Methomyl

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methomyl

EC Number:

240-815-0

EC Name:

Methomyl

Cas Number:

16752-77-5

Molecular formula:

C5H10N2O2S

IUPAC Name:

(E)-[1-(methylsulfanyl)ethylidene]amino N-methylcarbamate

Test material form:

solid: crystalline

Specific details on test material used for the study:

- Substance name: S-Methyl-N-(methyl-carbamoyl)-oxythioacetimidate
- Substance ID: Code 7-8-0-0
- Purity: >98%

Test animals

Species:

rat

Strain:

other: Sprague Dawley COBS® CD®

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Labs, Portage, Michigan
- Age at study initiation: 8-9 weeks
- Housing: The rats were initially housed in groups of four according to sex, then individually housed at the study start. After confirmation of mating, females were placed in plastic nesting boxes with bedding.
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: acetone, corn oil and diet

Details on exposure:

PREPARATION OF TEST SUBSTANCE IN DIET: A pre-measured amount of test substance was dissolved in 500 mL of acetone, and corn oil was added at a concentration so that it provided a constant 2% weight level of corn oil in the diet. The ration was weighed and placed in the Hobart planetary mixer for one hour. Test substance/acetone/corn oil mixture was slowly added to the ration with a small acetone rinse to achieve a quantitative transfer.

DIET PREPARATION
- Rate of preparation of diet (frequency): Once weekly
- Mixing appropriate amounts with (Type of food): Purina® certified Rodent Chow® #5002
- Storage of food: Stored in a light free vermin proof container and kept in a cool dry place.

Details on mating procedure:

- M/F ratio per cage: One male and one female
- Length of cohabitation: Until evidence of insemination (copulatory plug) or 15 days (designated as Day 0 of gestation). At this time the first female was removed and a second female placed in the breeding cage following the same procedure.
- Proof of pregnancy: Presence of copulatory plug
- After 15 days of unsuccessful pairing, the female of the F0 generation was considered barren, and placed in an individual cage; however, if there was evidence of weight gain, the rat was placed in a plastic nesting box. The female of the F1 generation was placed with a different male of the same treatment group.
- Further matings after two unsuccessful attempt: No
- After successful mating each pregnant female was placed in a plastic nesting box

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

for 2-generations

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

75 ppm

Doses / concentrations 2

Dose / conc.:

600 ppm

Doses / concentrations 3

Dose / conc.:

1 200 ppm

No. of animals per sex per dose:

F0 males: 13/group; F1 males: 20/group
F0 females: 26/group; F1 females: 40/group (for 0, 75, 600 ppm), 38 (for 1200 ppm)

Control animals:

yes, plain diet

Details on study design:

All generations had diet available in accordance to their treatment groups; females of the F0 generation from initiation of the study through Week 18 or until the F1 generation was weaned; F1 from birth through Week 30; and F2 from birth through Day 30. Males of the F0 and F1 generations were provided treatment diet until sacrifice.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Twice daily

BODY WEIGHT:
- Time schedule for examinations: Males were weighed at weekly intervals as were females that failed to mate. Females were weighed weekly until mating was confirmed; during gestation they were weighed on Day 3, and daily from Day 6 to Day 1 postpartum. After parturition, weekly weighing was resumed until sacrifice.

Sperm parameters (parental animals):

Parameters examined in [F0/F1] male parental generations:
Counts of sperm heads, which represent the nuclei of elongated spermatids, were performed with a hemacytometer and phase contrast microscope.

Litter observations:

At the time of delivery the following observations were made: litter size, sex of pups, number of live and stillborn. At birth and periodically when needed, pups were identified by applying basic fuschin to the base of the limbs following a specific numbering system. Animals were sexed and weighed on Days 1, 4, 7, 14 and 21 of lactation. Pups were observed daily for mortality, physical and behavioral abnormalities. On Day 4 of lactation any litters greater than ten pups were reduced to ten by the use of a table of random numbers and a balanced sex ratio was sought. After weaning (21 days) any remaining pups were weighed weekly.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: 13 per group (F0 and F1 generation adults)
- Maternal animals: 26 per group (F0 and F1 generation adults)

GROSS NECROPSY
- A complete gross examination was performed on all tissues.
The following organs were weighed: Brain, spleen, ovaries, liver, testes, uterus (F0 generation); Brain, spleen, ovaries, liver, testes, uterus, adrenals, heart, kidneys (F1 generation).

HISTOPATHOLOGY
F0 generation: Tissues examined: Bone marrow smears (femoral), brain (3 levels, including entire brainstem), liver, ovaries, sciatic nerve, testes, uterus, any lesions or tumors.
F1 generation: Brain (3 levels, including entire brainstem), pituitary, eyes, thyroids, parathyroids, salivary glands (submaxillary), larynx, esophagus, trachea, lung, heart, thymus, liver, kidneys, ovaries, urinary bladder, adrenals, spleen, stomach, small intestine (3 levels), large intestine (3 levels), lymph nodes, pancreas, skin of back, mammary gland, bone marrow smears (femoral), skeletal muscle, sciatic nerve, spinal cord, testes, prostate, uterus, any gross lesions or tumors.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Increased activity, pilo erection, depressed righting reflex and myoclonic body tics were more frequent and prominent in the F0 rats as the dosage level increased. This increase was initially noted during study week one through week three, thereafter this incidence was infrequent and sporadic. There were no apparent dose related clinical observations in the F0 rats. Alopecia was noted in all groups of both generations with increased severity in the 1200 ppm group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female rat in the 600 ppm group, died during the 18th week of the F0 generation. Pathological examination attributed the cause of death to chronic myocarditis. Another female was sacrificed moribund resulting from a malignant lymphosarcoma.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The F0 generation body weights of the 75 ppm group were not affected by treatment when compared to the control group. Body weights, however, of the males were statistically significantly increased from weeks six to fourteen but this was not considered to be biologically meaningful. The body weights of the males in the 600 ppm and the males and females in the 1200 ppm groups were statistically significantly decreased from week two until mating when compared to the controls. F0 generation gestation body weights of the 75 ppm group were comparable to the control group. The gestation body weights of the 600 ppm and the 1200 ppm groups were statistically significantly decreased in a dose related manner. The F0 lactation body weights were initially significantly less for the dams of the 75 ppm group when compared to the control group. These weights remained decreased though not significant until sacrifice. The lactation body weights of the 600 ppm and 1200 ppm group paralleled that of the gestation body weights.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The F0 generation groups of 600 ppm and 1200 ppm for both males and females, had decreased food consumption when compared to the controls, however, this decrease was only occasionally significant. Food consumption for the 75 ppm group generally paralleled that of the control group.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The only effect on hematology was a decrease in red blood cell count, hemoglobin and hematocrit in high dose females, which suggests a slight anemia. There was a decrease in mid-dose females also, however, all mean values were within normal ranges for this laboratory.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test substance related effects on red blood cell and plasma cholinesterase.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant test material related changes noted grossly and histopathologically in tissues from the F0 generation (adults).

Reproductive function / performance (P0)

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

The fertility and mean gestation length were comparable for all treatment groups to the control in the F0 generation.

Effect levels (P0)

Key result

Remarks on result:

other: Significant body weight and food consumption changes were observed

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Alopecia was noted in all groups of both generations (F0 and F1) with increased severity in the 1200 ppm group.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Litter size for the F1 generation was slightly reduced in the treatment groups when compared to the control. The mean number of dead pups at birth for the treated groups was comparable to the control group for the F1 generation. The mean number of live pups and pup survival for the remainder of the lactation period for both the 75 ppm and the 600 ppm groups was comparable to the control group for both generations. The mean number of live pups at birth in the 1200 ppm group was statistically significantly decreased when compared to the control group and pup survival was significantly reduced for the remainder of the lactation period. The reduced pup survival occurred predominately during the first 4 days of lactation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Following selection of the F1 generation body weights for females of the 600 ppm group and males/females of the 1200 ppm group were significantly decreased when compared to their respective controls. The males in the 600 ppm group also had decreased body weights and this reduction became significant in the second week after selection and remained so throughout the study. The males and females of the 600 ppm and 1200 ppm group remained statistically significantly less throughout gestation and lactation until sacrifice. Rats in the 75 ppm group weighed slightly less than those of the controls following selection. These decreases however were sporadically significant throughout the generation.

Pups in the 75 ppm group of the F1 generation were slightly lighter in body weight at birth. However, these weights became statistically significantly reduced by the time of weaning. Pup body weights for both generations in the 600 and 1200 ppm groups were statistically significantly reduced from birth to weaning.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the F1 generation food consumption was statistically significantly less for the males and females of the 600 ppm and the 1200 ppm. Both the males and females of the 75 ppm consumed slightly less than the controls and this decrease was only occasionally significant.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Some significant changes in organ weights and relative weights were noted. However, no histopathological changes were noted to explain these variations.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant test material related changes noted grossly and histopathologically in tissues from the F1 generation (adults) or the F1 (pups).

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant test material related changes noted grossly and histopathologically in tissues from the F1 generation (adults) or the F1 (pups)

Other effects:

no effects observed

Description (incidence and severity):

The fertility and mean gestation length were comparable for all treatment groups to the control in the F1 generation. No meaningful differences were noted in any of the groups in spermatogenesis when compared with the controls.

Effect levels (F1)

Key result

Remarks on result:

other: Dosage of 1200 ppm produced significant reduction in body weight, reduced pup survival; dose of 600 ppm produced reduction of body weight in pups in 2 generations but no consistent deleterious effects.

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Litter size for the F2 generation was slightly reduced in the treatment groups when compared to the control and this decrease was statistically significant in the 600 ppm group of the F2 generation. The mean number of dead pups at birth for the treated groups was comparable to the control group for the F2 generation except for a statistically significant increase in the number of dead pups in the 1200 ppm group of the F2 generation. The mean number of live pups in both the 75 ppm and the 600 ppm groups were statistically significantly reduced at birth in the F2 generation. This decrease may not be meaningful as the control value was considerably greater than that of the F1 value. The mean number of live pups in the 600 ppm group remained significantly decreased on Day 4 prior to culling. The mean number of live pups and pup survival for the remainder of the lactation period for both the 75 ppm and the 600 ppm groups was comparable to the control group for both generations. The mean number of live pups at birth in the 1200 ppm group was statistically significantly decreased when compared to the control group and pup survival was significantly reduced for the remainder of the lactation period. The reduced pup survival occurred predominately during the first 4 days of lactation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The F2 pup body weights of the 75 ppm group were comparable at birth and remained comparable through weaning and until sacrifice. Pup body weights for both generations in the 600 and 1200 ppm groups were statistically significantly reduced from birth to weaning.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Some significant changes in organ weights and relative weights were noted. However, no histopathological changes were noted to explain these variations.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant test material related changes noted grossly and histopathologically in tissues from the F2 generation (pups).

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant test material related changes noted grossly and histopathologically in tissues from the F2 generation (pups).

Effect levels (F2)

Key result

Remarks on result:

other: Dosage of 1200 ppm produced significant reduction in body weight, reduced pup survival; dose of 600 ppm produced reduction of body weight in pups in 2 generations but no consistent deleterious effects.

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Dosage of 1200 ppm produced significant reduction in body weight in parental rats and offspring, reduced pup survival; no pathological or histopathological changes found; dose of 600 ppm produced reduction of body weight in parents and pups in 2 generations but no consistent deleterious effects; at 75 ppm no effect on body weight or pup survival. The fertility and mean gestation length were comparable for all treatment groups to the control in the F0 and F1 generations.

Executive summary:

The test substance was administered in the diet during the course of this study. This study was designed to meet the criteria for a two generation reproduction study in rats as document in EPA guideline 83-4. The effects of the test substance on the reproductive performance was evaluated by monitoring fertility, gestation, parturition, lactation, pup growth rate and survival of rats over two generations.

The test substance was administered in the diet continuously for two generations to rats at 75, 600 or 1200 ppm. The diets were analyzed for test substance concentration and all rats were observed for signs of toxicity. Gross and histopathological examinations of tissues from parental rats and their offspring were performed.

A dosage level of 1200 ppm produced significant reduction in body weight in parental rats and their offspring and reduced pup survival, however, no gross pathological or histopathological changes were noted. Rats and their offspring receiving 600 ppm were significantly lighter in body weight throughout the two generations without any consistent deleterious effects observed.

The test substance fed at 75 ppm during the F0 generation had no effect on body weight or pup survival. During the initial weeks of the F1 generation the amount of test substance consumed in this group was approximately twice that in the F0 generation based on a mg/kg/day level and decreased body weights resulted. These decreased body weights were minimal and no other signs of toxicity were observed. The fertility and mean gestation length were comparable for all treatment groups to the control in the F0 and F1 generations.

The fertility and mean gestation length were comparable for all treatment groups to the control in the F0 and F1 generations. No meaningful differences were noted in any of the groups in spermatogenesis when compared with the controls.