed by the Jonckheere test for trend (p <0.05). Increases in the incidences Of clinical observations were evaluated by the Cochran-Armitage test for trend.
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Pupillary constriction, cataracts were considered to be non test substance related and spontaenous. Mucoidal feces, blood in feces, diarrhea, injured leg were considered to be related to stress occured during dosing and washing procedures.
Other clinical signs including pallor of the eyes, raised area on the rib(s), conjunctivitis, appears not to be eating, shivering, and weakness were not considered toxicologically important. These signs were observed sporadically or with similar frequency in both controls and treated animals. - Dermal irritation:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Slight or mild erythema, purplish areas, epidermal scaling, eschar, scratches, cuts, nicks, raw areas, bruises, tape burn, scars, sloughing, and scabs were observed sporadically in male and female rabbits at all dosages. These observations, although suggestive of skin trauma, exhibited no dose-response relationship and/or were attributed to mechanical irritation resulting from the daily bandaging, unwrapping, and washing regimen.
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No significant differences in mean body weights or mean body weight gains occurred during the study.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No significant differences in food consumption occurred during the study.
- Food efficiency:
- no effects observed
- Description (incidence and severity):
- No significant differences in food efficiency occurred during the study.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Decreases in cholinesterase activity measured in brain, erythrocytes, or plasma attributable to the test substance were not demonstrated under the conditions of this study. There were some statistically significant differences between treated and control male and female groups for brain cholinesterase activity but in the absence of a meaningful dose response these were not considered to be biologically adverse.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test substance-related gross lesions were observed at necropsy. Discoloration noted in the eyes of a female rabbit in the 90 mg/kg/day treatment group was considered to be spontaneous since no other rabbits exhibited this lesion.
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 90 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: highest dose tested
- Key result
- Dose descriptor:
- LOEL
- Effect level:
- > 90 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Critical effects observed:
- no
- Conclusions:
- Dermal NOEL (Rabbit): 90 mg/kg (highest dose tested)
- Executive summary:
The test substance was applied to the shaved, intact skin of male and female New Zealand White rabbits for 21 consecutive days to evaluate the toxicity of the test substance and to evaluate the potential for cholinesterase inhibition in the blood and brain following the guideline U.S. EPA OPP 82-2. The daily exposure period was approximately 6 hours. Four groups of 6 male and 6 female rabbits were treated dermally with 15, 30, 45, or 90 mg/kg of test substance per day. A vehicle control group of 6 male and 6 female rabbits was similarly treated with deionized water only. Body weights and food consumption were measured. Clinical observations and dermal effects were recorded before treatment and after test substance removal each day during the study. On test day 21, blood was collected from each rabbit approximately 1 hour after the exposure period to determine erythrocyte and plasma cholinesterase activity. Following blood collection, all rabbits were sacrificed, a gross necropsy was performed, and the brains were collected, weighed, and later analyzed to determine brain cholinesterase activity.
No mortalities occurred during the study. There were no significant differences in mean body weight, mean body weight gains, food consumption, or food efficiency. No clinical signs of toxicity attributable to dermal exposure to test substance occurred during the study.
No test substance-related gross lesions were observed in any dose group during necropsy. Decreases in cholinesterase activity measured in brain, erythrocytes, or plasma attributable to the test substance were not demonstrated under the conditions of this study. There were some statistically significant differences between treated and control male and female groups for brain cholinesterase activity but in the absence of a meaningful dose response these were not considered to be biologically adverse.
The no-observed-effect-level was determined to be 90 mg/kg, the highest dose tested, for both male and female rabbits.
- Endpoint:
- short-term repeated dose toxicity: dermal
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- Ethanimidothioic acid, N-[[(methyl amino)-carbonyl]oxy]-, methyl ester
DPX-X1179-394
Lot #: T00620-06
Purity: 98.35% - Species:
- rabbit
- Strain:
- New Zealand White
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Hare Marland, Hewitt, New Jersey
- Age at study initiation: Approximately 10 weeks old
- Weight at study initiation: 2.3-2.4 Kg
- Housing: The rabbits were housed singly in suspended, stainless steel, wire-mesh cages.
- Diet: Purina certified high fiber rabbit chow # 5325, ad libitum
- Water: ad libitum
- Acclimation period: Quarantined for 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature: 20±2°C
- Humidity: 40-60%
- Photoperiod (hrs dark / hrs light): 12/12 - Type of coverage:
- occlusive
- Vehicle:
- other: deionized water
- Details on exposure:
- TEST SITE
- Area of exposure: Approximately 190 cm²
- Type of wrap if used: Wrapped with successive layers of plastic film, stretch gauze bandage and elastic adhesive bandage
REMOVAL OF TEST SUBSTANCE
- Approximately 6 hours after treatment, the rabbits were removed from their cages and the wrappings were removed. The test site of each rabbit was gently washed with warm water to remove excess test substance and the skin was gently patted dry.
TEST MATERIAL
- Amount applied: 5 mL
- Constant volume or concentration used: Yes
- For solids, paste formed: Yes
VEHICLE
- Amount applied: 5 mL
USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes - Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 6 hours
- Frequency of treatment:
- daily for 21 days
- Dose / conc.:
- 5 mg/kg bw/day
- Dose / conc.:
- 50 mg/kg bw/day
- Dose / conc.:
- 500 mg/kg bw/day
- No. of animals per sex per dose:
- 5 per sex in 5 and 50 mg/kg groups
10 per sex in 500 mg/kg group - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: The results of the range finding studies indicated that an effect on cholinesterase activity could occur at a dosage as low as 50 mg/kg. Therefore, based on these previous studies and the results of the range finding studies, dosages of 5, 50 and 500 mg/kg were selected for the main study.
- Range finding study 1: Male and female rabbits were treated with 5, 50, 200 or 500 mg/kg (one rabbit at each dosage level) of the test substance for 5 consecutive days. No dermal irritation was observed throughout the range finding study. The cholinesterase activity measured at day 3 indicated an effect at all dosage levels, although no clear dose-response relationship was evident. The red blood cell activity at day 3 was lower than normally observed for rabbits, but was considered related to an unknown problem with the assay. At the day 5 evaluation, plasma cholinesterase activity was lower only in animals treated with 50 mg/kg and greater. The red blood cell activity was considered unaffected by treatment with the test substance. However, the results overall suggested a slight effect at all dose levels, although a very marginal effect at 5 mg/kg.
- Range finding study 2: Male and female rabbits were treated with 2, 5, 25 or 50 mg/kg (one rabbit at each dosage level) of the test substance for 5 consecutive days. In this second study, the results were less variable and suggested only a marginal effect on cholinesterase activities at 50 mg/kg. The results at 2, 5 and 25 mg/kg were considered comparable to the pre-test evaluation. - Observations and examinations performed and frequency:
- OBSERVATIONS: Prior to each treatment the rabbits were examined for clinical signs of toxicity including dermal irritation. Approximately 1 hour after unwrapping, the animals were observed for dermal irritation and clinical signs of toxicity.
BODY WEIGHT: Rabbits were weighed twice weekly (at 3-4 day intervals) prior to treatment during the dosing period, and weekly during the 14-day recovery phase of the study.
FOOD CONSUMPTION: The amount of food consumed by each group was determined weekly. From this data, individual food consumption (grams per rabbit) was calculated.
FOOD EFFICIENCY:Food consumption and body weight data were used to calculate mean individual daily food consumption and group mean food efficiency values.
HAEMATOLOGY
- Time schedule for collection of blood: 1 hour (day 21) and 14 days (day 35) after the last treatment
- Anaesthetic used for blood collection: No
- How many animals: day 21 - 5 male and females from control and high dose group and all animals from other groups; day 35 - all surviving animals
- Parameters examined: The hematological parameters examined at each evaluation consisted of erythrocyte, leukocyte, differential leukocyte (control and high-dose animals only), and platelet counts and hemoglobin and hematocrit. Mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and mean corpuscular volume were calculated from the erythrocytic data.
CLINICAL CHEMISTRY
- Time schedule for collection of blood: 1 hour (day 21) and 14 days (day 35) after the last treatment
- How many animals: day 21 - 5 male and females from control and high dose group and all animals from other groups; day 35 - all surviving animals
- Parameters examined: Alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase activities, and concentrations of blood urea nitrogen, total serum protein, albumin, globulin (calculated), creatinine, cholesterol, bilirubin, glucose, calcium, sodium, potassium, chloride and phosphorous.
OTHER
Blood samples for plasma and whole blood cholinesterase activities were collected approximately 1 hour following the last treatment (at the end of the 6-hour treatment period). Blood samples for plasma and whole blood cholinesterase activities were again collected on test day 35. Red blood cell cholinesterase activity was calculated from the whole blood and plasma activities and the hematocrit. Brain cholinesterase activity was also measured on test days 21 and 35. - Sacrifice and pathology:
- Following blood collection on the day of the final treatment (test day 21), 5 male and 5 female rabbits from the control groups, 5 male and 5 female rabbits from the high-dose groups and all male and female rabbits from the low- and intermediate-dose groups were killed by barbiturate anesthesia and exsanguination for gross pathological examination. Surviving rabbits from the control and 500 mg/kg treatment groups were killed 14 days after the last treatment. The brain, adrenals, liver, spleen, kidneys and testes were weighed and relative organ weights (organ weight to final body weight ratio expressed as percent body weight) were calculated. Along with these organs, the thymus, bone and bone marrow (sternal), heart, lymph nodes (mandibular and mesenteric), thoracic aorta, trachea, lungs, salivary glands (submaxillary, sublingual and parotid), esophagus, stomach, small intestine (duodenum, jejunum and ileum), large intestine (cecum, colon and rectum), gall bladder, pancreas, urinary bladder, pituitary, thyroid, parathyroids, prostate, epididymides, ovaries, corpus and cervix uteri, vagina, spinal cord, sciatic nerve, eyes, skeletal muscle (thigh), treated and untreated dorsal skin and all gross lesions were processed to tissue blocks for all groups. These tissues were further processed to slides and examined microscopically from rabbits sacrificed by design in the control and high-dose groups, and from one high-dose female rabbit found dead. Treated and untreated skin, liver, kidneys, brain and gross lesions from rabbits in the low- and intermediate-dose groups were also examined microscopically.
- Statistics:
- Body weights, body weight gains, and organ weights were analyzed by a one-way analysis of variance. For body weights and body weight gains, the test for differences among group means (F-test) was significant, pairwise comparisons were made between control and test groups with the least significant difference (LSD) test. Organ weights were also examined by LSD and Dunnett's test. Clinical observations were analyzed by Fisher's Exact test with a Bonferroni correction. Clinical laboratory measurements were analyzed by a one-way analysis of variance and Bartlett's test. When the F-test was significant, comparisons were made with Dunnett's test. When the results of Bartlett's test were significant, the Kruskal-Wallis and Mann-Whitney U tests were employed. Tests for the comparison of means were considered significant at the alpha = 0.05.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- A statistically significantly greater incidence of hyperreactivity (increased reaction to stimuli, usually noise) was observed for male rabbits in the high-dose group compared to controls. Al though not significantly different from controls, the incidence of hyperreactivity in female rabbits was slightly increased. This greater incidence was considered to be related, in part, to the cholinesterase inhibition produced by the compound. There is, however, a background incidence of hyperreactivity observed in rabbits as evidenced by the incidence in the control and Iow- and intermediate-dose groups. The other clinical observations noted during the dosing and recovery phases were considered incidental and not compound related.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One female rabbit in the high-dose group was found dead during the study. The cause of death of this rabbit was related to a fracture at the thoracic- cervical junction of the vertebral column.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No meaningful differences between control rabbits and rabbits treated with the test substance were observed during the dosing or recovery periods with respect to body weights or body weight gains.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No compound-related effects on food consumption were observed during the study.
- Food efficiency:
- no effects observed
- Description (incidence and severity):
- No compound-related effects on food efficiency values were observed during the study.
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Plasma cholinesterase activities measured on test day 21 in male and female rabbits in the 500 mg/kg group were 36 and 55%, respectively, of the control mean. Brain cholinesterase levels were 48 and 68% of the control mean for male and female rabbits, respectively, in the high-dose group. The red blood cell activity was also lower in male and female rabbits in the 500 mg/kg group, but the decreases were considered slight and biologically insignificant. Plasma activity was 77% of the control mean in male rabbits in the 50 mg/kg group. Cholinesterase activities for female rabbits in the 50 mg/kg group were not different from control values. Following 14 days of recovery, cholinesterase activities were similar to control values. Other statistically significant results observed during the study for clinical pathology parameters were within the range of biological variation and were considered not to be compound related.
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- At the 21-day pathological evaluation, the mean absolute brain weight of low-dose male rabbits was statistically significantly lower compared to controls. Following 14 days of recovery, the mean absolute and relative spleen weights of high-dose female rabbits were statistically significantly lower than controls. In the absence of microscopic changes or a dose-related change, these organ weight effects were considered not to be biologically significant.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Gross observations were considered incidental and unrelated to compound administration.
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Microscopic observations were considered incidental and unrelated to compound administration.
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 5 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: decrease in plasma and brain cholinesterase activities at 50 mg/kg
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 50 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: decrease in plasma and brain cholinesterase activities at 500 mg/kg
- Key result
- Dose descriptor:
- LOEL
- Effect level:
- 50 mg/kg bw/day
- Sex:
- male
- Basis for effect level:
- haematology
- Key result
- Dose descriptor:
- LOEL
- Effect level:
- 500 mg/kg bw/day
- Sex:
- female
- Basis for effect level:
- haematology
- Critical effects observed:
- no
- Conclusions:
- Dermal NOEL (Rabbit, male): 5 mg/kg/day
Dermal NOEL (Rabbit, female): 50 mg/kg/day - Executive summary:
The purpose of this study was to evaluate the toxicity of the test substance when administered topically (dermally) to rabbits for approximately 21 consecutive days following the guidelines OECD 410 and U.S. EPA OPP 82-2. The test substance was applied to the clipped, intact skin of male and female White rabbits and occluded for approximately 6 hours daily for 21 consecutive days. Two groups of 5 male and 5 female rabbits each were treated dermally with 5 or 50 mg/kg, and one group of 10 male and 10 female rabbits was treated dermally with 500 mg/kg. The test material was moistened with approximately 5 mL of distilled water. A control group of 10 male and 10 female rabbits was treated with distilled water. Body weights were measured twice each week during the dosing period, and weekly during the recovery phase. The rabbits were examined daily for clinical signs of toxicity and mortality. Food consumption was measured weekly. Approximately 1 hour after the last 6-hour treatment (day 21), blood samples were collected from the auricular artery of each rabbit for a clinical laboratory evaluation. Five male and 5 female rabbits from each of the control and the 500 mg/kg groups, and all male and female rabbits from the 5 and 50 mg/kg groups were subsequently sacrificed for gross and microscopic examinations. Fourteen days following the last treatment, surviving rabbits in each of the control and high-dose groups were subjected to a clinical pathological examination and subsequently sacrificed for pathological examination.
No meaningful differences between control rabbits and rabbits treated with the test substance were observed during the dosing or recovery periods with respect to body weights or body weight gains.
No compound-related effects on food consumption or food efficiency values were observed during the study.
A greater incidence of hyperreactivity (increased reaction to stimuli, e.g. noise) was observed in the high-dose groups compared to the controls. A background incidence of hyperreactivity was evident in the control and in the low- and intermediate- dose groups, but the greater incidence in the high-dose groups was considered related, in part, to the cholinesterase inhibition produced by the compound. Other clinical observations noted during the study were considered unremarkable and unrelated to administration of the compound.
One female rabbit in the high-dose group was found dead during the study. The cause of death of this rabbit was related to a fracture at the thoracic- cervical junction of the vertebral column.
Plasma cholinesterase activities measured on test day 21 in male and female rabbits in the 500 mg/kg group were 36 and 55%, respectively, of the control mean. Brain cholinesterase levels were 48 and 68% of the control mean for male and female rabbits, respectively, in the high-dose group. The red blood cell activity was also lower in male and female rabbits in the 500 mg/kg group, but the decreases were considered slight and biologically insignificant. Plasma activity was 77% of the control mean in male rabbits in the 50 mg/kg group. Cholinesterase activities for female rabbits in the 50 mg/kg group were not different from control values. Following 14 days of recovery, cholinesterase activities were similar to control values. Other statistically significant results observed during the study for clinical pathology parameters were within the range of biological variation and were considered not to be compound related.
No compound-related effects on organ weights were observed. No gross or microscopic compound-related pathological changes were observed at the end of the dosing phase or after a 14-day recovery period.
In summary, compound-related effects on plasma and brain cholinesterase activities were observed in male and female rabbits in the 500 mg/kg groups and in male rabbits in the 50 mg/kg group. Consistent with the cholinesterase inhibition in rabbits in the high-dose groups was a slight increase in hyperreactivity. Under the conditions of this study, a no-observable-effect level (NOEL) for the administration of the test substance to intact skin of male rabbits was 5 mg/kg/day. In female rabbits, the NOEL was 50 mg/kg/day.
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 50 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rabbit
- Quality of whole database:
- Rabbit 21-day using porous, semi-occlusive (gauze) wrapping: NOAEL is 90 mg/kg bw/day. No adverse effect observed.
Rabbit 21-day using a non-porous, occlusive (plastic) wrapping. NOAEL is 50 mg/kg bw/day (males and females) based on an increased incidence of hyperreactivity and biologically significant decreases in plasma and brain cholinesterase activities at 500 mg/kg bw/day. - System:
- other: significant decreases in plasma and brain cholinesterase activities
Repeated dose toxicity: dermal - local effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: dermal
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- Methomyl technical
Lot #: DPX-X1179-512
Purity: 98.6% - Species:
- rabbit
- Strain:
- New Zealand White
- Remarks:
- HM:(NZW)fBR
- Details on species / strain selection:
- Rabbits are considered an acceptable species suited for conducting dermal absorption studies. Their use is designated as appropriate according to the testing guidelines for toxicological evaluation of pesticides by the dermal route of exposure. In addition, rabbits were used as the test species in the previous study, and use of the same species for this new study facilitated comparison of results. The New Zealand White strain was chosen because extensive background information is available from the literature, the supplier, and previous studies conducted at the test facility.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Hare Marland
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Approximately 10 weeks old
- Weight at study initiation: 1603.6-1973.9 g
- Housing: The rabbits were housed singly in suspended, stainless steel, wire-mesh cages
- Diet: Purina certified high fiber rabbit chow # 5325, approximately 125 g per day
- Water: ad libitum
- Acclimation period: Quarantined for 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature: 20±1°C
- Humidity: 40-60%
- Photoperiod (hrs dark / hrs light): 12/12 - Type of coverage:
- semiocclusive
- Vehicle:
- other: deionized water
- Details on exposure:
- TEST SITE
- Area of exposure: Approximately 190 cm²
- % coverage: 10% of total body surface area
- Type of wrap if used: Wrapped with successive layers of porous dressing (stretch gauze and adhesive bandage)
REMOVAL OF TEST SUBSTANCE
- After an exposure period of approximately 6 hours, the bandages were removed and the test sites were washed with Ivory® soap and warm tap water, and the skin was patted dry.
TEST MATERIAL
- Amount applied: 1 mL
- Constant volume or concentration used: yes
- For solids, paste formed: yes
VEHICLE
- Amount applied: 1 mL
USE OF RESTRAINERS FOR PREVENTING INGESTION: yes - Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 6 hours
- Frequency of treatment:
- daily for 21 days
- Dose / conc.:
- 15 mg/kg bw/day
- Dose / conc.:
- 30 mg/kg bw/day
- Dose / conc.:
- 45 mg/kg bw/day
- Dose / conc.:
- 90 mg/kg bw/day
- No. of animals per sex per dose:
- 6
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: In a previous 21-day dermal study conducted with the LOEL was reported to be 50 mg/kg and the NOEL to be 5 mg/kg. Based on the results of the previous study and the pilot study, the dosages for this 21-day dermal study were selected.
- Pilot study: A dermal pilot study was conducted with the test substance to compare the sensitivity of the assay presently used to determine cholinesterase activity to the assay used in the previous study and to determine the washing procedures to be used in the main study. Additionally, the results of pilot study were used to determine the highest dosage tested in the main study. Before exposure, approximately 0.5 mL of blood was collected from the jugular vein of 4 male rabbits. Baseline cholinesterase activity in plasma and red blood cells was assessed.
Each rabbit was treated once dermally at a dosage of 100, 250, or 500 mg/kg. The test site was covered with a porous wrap. A control animal was similarly treated with deionized water only. After an exposure period of approximately 6 hours, the bandages were removed. The test site of each rabbit was washed twice with warm water. The test site was wiped with a dry swab after each washing; the swabs were analyzed for the presence of test substance to determine if the method of washing removed all of the test substance. After washing, the skin was patted dry. After the exposure period, the rabbits were examined for clinical signs of toxicity and dermal response.
Approximately 1 hour after the end of the exposure period, approximately 0.5 mL of blood was collected from the jugular vein of the rabbits. The rabbits were then euthanized by injection with a barbiturate into the auricular blood vessel and exsanguinated. Brains were collected and weighed, frozen at approximately -70°C, and analyzed for brain cholinesterase activity. Cholinesterase was evaluated in plasma and red blood cells.
No clinical signs of toxicity or dermal irritation were observed. The rabbit dosed at 500 mg/kg exhibited inhibition in plasma (56% of control) and brain (61% of control) cholinesterase activity.
Analyses of the swabs indicated there was residual test substance on the skin of the rabbits after washing with water. Therefore, the rabbits were washed with Ivory soap and warm water during the main study. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Rabbits were checked daily for mortality and for signs of illness, injury, or abnormal behavior.
OBSERVATIONS: Before treatment and after test substance removal each day, the rabbits were observed for the following clinical signs of toxicity indicative of cholinergic effects: salivation, excessive urination, convulsions, tremors, muscle fasciculations, diarrhea, hyperactivity, and hyperreactivity.
DERMAL IRRITATION: The animals were also observed for dermal irritation before treatment and after unwrapping each day. The Draize Scale was used to score skin responses. Adjacent areas of untreated skin were used for comparison.
BODY WEIGHT: The rabbits were weighed 2 times each week (at approximately 3-4 day intervals) during the dosing period.
FOOD CONSUMPTION and EFFICIENCY: The amount of food consumed by each rabbit was determined weekly. From these determinations and body weight data, individual daily food consumption and mean food efficiency were calculated.
CLINICAL CHEMISTRY: One hour following the last exposure (day 21), approximately 0.5-1 mL blood was collected from the jugular vein of all study rabbits and evaluated for cholinesterase activity in plasma and red blood cells. - Sacrifice and pathology:
- Following blood collection on day 21, all rabbits were euthanized by injection with a barbiturate into the auricular blood vessel and then exsanguinated. The rabbits were given a gross pathological examination. Brains were collected and weighed, frozen at approximately -70°C, and analyzed for brain cholinesterase activity. The remaining tissues were discarded without microscopic evaluation.
- Statistics:
- Body weights, body weight gains, and pathology data were analyzed by a one-way analysis of variance. Pairwise comparisons between test and control groups were made with Dunnett's test. Cholinesterase measurements were analyzed by the Jonckheere test for trend (p <0.05). Increases in the incidences Of clinical observations were evaluated by the Cochran-Armitage test for trend.
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Pupillary constriction, cataracts were considered to be non test substance related and spontaenous. Mucoidal feces, blood in feces, diarrhea, injured leg were considered to be related to stress occured during dosing and washing procedures.
Other clinical signs including pallor of the eyes, raised area on the rib(s), conjunctivitis, appears not to be eating, shivering, and weakness were not considered toxicologically important. These signs were observed sporadically or with similar frequency in both controls and treated animals. - Dermal irritation:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Slight or mild erythema, purplish areas, epidermal scaling, eschar, scratches, cuts, nicks, raw areas, bruises, tape burn, scars, sloughing, and scabs were observed sporadically in male and female rabbits at all dosages. These observations, although suggestive of skin trauma, exhibited no dose-response relationship and/or were attributed to mechanical irritation resulting from the daily bandaging, unwrapping, and washing regimen.
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No significant differences in mean body weights or mean body weight gains occurred during the study.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No significant differences in food consumption occurred during the study.
- Food efficiency:
- no effects observed
- Description (incidence and severity):
- No significant differences in food efficiency occurred during the study.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Decreases in cholinesterase activity measured in brain, erythrocytes, or plasma attributable to the test substance were not demonstrated under the conditions of this study. There were some statistically significant differences between treated and control male and female groups for brain cholinesterase activity but in the absence of a meaningful dose response these were not considered to be biologically adverse.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test substance-related gross lesions were observed at necropsy. Discoloration noted in the eyes of a female rabbit in the 90 mg/kg/day treatment group was considered to be spontaneous since no other rabbits exhibited this lesion.
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 90 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: highest dose tested
- Key result
- Dose descriptor:
- LOEL
- Effect level:
- > 90 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Critical effects observed:
- no
- Conclusions:
- Dermal NOEL (Rabbit): 90 mg/kg (highest dose tested)
- Executive summary:
The test substance was applied to the shaved, intact skin of male and female New Zealand White rabbits for 21 consecutive days to evaluate the toxicity of the test substance and to evaluate the potential for cholinesterase inhibition in the blood and brain following the guideline U.S. EPA OPP 82-2. The daily exposure period was approximately 6 hours. Four groups of 6 male and 6 female rabbits were treated dermally with 15, 30, 45, or 90 mg/kg of test substance per day. A vehicle control group of 6 male and 6 female rabbits was similarly treated with deionized water only. Body weights and food consumption were measured. Clinical observations and dermal effects were recorded before treatment and after test substance removal each day during the study. On test day 21, blood was collected from each rabbit approximately 1 hour after the exposure period to determine erythrocyte and plasma cholinesterase activity. Following blood collection, all rabbits were sacrificed, a gross necropsy was performed, and the brains were collected, weighed, and later analyzed to determine brain cholinesterase activity.
No mortalities occurred during the study. There were no significant differences in mean body weight, mean body weight gains, food consumption, or food efficiency. No clinical signs of toxicity attributable to dermal exposure to test substance occurred during the study.
No test substance-related gross lesions were observed in any dose group during necropsy. Decreases in cholinesterase activity measured in brain, erythrocytes, or plasma attributable to the test substance were not demonstrated under the conditions of this study. There were some statistically significant differences between treated and control male and female groups for brain cholinesterase activity but in the absence of a meaningful dose response these were not considered to be biologically adverse.
The no-observed-effect-level was determined to be 90 mg/kg, the highest dose tested, for both male and female rabbits.
- Endpoint:
- short-term repeated dose toxicity: dermal
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- Ethanimidothioic acid, N-[[(methyl amino)-carbonyl]oxy]-, methyl ester
DPX-X1179-394
Lot #: T00620-06
Purity: 98.35% - Species:
- rabbit
- Strain:
- New Zealand White
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Hare Marland, Hewitt, New Jersey
- Age at study initiation: Approximately 10 weeks old
- Weight at study initiation: 2.3-2.4 Kg
- Housing: The rabbits were housed singly in suspended, stainless steel, wire-mesh cages.
- Diet: Purina certified high fiber rabbit chow # 5325, ad libitum
- Water: ad libitum
- Acclimation period: Quarantined for 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature: 20±2°C
- Humidity: 40-60%
- Photoperiod (hrs dark / hrs light): 12/12 - Type of coverage:
- occlusive
- Vehicle:
- other: deionized water
- Details on exposure:
- TEST SITE
- Area of exposure: Approximately 190 cm²
- Type of wrap if used: Wrapped with successive layers of plastic film, stretch gauze bandage and elastic adhesive bandage
REMOVAL OF TEST SUBSTANCE
- Approximately 6 hours after treatment, the rabbits were removed from their cages and the wrappings were removed. The test site of each rabbit was gently washed with warm water to remove excess test substance and the skin was gently patted dry.
TEST MATERIAL
- Amount applied: 5 mL
- Constant volume or concentration used: Yes
- For solids, paste formed: Yes
VEHICLE
- Amount applied: 5 mL
USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes - Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 6 hours
- Frequency of treatment:
- daily for 21 days
- Dose / conc.:
- 5 mg/kg bw/day
- Dose / conc.:
- 50 mg/kg bw/day
- Dose / conc.:
- 500 mg/kg bw/day
- No. of animals per sex per dose:
- 5 per sex in 5 and 50 mg/kg groups
10 per sex in 500 mg/kg group - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: The results of the range finding studies indicated that an effect on cholinesterase activity could occur at a dosage as low as 50 mg/kg. Therefore, based on these previous studies and the results of the range finding studies, dosages of 5, 50 and 500 mg/kg were selected for the main study.
- Range finding study 1: Male and female rabbits were treated with 5, 50, 200 or 500 mg/kg (one rabbit at each dosage level) of the test substance for 5 consecutive days. No dermal irritation was observed throughout the range finding study. The cholinesterase activity measured at day 3 indicated an effect at all dosage levels, although no clear dose-response relationship was evident. The red blood cell activity at day 3 was lower than normally observed for rabbits, but was considered related to an unknown problem with the assay. At the day 5 evaluation, plasma cholinesterase activity was lower only in animals treated with 50 mg/kg and greater. The red blood cell activity was considered unaffected by treatment with the test substance. However, the results overall suggested a slight effect at all dose levels, although a very marginal effect at 5 mg/kg.
- Range finding study 2: Male and female rabbits were treated with 2, 5, 25 or 50 mg/kg (one rabbit at each dosage level) of the test substance for 5 consecutive days. In this second study, the results were less variable and suggested only a marginal effect on cholinesterase activities at 50 mg/kg. The results at 2, 5 and 25 mg/kg were considered comparable to the pre-test evaluation. - Observations and examinations performed and frequency:
- OBSERVATIONS: Prior to each treatment the rabbits were examined for clinical signs of toxicity including dermal irritation. Approximately 1 hour after unwrapping, the animals were observed for dermal irritation and clinical signs of toxicity.
BODY WEIGHT: Rabbits were weighed twice weekly (at 3-4 day intervals) prior to treatment during the dosing period, and weekly during the 14-day recovery phase of the study.
FOOD CONSUMPTION: The amount of food consumed by each group was determined weekly. From this data, individual food consumption (grams per rabbit) was calculated.
FOOD EFFICIENCY:Food consumption and body weight data were used to calculate mean individual daily food consumption and group mean food efficiency values.
HAEMATOLOGY
- Time schedule for collection of blood: 1 hour (day 21) and 14 days (day 35) after the last treatment
- Anaesthetic used for blood collection: No
- How many animals: day 21 - 5 male and females from control and high dose group and all animals from other groups; day 35 - all surviving animals
- Parameters examined: The hematological parameters examined at each evaluation consisted of erythrocyte, leukocyte, differential leukocyte (control and high-dose animals only), and platelet counts and hemoglobin and hematocrit. Mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and mean corpuscular volume were calculated from the erythrocytic data.
CLINICAL CHEMISTRY
- Time schedule for collection of blood: 1 hour (day 21) and 14 days (day 35) after the last treatment
- How many animals: day 21 - 5 male and females from control and high dose group and all animals from other groups; day 35 - all surviving animals
- Parameters examined: Alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase activities, and concentrations of blood urea nitrogen, total serum protein, albumin, globulin (calculated), creatinine, cholesterol, bilirubin, glucose, calcium, sodium, potassium, chloride and phosphorous.
OTHER
Blood samples for plasma and whole blood cholinesterase activities were collected approximately 1 hour following the last treatment (at the end of the 6-hour treatment period). Blood samples for plasma and whole blood cholinesterase activities were again collected on test day 35. Red blood cell cholinesterase activity was calculated from the whole blood and plasma activities and the hematocrit. Brain cholinesterase activity was also measured on test days 21 and 35. - Sacrifice and pathology:
- Following blood collection on the day of the final treatment (test day 21), 5 male and 5 female rabbits from the control groups, 5 male and 5 female rabbits from the high-dose groups and all male and female rabbits from the low- and intermediate-dose groups were killed by barbiturate anesthesia and exsanguination for gross pathological examination. Surviving rabbits from the control and 500 mg/kg treatment groups were killed 14 days after the last treatment. The brain, adrenals, liver, spleen, kidneys and testes were weighed and relative organ weights (organ weight to final body weight ratio expressed as percent body weight) were calculated. Along with these organs, the thymus, bone and bone marrow (sternal), heart, lymph nodes (mandibular and mesenteric), thoracic aorta, trachea, lungs, salivary glands (submaxillary, sublingual and parotid), esophagus, stomach, small intestine (duodenum, jejunum and ileum), large intestine (cecum, colon and rectum), gall bladder, pancreas, urinary bladder, pituitary, thyroid, parathyroids, prostate, epididymides, ovaries, corpus and cervix uteri, vagina, spinal cord, sciatic nerve, eyes, skeletal muscle (thigh), treated and untreated dorsal skin and all gross lesions were processed to tissue blocks for all groups. These tissues were further processed to slides and examined microscopically from rabbits sacrificed by design in the control and high-dose groups, and from one high-dose female rabbit found dead. Treated and untreated skin, liver, kidneys, brain and gross lesions from rabbits in the low- and intermediate-dose groups were also examined microscopically.
- Statistics:
- Body weights, body weight gains, and organ weights were analyzed by a one-way analysis of variance. For body weights and body weight gains, the test for differences among group means (F-test) was significant, pairwise comparisons were made between control and test groups with the least significant difference (LSD) test. Organ weights were also examined by LSD and Dunnett's test. Clinical observations were analyzed by Fisher's Exact test with a Bonferroni correction. Clinical laboratory measurements were analyzed by a one-way analysis of variance and Bartlett's test. When the F-test was significant, comparisons were made with Dunnett's test. When the results of Bartlett's test were significant, the Kruskal-Wallis and Mann-Whitney U tests were employed. Tests for the comparison of means were considered significant at the alpha = 0.05.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- A statistically significantly greater incidence of hyperreactivity (increased reaction to stimuli, usually noise) was observed for male rabbits in the high-dose group compared to controls. Al though not significantly different from controls, the incidence of hyperreactivity in female rabbits was slightly increased. This greater incidence was considered to be related, in part, to the cholinesterase inhibition produced by the compound. There is, however, a background incidence of hyperreactivity observed in rabbits as evidenced by the incidence in the control and Iow- and intermediate-dose groups. The other clinical observations noted during the dosing and recovery phases were considered incidental and not compound related.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One female rabbit in the high-dose group was found dead during the study. The cause of death of this rabbit was related to a fracture at the thoracic- cervical junction of the vertebral column.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No meaningful differences between control rabbits and rabbits treated with the test substance were observed during the dosing or recovery periods with respect to body weights or body weight gains.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No compound-related effects on food consumption were observed during the study.
- Food efficiency:
- no effects observed
- Description (incidence and severity):
- No compound-related effects on food efficiency values were observed during the study.
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Plasma cholinesterase activities measured on test day 21 in male and female rabbits in the 500 mg/kg group were 36 and 55%, respectively, of the control mean. Brain cholinesterase levels were 48 and 68% of the control mean for male and female rabbits, respectively, in the high-dose group. The red blood cell activity was also lower in male and female rabbits in the 500 mg/kg group, but the decreases were considered slight and biologically insignificant. Plasma activity was 77% of the control mean in male rabbits in the 50 mg/kg group. Cholinesterase activities for female rabbits in the 50 mg/kg group were not different from control values. Following 14 days of recovery, cholinesterase activities were similar to control values. Other statistically significant results observed during the study for clinical pathology parameters were within the range of biological variation and were considered not to be compound related.
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- At the 21-day pathological evaluation, the mean absolute brain weight of low-dose male rabbits was statistically significantly lower compared to controls. Following 14 days of recovery, the mean absolute and relative spleen weights of high-dose female rabbits were statistically significantly lower than controls. In the absence of microscopic changes or a dose-related change, these organ weight effects were considered not to be biologically significant.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Gross observations were considered incidental and unrelated to compound administration.
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Microscopic observations were considered incidental and unrelated to compound administration.
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 5 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: decrease in plasma and brain cholinesterase activities at 50 mg/kg
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 50 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: decrease in plasma and brain cholinesterase activities at 500 mg/kg
- Key result
- Dose descriptor:
- LOEL
- Effect level:
- 50 mg/kg bw/day
- Sex:
- male
- Basis for effect level:
- haematology
- Key result
- Dose descriptor:
- LOEL
- Effect level:
- 500 mg/kg bw/day
- Sex:
- female
- Basis for effect level:
- haematology
- Critical effects observed:
- no
- Conclusions:
- Dermal NOEL (Rabbit, male): 5 mg/kg/day
Dermal NOEL (Rabbit, female): 50 mg/kg/day - Executive summary:
The purpose of this study was to evaluate the toxicity of the test substance when administered topically (dermally) to rabbits for approximately 21 consecutive days following the guidelines OECD 410 and U.S. EPA OPP 82-2. The test substance was applied to the clipped, intact skin of male and female White rabbits and occluded for approximately 6 hours daily for 21 consecutive days. Two groups of 5 male and 5 female rabbits each were treated dermally with 5 or 50 mg/kg, and one group of 10 male and 10 female rabbits was treated dermally with 500 mg/kg. The test material was moistened with approximately 5 mL of distilled water. A control group of 10 male and 10 female rabbits was treated with distilled water. Body weights were measured twice each week during the dosing period, and weekly during the recovery phase. The rabbits were examined daily for clinical signs of toxicity and mortality. Food consumption was measured weekly. Approximately 1 hour after the last 6-hour treatment (day 21), blood samples were collected from the auricular artery of each rabbit for a clinical laboratory evaluation. Five male and 5 female rabbits from each of the control and the 500 mg/kg groups, and all male and female rabbits from the 5 and 50 mg/kg groups were subsequently sacrificed for gross and microscopic examinations. Fourteen days following the last treatment, surviving rabbits in each of the control and high-dose groups were subjected to a clinical pathological examination and subsequently sacrificed for pathological examination.
No meaningful differences between control rabbits and rabbits treated with the test substance were observed during the dosing or recovery periods with respect to body weights or body weight gains.
No compound-related effects on food consumption or food efficiency values were observed during the study.
A greater incidence of hyperreactivity (increased reaction to stimuli, e.g. noise) was observed in the high-dose groups compared to the controls. A background incidence of hyperreactivity was evident in the control and in the low- and intermediate- dose groups, but the greater incidence in the high-dose groups was considered related, in part, to the cholinesterase inhibition produced by the compound. Other clinical observations noted during the study were considered unremarkable and unrelated to administration of the compound.
One female rabbit in the high-dose group was found dead during the study. The cause of death of this rabbit was related to a fracture at the thoracic- cervical junction of the vertebral column.
Plasma cholinesterase activities measured on test day 21 in male and female rabbits in the 500 mg/kg group were 36 and 55%, respectively, of the control mean. Brain cholinesterase levels were 48 and 68% of the control mean for male and female rabbits, respectively, in the high-dose group. The red blood cell activity was also lower in male and female rabbits in the 500 mg/kg group, but the decreases were considered slight and biologically insignificant. Plasma activity was 77% of the control mean in male rabbits in the 50 mg/kg group. Cholinesterase activities for female rabbits in the 50 mg/kg group were not different from control values. Following 14 days of recovery, cholinesterase activities were similar to control values. Other statistically significant results observed during the study for clinical pathology parameters were within the range of biological variation and were considered not to be compound related.
No compound-related effects on organ weights were observed. No gross or microscopic compound-related pathological changes were observed at the end of the dosing phase or after a 14-day recovery period.
In summary, compound-related effects on plasma and brain cholinesterase activities were observed in male and female rabbits in the 500 mg/kg groups and in male rabbits in the 50 mg/kg group. Consistent with the cholinesterase inhibition in rabbits in the high-dose groups was a slight increase in hyperreactivity. Under the conditions of this study, a no-observable-effect level (NOEL) for the administration of the test substance to intact skin of male rabbits was 5 mg/kg/day. In female rabbits, the NOEL was 50 mg/kg/day.
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
Additional information
Based on the results of four short-term feeding studies, the effects of methomyl are characterized primarily by minimal decreases in body weight parameters, nutritional status, and changes in haematology parameters. Findings included evidence of cholinesterase inhibition in rats and mice. In rats and mice, females and males were equally sensitive to the effects of methomyl. In these studies, test-substance-induced haematology changes, such as reticulocytosis were of minimal to mild clinical severity and were associated with evidence of regeneration (reversibility). No effects were observed in dogs.
Two 21-day dermal toxicity studies were conducted in rabbits. In the first study, statistically significant and biologically relevant decreases in plasma and brain cholinesterase activities, as well as increased incidences of hyper-reactivity, were observed in male and female rabbits at the high dose (500 mg/kg bw/day). A statistically significant decrease in plasma cholinesterase activity in the 50 mg/kg bw/day males was not considered biologically relevant. A second 21-day study was subsequently conducted to more precisely define the NOEL in rabbits by the dermal route of exposure. In this study, no biologically significant changes in plasma, RBC, and brain cholinesterase activities were observed in males or females at methomyl doses ranging from 15 to 90 mg/kg bw/day, thus confirming the lack of effects at 50 mg/kg bw/day in the previous study. The test sites in this second study were wrapped with a porous, semi-occlusive dressing, which for the purposes of assessing risk to workers, more closely resembles actual dermal exposure conditions. The results of the dermal toxicity studies suggest that methomyl is poorly absorbed by the dermal route. This conclusion is further supported by the high dermal LD50 for methomyl (>2000 mg/kg bw).
The NOAELs for chronic toxicity in rats were based on lower body weight gain and a mild haematology effect characterised by lower RBC count, haemoglobin, and haematocrit. In mice, the NOAEL levels for chronic toxicity were based on decreased survival and mild transient haematology changes, which were not present after 26 weeks. These changes were characterised by lower RBC count, haemoglobin, and haematocrit. The decreased survival due to systemic toxicity, resulted in the dietary concentration being reduced at Week 39. Systemic toxicity resulting in mortality was also observed in a 2-year dog study at dietary concentrations of 1000 ppm. In addition, haematology changes characterised by decreased RBC counts, haemoglobin, and haematocrit were also present in 1000 ppm dogs and histopathology changes in kidney and spleen characterised by pigment disposition, epithelial swelling (kidney), or extramedullary haematopoiesis (spleen) at 400 ppm.
Justification for classification or non-classification
Under the criteria of CLP Regulation [EC] No. 1272/2008, STOT RE may be assigned on the basis of a substance demonstrating evidence of significant or severe specific organ toxicity in a 90-day oral study at or below a guidance value of 100 mg/kg bw/day (basis of Category 2). This guidance value is adjusted in accordance with the Haber’s rule for studies of different durations. ‘Significant’ toxicity is taken to mean changes that clearly indicate functional disturbance or morphological changes that are toxicologically relevant. ‘Severe’ toxicity is considered to be more profound or serious and indicates changes that are of a considerably adverse nature with a significant impact on health. There is no evidence for any adverse findings or serious target organ toxicity in 90-day repeat dosing studies in rats, mice or dogs that meet the criteria of CLP Regulation [EC] No. 1272/2008 for STOT RE. The primary effects in these feeding studies were reductions in body weight, effects on hemoglobin or effects on cholinesterase activity. Similar effects were observed in longer term studies, as well as some pathology effects in kidney and spleen in a 2-year feeding study in dogs. Since these effects were considered minimal to mild and were reversible, the test substance is not classified for STOT RE according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
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