Methomyl

Neurotoxicity

Description of key information

Study Type	Species	Findings	Guideline	Reliability
Acute Oral Gavage	Rat	NOEL = 0.25 mg/kg LOEL = 0.5 mg/kg reversible inhibition of cholinesterase at dose levels of 0.5 mg/kg and above	EPA OPP 81-8	1
Acute Oral Gavage	Pre-weanling rat pups and adults	Brain cholinesterase in preweanling pups was mildly more sensitive to inhibition than adult brain cholinesterase at high doses. Difference in RBC cholinesterase inhibition between pups and adults was minimal.	no guideline	2
Acute Oral	Rat	LOEL 3 mg/kg Biologically adverse inhibition of blood (plasma and red blood cell) and brain cholinesterase activity 30 minutes after treatment. Recovery of effects by 2-3 hours post-exposure.	no guideline	2
Acute Oral	Rat	LOEL = 60 mg/kg based on lowered cholinesterase activity in whole blood	no guideline	2
Acute Oral Feed study	Rat	NOEL = 30 ppm LOEL = 60 ppm based on the increased incidence of a diminished response to a tail-pinch	U.S. EPA, Neurotoxicity. Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, addendum 10, Series 81, 82, and 83: National Technical Information Service, PB 91-154616, 1991	1
Acute Dermal	Rat	LOEL = 25 mg/kg Decreased plasma cholinesterase levels at 25 and 100 mg test substance. There was no effect on the red blood cell cholinesterase activity of the groups of rats treated with 25 mg or 100 mg methomyl for 24 or 72 hours.	no guideline	2
28-Day Feeding Study	Rat	LOEL = 800 ppm based on brain cholinesterase activity inhibition	no guideline	2
90-Day Feeding Study	Rat	NOEL = 150 ppm LOEL = 1500 ppm based on reduced body weights, reduced food consumption, reduced food efficiency, decreased forelimb and hindlimb grip strength, inhibition of brain cholinesterase, and clinical signs of toxicity	EPA 82-7	1
5-Month Feeding Study	Rat	no effect on blood cholinesterase activity after 5-months of administration via feed. NOEL = 800 ppm	no guideline	2
79-Day Feeding study	Rat	NOEL = 400 ppm LOEL = 800 ppm decreased blood cholinesterase levels	no guideline	2
75-Day Feeding study	Dog	LOEL = 250 ppm salivation, plasma and/or erythrocyte cholinesterase activity inhibition	no guideline	2
60-Day Feeding study	Rat	NOEL = 800 ppm no effect on cholinesterase levels after 60 days of exposure	no guideline	2
Acute intradermal injection	Guinea pig	LOEL = 1 mg local fasciculations observed	no guideline	2
Acute eye contact	Rabbit	LOEL = 10 mg local and systemic effects typical of anticholinesterase activity	no guideline	2
Acute oral	Hen	NOEL–28 mg/kg id not produce clinical signs of wing or leg weakness in hens	no guideline	2
Acute Inhalation	Rat	MTD = 150 mg/m3 based on impaired muscle coordination and intermittent convulsions observed at the 195 mg/m³	OECD 403, OPPTS 870.1300	1
In vitro	Human and Rat red blood cells	The enzyme from human RBCs was approximately six times more sensitive to inhibition by test substance than was that from the rat. The I50 values were 0.265E-5 and 1.56E-5 M, respectively. Acetylcholinesterase from the rat regenerates approximately 1.4 times faster than the human enzyme; regeneration half-lives were 26.6 and 38.0 minutes, respectively	no guideline	2

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: U.S. EPA Pesticide Assessment Guidelines Subdivision F, 82-7

Deviations:

yes

Remarks:

Test substance was administered in the diet and that brain cholinesterase activity was assessed in homogenates of whole brain only.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Substance name: Methomyl Technical
- Substance ID: DPX-X1179
- Lot#: X1179-512
- Purity: 98.6%

Species:

rat

Strain:

other: Crl:CD®BR

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: diet

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

91 days

Frequency of treatment:

Daily

Dose / conc.:

20 ppm

Dose / conc.:

50 ppm

Dose / conc.:

150 ppm

Dose / conc.:

1 500 ppm

No. of animals per sex per dose:

42

Control animals:

yes, plain diet

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related and statistically significant clinical observations were observed in the male and female 1500 ppm rats throughout the study. Tremors were observed in 32/42 1500 ppm males and 23/42 1500 ppm females during test days 0–27 or 0-28, respectively. The incidence of tremors continued to be statistically significant for the 1500 ppm males (2/32) during test days 28-49 and for the 1500 ppm females (2/32) during test days 29-55. Tremors were not observed in the 1500 ppm males or females after test day 55 and tremors were not observed in the 20, 50, or 150 ppm concentration groups at any time during the study. The 1500 ppm male rats also exhibited statistically increased incidences of aggressive behavior (4/22 for test days 50-85) and statistically increased incidences of hyperreactivity (5/22 for test days 50-85). A statistically significant increase in the incidence of alopecia was observed in the 1500 ppm females (14/32 for test days 29-55; 12/22 for test days 56-86; and 6/12 for test days 87-93).
No other test substance-related clinical observations occurred during the study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test substance-related mortality occurred during the study. One male rat in the 50 ppm group was found dead on test day 31. The only finding noted on the gross necropsy of this rat was that severe autolysis was present. The cause of the rat’s death is unknown but it is not considered to be test substance-related since no abnormalities were observed for this rat at any time during the study including the gross necropsy and since the rat was in an intermediate dose level which produced no adverse effects on any parameter throughout the study. One male rat in the 20 ppm group was sacrificed in extremis on test day 89 because of an injury to the mouth and nose that was interfering with the rat’s ability to eat. Gross necropsy of this rat revealed that the rat had a fractured nose.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant and test substance-related decreases in body weight and body weight gain occurred in the 1500 ppm male and female rats. The body weights of the 1500 ppm male and female rats were statistically decreased from the control value on each weigh day from test Day 7 through test Day 91. The body weights of the male and female 1500 ppm male rats were decreased 24% and 19%, respectively, on test Day 91.

The body weight gains of the 1500 ppm male rats were significantly decreased from the control value for test Days 0-7, 7-14, 14-21, 21-28, 35-42, 42-49, 63-70, and for the overall interval of test Days 0-91. The body weight gains of the 1500 ppm female rats were significantly decreased from the control group for test Days 0-7, 42-49, and the overall interval of 0-91.
The body weights and body weight gains of the 20, 50, and 150 ppm male and female rats were unaffected by the test substance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significant and test substance-related decreases in the mean daily food consumption of the 1500 ppm male and female rats occurred throughout the study. The mean daily food consumption of the 1500 ppm male rats was significantly decreased from the control value every week of the study and for the overall interval of test Days 0-91. The mean daily food consumption of the 1500 ppm female rats was significantly decreased from the control value on test Days 0-7, 7-14, 14-21, 21-28, 28-35, 35-42, 42-49, 84-91, and for the overall interval of test Days 0-91.
The mean daily food consumption of the 20, 50, and 150 ppm male and female rats were unaffected by the test substance.

The mean daily intake values of the test substance for the 0, 20, 50, 150, and 1500 ppm males over the 91-day test period were 0, 1.29, 3.14, 9.42, and 94.9 mg/kg/day, respectively. The mean daily intake values of the test substance for the 0, 20, 50, 150, and 1500 ppm females over the 91-day test period were 0, 1.48, 3.85, 11.2, and 113 mg/kg/day, respectively.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The mean food efficiency of the 1500 ppm male rats was significantly decreased from the control value on test Days 0-7, 7-14, 21-28, 35-42, 63-70, and for the overall interval of test Days 0-91. The mean food efficiency of the 1500 ppm female rats was significantly decreased from the control value on test Days 0-7, 7-14, and for the overall interval of test Days 0-91.
The food efficiency values of the 20, 50, and 150 ppm male and female rats were unaffected by the test substance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Brain Cholinesterase Activity: Male and female rats in the 1500 ppm groups exhibited minimal to mild, test substance-related inhibition of brain cholinesterase activity. This effect was most pronounced and most consistent in the male 1500 ppm group at the week-8 sampling time, when the mean brain cholinesterase activity value was 81% of the control group mean (statistically significant). At the week-4 and week-13 sampling times in the 1500 ppm male group, mean brain cholinesterase activity was 92% and 95% of the control group mean, respectively, and the differences were not statistically significant. At the week-13 sampling time, one male rat in the 1500 ppm group had brain cholinesterase activity which was less than 50% of the control group mean.

Male and female rats in the 1500 ppm groups exhibited minimal to mild, test substance-related inhibition of brain cholinesterase activity. This effect was most pronounced and most consistent in the male 1500 ppm group at the week-8 sampling time, when the mean brain cholinesterase activity value was 81% of the control group mean (statistically significant). At the week-4 and week-13 sampling times in the 1500 ppm male group, mean brain cholinesterase activity was 92% and 95% of the control group mean, respectively, and the differences were not statistically significant. At the week-13 sampling time, one male rat in the 1500 ppm group had brain cholinesterase activity which was less than 50% of the control group mean.
The test substance-related effects on brain cholinesterase activity in the 1500 ppm male and female groups correspond to cholinergic signs which were observed in these groups during the early part of the study.

Red Blood Cell Cholinesterase Activity: There was no evidence for inhibition of red blood cell cholinesterase activity in any group of male or female rats during this study.

Plasma Cholinesterase Activity: There was some evidence for mild, test substance-related inhibition of plasma cholinesterase activity in 1500 ppm males, especially at the 8-week sampling time. Although the mean value of this group was not significantly lower than the mean value of the control group at any sampling time, at the 8-week sampling time two rats had plasma cholinesterase activity which was 65% or less compared to the control group mean. None of the other male groups or any group of females had inhibition of plasma cholinesterase activity during this study. Lack of evidence for inhibition of plasma cholinesterase activity can be attributed to the aforementioned mildness of the effects and the inherent biological variability of the plasma cholinesterase activity.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Forelimb and Hindlimb Grip Strength: Statistically significant and test substance-related decreases in forelimb and hindlimb grip strength occurred in the 1500 ppm males on week 13. Female rats at 1500 ppm were unaffected by the test substance. The scores for 20, 50, and 150 ppm male and female rats were comparable with the control group values at each test period during the study.

Hindlimb Foot Splay: There were no toxicologically remarkable changes in hindlimb foot splay for any male or female rats at any concentration level.

Other Functional Observational Battery Parameters: Statistically significant and test substance-related findings were observed in the 1500 ppm males and females. The findings that were common to both males and females included difficulty removing the rats from their cages, difficulty handling the rats, ptosis, and abnormal pupillary responses. On Week 4, difficulty removing the rat from their home cage was observed in 3/12 of the 1500 ppm males and difficulty handling the rat was scored for 4/12 of the 1500 ppm male rats. The difficult handling of the rats continued on Week 8 in 3/12 of the 1500 ppm males. The 1500 ppm females were difficult to remove (2/12) and difficult to handle (2/12) on Week 8.
Ptosis (slightly closed eyelids) was observed in both the 1500 ppm males and females. On Week 4, ptosis was observed in 2/12 females when the rats were removed from the home cage and in 2/12 females during the open field assessment. Ptosis was also evident on Week 8 in the 1500 ppm males and females during the removal from cage assessment (3/12 and 2/12, respectively) and in the 1500 ppm males during the open field assessment (2/12).
Abnormal pupillary responses were observed on Weeks 4, 8, and 13 for the 1500 ppm males and females. On Week 4, the behavior was observed in 5/12 males and 4/12 females. On Week 8, the abnormal responses were recorded in 6/12 males and 2/12 females and on week 13, 5/12 males and 4/12 females at the 1500 ppm concentration group were scored with abnormal pupillary responses.
Statistically significant and test substance-related findings limited to the 1500 ppm females included low arousal and abnormal gait. On Week 4, 3/12 females from the 1500 ppm concentration group were scored as having a low arousal. In addition, abnormal gait was scored for the 1500 ppm females (4/12) on Week 8. These rats were described as having a high carriage.

Motor Activity: There were no test substance-related effects on either parameter measured in the motor activity test: duration of movement and number of movements.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related gross lesions observed in neuropathology rats in this study.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related microscopic observations in neuropathology rats in this study. The microscopic lesions that were observed were predominantly minimal changes (axon/myelin degeneration) that typically occurred sporadically in only one or two individual nerve fibers in any given anatomic location. Lesions observed in the high concentration (1500 ppm) males and females were essentially the same in character and severity as those observed in the control (0 ppm) males and females, and have been described as occurring spontaneously in the rat. Thus, lesions observed microscopically in this study were judged not to be test substance-related.

Key result

Dose descriptor:

NOEL

Effect level:

150 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

Dose descriptor:

LOEL

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: based on reduced body weights, reduced food consumption, reduced food efficiency, decreased forelimb and hindlimb grip strength, inhibition of brain cholinesterase, and clinical signs of toxicity in the 1500 ppm male and/or female rats.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 500 ppm

System:

nervous system

Treatment related:

yes

Conclusions:

NOEL (Rat): 150 ppm (9.42 mg/kg/day for males and 11.2 mg/kg/day for females)

Executive summary:

The purpose of this study was to assess the potential neurotoxicity of subchronic exposure to test substance in adult rats by neurobehavioral, neuropathological, and neurochemical tests. The study was conducted according to U.S. EPA guideline, subdivision F, 82-7. The test substance was administered by ingestion in the diet to adult male and female rats (42 rats/concentration) at concentrations of 0, 20, 50, 150, or 1500 ppm for approximately 91 days. The mean daily intake values of test substance for the 0, 20, 50, 150, and 1500 ppm males over the 91-day test period were 0, 1.29, 3.14, 9.42, and 94.9 mg/kg/day, respectively. The mean daily intake values of test substance for the 0, 20, 50, 150, and 1500 ppm females over the 91-day test period were 0, 1.48, 3.85, 11.2, and 113 mg/kg/day, respectively. Chemical analysis verified that the test substance was homogeneously distributed in the diets and that the diets were at expected concentrations. The test substance was stable in the diets for the period of time that covered the feeding and storage conditions in the study.

All rats/concentration of each gender were evaluated for clinical observations, body weights, and food consumption weekly throughout the study. Twelve rats/gender/concentration were designated as the neurobehavioral subgroup and these rats were evaluated with a functional observational battery (FOB) and motor activity (MA) test prior to test substance exposure (baseline) and again during Weeks 4, 8, and 13. Six neurobehavioral rats/gender/concentration underwent in situ perfusion following Week 13. Tissues from all rats were saved but microscopic evaluation was conducted on the rats in the 0 and 1500 ppm concentration groups only. Thirty rats/gender/concentration were designated as the clinical pathology subgroup. Cholinesterase activities in plasma and red blood cells were measured for the first set of 10 clinical pathology rats/gender/concentration prior to compound administration to establish baseline levels. Cholinesterase activities in brain tissue, plasma, and red blood cells were measured again in the first set of clinical pathology rats during Week 4, in the second set during Week 8, and in the third set during Week 13.

No test substance-related mortality occurred during the study. Clinical signs of toxicity were observed in the 1500 ppm male and female rats. Both the 1500 ppm males and females exhibited tremors during the early portion of the study. The 1500 ppm males also exhibited increased incidences of aggressive behavior and hyperreactivity. An increased incidence of alopecia was observed in the 1500 ppm females. Statistically significant and test substance-related decreases in body weight, body weight gain, food consumption, and food efficiency occurred in the 1500 ppm male and female rats.

During the functional observational battery, the 1500 ppm males and females exhibited increased incidences of difficult removal from their home cages, difficult handling, ptosis, and abnormal pupillary responses. The 1500 ppm females also had decreased incidences of defecation and urination, and increased incidences of low arousal and abnormal gait. Statistically significant and test substance-related decreases in forelimb and hindlimb grip strength occurred in the 1500 ppm males on Week 13. The forelimb and hindlimb grip strength scores of the female rats at all concentration levels were unaffected by the test substance. There were no statistically significant or toxicologically remarkable changes in hindlimb foot splay for male or female rats during the study. The two dependent variables measured in the motor activity test, duration of movements and number of movements, were unaffected by the test substance. Neuropathological evaluation revealed no findings related to the test substance.

Male and female rats in the 1500 ppm concentration groups exhibited minimal to mild, test substance-related inhibition of brain cholinesterase activity. There was no evidence for inhibition of red blood cell cholinesterase activity in any group of male or female rats during the study. There was some evidence of mild, test substance-related inhibition of plasma cholinesterase activity in the 1500 ppm males during the Week 8 assessment.

Under the conditions of this study, the NOEL for the test substance in male and female rats is 150 ppm based on reduced body weights, reduced food consumption, reduced food efficiency, decreased forelimb and hindlimb grip strength, inhibition of brain cholinesterase, and clinical signs of toxicity (tremors, aggressive behavior, hyperreactivity, abnormal pupil responses) in the 1500 ppm male and/or female rats.

Reference 2

Endpoint:

neurotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

other: U.S. EPA, Neurotoxicity. Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, addendum 10, Series 81, 82, and 83: National Technical Information Service, PB 91-154616, 1991

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Substance name: Methomyl Technical
- Substance ID: DPX-X1179
- Lot#: X1179-394
- Purity: 98.35%

Species:

rat

Strain:

other: Crl:CD®(SD)BR

Sex:

male

Route of administration:

oral: feed

Vehicle:

other: diet

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

2 hours

Frequency of treatment:

Once

Dose / conc.:

30 ppm

Remarks:

0.953 mg/kg bw

Dose / conc.:

60 ppm

Remarks:

1.88 mg/kg bw

Dose / conc.:

120 ppm

Remarks:

3.74 mg/kg bw

Dose / conc.:

360 ppm

Remarks:

9.98 mg/kg bw

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weights of the rats were within ±15% of the mean amongst the dose groups. To achieve the expected dietary intake of the test substance for each treatment group, the rats should have weighed 300 grams and eaten 10 grams of their respective diet. However, the body weights of the rats ranged from 272.8 g to 348.7 grams. The actual body weights resulted in higher than expected intakes of test substance for the rats lighter than 300 grams that consumed the entire 10 grams of diet and lower than expected intakes for the rats heavier than 300 grams. Intakes at 30, 60, and 120 ppm groups varied by less than 12% from the mean for the group, while animals in 360 ppm group varied by as much as 30%.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumed per rat within the 2-hour feeding period for the 0, 30, 60, 120, and 360 ppm groups was 10.1, 9.9, 9.8, 9.8, and 8.6 grams, respectively. The mean intake of the test substance for male rats fed diets containing 0, 30, 60, 120, and 360 ppm of test substance was 0, 0.953, 1.88, 3.74, and 9.98 mg of test substance per kg of body weight, respectively. The lower than expected mean intake for the animals in the 360 ppm group was attributed to the animals not consuming all of their feed in the allotted time, most likely due to a palatability problem.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Plasma, erythrocyte, and brain cholinesterase activity were decreased in the 120 and 360 ppm groups (statistically significant). The mean values for these parameters exhibited a dose-response relationship and the changes were unequivocally compound-related. Furthermore, since the mean values were consistently decreased by more than 20% compared to the control group mean (except CHE-P was decreased only 17%), the changes in cholinesterase activity were considered to be biologically adverse.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

During manipulations, no significant findings were observed with respect to responses to the auditory stimulus; only one rat in the 360 ppm group did not react to the auditory stimulus. However, seven out of ten rats from the 360 ppm group exhibited no reaction to the approach-and-touch stimulus; this finding was statistically significant. The incidences of no reaction to the tail-pinch in the 60, 120, and 360 ppm groups were statistically significant.
The low arousal and no reaction to approach-and-touch stimulus observed in the 360 ppm dose group and the lack of reaction to the tail-pinch in the 60, 120, and 360 ppm dose groups were considered to be adverse, compound-related effects because the findings were 1) statistically significant, 2) exhibited a dose-response relationship, and 3) the pattern of findings can potentially reflect a reduced ability to adapt to unexpected, noxious stimuli in the environment.

Key result

Dose descriptor:

NOEL

Effect level:

30 ppm

Based on:

test mat.

Sex:

male

Remarks on result:

other: Based on the increased incidence of a diminished response to a tail-pinch noted during conduct of the modified functional observational battery in the 60 ppm dose group

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

60 ppm

System:

nervous system

Treatment related:

yes

Dose response relationship:

yes

Conclusions:

NOEL (Rat): 30 ppm (0.953 mg/kg bw) based on the increased incidence of a diminished response to a tail-pinch noted during conduct of the modified functional observational battery in the 60 ppm dose group

Executive summary:

The purpose of this study was to investigate the acute dietary toxicity of the test substance when incorporated into chow and fed to male rats for an approximate 2-hour feeding period. Approximately 10 grams of diet containing 0, 30, 60, 120, or 360 ppm of test substance was presented for consumption to young adult male rats (10 per group) for approximately 2 hours. These concentrations were expected to result in a dietary intake of 0, 1, 2, 4, or 12 mg of test substance per kg of body weight.

During the 18 days preceding the day of exposure, the rats were preconditioned to eat within a 2-hour time frame. The rats were weighed and observed for clinical signs of toxicity prior to administration of the feed. During exposure, a cage-side observation for clinical signs of toxicity was conducted. Approximately 1 hour after the end of the 2-hour feeding period, a modified functional observational battery (MFOB) was conducted on each rat. Immediately after the modified FOB, blood was collected from the orbital sinus of each rat for determination of plasma and erythrocyte cholinesterase activities (CHE-P and CHE-R, respectively). The rats were then sacrificed, and brains were weighed and collected for determination of brain cholinesterase (CHE-B) activity.

The mean intake of test substance for male rats fed diets containing 0, 30, 60, 120, and 360 ppm was 0, 0.953, 1.88, 3.74, and 9.98 mg of test substance per kg of body weight, respectively. The lower than expected mean intake of test substance for the animals in the 360 ppm group was attributed to the animals not consuming all of their feed within the allotted time period.

Plasma, erythrocyte, and brain cholinesterase activities were significantly decreased in the 120 and 360 ppm groups. The mean values for these parameters exhibited a dose-response relationship and the changes were considered compound-related and biologically adverse.

No clinical signs of toxicity were observed during the cage-side observations conducted during exposure. During the modified FOB, the rats were evaluated in-cage, in free-roaming space, and during manipulations. No statistically significant findings were observed during the in-cage observations. In free-roaming space, no abnormal findings were noted with respect to righting reflex, labored breathing, convulsions, coordination, ability to locomote, gait characteristics, vocalizations, or palpebral closure. A statistically significant difference from control animals was noted in the 360 ppm group in arousal. Seven out of 10 rats exhibited low arousal, which was defined as some exploratory movements with periods of immobility. During manipulations, no significant findings were observed in reaction to auditory stimulus. However, 7 out of 10 rats from the 360 ppm group exhibited no reaction to the approach-and-touch stimulus; this finding was statistically significant. Another statistically significant observation noted in the 60 ppm, 120 ppm, and 360 ppm groups was no reaction to a tail-pinch stimulus. These significant findings were considered to be adverse effects of exposure to the test substance.

The NOEL for this study was 30 ppm or 1 mg/kg bw for male rats. The NOEL was based on the increased incidence of a diminished response to a tail-pinch noted during conduct of the modified functional observational battery in the 60 ppm dose group.

Reference 3

Endpoint:

neurotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline available

Principles of method if other than guideline:

The purpose of this study was to determine the relative sensitivity of preweanling rat pups and adult rats to inhibition and recovery of cholinesterase activity following administration of a single oral dose of test substance. In order to accomplish this goal, 3 subsets of experiments were conducted. In Subset 1, the time to peak inhibition and recovery of cholinesterase activity in pups was evaluated. In Subset 2, the dose response of cholinesterase activity inhibition was determined at the time of peak inhibition in pups. In Subset 3, the inhibition of cholinesterase activity in adults administered different dosages at the time of peak effect and after approximately 4 hours of recovery time was evaluated in adult male and female rats.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Substance name: Methomyl Technical
- Substance ID: DPX-X1179
- Lot#: X1179-512
- Purity: 98.8%

Species:

rat

Strain:

other: Crl:CD®(SD)IGS BR

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

NanoPure®

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Single dose

Dose / conc.:

0.3 mg/kg bw (total dose)

Remarks:

Subset 1 [11-day old (preweanling) rat pups]

Dose / conc.:

0.1 mg/kg bw (total dose)

Remarks:

Subset 2 (11-day old rat pups)

Dose / conc.:

0.2 mg/kg bw (total dose)

Remarks:

Subset 2 (11-day old rat pups)

Dose / conc.:

0.3 mg/kg bw (total dose)

Remarks:

Subset 2 (11-day old rat pups)

Dose / conc.:

0.4 mg/kg bw (total dose)

Remarks:

Subset 2 (11-day old rat pups)

Dose / conc.:

0.3 mg/kg bw (total dose)

Remarks:

Subset 3 [42-day old (young adult) rats]

Dose / conc.:

0.5 mg/kg bw (total dose)

Remarks:

Subset 3 [42-day old (young adult) rats]

Dose / conc.:

0.75 mg/kg bw (total dose)

Remarks:

Subset 3 [42-day old (young adult) rats]

No. of animals per sex per dose:

Subset 1: 5
Subset 2 and 3: 10

Control animals:

yes, concurrent vehicle

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Subset 1: The lowest RBC and brain cholinesterase activities occurred at the first time point (30 minutes after dosing) for males. Female RBC cholinesterase activities were similar at both the 30 and 60 minute time points. For male pups, RBC and brain cholinesterase activities were similar to controls at 90 minutes after dosing. For female pups, RBC and brain cholinesterase activities were similar to controls by 180 and 60 minutes, respectively, after dosing.
Subset 2: Both RBC and brain cholinesterase activities decreased in a dose-dependent pattern across the range of doses tested. For both parameters, activities were similar between males and females at a given dose. At 0.4 mg/kg, the highest dose tested, RBC cholinesterase was inhibited 49% in males and females while brain cholinesterase was inhibited 41 and 42% for males and females, respectively. For a given dose and sex, the magnitude of inhibition was similar for RBC and brain cholinesterase activities.
Subset 3: At 30 minutes post-dosing, in adult males, RBC cholinesterase was more sensitive to inhibition than brain cholinesterase (41% vs. 19% for RBC and brain, respectively, at the 0.75 mg/kg dose). However, in adult females, brain cholinesterase was similar or minimally more sensitive to inhibition (25% and 29% for RBC and brain, respectively, at the 0.75 mg/kg dose). These apparent sex differences in adults are likely the result of the marginal inhibition present at the low doses administered and the variability in cholinesterase measurements, rather than a true biological difference.
At 240 minutes after dosing, RBC and brain cholinesterase activities of treated male and female rats, were similar to the cholinesterase activity of concurrent control animals. Any apparent changes in this study at 240 minutes after dosing were considered to represent normal variability in RBC and brain cholinesterase measurements.

Key result

Based on:

test mat.

Sex:

male/female

Remarks on result:

other:

Remarks:

Red blood cell and brain cholinesterase activities were marginally decreased in pre-weanling male and female pups at 30 minutes after dosing with the test substance and returned to baseline by 240 minutes post dose. For pups, the magnitude of inhibition was similar for RBC and brain cholinesterase activities. RBC cholinesterase was inhibited to a greater extent than brain cholinesterase for adult males but inhibited to a lesser extent for adult females. Difference in RBC cholinesterase inhibition between pups and adults was minimal. Mean values and linear trend lines for brain cholinesterase inhibition were greater in pups than adults for both males and females. In addition, there was no significant overlap in inhibition values for pups and adults dosed with either the common dose (0.3 mg/kg) or a near common dose (0.4 mg/kg in pups and 0.5 mg/kg in adults). However, at the lowest dose administered to pups (0.1 mg/kg), the range of values for brain cholinesterase inhibition overlapped with the linear trend line in dose response for brain cholinesterase in adults.

Conclusions:

Brain cholinesterase in preweanling pups is mildly more sensitive to inhibition than adult brain cholinesterase at high doses. Difference in RBC cholinesterase inhibition between pups and adults was minimal in this study.

Executive summary:

The purpose of this study was to determine the relative sensitivity of male and female preweanling rat pups (lactation day 11) and young adult rats to inhibition and recovery of cholinesterase activity following administration of a single oral dose of the test substance. In order to accomplish this goal, 3 subsets of experiments were conducted. In Subset 1, the time to peak inhibition and recovery of cholinesterase activity in pups were evaluated. In Subset 2, the dose response of cholinesterase activity inhibition was determined at the time of peak inhibition in pups. In Subset 3, dose responses at peak inhibition and at recovery were evaluated in young adult rats. The dosages of test substance for pups and adults, and peak and recovery time points for young adults were selected based on data from previously conducted method development studies and previously conducted studies in adult rats.

Subset 1: Groups of approximately 5 male and 5 female 11-day old (preweanling) rat pups were administered a single oral gavage dose of 0 or 0.3 mg/kg test substance in NanoPure® water. Approximately 30, 60, 90, 120, 180, 240, or 360 minutes after dose administration, the pups were euthanized, and blood and brains were collected for determination of cholinesterase activity. Groups of control rats were administered a single oral dose of NanoPure® water; approximately 60, 120, or 240 minutes after dose administration, the pups were euthanized, and blood and brains were collected for determination of cholinesterase activity.

Subset 2: Groups of approximately 10 male and 10 female 11-day old rat pups were administered a single oral gavage dose of 0, 0.1, 0.2, 0.3, or 0.4 mg/kg test substance in NanoPure® water. Approximately 30 minutes after dose administration, the pups were euthanized, and blood and brains were collected for determination of cholinesterase activity.

Subset 3: Groups of 10 male and 10 female 42-day old (young adult) rats were administered a single oral gavage dose of 0, 0.3, 0.5, or 0.75 mg/kg test substance in NanoPure® water. Approximately 30 or 240 minutes after dose administration, the rats were euthanized, and blood and brains were collected for determination of cholinesterase activity.

The results of the study are as follows:

In untreated preweanling pups, RBC (red blood cell) cholinesterase activities (U/L) were higher and brain cholinesterase activities (U/g) were lower compared to the corresponding activities in untreated adults.

The greatest degree of cholinesterase inhibition in preweanling male and female pups dosed with 0.3 mg/kg generally occurred at 30 minutes after dosing. Based on this and previous studies in adult rats, 30 minutes was chosen as the sampling time for peak effects in subsequent dose-response studies.

RBC and brain cholinesterase activities were decreased in preweanling male and female pups at 30 minutes after dosing with test substance, and returned to baseline by 240 minutes post dose. For pups, the magnitude of inhibition was similar for RBC and brain cholinesterase activities.

RBC and brain cholinesterase activities were only marginally affected in male and female adults at 30 minutes after dosing with methomyl, and returned to baseline by 240 minutes after dosing. RBC cholinesterase was inhibited to a greater extent than brain cholinesterase for adult males but inhibited to a lesser extent for adult females. These apparent sex differences in adults are likely the result of the marginal inhibition present at the low doses administered and the variability in cholinesterase measurements.

Mean values and linear trend lines for RBC cholinesterase inhibition were greater in pups than adults for both males and females. However, individual RBC cholinesterase inhibition values overlapped for pups and adults dosed with either the common dose (0.3 mg/kg) or a near common dose (0.4 mg/kg in pups and 0.5 mg/kg in adults). As a result of the inherent variability of this parameter, difference in RBC cholinesterase inhibition between pups and adults was minimal in this study.

Mean values and linear trend lines for brain cholinesterase inhibition were greater in pups than adults for both males and females. In addition, there was no significant overlap in inhibition values for pups and adults dosed with either the common dose (0.3 mg/kg) or a near common dose (0.4 mg/kg in pups and 0.5 mg/kg in adults). These results suggest that brain cholinesterase in preweanling pups is mildly more sensitive to inhibition than adult brain cholinesterase at high doses. However, at the lowest dose administered to pups (0.1 mg/kg), the range of values for brain cholinesterase inhibition overlapped with the linear trend line in dose response for brain cholinesterase in adults.

Reference 4

Endpoint:

neurotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 81-8 (Neurotoxicity Screening Battery)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Substance name: Methomyl Technical
- Substance ID: DPX-X1179
- Lot#: X1179-512
- Purity: 98.6%

Species:

rat

Strain:

other: Crl:CD®BR

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

commercially supplied bottled water (HPLC grade)

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Single dose

Dose / conc.:

0.25 mg/kg bw (total dose)

Dose / conc.:

0.5 mg/kg bw (total dose)

Dose / conc.:

0.75 mg/kg bw (total dose)

Dose / conc.:

2 mg/kg bw (total dose)

No. of animals per sex per dose:

52

Control animals:

yes, concurrent vehicle

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

On test Day 1, post-dosing clinical observations were recorded at the anticipated time of peak signs (approximately 30 minutes post-dosing) for the clinical pathology subgroup rats only. Male (5/40) and female (5/40) rats in the 2 mg/kg clinical pathology subgroup exhibited tremors on test Day 1. These incidence values were significantly different from control and are regarded as effects of the test substance. One female clinical pathology rat in the 0.75 mg/kg dose group exhibited tremors on test Day 1. This incidence was not significantly different from the control group. Other clinical findings on test Day 1 included one female rat in the 2 mg/kg clinical pathology subgroup that exhibited salivation and another female rat in this subgroup that had a clear discharge from the eye (lacrimation). Tremors, salivation, and lacrimation are known effects of cholinesterase inhibition and are regarded as adverse effects of the test substance at the 2 mg/kg dose level. Tremors and salivation were not observed in any animal after test Day 1.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test substance-related mortality was observed in the study. One female rat in the 0.75 mg/kg was sacrificed in extremis on test Day 2. Gross pathology revealed a perforation of the esophagus which probably occurred during the gavage procedure on test Day 1.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Male and female rats dosed with 0.5 mg/kg and above exhibited inhibition of cholinesterase activity at the test Day 1 sampling time. By the Day 2 sampling time cholinesterase activity was comparable to controls at all dose levels. These data indicate that the test substance caused biologically adverse inhibition of cholinesterase activity immediately after treatment at 0.5 mg/kg and above, followed by rapid recovery from inhibition.
At the Day 1 sampling time, mean plasma, RBC, and brain cholinesterase activities were decreased in the 0.75 and 2.0 mg/kg male and female groups. In these groups, mean blood and brain cholinesterase activities were decreased to 77% or less of the control group mean and the changes were statistically significant (except for the decrease in RBC cholinesterase activity in the 0.75 mg/kg male group).
At 0.5 mg/kg, mean brain cholinesterase activity was decreased to 81% and 80% of the control group mean in males and females, respectively at the Day 1 sampling time, and the changes were statistically significant. In addition, plasma and RBC cholinesterase activities in 0.5 mg/kg females were 69% and 75% of the control group mean, respectively, and the change in RBC cholinesterase activity was statistically significant. Plasma and RBC cholinesterase activities in 0.5 mg/kg males were 87% and 95% of control, respectively (not statistically significant).
The changes in cholinesterase activity in the 2.0 mg/kg groups were accompanied by neurobehavioral signs of excessive cholinergic stimulation. These results indicate that, when brain cholinesterase activity was inhibited to approximately 50% of control values, clinical signs of excessive cholinergic stimulation occurred (tremors, salivation, and lacrimation). In the 0.75 mg/kg groups there was only one female rat which exhibited mild neurobehavioral signs of excessive cholinergic stimulation (tremors) when brain cholinesterase activity was inhibited to 75% of controls. In the 0.5 mg/kg groups, however, no cholinergic signs were observed when brain cholinesterase activity was 81% and 80% of controls in males and females, respectively. Therefore, the magnitude of the brain cholinesterase changes observed in the 0.5 mg/kg groups are considered to be of equivocal biological importance.
At 0.25 mg/kg, there were no statistically significant or biologically adverse changes in blood or brain cholinesterase activity in males or females.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Forelimb grip strength: There were no statistically significant or toxicologically remarkable changes in forelimb grip strength of male and female rats at any dose level.
Hindlimb grip strength: The increased grip strength values in the 0.25 and 0.5 mg/kg dose groups are not considered to be related to treatment. All other hindlimb grip strength values in male rats were comparable to control during the study. No statistically significant or toxicologically remarkable findings were observed in the female rats.
Foot splay: Mean foot splay values were unaffected by the test substance in male and female rats at all time periods tested.
Other FOB findings: There were no toxicologically significant or toxicologically remarkable findings in male or female rats during the baseline FOB assessment.
Findings consistent with known effects of cholinesterase inhibitors were observed in 2 mg/kg male rats on test Day 1. Four of 12 male rats in the 2 mg/kg group exhibited tremors on Day 1, and lacrimation was identified in 1/12 male rats. Tremors and lacrimation observed in the 2 mg/kg male neurotoxicity rats are considered effects of the test substance.
The incidence levels of all other FOB parameters were comparable among the dose groups on test Day 1.
Motor activity: No test substance or toxicologically important findings in motor activity on test Days 1, 8, and 15

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related gross lesions observed in neuropathology rats in this study.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related microscopic observations in neuropathology rats in this study. The microscopic lesions that were observed were minimal changes (axon/myelin degeneration) that typically occurred sporadically in only one or two individual nerve fibers in any given anatomic location.

Key result

Dose descriptor:

NOEL

Effect level:

0.25 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Dose descriptor:

LOEL

Effect level:

0.5 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.5 mg/kg bw (total dose)

System:

nervous system

Treatment related:

yes

Dose response relationship:

yes

Conclusions:

NOEL (rat): 0.25 mg/kg based on reversible inhibition of cholinesterase at dose levels of 0.5 mg/kg and above

Executive summary:

The purpose of this study was to assess the potential neurotoxicity from acute exposure to test substance in adult rats by neurobehavioral and neuropathological tests, and cholinesterase activity. The neurotoxicity from acute oral exposure to the test substance had not been previously studied through the use of a functional observational battery (FOB), motor activity (MA) test, or neuropathology (NP) tests.

The study was conducted following the guideline, EPA OPP 81-8.

The test substance was administered once via gavage on test Day 1 to adult male and female rats (52 rats/dose) at dosages of 0, 0.25, 0.5, 0.75, or 2 mg/kg. Chemical analysis verified that the concentrations of the test substance in the dose solutions ranged from 92.4 to 104.5% of the nominal value. Twelve rats/group were designated as the neurotoxicity subgroup and clinical observations, body weights, and food consumption were recorded periodically throughout the test period. Functional observational battery and motor activity tests were conducted prior to exposure, approximately 30 minutes post-dosing on test Day 1, and again on test Days 8 and 15. Six rats/gender/group for the neurotoxicity subgroup were sacrificed and tissues were fixed with in situ perfusion techniques on test Day 16. Tissues from the control and high level rats were processed and examined for neuropathological effects. Forty rats/gender/group were designated as the clinical pathology subgroup. Cholinesterase activity in plasma and erythrocytes were measured for the first ten clinical pathology rats/group prior to compound administration to establish baseline levels. Cholinesterase activity in brain tissue, plasma, and erythrocytes were measured again in the same ten animals/group at approximately 30 minutes post-dosing on test Day 1. Cholinesterase activity was measured in the second ten clinical pathology rats/group on test Day 2. The last twenty clinical pathology rats/group were dosed and evaluated for clinical signs on test Days 1 and 2; however, they were not needed for cholinesterase assessments on test Days 8 and 15 and consequently were removed from the study.

Body weights and food consumption of male and female neurotoxicity subgroup rats were unaffected by the test substance. Mean body weight gains of the males were unaffected by the test substance. Body weight gains were decreased in the female rats administered 2 mg/kg during the interval of test Days 2-8 but had returned to control values during the interval of test Days 8-15. The functional observational battery test on test Day 1 revealed tremors and lacrimation in the male rats administered 2 mg/kg. No compound-related effects were observed on test Days 8 and 15. There were no compound-related effects on forelimb grip strength, hindlimb grip strength, and hindlimb foot splay. Motor activity scores of treated rats were unaffected by the test substance. Neuropathological evaluation revealed no adverse findings related to the test substance.

Clinical signs of toxicity were observed in male and female animals of the clinical pathology subgroup which had received 2 mg/kg of the test substance. These signs included tremors, salivation, and/or lacrimation approximately 30 minutes post-dosing on test Day 1. No clinical signs of toxicity were observed on test Day 2. Inhibition of blood and brain cholinesterase activity was detected in males and females administered 0.5, 0.75, and 2 mg/kg. The inhibition was observed on test Day 1 approximately 30 minutes after dose administration. All cholinesterase activity levels were comparable with the control values on test Day 2.

Under the conditions of this study, the NOEL for the test substance in male and female rats is 0.25 mg/kg based on reversible inhibition of cholinesterase at dose levels of 0.5 mg/kg and above.

Reference 5

Endpoint:

neurotoxicity: short-term 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:

32 male and female rats were divided into 4 groups of 8 on the basis of the erythrocyte and plasma cholinesterase activity (AChE) in blood taken from the tail. 8 additional rats were assigned to each group for assay of brain AChE 14 days after the test began. 7 days after the initial AChE measurements, the test substance was added to the rat's diet at levels of 0, 100, 400, and 800 ppm. 1, 7, 14, 21, and 28 days later the measurements were repeated on the eight rats in each group. on the 29th day the rats were sacrificed to measure AChE in the brain.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Substance ID: INX-1179-255
- Purity: > 99 < 100%

Species:

rat

Strain:

other: ChR-CD

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: diet

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

100 ppm

Dose / conc.:

400 ppm

Dose / conc.:

800 ppm

No. of animals per sex per dose:

8

Control animals:

yes, plain diet

Details on results:

There was a slight depression in erythrocyte (8%), plasma (13%), and brain (3%) AChE of male rats fed 800 ppm test substance when compared to the controls, but this difference was not statistically significant. A similar depression in erythrocyte (4%) and plasma (8%) AChE was found in the females.
The brain AChE was lower in the females fed 800 ppm test substance after 15 days on test and in the females fed 100, 400, and 800 ppm after 29 days on test. The statistical analysis, however, indicated that only the brain AChE of the females fed 800 ppm was significantly different than the controls.

Key result

Dose descriptor:

LOEL

Effect level:

800 ppm

Remarks on result:

other: Cholinesterase activity was inhibited in the brain of female rats fed 800 ppm test substance

Conclusions:

Cholinesterase activity was inhibited in the brain of female rats fed 800 ppm test substance. No inhibition of erythrocyte, plasma and brain cholinesterase activity was found in rats fed 100 or 400 ppm test substance.

Executive summary:

32 male and female rats were divided into 4 groups of 8 on the basis of the erythrocyte and plasma cholinesterase activity (AChE) in blood taken from the tail. 8 additional rats were assigned to each group for assay of brain AChE 14 days after the test began. 7 days after the initial AChE measurements, the test substance was added to the rat's diet at levels of 0, 100, 400, and 800 ppm. 1, 7, 14, 21, and 28 days later the measurements were repeated on the eight rats in each group. on the 29th day the rats were sacrificed to measure AChE in the brain.

There was a slight depression in erythrocyte (8%), plasma (13%), and brain (3%) AChE of male rats fed 800 ppm test substance when compared to the controls, but this difference was not statistically significant. A similar depression in erythrocyte (4%) and plasma (8%) AChE was found in the females.

The brain AChE was lower in the females fed 800 ppm test substance after 15 days on test and in the females fed 100, 400, and 800 ppm after 29 days on test. The statistical analysis, however, indicated that only the brain AChE of the females fed 800 ppm was significantly different than the controls.

Reference 6

Endpoint:

neurotoxicity: 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:

Six groups of 10 male and 10 female rats were fed 0, 50, 100, 200, 400, and 800 ppm of the test substance mixed in basal diet and corn oil. At the end of five months, peripheral blood was drawn from groups fed 0, 400, and 800 ppm and analyzed for cholinesterase activity. The rats were then anesthetized with chloroform, and a sample of blood drawn by cardiac punture and analyzed for cholinesterase activity.

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: INX-1179
- Purity: Not reported

Species:

rat

Strain:

other: ChR-CD

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: basal diet and corn oil

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

5 months

Frequency of treatment:

daily

Dose / conc.:

50 ppm

Dose / conc.:

100 ppm

Dose / conc.:

200 ppm

Dose / conc.:

400 ppm

Dose / conc.:

800 ppm

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on results:

It was shown previously that 800 ppm test substance when fed to rats for 79 days inhibited blood cholinesterase activity. This effect occurred early in the males and later in the females. With continued feeding, however, the apparent effect disappeared. At the end of 5 months, no effect on cholinesterase activity was evident when blood from the rats was assayed for colorimetric method of Ellman or the pH-stat method of Casterline and Williams.

Key result

Dose descriptor:

NOEL

Effect level:

800 ppm

Remarks on result:

other: At the end of 5 months, no effect on cholinesterase activity was evident

Conclusions:

At the end of 5 months, no effect on cholinesterase activity was evident in rats

Executive summary:

Six groups of 10 male and 10 female rats were fed 0, 50, 100, 200, 400, and 800 ppm of the test substance mixed in basal diet and corn oil. At the end of five months, peripheral blood was drawn from groups fed 0, 400, and 800 ppm and analyzed for cholinesterase activity by Ellman method. The rats were then anesthetized with chloroform, and a sample of blood drawn by cardiac punture and analyzed for cholinesterase activity by the pH-stat method of Casterline and Williams.

It was shown previously that 800 ppm test substance when fed to rats for 79 days inhibited blood cholinesterase activity. This effect occurred early in the males and later in the females. With continued feeding, however, the apparent effect disappeared. At the end of 5 months, no effect on cholinesterase activity was evident when blood from the rats was assayed for colorimetric method of Ellman or the pH-stat method of Casterline and Williams.

Reference 7

Endpoint:

neurotoxicity: sub-chronic 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:

Six groups of 10 male and 10 female rats were fed 0, 50, 100, 200, 400, and 800 ppm of the test substance mixed in basal diet and corn oil. Peripheral blood was drawn and analyzed for cholinesterase activity at the end of 4, 11, 32, 43, and 60 days.

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: INX-1179
- Purity: Not reported

Species:

rat

Strain:

other: ChR-CD

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: basal diet and corn oil

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

60 days

Frequency of treatment:

daily

Dose / conc.:

50 ppm

Dose / conc.:

100 ppm

Dose / conc.:

200 ppm

Dose / conc.:

400 ppm

Dose / conc.:

800 ppm

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The cholinesterase activity of the blood was lower (p >0.05) in males fed 800 ppm after 11 days and in the females fed 800 ppm after 60 days. Slightly, but not significantly, lower cholinesterase activity occurred in males fed 800 ppm test substance after 32 and 43 days, and in females after 11 days. Rats with abnormally lowered cholinesterase activity (-2 SD from control mean) were found most frequently in these groups. The animals affected had cholinesterase activities 25-40% below normal. From these results, it was concluded that the addition of 800 ppm test substance to the diet results in an effect on blood cholinesterase activity of rats.

Key result

Dose descriptor:

NOEL

Effect level:

800 ppm

Remarks on result:

other: Dose of 800 ppm resulted in an effect on blood cholinesterase activity in rats.

Conclusions:

Dose of 800 ppm resulted in an effect on blood cholinesterase activity in rats

Executive summary:

Peripheral blood was drawn from 6 groups of 10 male and 10 female rats, and analyzed for cholinesterase activity. The rats were approximately 60 days of age and weighed approximately 150 g for males and 100 g for females. They were then fed 0, 50, 100, 200, 400, and 800 ppm of the test substance mixed in basal diet and corn oil. Peripheral blood was drawn and analyzed for cholinesterase activity at the end of 4, 11, 32, 43, and 60 days. The control group was analyzed first, then the high level down through the groups until no inhibition was found. All six groups were analyzed at the 60-day sampling periods.

The cholinesterase activity of the blood was lower (p >0.05) in males fed 800 ppm after 11 days and in the females fed 800 ppm after 60 days. Slightly, but not significantly, lower cholinesterase activity occurred in males fed 800 ppm test substance after 32 and 43 days, and in females after 11 days. Rats with abnormally lowered cholinesterase activity (-2 SD from control mean) were found most frequently in these groups. The animals affected had cholinesterase activities 25-40% below normal. From these results, it was concluded that the addition of 800 ppm test substance to the diet results in an effect on blood cholinesterase activity of rats.

Reference 8

Endpoint:

neurotoxicity: sub-chronic 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 purpose of the study was to characterize and evaluate the toxicity of the test substance in dogs. One female and one male dog received the test substance at dietary levels ranging from 250 to 4000 ppm of the basal diet. Daily records of appearance, behaviour, and signs of substance effect were recorded weekly.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Substance name: Insecticide 1179
- Purity: 100%

Species:

dog

Strain:

Beagle

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: basal diet

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

75 days

Frequency of treatment:

daily

Dose / conc.:

250 ppm

Dose / conc.:

500 ppm

Dose / conc.:

1 000 ppm

Dose / conc.:

2 000 ppm

Dose / conc.:

2 500 ppm

Dose / conc.:

4 000 ppm

No. of animals per sex per dose:

1

Control animals:

yes, plain diet

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Except for an instance of profuse salivation observed in the male dog on the 28th day, dietary levels of test substance ranging from 250 to 2000 ppm, administered during a 28-day period, did not produce any compound related signs in the two dogs. On the 29th day the level of test substance was increased to 4000 ppm of the diet of each dog. Signs of compound effect were observed in the male dog during this day. These signs consisted of copious salivation, slightly labored respiration, a crouching gait, and miosis. The female dog showed only slight salivation. On the 30th day the male dog appeared normal. Repeated episodes of emesis were observed in the female dog during the next two days. Dietary feeding of the test substance at 2500 ppm was resumed and continued at this level for 35 consecutive days. During this 35-day period, both dogs exhibited fair appetite and normal elimination. The male dog displayed dry nasal skin and lost 1.4 kg of body weight; the female dog maintained body weight.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 4000 ppm, the male dog lost 0.4 kg of body weight and the female lost 0.7 kg. After being fed 4000 ppm for two days, the dogs were placed on a control diet to which no test substance added. The dogs were maintained on this basal diet for 10 days. The male dog maintained body weight and the female gained 0.8 kg. Dietary feeding of the test substance at 2500 ppm was resumed and continued at this level for 35 consecutive days. During this 35-day period the male dog lost 1.4 kg of body weight; the female dog maintained body weight.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Slight inhibition of the plasma and erythrocyte cholinesterase activity was evident in the male dog on the 29th day. This finding coincided with the increase in dietary level to 4000 ppm and the appearance of clinical signs. On the 30th day, plasma and erythrocyte cholinesterase activity were normal.

Key result

Dose descriptor:

LOEL

Effect level:

250 ppm

Remarks on result:

other:

Remarks:

At 250-2000 ppm dogs did not elicit test substance related related signs, other than salivation, or plasma or erythrocyte cholinesterase activity inhibition; at 2500 ppm showed weight loss; at 4000 ppm showed salivation, labored respiration, other signs, and slight decrease in plasma or erythrocyte cholinesterase activity.

Conclusions:

At 250-2000 ppm dogs did not elicit test substance related signs, other than salivation, or plasma or erythrocyte cholinesterase activity inhibition; at 2500 ppm showed weight loss; at 4000 ppm showed salivation, labored respiration, other signs, and slight decrease in plasma or erythrocyte cholinesterase activity.

Executive summary:

The purpose of the study was to characterize and evaluate the toxicity of the test substance in dogs. One female and one male dog received the test substance at dietary levels ranging from 250 to 4000 ppm of the basal diet. Daily records of appearance, behaviour, and signs of substance effect were recorded weekly.

Except for one instance of salivation in the male dog at 2000 ppm, dietary feeding of the test substance at levels ranging from 250 to 2000 ppm did not elicit any test substance related signs in one male and one female dog. No inhibition or plasma and erythrocyte cholinesterase activity noted in either animal.

Dietary feeding of the test substance at a level of 4000 ppm for two days produced salivation, slightly labored respiration, a crouching gait, and miosis in the male dog, and salivation and emesis In the female. Both animals lost weight at this Level. There was a slight transient decrease in the plasma and erythrocyte cholinesterase levels in the male dog.

Dietary feeding of test substance at a level of 2500 ppm for 35 days caused body weight loss in the male dog; no signs of test substance effect were seen in the female. No cholinesterase data are available for this period.

Reference 9

Endpoint:

neurotoxicity: sub-chronic 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:

Six groups of 10 male and 10 female rats were fed 0, 50, 100, 200, 400, and 800 ppm of the test substance mixed in basal diet and corn oil. Peripheral blood was drawn and analyzed for cholinesterase activity at the end of 4, 11, 32, 43, 60 and 79 days.

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: INX-1179
- Purity: Not reported

Species:

rat

Strain:

other: ChR-CD

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: basal diet and corn oil

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

79 days

Frequency of treatment:

daily

Dose / conc.:

50 ppm

Dose / conc.:

100 ppm

Dose / conc.:

200 ppm

Dose / conc.:

400 ppm

Dose / conc.:

800 ppm

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The cholinesterase activity of the blood was lower in males fed 800 ppm after 11 days and in the females fed 800 ppm after 60 and 79 days. Slightly, but not significantly, lower cholinesterase activity occurred in males fed 800 ppm test substance after 32 and 43 days, and in females after 11 days. Rats with abnormally lowered cholinesterase activity were found most frequently in these groups. The animals affected had cholinesterase activities 25-40% below normal. Decreased cholinesterase activity was not observed more than once in any individual female rat fed 400, 200, or 0 ppm in the diet. All decreased cholinesterase activity observed in the male rats fed 400 ppm occurred in one rat. From these results, it was concluded that the addition of 800 ppm test substance to the diet results in an effect, and 400 ppm results in no effect on blood cholinesterase activity of rats at the end of 79 days.

Key result

Remarks on result:

other: Dose of 400 ppm resulted in no effect on blood cholinesterase; dose of 800 ppm leads to lower cholinesterase activity at the end of 79 days in rats

Conclusions:

Dose of 400 ppm resulted in no effect on blood cholinesterase; dose of 800 ppm leads to lower cholinesterase activity at the end of 79 days in rats.

Executive summary:

Peripheral blood was drawn from 6 groups of 10 male and 10 female rats, and analyzed for cholinesterase activity. The rats were approximately 60 days of age and weighed approximately 150 g for males and 100 g for females. They were then fed 0, 50, 100, 200, 400, and 800 ppm of the test substance mixed in basal diet and corn oil. Peripheral blood was drawn and analyzed for cholinesterase activity at the end of 4, 11, 32, 43, 60 and 79 days. The control group was analyzed first, then the high level down through the groups until no inhibition was found. All six groups were analyzed at the 60- and 79-day sampling periods.

The cholinesterase activity of the blood was lower in males fed 800 ppm after 11 days and in the females fed 800 ppm after 60 and 79 days. Slightly, but not significantly, lower cholinesterase activity occurred in males fed 800 ppm test substance after 32 and 43 days, and in females after 11 days. Rats with abnormally lowered cholinesterase activity were found most frequently in these groups. The animals affected had cholinesterase activities 25-40% below normal. Decreased cholinesterase activity was not observed more than once in any individual female rat fed 400, 200, or 0 ppm in the diet. All decreased cholinesterase activity observed in the male rats fed 400 ppm occurred in one rat. From these results, it was concluded that the addition of 800 ppm test substance to the diet results in an effect, and 400 ppm results in no effect on blood cholinesterase activity of rats at the end of 79 days.

Reference 10

Endpoint:

neurotoxicity: acute 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 this study was to determine the length of time needed for recovery from inhibition of cholinesterase activity. The test substance was administered once via gavage on test Day 1 to adult male and female rats (40/gender/dose) at dosages of 0 or 3 mg/kg. Clinical signs of toxicity were evaluated in all rats at 30 minutes post-dosing, and in 10 rats/gender/dose at 2 hours, 3 hours, and 4 hours post-dosing, respectively. Following the clinical signs inventory, blood was collected from 10 rats/gender/dose at 30 minutes, 2 hours, 3 hours, and 4 hours post-dosing. Immediately after blood collection at each time point, the designated rats were sacrificed, brains were removed, and frozen for subsequent analysis of acetylcholinesterase activity.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Substance name: Methomyl Technical
- Substance ID: DPX-X1179
- Lot#: X1179-512
- Purity: 98.6%

Species:

rat

Strain:

other: Crl:CD®BR

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

other: deionized water

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Once

Frequency of treatment:

Once

Dose / conc.:

3 mg/kg bw (total dose)

No. of animals per sex per dose:

40

Control animals:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related tremors were observed in males (17/40) and females (6/40) dosed with 3 mg/kg. In addition, although the incidence was not statistically significant, one male was also observed to have clear discharge from both eyes. This observation is most likely indicative of lacrimation. Both tremors and lacrimation were consistent with inhibition of cholinesterase activity and were considered to be test-substance related. These signs were observed at the 0.5 hour evaluation; however, by two hours after dosing, no clinical signs of toxicity were evident. Therefore, recovery of test-substance-induced clinical signs of toxicity occurred within 2 hours following dosing.

Mortality:

no mortality observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the first sampling time, 30 minutes after treatment with 3 mg/kg of the test substance, male and female rats had moderately severe inhibition of erythrocyte, plasma, and brain cholinesterase activity (except plasma cholinesterase was not statistically significantly decreased in females). In males, mean erythrocyte and plasma cholinesterase activity were 44 and 73% of the control means, respectively, and mean brain cholinesterase activity was 54% of the control mean. In females, mean erythrocyte and plasma cholinesterase activity were 59 and 90% of the control means, respectively, and mean brain cholinesterase activity was 61% of the control mean.

At the second sampling time, 2 hours after treatment with 3 mg/kg of the test substance, males had minimal, statistically significant decreases in blood and brain cholinesterase activity. Mean erythrocyte and plasma cholinesterase activity were 76 and 81% of the control means, respectively, and mean brain cholinesterase activity was 84% of the control mean. These differences may reflect residual test substance-related inhibition of cholinesterase activity. However, the changes were not associated with any clinical signs of cholinesterase inhibition. In females at the 2-hour sampling time, blood cholinesterase activity was not significantly different from control. No test substance treated effect in brain cholinesterase activity.
At the thrid and fourth sampling times, there no test substance related effects in blood or cholinesterase activities.
These results demonstrate biologically adverse inhibition of blood and brain cholinesterase activity 30 minutes after treatment with 3 mg/kg of test substance. By 2 hours after treatment, males had residual inhibition of blood and brain cholinesterase activity. In females and males, recovery from cholinesterase activity inhibition was complete at the 2-hour and 3-hour sampling times, respectively. Brain cholinesterase activity was significantly lower than controls but the magnitude of the change was within the range observed in the control groups in this study and the difference was considered to be due to biological and analytical variation.

Key result

Dose descriptor:

LOEL

Effect level:

3 mg/kg bw/day

Remarks on result:

other:

Remarks:

Male and female rats treated with 3 mg/kg of the test substance had biologically adverse inhibition of blood (plasma and red blood cell) and brain cholinesterase activity 30 minutes after treatment. By 2 hours after treatment, males had minimal residual inhibition of blood and brain cholinesterase activity. In females and males, recovery from cholinesterase activity inhibition was complete at the 2-hour and 3-hour sampling times, respectively.

Conclusions:

Male and female rats treated with 3 mg/kg of the test substance had biologically adverse inhibition of blood (plasma and red blood cell) and brain cholinesterase activity 30 minutes after treatment. By 2 hours after treatment, males had minimal residual inhibition of blood and brain cholinesterase activity. In females and males, recovery from cholinesterase activity inhibition was complete at the 2-hour and 3-hour sampling times, respectively.

Executive summary:

The objective of this study was to determine the length of time needed for recovery from inhibition of cholinesterase activity. The test substance was administered once via gavage on test Day 1 to adult male and female rats (40/gender/dose) at dosages of 0 or 3 mg/kg. Clinical signs of toxicity were evaluated in all rats at 30 minutes post-dosing, and in 10 rats/gender/dose at 2 hours, 3 hours, and 4 hours post-dosing, respectively. Following the clinical signs inventory, blood was collected from 10 rats/gender/dose at 30 minutes, 2 hours, 3 hours, and 4 hours post-dosing. Immediately after blood collection at each time point, the designated rats were sacrificed, brains were removed, and frozen for subsequent analysis of acetylcholinesterase activity.

The test substance produced clinical signs indicative of cholinesterase inhibition within 30 minutes post-dosing. The predominant clinical observations were tremors, although signs of salivation and lacrimation also occurred. Recovery from test substance-induced clinical signs of cholinesterase inhibition occurred within 2 hours post-dosing.

Male and female rats treated with 3 mg/kg of the test substance had biologically adverse inhibition of blood (plasma and red blood cell) and brain cholinesterase activity 30 minutes after treatment. By 2 hours after treatment, males had minimal residual inhibition of blood and brain cholinesterase activity. In females and males, recovery from cholinesterase activity inhibition was complete at the 2-hour and 3-hour sampling times, respectively. At the 2-hour and 3-hour sampling times, brain cholinesterase activity was slightly lower than controls but the magnitude of the change was within the range observed in the control groups in this study and the difference was considered to be due to biological and analytical variation.

Reference 11

Endpoint:

neurotoxicity: acute 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:

Peripheral blood was drawn from each rat (male) in two groups of 10 and analyzed for cholinesterase activity. Approximately two hours later, each rat in each group was given, by intragastric intubations, 60 mg/kg of the test substance as a 1% suspension in peanut oil. Immediately after the dosing, each of the 10 rats in one group was given 1.30 ml of physiological saline by intraperitoneal Injection (IP); the other group was given IP 50 mg/kg atropine sulfate, as a 1% solution in physiological saline. 5 minutes after each dose of the test substance had been administered, peripheral blood was taken from the tail vein of each rat, wherever possible, for cholinesterase activity measurements. 26 hours later, blood was again taken the tail vein of those rats that survived for cholinesterase activity measurements. Surviving animals were sacrificed one week later.

GLP compliance:

no

Specific details on test material used for the study:

- Substance ID: INX-1179-16
- Purity: Not reported

Species:

rat

Strain:

other: ChR-CD

Sex:

male

Route of administration:

oral: gavage

Vehicle:

other: Peanut oil

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Once

Frequency of treatment:

Once

Dose / conc.:

60 mg/kg bw (total dose)

No. of animals per sex per dose:

10 males

Control animals:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals receiving only the test substance: Chewing motions, salivation, bulging eyes, lacrimation, fasciculations.
Animals receiving atropine sulfate dose immediately after the test substance administration: Same observations but milder and lasting about 2 1/2 hours; weight loss 1-2 days.

Mortality:

mortality observed, treatment-related

Description (incidence):

All animals died which received only the test substance.
2/10 animals died which received the atropine sulfate dose immediately after the test substance administration.

Key result

Dose descriptor:

LOEL

Effect level:

60 mg/kg bw/day

Remarks on result:

other: At 60 mg/kg lowered cholinesterase activity of whole blood (rat) to 50%. Atropine sulfate appears to be an antidote for the test substance administered immediately after exposure.

Conclusions:

The test substance, when administered orally to male rats in a single doge of 60 mg/kg (ca. twice the ALD), lowered the cholinesterase activity of whole blood to 50% of the pre-exposure value within five minutes. Atropine sulfate appears to be an antidote for the test substance administered immediately after exposure. Although it did not prevent the lowering of the blood cholinesterase activity of the rats, it did afford some protection against the toxic and lethal effects of the test substance.

Executive summary:

Peripheral blood was drawn from each rat (male) in two groups of ten and analyzed for cholinesterase activity. Approximately two hours later, each rat in each group was given, by intragastric intubations, 60 mg/kg of the test substance as a 1% suspension in peanut oil. Immediately after the dosing, each of the ten rats in one group was given 1.30 ml of physiological saline by intraperitoneal Injection (IP); the other group was given IP 50 mg/kg atropine sulfate, as a 1% solution in physiological saline. 5 minutes after each dose of the test substance had been administered, peripheral blood was taken from the tail vein of each rat, wherever possible, for cholinesterase activity measurements. 26 hours later, blood was again taken the tail vein of those rats that survived for cholinesterase activity measurements. Surviving animals were sacrificed one week later.

All animals died which received only the test substance. 2/10 animals died which received the atropine sulfate dose immediately after the test substance administration. The test substance, when administered orally to male rats in a single doge of 60 mg/kg (ca. twice the ALD), lowered the cholinesterase activity of whole blood to 50% of the pre-exposure value within five minutes. Atropine sulfate appears to be an antidote for the test substance administered immediately after exposure. Although it did not prevent the lowering of the blood cholinesterase activity of the rats, it did afford some protection against the toxic and lethal effects of the test substance.

Reference 12

Endpoint:

neurotoxicity: 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, as a 5% suspension in acetone:water (1:10) was administered in single doses directly into the crop of ten sex-linked hens. 10 hens were dosed with the LD50 dose with a 22-day Recovery period. Each of the other 4 hens were dosed with 60, 90, 120, 200 mg/kg of test substance. These hens were pre-treated subcutaneously with 10 mg/kg atropine sulfate and had a 21-day recovery period.

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: INX-1179-68
- Purity: Not reported

Species:

hen

Strain:

other: cross between a Barred Rock female and a Rhode Island Red male

Sex:

female

Route of administration:

other: oral

Vehicle:

other: acetone:water (1:10)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

single dose (with 22 and 21-day recovery period)

Frequency of treatment:

Single dose

Dose / conc.:

28 mg/kg bw (total dose)

Remarks:

LD50 dose (22-day recovery period)

Dose / conc.:

60 mg/kg bw (total dose)

Remarks:

pre-treatment subcutaneously with 10 mg/kg atropine sulfate and a 21-day recovery period

Dose / conc.:

90 mg/kg bw (total dose)

Remarks:

pre-treatment subcutaneously with 10 mg/kg atropine sulfate and a 21-day recovery period

Dose / conc.:

120 mg/kg bw (total dose)

Remarks:

pre-treatment subcutaneously with 10 mg/kg atropine sulfate and a 21-day recovery period

Dose / conc.:

200 mg/kg bw (total dose)

Remarks:

pre-treatment subcutaneously with 10 mg/kg atropine sulfate and a 21-day recovery period

No. of animals per sex per dose:

10 (28 mg/kg)
1 (60, 90, 120, 200 mg/kg)

Control animals:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

28 mg/kg dose with 22-day recovery period:
Fatal cases: Salivation and respiratory disorders; died within ten minutes
Survivors: Lacrimation and salivation with occasional convulsions lasting up to 1 hour after dosing; egg production reduced during third day through tenth day; no evidence of wing or leg paralysis.

21-Day recovery period with all the other doses:
Moderate convulsions but no salivation; reduction in egg production; the hen given 200 mg/kg did not eat for three days; no evidence of wing or leg paralysis

Mortality:

mortality observed, treatment-related

Description (incidence):

28 mg/kg: 4/10 hens died
All other doses: No hens died

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Description (incidence and severity):

No abnormalities detected in sciatic nerves

Key result

Dose descriptor:

NOEL

Effect level:

28 mg/kg bw/day

Remarks on result:

other: LD50 dose of 28 mg/kg and higher doses did not produce clinical signs of wing or leg weakness in hens during a 21-22 day recovery period; no abnormalities in sciatic nerves.

Conclusions:

LD50 dose of 28 mg/kg and higher doses did not produce clinical signs of wing or leg weakness in hens during a 21-22 day recovery period; no abnormalities in sciatic nerves.

Executive summary:

The test substance, as a 5% suspension in acetone:water (1:10) was administered in single doses directly into the crop if ten sex-linked hens. 10 hens were dosed with the LD50 dose of 28 mg/kg with a 22-day Recovery period. Each of the other 4 hens were dosed with 60, 90, 120, 200 mg/kg of the test substance. These hens were pre-treated subcutaneously with 10 mg/kg atropine sulfate and had a 21-Day recovery period.

The LD50 dose as well as doses higher than the LD50 which were attained with birds pre-treated with atropine sulfate, did not produce clinical signs of wing or leg weakness during a 21-22 day recovery period.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.25 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Human (capsule): Acute oral toxicity study, 0.1 mg/kg bw (males) based on increased saliva secretion and decreased plasma and RBC cholinesterase activities at 0.2 mg/kg bw

Rat (gavage): Acute oral neurotoxicity study,
0.25 mg/kg bw (males and females) based on reversible cholinesterase inhibition
at 0.5 mg/kg bw.

Rat (diet): Acute oral neurotoxicity study, 30 ppm (1 mg/kg bw males) based on diminished tail pinch response at 60 ppm.

Rat (diet): 90-day oral neurotoxicity study, 150 ppm (males and females) equivalent to 9.42 mg/kg bw/day (males) and 11.2 mg/kg bw/day (females) based on reduced body weight, food consumption food efficiency, decreased grip strength, and clinical signs and biochemical evidence of cholinesterase inhibition at 1500 ppm.

Effect on neurotoxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

neurotoxicity: acute inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OPPTS Guideline 870.1300 (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 403 (September 2009)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Substance name: Methomyl Technical
- Substance ID: DPX-X1179
- Lot#: X1179-571
- Purity: 99.4%

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Route of administration:

inhalation: aerosol

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.3 - <= 1.8 other: microns

Remarks on MMAD:

Mean GSD range: 1.69-2.08

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Phase I and II: 6 hours nose-only exposure
Phase IIIA and IIIB: 3 hours nose-only expsoure

Frequency of treatment:

Single exposure

Dose / conc.:

100 mg/m³ air

Remarks:

Phase I

Dose / conc.:

150 mg/m³ air

Remarks:

Phase I

Dose / conc.:

200 mg/m³ air

Remarks:

Phase I

Dose / conc.:

150 mg/m³ air

Remarks:

Phase II

Dose / conc.:

33 mg/m³ air

Remarks:

Phase IIIA

Dose / conc.:

67 mg/m³ air

Remarks:

Phase IIIA

Dose / conc.:

100 mg/m³ air

Remarks:

Phase IIIA

Dose / conc.:

5 mg/m³ air

Remarks:

Phase IIIB

Dose / conc.:

10 mg/m³ air

Remarks:

Phase IIIB

Dose / conc.:

20 mg/m³ air

Remarks:

Phase IIIB

Dose / conc.:

35 mg/m³ air

Remarks:

Phase IIIB

No. of animals per sex per dose:

Phase I: 3/sex/conc.
Phase II: 30/sex/conc.
Phase IIIA and IIIB: 10 males/conc.

Control animals:

yes, concurrent vehicle

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Phase I: Clinical observations during exposure consisted of lacrimation and salivation for the 100, 146, and 195 mg/m³ groups and tail twitching and decreased respiration for the 195 mg/m³ group. Significant clinical observations following exposure included unkempt appearance, intermittent tremors, hypoactivity, body and extremities cool to touch, lacrimation, and salivation for the 100, 146, and 195 mg/m³ groups; impaired muscle coordination, intermittent convulsions, and swollen facial area for the 195 mg/m³ group; ataxia for the 146 mg/m³ group; and prostration for the 100 mg/m³ group. Animals in all test substance groups were noted with clear, red, and/or yellow material around the nose, eye(s), mouth, head, facial area, neck, forelimb(s), hindlimb(s), trunk, rump, and/or urogenital area following exposure.

Phase II: Animals sacrificed 3 hours after initiation of the 136 mg/m³ test substance exposure demonstrated clinical observations that consisted of lacrimation, salivation, tail twitching, and/or labored breathing (1 female control [0 mg/m3] rat demonstrated lacrimation and salivation). For animals sacrificed 6 hours after initiation of the 136 mg/m³ exposure, clinical observations consisted of decreased respiration, lacrimation, and salivation. Animals sacrificed 7 hours after initiation of the exposure (1 hour post-exposure) demonstrated clinical observations that consisted of body cool to touch, extremities cool to touch, and salivation. Animals sacrificed 8 hours (2 hours post-exposure) after initiation of the 136 mg/m³ test substance exposure demonstrated extremities cool to touch. The animals sacrificed 10 hours (4 hours post-exposure) after initiation of the 136 mg/m³ exposure demonstrated clinical observations that included labored respiration, red material around the nose, head, and mouth, and yellow material on the forelimb(s), hindlimb(s), trunk, rump, and urogenital area.

Phase IIIA/IIIB: Significant clinical observations during exposure included lacrimation and salivation for the 5.6, 14, 19, 31, and 105 mg/m³ test substance groups while tail twitching was observed in animals exposed to 68 (1 male) and 105 mg/m³ (2 males) test substance.

Mortality:

no mortality observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Phase II: Male and female rats demonstrated a rapid reduction of RBC ChE activity. One hour after the initiation of the 136 mg/m³ exposure both male and females demonstrated a 73% RBC ChE inhibition level from control animals. RBC ChE inhibition peaked 3 hours following the initiation of the exposure where males and females demonstrated 83 and 84% RBC ChE inhibition from control animals, respectively. At the end of the 6 hour exposure period to 136 mg/m³ test substance exposed males and females demonstrated 77 and 75% RBC ChE inhibition from control animals, respectively. 7 hours following the initiation of the 136 mg/m³ exposure (1 hour post-exposure), males and females demonstrated 66 and 76% RBC ChE inhibition from control animals. 8 hours following the initiation of the 136 mg/m³ exposure (2 hours post-exposure) RBC ChE inhibition in males and females demonstrated a 51 and 52% RBC ChE inhibition from control animals and by 10 hours following the initiation of the exposure (4 hours post-exposure) RBC ChE was 16 and 20% inhibited from control animals, respectively.
Similar to the RBC ChE, both male and female rats exposed to test substance demonstrated a rapid reduction in brain ChE activity. One hour after the initiation of the 136 mg/m³ exposure both male and females demonstrated a 65% brain ChE inhibition level from control animals. Brain ChE inhibition peaked 3 hours following the initiation of the exposure where males and females demonstrated 69% brain ChE inhibition from control animals. At the end of the 6 hour exposure period to 136 mg/m³ test substance males and females demonstrated 66 and 68% brain ChE inhibition from control animals, respectively. 7 hours following the initiation of the 136 mg/m³ exposure (1 hour post-exposure), males and females demonstrated 53 and 61% brain ChE inhibition from control animals. 8 hours following the initiation of the 136 mg/m³ exposure (2 hours post-exposure) brain ChE inhibition in males and females demonstrated a 42 and 56% RBC ChE inhibition from control animals and by 10 hour following the initiation of the exposure (4 hours post-exposure) brain ChE was 19 and 25% inhibited from control animals, respectively.

Phase IIIA/IIIB: Male rats demonstrated a concentration-dependent reduction in RBC ChE activity that ranged from 16 to 93% for the 5.6 to 105 mg/m³ test substance groups, respectively. Similarly, there was a concentration-dependent reduction in brain ChE that ranged from 13 to 66% for the 5.6 to 105 mg/m³ test substance groups, respectively.

Key result

Dose descriptor:

other: MTD

Effect level:

150 mg/m³ air

Remarks on result:

other:

Remarks:

The maximum tolerated exposure concentration in male and female rats was approximately 150 mg/m³ based upon impaired muscle coordination and intermittent convulsions observed at the 195 mg/m³ test substance exposure concentration for 6 hours. The time to peak RBC and brain ChE inhibition from control animals was 3 hours following the initiation of a 6 hour, 136 mg/m³ test substance exposure. RBC and brain ChE inhibition from control animals demonstrated a robust concentration-response relationship where exposure ranging from 5.6 to 105 mg/m³, resulted in 16 to 93% reduction and 13 to 66% reduction in RBC and brain ChE activity, respectively.

Key result

Critical effects observed:

yes

System:

nervous system

Treatment related:

yes

Dose response relationship:

yes

Conclusions:

The time to peak RBC and brain ChE inhibition from control animals was 3 hours following the initiation of a 6 hour, 136 mg/m³ test substance exposure. RBC and brain ChE inhibition from control animals demonstrated a robust concentration-response relationship where exposure ranging from 5.6 to 105 mg/m³, resulted in 16 to 93% reduction and 13 to 66% reduction in RBC and brain ChE activity, respectively.

Executive summary:

The objective of this study was to determine the effect of the test substance on red blood cell (RBC) and brain cholinesterase (ChE) activity in Sprague Dawley rats. The study was conducted following U.S. EPA OPPTS 870.1300 and OECD guideline 403.

The study was conducted in 4 phases, a range-finding/maximum tolerable concentration phase (Phase I), a time to peak cholinesterase inhibition effect phase (Phase II), and a definitive study examining the concentration-response curve of test substance exposure concentration on RBC and brain cholinesterase inhibition at the time of peak effect (Phases IIIA and IIIB). Filtered air or test substance was administered to male and female Crl:CD(SD) albino rats via nose-only inhalation exposure as an aerosol for a single, 6-hour period at target concentrations of 0, 100, 150, and 200 mg/m³ for Phase I. To determine the time of peak cholinesterase inhibition effect, rats were exposed to target concentrations of 0 or 150 mg/m³ for 6 hours with groups of 5 males and 5 females sacrificed at 1, 3, 6, 7, 8, or 10 hours after the initiation of the exposure for RBC and brain cholinesterase activity (Phase II). To establish the concentration-response curve at the time of peak ChE inhibition, filtered air or test substance was administered to male rats for a single, 3-hour period (time of peak cholinesterase inhibition) at target concentrations of 0, 33, 67, and 100 mg/m³ for Phase IIIA and at target concentrations of 0, 5.0, 10, 20, and 35 mg/m³ for Phase IIIB.

The animals were observed twice daily for mortality and morbundity. Clinical observations were performed during exposure for all phases and following exposure for Phase I and Phase II. Animals were euthanized by CO2 inhalation. Blood samples and whole brains were collected at 1, 3, 6, 7, 8, and 10 hours after initiation of exposure for Phase II and at 3 hours after initiation of exposure for Phase IIIA and IIIB.

Phase I: Animals were exposed to actual concentrations of 100, 146, or 195 mg/m³ for 6 hours. The mass median aerodynamic diameters (MMAD) ranged from 1.4 to 1.6 μm and the geometric standard deviations (GSD) ranged from 1.86 to 2.08 for the Phase I exposures. All animals survived to study termination. Clinical observations during exposure consisted of lacrimation and salivation for the 100, 146, and 195 mg/m³ groups and tail twitching and decreased respiration for the 195 mg/m³ group. Significant clinical observations following exposure included unkempt appearance, intermittent tremors, hypoactivity, body and extremities cool to touch, lacrimation, and salivation for the 100, 146, and 195 mg/m³ groups; impaired muscle coordination, intermittent convulsions, and swollen facial area for the 195 mg/m³ group; ataxia for the 146 mg/m3 group; and prostration for the 100 mg/m³ group. Animals in all test substance groups were noted with clear, red, and/or yellow material around the nose, eye(s), mouth, head, facial area, neck, forelimb(s), hindlimb(s), trunk, rump, and/or urogenital area following exposure. Animals in the control group were noted with red material around the right eye and yellow material urogenital area.

Phrase II: Animals were exposed to mean actual concentrations of 0 or 136 mg/m³ for 6 hours. The MMAD (GSD) for this exposure ranged from 1.3 to 1.4 μm (1.97 to 1.99). Animals sacrificed 3 hours after initiation of the 136 mg/m³ test substance exposure demonstrated clinical observations that consisted of lacrimation, salivation, tail twitching, and/or labored breathing (1 female control [0 mg/m3] rat demonstrated lacrimation and salivation). For animals sacrificed 6 hours after initiation of the 136 mg/m³ exposure, clinical observations consisted of decreased respiration, lacrimation, and salivation. Animals sacrificed 7 hours after initiation of the exposure (1 hour post-exposure) demonstrated clinical observations that consisted of body cool to touch, extremities cool to touch, and salivation. Animals sacrificed 8 hours (2 hours post-exposure) after initiation of the 136 mg/m³ test substance exposure demonstrated extremities cool to touch. The animals sacrificed 10 hours (4 hours post-exposure) after initiation of the 136 mg/m³ exposure demonstrated clinical observations that included labored respiration, red material around the nose, head, and mouth, and yellow material on the forelimb(s), hindlimb(s), trunk, rump, and urogenital area.

Male and female rats demonstrated a rapid reduction of RBC ChE activity. One hour after the initiation of the 136 mg/m³ exposure both male and females demonstrated a 73% RBC ChE inhibition level from control animals. RBC ChE inhibition peaked 3 hours following the initiation of the exposure where males and females demonstrated 83% and 84% RBC ChE inhibition from control animals, respectively. At the end of the 6 hour exposure period to 136 mg/m³ test substance males and females demonstrated 77 and 75% RBC ChE inhibition from control animals, respectively. Seven hours following the initiation of the 136 mg/m³ exposure (1 hour post-exposure), males and females demonstrated 66 and 76% RBC ChE inhibition from control animals, respectively. Eight hours following the initiation of the 136 mg/m³ exposure (2 hours post-exposure) RBC ChE inhibition in males and females demonstrated a 51 and 52% RBC ChE inhibition from control animals and by 10 hours following the initiation of the exposure (4 hours post-exposure) RBC ChE was 16 and 20% inhibited from control animals, respectively.

Similar to the RBC ChE, both male and female rats exposed to test substance demonstrated a rapid reduction in brain ChE activity. One hour after the initiation of the 136 mg/m³ exposure both male and females demonstrated a 65% brain ChE inhibition level from control animals. Brain ChE inhibition peaked 3 hours following the initiation of the exposure where males and females demonstrated 69% brain ChE inhibition from control animals. At the end of the 6 hour exposure period to 136 mg/m³ test substance males and females demonstrated 66 and 68% brain ChE inhibition from control animals, respectively. 7 hours following the initiation of the 136 mg/m³ exposure (1 hour post-exposure), males and females demonstrated 53 and 61% brain ChE inhibition from control animals, respectively. 8 hours following the initiation of the 136 mg/m³ exposure (2 hours post-exposure) brain ChE inhibition in males and females demonstrated a 42 and 56% RBC ChE inhibition from control animals, respectively, and by 10 hours following the initiation of the exposure (4 hours post-exposure) brain ChE was 19 and 25% inhibited from control animals, respectively.

Phase IIIA/IIIB: Since no sex difference in RBC or brain ChE inhibition levels were observed between male and female rats in Phases I or II, groups of 10 male rats were used for Phase IIIA/IIIB of this study. Male rats were exposed to actual concentrations of 0, 5.6, 14, 19, 31, 36, 68, or 105 mg/m³ for 3 hours, the time to peak cholinesterase inhibition. The mass median aerodynamic diameters (MMAD) ranged from 1.3 to 1.8 μm and the geometric standard deviations (GSD) ranged from 1.69 to 2.02 for the Phase IIIA/IIIB exposures. Significant clinical observations during exposure included lacrimation and salivation for the 5.6, 14, 19, 31, and 105 mg/m³ test substance groups while tail twitching was observed in animals exposed to 68 (1 male) and 105 mg/m³ (2 males) test substance. Rats demonstrated a concentration-dependent reduction in RBC ChE activity that ranged from 16 to 93% from control animals for the 5.6 to 105 mg/m³ test substance groups, respectively. Similarly, there was a concentration-dependent reduction in brain ChE from control animals that ranged from 13 to 66% for the 5.6 to 105 mg/m³ test substance groups, respectively.

In conclusion, the maximum tolerated exposure concentration in male and female rats was approximately 150 mg/m³ based upon impaired muscle coordination and intermittent convulsions observed at the 195 mg/m³ test substance exposure for 6 hours. The time to peak RBC and brain ChE inhibition from control animals was 3 hours following the initiation of a 6 hour, 136 mg/m³ test substance exposure. RBC and brain ChE inhibition from control animals demonstrated a robust concentration-response relationship where exposure ranging from 5.6 to 105 mg/m³, resulted in 16 to 93% reduction and 13 to 66% reduction in RBC and brain ChE activity, respectively.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

5.6 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Rat acute inhalation toxicity study. NOAEC 5.6 mg/m3 based on brain cholinesterase inhibition (most sensitive endpoint) of >20% observed at 14 mg/m3 and above.

Effect on neurotoxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

neurotoxicity: acute dermal

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:

Rats were treated with 100, 25 mg of the test substance in acetone on a 25 cm² area on their backs. Five rats from each group of 100 and 25 mg were sent for blood analysis 24 hours post treatment and the remaining five from each group of 100 and 25 mg at 72 hours post treatment. Plasma and red blood cell cholinesterase activity was determined.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Substance name: Methomyl
- Substance ID: INX-1179-225
- Purity: 99-100%

Species:

rat

Strain:

other: Crl:CD®

Sex:

male

Route of administration:

dermal

Vehicle:

acetone

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

24 and 72 hours

Frequency of treatment:

Single dose

Dose / conc.:

100 other: mg

Dose / conc.:

25 other: mg

No. of animals per sex per dose:

5 males (2 groups per dose level)

Control animals:

yes, concurrent no treatment

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In the groups treated with 25 mg for 24 and 72 hours, plasma cholinesterase was lower than the controls, but in the groups treated with 100 mg only those exposed for 24 hours were significantly lower than the controls. There was no effect on the red blood cell cholinesterase activity of the groups of rats treated with 25 mg or 100 mg methomyl for 24 or 72 hours.

Key result

Dose descriptor:

LOEL

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Sex:

male

Remarks on result:

other: Plasma cholinesterase levels were lower than in controls in rats treated with 25 or 100 mg test substance.

Conclusions:

Plasma cholinesterase levels were lower than in controls in rats treated with 25 or 100 mg test substance. There was no effect on the red blood cell cholinesterase activity of the groups of rats treated with 25 mg or 100 mg methomyl for 24 or 72 hours.

Executive summary:

Doses of 0 (control), 25 or 100 mg test substance were applied to the skin of two groups of five rats for 24 hours and to two other groups for 72 hours. Red blood cell and plasma cholinesterase activities were measured in blood taken from the tail at the end of the exposure periods.

In the groups treated with 25 mg for 24 and 72 hours, plasma cholinesterase was lower than the controls, but in the groups treated with 100 mg only those exposed for 24 hours were significantly lower than the controls. There was no effect on the red blood cell cholinesterase activity of the groups of rats treated with 25 mg or 100 mg methomyl for 24 or 72 hours.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

25 mg/kg bw/day

Study duration:

subacute

Mode of Action Analysis / Human Relevance Framework

Methomyl is a cholinesterase inhibitor. The inhibition effects are reversible in rats within 2 -3 hours after dosing. The NOAEL for acute neurotoxicity was 0.25 mg/kg bw based on reversible dose-related braincholinesterase activity. Methomyl did not show any evidence of delayed toxicity.

The NOAEL for repeated dose neurotoxicity was 9.42 mg/kg bw/day. Reduced body weight and food consumption, clinical signs, brain cholinesterase inhibition and effects on FOB parameters at the next highest dose were observed in rats.

In a human volunteer study, a statistically significant decrease in RBC cholinesterase activity at doses of 0.2 and 0.3 mg/kg bw was registered within 1.75 hours post dosing; a single occurrence of a mild

headache at a dose of 0.3 mg/kg bw and quantitatively increased salivation at 0.2 and 0.3 mg/kg bw were reported. Red blood cholinesterase activity was depressed by 19% at 0.1 mg/kg bw at 1.25 hour

post dosing. Plasma cholinesterase activity was depressed at 0.2 mg/kg bw and above. The NOAEL was considered to be 0.1 mg/kg.

Justification for classification or non-classification

Methomyl is a cholinesterase inhibitor and as such animals exposed to methomyl have shown cholinesterase inhibition in both blood and brain cholinesterase levels. Animals have also exhibited clinical signs indicative of cholinesterase inhibition. These have included hypoactivity, tremors, prone posture, piloerection, fasciculations, lacrimation, salivation, and stained face. In acute inhalation studies, lethargy, ocular and nasal discharge, diarrhea, abnormal gait or mobility, tremors, muscle fasciculations, and hunched or low posture were noted. These effects were rapidly reversible, and similar across species. Similar effects on blood cholinesterase have been observed in a human volunteer study conducted with a methomyl formulation (Lannate SP). Clinical observations noted in humans included increased salivation and a single occurrence of reported headache. Based on the transient central nervous system effects (consistent with cholinesterase inhibitors) observed in acute animal studies via the oral and inhalation routes and the results from a human volunteer study, a classification of STOT SE Category 3 is warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. The criteria for Category 3 states thatthe effects observed could adversely alter human function for a short duration after exposure and from which humans may recover in a reasonable period without leaving significant alteration of structure or function.