Methomyl

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Study Type	Species	Findings	Guideline	Reliability
2-Generation Feeding Study	Rat	NOEL = 75 ppm (parental and offspring). LOEL = 600 ppm based on reduction in body weights in parents and pups. No adverse effects on reproduction or development	EPA OPP 83-4	1
3-Generation Feeding Study	Rat	Parental and offspring NOAEL = 4.6 mg/kg bw/day. Reproductive NOAEL = 80 mg/kg bw/day	No guideline followed	2

Link to relevant study records

Reference

Reference 1

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

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes

Limit test:

no

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%

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

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

Dose / conc.:

75 ppm

Dose / conc.:

600 ppm

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.

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.

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: 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.

Key result

Remarks on result:

other: Significant body weight and food consumption changes were observed

Critical effects observed:

no

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.

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.

Critical effects observed:

no

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).

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.

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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.

Reference 2

Endpoint:

three-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was fed to male and female rats for approximately three months at levels of 0, 0. 005 and 0.01% of a nutritionally complete diet. After completion of the feeding period, a reproduction study was conducted with 10 male and 20 female rats within each group in which F1A and F1B litters were cast. 10 male and 20 female weanling rats were selected from the F1B litter in each group and continued on their respective diets for three months, at which time they were bred within groups to produce F2A and F2B litters. This same procedure was followed with the F2B litter to produce the F3A and F3B litters. 10 male and 10 female weanling rats of the F3B litter from the control and each test group were subjected to a histopathological evaluation following gross necropsy.

GLP compliance:

no

Specific details on test material used for the study:

- Substance name: Lannate methomyl insecticide
- Lot#: H-4429, H-5116
- Purity: Not reported

Species:

rat

Strain:

other: Charles River Caesarean-derived albino

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: feed

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

First generation parents, F1B litter, F2B litter: Three months

Frequency of treatment:

daily

Dose / conc.:

0.005 other: %

Dose / conc.:

0.01 other: %

No. of animals per sex per dose:

10 male and 20 female rats

Control animals:

yes, plain diet

Key result

Dose descriptor:

NOAEL

Effect level:

4.6 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Effect level:

4.6 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Effect level:

4.6 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Effect level:

4.6 mg/kg bw/day

Key result

Reproductive effects observed:

no

Conclusions:

There were no effects on parents or reproduction and fertility. Pup weight and food consumption were reduced in F3 pups. The parental and offspring NOAEL was 4.6 mg/kg bw/day and the reproductive NOAEL 80 mg/kg bw/day.

Executive summary:

The test substance was fed to male and female rats for approximately three months at levels of 0, 0.005 and 0.01% of a nutritionally complete diet. After completion of the feeding period, a reproduction study was conducted with 10 male and 20 female rats within each group in which F1A and F1B litters were cast. 10 male and 20 female weanling rats were selected from the F1B litter in each group and continued on their respective diets for three months, at which time they were bred within groups to produce F2A and F2B litters. This same procedure was followed with the F2B litter to produce the F3A and F3B litters. 10 male and 10 female weanling rats of the F3B litter from the control and each test group were subjected to a histopathological evaluation following gross necropsy. The indices and litter data obtained for the test groups were comparable with those for the control group. The average weaning weights of the second (F2) and third (F3) generation pups fed 0.01% the test substance were slightly lower than those of the controls, but the difference was statistically significant only with the F2B litter; these differences were not considered related to administration of the test substance. A post-weaning growing phase was conducted with female pups from an additional third generation litter (F3C) The test litters were fed the test substance for nine weeks and received only control feed from Week 10 through Week 10. Body weight gains from Week 0 through Week 9 for the litter fed 0.01% the test substance were significantly lower than those for the control litter; however,

the decreased growth in the 0.01% test litter was not considered related to administration of the test substance, but was attributed to reduced food Intake; food consumption through the 14-week growing phase for the 0.01% test Litter was significantly lower than that for the control litter; and body weight gains in the test litter remained lower than the control after the test substance was withdrawn, and the pups were fed the control diet only. No test substance-related gross or histopathologic changes were observed in any of the tissues examined from F3B weanling rats. Organ/body weight and brain/body weight ratios were determined. The organ weight values for weanlings in the test groups showed no test substance-related differences from control values. The test substance was fed to rats at dietary levels of 0.005 and 0.01% for three generations without any adverse effect upon reproduction and without any gross or histologic evidence of test substance-related congenital anomalies.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Species:

rat

Quality of whole database:

Rat (diet) 2-generation reproductive study, 75 ppm equivalent to 4.6 mg/kg bw/day based on body weight effects in males and females at 600 ppm.
Repro/Fertility: >1200 ppm No adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The two-generation reproduction study in rats was designed to meet the criteria 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.

Effects on developmental toxicity

Description of key information

Study Type	Species	Findings	Guideline	Reliability
Developmental Feeding Study	Rat	Maternal NOAEL = 100 ppm (9.4 mg/kg bw/d). Fetal NOAEL = 400 ppm (33.9 mg/kg bw/d) Not embryotoxic or teratogenic	no guideline followed	2
Developmental Gavage Study	Rabbit	Maternal NOEL = 6 mg/kg. Fetal NOEL = 16 mg/kg Not embryotoxic or teratogenic	no guideline followed	2

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental 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

Qualifier:

no guideline followed

Principles of method if other than guideline:

The purpose of this study was to determine the toxicity and the teratogenic potential of the test substance in the rabbit. Solutions of the test substance in deionized water were administered by gavage on days 7 through 19 of presumed gestation to artificially inseminated rabbits at dosages of 0 (vehicle), 2, 6 and 16 mg/kg/day at a dosage volume of 5 mL/kg/day. Rabbits were observed for signs of agent effect, abortion or delivery. Body weights were recorded. Does which aborted were killed, examined, and observed uterine contents were recorded. Does which were found dead were similarly examined. On day 29 of presumed gestation, surviving female rabbits were killed, the abdomen of each was opened, and the uterus wag examined. Each fetus or delivered pup was weighed and subsequently examined for gender and for gross external, visceral and skeletal alterations.

GLP compliance:

yes

Specific details on test material used for the study:

Methomyl
Lot#: INX-1179-255
Purity: 98.7%

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dutchland Laboratories, Inc., Swampbridge Road, Box 139A, Denver, Pennsylvania
- Age at study initiation: Approximately 28 weeks
- Weight at study initiation: 3.14 to 4.71 Kg
- Housing: Female rabbits were housed in vertical order according to dosage and insemination groups. Housing units consisted of eight individual stainless steel cages (5 square feet of floor area with 16 inch height per individual cage).
- Diet: Certified rabbit chow #5322, approximately 180 g each per day
- Water: ad libitum
- Acclimation period: Approximately 7 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 17.7-22.2°C
- Humidity: 30-80%
- Air changes: 12-15 per hr
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Solutions of the test substance in reverse osmosis processed deionized water (R.O. water) a were prepared daily prior to use. Concentrations, calculated on the basis of the active ingredient were selected to provide 5 mL/kg dosage volume to rabbits in all dosage groups.

VEHICLE
- Concentration in vehicle: 0.4, 1.2 and 3.2 mg/mL for dosages of 2, 6 and 16 mg/kg respectively

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: Artificial insemination
- Verification of same strain and source of both sexes: yes
- Day of artificial insemination is referred to as day 0 of pregnancy

Duration of treatment / exposure:

GD 7 to GD 19

Frequency of treatment:

daily

Dose / conc.:

2 mg/kg bw/day

Dose / conc.:

6 mg/kg bw/day

Dose / conc.:

16 mg/kg bw/day

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages evaluated in this study were selected on the basis of an initial toxicity assessment and a pilot evaluation.
In the initial toxicity assessment, one female rabbit (not inseminated) was administered a single 24 mg/kg/day dosage of the test substance to determine if a rabbit could tolerate the agent at the maximum dosage proposed for the preliminary study. In the preliminary study four artificially-inseminated rabbits were assigned to each dosage group, and the test substance was administered at dosages of 8, 16 or 24 mg/kg/day on days 7 through 19 of presumed gestation.
The 24 mg/kg/day dosage was lethal. Two rabbits in this dosage group died on day 7 of presumed gestation at approximately 10 and 30 minutes post dosage. Dosage-dependent clinical signs observed for surviving rabbits included ataxia, decreased motor activity, body jerks, tremors, vocalization (16 and 24 mg/kg/ day dosage groups), salivation, clonic convulsions, dyspnea, pin-point pupils, tales and reluctance to use hindlimbs (24 mg/kg/day dosage group). Pregnancy occurred in 4, 2, 1 and 3 rabbits in the control, low, middle and high dosage groups, respectively. Of these, one control rabbit aborted, and two high dosage group rabbits died. Maternal body weight and caesarean-sectioning data were inconclusive. The single surviving pregnant rabbit in the high dosage group had a larger number of resorptions and fewer live fetuses, as compared with control group rabbits, a possible agent-related effect. All fetuses from litters of does given test substance appeared normal at gross external examination. Two fetuses, one from each of two control litters, were hydrocephalic (domed heads).

Maternal examinations:

OBSERVATIONS: Female rabbits were observed for physical signs and/or general appearance several times during the prestudy period and on days 0, 3 and 6 of presumed gestation. Observations for physical signs of agent effect, abortion and/or viability were made several times each day during the administration period (days 7 through 19 of presumed gestation) and daily during the post administration period (days 20 through 29 of presumed gestation).


BODY WEIGHT
- Time schedule for examinations: Day after arrival, once weekly during the acclimation period, on days 0 and 6 of presumed gestation, and daily during the administration period (days 7 through 19 of presumed gestation) and Post administration period (days 20 through 29 of presumed gestation) .

FOOD CONSUMPTION: During the study rabbits were observed for anorexia.

POST-MORTEM EXAMINATIONS
- Sacrifice on gestation day # 29
- Organs examined: Lungs, uterus and gross lesions

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Weight of each live fetus
- Number of corpora lutea
- Number of implantations
- Number and location of early and late resorptions
- Number and location of all live and dead fetuses
- Other: Any gross anomaly

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]

Statistics:

Maternal physical sign data and the incidence of pregnancy and abortion were analyzed using Fisher's exact test.
The one-way analysis of variance was used to evaluate average body weights of rabbits assigned to the four dosage groups on days 0 and 6 of presumed gestation. Maternal body weight data for days 0, 7, 11, 15, 20, 24, 29 and 29C (corrected) of pregnancy a were analyzed using Bartlett's test of homogeneity of variances, and either the one-way analysis of variance or the Mann-Whitney U test. If Bartlett's test was positive (P ≤0.05), then the Mann-Whitney U test was used to analyze the data by testing each group individually against the control dosage group.
If Bartlett's test was negative (P >0.05), then the one-way analysis of variance was used to analyze the data. If the F-ratio was positive (P ≤0.05), then Dunnett's test was used to test each group individually against the control dosage group.
Data obtained during Caesarean-sectioning and for malformations and variations was evaluated using Jonckheere's test with the population of affected fetuses per litter considered to be the basic experimental unit. If Jonckheere's test was positive (P ≤0.05), and if there were fewer than 75% ties in a group, then the Mann-Whitney U test was used to analyze the data by testing each group individually against the control dosage group. If Jonckheere' s test was positive (P ≤0.05), and if there were more than 75% ties in a group, then Fisher's exact test was used with fixed point censoring to evaluate the data.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

CNS stimulation occurred for high dosage group rabbits and was considered to be an effect of the test substance. Dosage-dependent increases (P =0.047 to P <0.0001) in incidence of tremors, hyperactivity, body jerks, excess salivation, aggressive behavior (stamping of hind feet), hyperpnea, and convulsions were observed for high dosage group rabbits, as compared with control. Dosage-dependent increased incidences were also observed for ataxia, impaired or lost righting reflex, self-inflicted or spontaneous lesions, dyspnea, hyper sensitivity, pin-point pupils and vocalization. These incidences were not significantly different (P >0.05) from those observed for control rabbits.

Mortality:

mortality observed, treatment-related

Description (incidence):

Test substance related deaths occurred for one rabbit in the 6 mg/kg/day dosage group and 7 (P =0.003) rabbits in the 16 mg/kg/ day dosage group. These deaths generally followed observed evidence of central nervous system (CNS) stimulation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A small, dosage-related increase (P <0.05) in maternal body weight gain occurred between days 0 and 29 of gestation; however, only the gain of the high dosage group differed significantly from the control value. This difference was due to the decreased incidence of anorexia of the dams of the high dosage group during the latter portion of gestation.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Postmortem observations included a perforated stomach and evidence of excess salivation in the middle dosage group rabbit. Excess salivation was also observed for high dosage group rabbits, and the incidence was significantly increased (P =0.008), as compared with the control group.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

As compared with the control group, the incidence of anorexia was significantly decreased (P =0.009) for rabbits in the high dosage group. This observation was considered to be a rebound in feed consumption after the dosage period. During the last part of gestation, spontaneous anorexia did occur among the control, low and middle dosage group rabbits.

Number of abortions:

no effects observed

Description (incidence and severity):

The incidence of pregnancy and number of corpora lutea per dam were similar for all dosage groups.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The incidence of implantations per dam were similar for all dosage groups.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The incidence of resorptions per dam were similar for all dosage groups.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The incidence of pregnancy per dam were similar for all dosage groups.

Remarks on result:

other: maternal toxicity was expressed as death and CNS stimulation

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A dosage-related increase in average fetal body weight occurred; the difference from the control value was statistically significant only for the middle (P =0.025) and high (P =0.003) dosage groups. This increase in fetal body weight was not considered to be an adverse effect, since it was not observed to be due to the presence of edema. It probably was due to increased feed consumption of the dams during the latter part of gestation.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Administration of 2, 6 and 16 mg/kg/day dosages of the test substance to does did not result in adverse effects on embryo/ fetal viability or fetal sex ratio. Similarly, no gross external, soft tissue or skeletal malformations, developmental variations or variations due to retarded development observed for fetuses were attributed to administration of the test substance to the does.

Key result

Remarks on result:

other: Administration of aqueous solutions of the test substance to pregnant rabbits at dosages of 2, 6 and 16 mg/kg/day on days 7 through 19 of gestation was neither teratogenic nor embryo-fetal toxic, even in the presence of maternal toxicity.

Key result

Developmental effects observed:

no

Conclusions:

Administration of aqueous solutions of the test substance to pregnant rabbits at dosages of 2, 6 and 16 mg/kg/day on days 7 through 19 of gestation was neither teratogenic nor embryo-fetal toxic, even in the presence of maternal toxicity.

Executive summary:

The purpose of this study was to determine the embryofetal toxicity and the teratogenic potential of the test substance in the rabbit. Solutions of the test substance in reverse osmosis processed deionized water were administered by gavage on days 7 through 19 of presumed gestation to artificially-inseminated rabbits which had been randomly assigned to each of four dosage groups (20 rabbits/group).

The test substance as administered to rabbits at dosages of 0 (vehicle), 2, 6 and 16 mg/kg/day (calculated as the active ingredient) at a dosage volume of 5 mL/kg/day.

Agent-related deaths occurred for one rabbit in the 6 mg/kg/day dosage group and seven (P =0.003) rabbits in the 16 mg/kg/ day dosage group. These deaths generally followed observed evidence of central nervous system (CNS) stimulation.

One low dosage group rabbit aborted on day 27 of gestation, and one middle dosage group rabbit delivered on day 29 of gestation. These occurrences were considered to be spontaneous.

CNS stimulation occurred for high dosage group rabbits and was considered to be an effect of the test agent. Dosage-dependent increases (P =0.047 to P <0.0001) in incidence of tremors, hyperactivity, body jerks, excess salivation, aggressive behavior (stamping of hind feet), hyperpnea, and convulsions were observed for high dosage group rabbits, as compared with control. Dosage-dependent increased incidences were also observed for ataxia, impaired or lost righting reflex, self-inflicted or spontaneous lesions, dyspnea, hyper sensitivity, pin-point pupils and vocalization. These incidences were not significantly different (P >0.05) from those observed for control rabbits.

Postmortem observations included a perforated stomach and evidence of excess salivation in the middle dosage group rabbit. Excess salivation was also observed for high dosage group rabbits, and the incidence was significantly increased (P =0.008), as compared with the control group.

As compared with the control group, the incidence of anorexia was significantly decreased (P =0.009) for rabbits in the high dosage group. This observation was considered to be a rebound in feed consumption after the dosage period. During the last part of gestation, spontaneous anorexia did occur among the control, low and middle dosage group rabbits.

A small, dosage-related increase (P <0.05) in maternal body weight gain occurred between days 0 and 29 of gestation; however, only the gain of the high dosage group differed significantly from the control value. This difference was due to the decreased incidence of anorexia of the dams of the high dosage group during the latter portion of gestation.

A significant decrease (P =0.024) in maternal body weight was observed among the dams in the 2 mg/kg/day dosage group on days 0 to 7, as compared with that for the control group. This decrease was not test substance-related, since it occurred before the dosage period.

The incidence of pregnancy and number of corpora lutea, implantations and resorptions per dam were similar for all dosage groups.

A dosage-related increase in average fetal body weight occurred; the difference from the control value was statistically significant only for the middle (P =0.025) and high (P =0.003) dosage groups. This increase in fetal body weight was not considered to be an adverse effect, since it was not observed to be due to the presence of edema. It probably was due to increased feed consumption of the dams during the latter part of gestation.

Administration of 2, 6 and 16 mg/kg/day dosages of the test substance to does did not result in adverse effects on embryo/ fetal viability or fetal sex ratio. Similarly, no gross external, soft tissue or skeletal malformations, developmental variations or variations due to retarded development observed for fetuses were attributed to administration of the test substance to the does.

On the basis of these data, administration of aqueous solutions of the test substance to pregnant rabbits at dosages of 2, 6 and 16 mg/kg/day on days 7 through 19 of gestation was neither teratogenic nor embryo-fetal toxic, even in the presence of maternal toxicity. The maternal toxicity was expressed as death and CNS stimulation.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was conducted to evaluate the teratogenic potential of the test substance when fed to pregnant rats during the period of fetal organogenesis. 25 pregnant females were assigned to each group. During the treatment period (days 6 through 15 of gestation) the diets containing 50, 100 and 400 ppm of the test substance were administered along with a concurrent control diet group. All rats were sacrificed by chloroform inhalation on the day 21 of gestation. At the time of sacrifice, the abdominal wall of the female was opened and both ovaries and uterus were removed and inspected. The uterus was then opened, and the fetuses removed and examined. About one half of the fetuses from each litter were preserved for subsequent examination to detect skeletal abnormalities. The remaining fetuses were examined for visceral and neural anomalies.

GLP compliance:

no

Specific details on test material used for the study:

S-methyl N-[(methyl-carbamoyl)oxy]-thioacetimidate
Lot #: INX-1179-255 (N.B. 7452-168)
Purity: >99%

Species:

rat

Strain:

other: ChR-CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., North Wilmington, Massachusetts
- Age at study initiation: Animals were received as 2 to 3 days pregnants
- Weight: 200-205 g on GD 6
- Housing: Housed individually in suspended stainless steel wire mesh cages
- Diet: Ground Purina Laboratory Chow, ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 22-23°C
- Humidity: 50%

Route of administration:

oral: feed

Vehicle:

other: diet + 1% mazola corn oil

Details on exposure:

During the treatment period (days 6 through 15 of gestation) the diets containing test substance (Ground Purina Laboratory Chow + 1% mazola corn oil + test substance) were administered.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

GD 6 to GD 15

Frequency of treatment:

daily

Dose / conc.:

50 ppm

Remarks:

equivalent to 4.9 mg/kg/day

Dose / conc.:

100 ppm

Remarks:

equivalent to 9.4 mg/kg/day

Dose / conc.:

400 ppm

Remarks:

equivalent to 33.9 mg/kg/day

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Maternal examinations:

OBSERVATIONS: Rats were observed daily for clinical signs of toxicity and changes in behavior.

BODY WEIGHT
- Time schedule for examinations: Day of arrival, and on gestation days 6, 10, 16 and 21

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption is calculated as gram per rat per day
- Compound intake is calculated as mg/kg/day

POST-MORTEM EXAMINATIONS
- Sacrifice on gestation day # 21
- Organs examined: Ovaries and uterus

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Weight of each live fetus
- Number of corpora lutea
- Number of implantations
- Number and location of resorptions
- Number and location of all live and dead fetuses
- Crown rump length of each live fetus (to 0.01 cm)
- Other: Any gross anomaly which could be observed under a long focal length lens of 2.5X

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]

Statistics:

The Fisher Exact probability test was used to evaluate the incidence of resorptions and abnormalities among litters. Maternal and fetal body weights and fetal crown-rump measurements were treated statistically by analysis of variance and least significant difference (LSD) tests. The number of corpora lutea, implantations and live fetuses per litter were analyzed by the Wilcoxon rank sum test. In all cases, two tailed significance tests were performed, and significance was judged at the 0.05 probability level.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were observed in any of the test groups during any part of the study.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The presence of 400 ppm test substance in the diet of pregnant rats resulted in a significantly lower mean body weight when compared to the controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the exposure period, rats receiving 100 ppm and 400 ppm of the test substance in their diets consumed less food than did those in the control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At necropsy a small area of the liver of one female receiving 100 ppm of the test substance was fibrotic in appearance but the weight was within normal limits. No gross pathological changes were observed in the ovaries and uterus or vital organs and tissues of other treated females.

Total litter losses by resorption:

no effects observed

Details on maternal toxic effects:

Parameters measuring the outcome of pregnancy and fetal development, including the number of litters with partial and total resorptions, and the body measurements of the fetuses were not different from those of the concurrent controls.

Remarks on result:

other: The presence of 400 ppm test substance in the diet of pregnant rats resulted in a significantly lower mean body weight when compared to the controls.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Only two fetuses, each one from a different litter, were found with multiple malformations. Both were from litters exposed to the intermediate level (100 ppm) of the test substance. They are considered as spontaneous congenital malformations of genetic origin and are seen in this strain of rat at about the same frequency as in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No malformations or major abnormalities of the skeletal system were noted. The incidence of minor anomalies and common variations are biological and are related to chronological age of the fetuses (delayed ossification and bipartite centra) or to their genetic background (wavy and 14th full or rudimentary ribs) and not to the treatment.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Most anomalies and malformations found in the soft-tissue sectioning were isolated occurrences with two exceptions; apparent hydronephrosis and peliosis of the liver. Apparent hydronephrosis was found in fetuses of all groups at approximately the same rate. Peliosis of the liver occurred in all but the highest level in numbers similar to the control. No major anomalies were found in the group receiving the highest level (400 ppm).

Key result

Remarks on result:

other: Under the conditions of this test, the test substance was not embryotoxic or teratogenic when fed to the rats.

Key result

Developmental effects observed:

no

Conclusions:

Under the conditions of this test, the test substance was not embryotoxic or teratogenic when fed to the rats.

Executive summary:

The purpose of this study was to evaluate the teratogenic potential of the test substance when fed to pregnant rats during the period of fetal organogenesis. 25 pregnant females were assigned to each group. During the treatment period (days 6 through 15 of gestation) the diets containing 50, 100 and 400 ppm of the test substance were administered along with a concurrent control diet group. All rats were sacrificed by chloroform inhalation on the day 21 of gestation. At the time of sacrifice, the abdominal wall of the female was opened and both ovaries and uterus were removed and inspected. The uterus was then opened, and the fetuses removed and examined. About one half of the fetuses from each litter were preserved for subsequent examination to detect skeletal abnormalities. The remaining fetuses were examined for visceral and neural anomalies.

In the group fed 400 ppm to pregnant rats resulted in decreased food consumption and body weight gains. Body weight gains were significantly lower. Only a slight effect on food consumption was noted at the intermediate level (100 ppm). With the exception of one female at the intermediate level with liver fibrosis, no clinical signs of toxicity, changes in behavior, or gross pathological changes in organs or tissues were observed in treated rats. The outcome of pregnancy measured by the number of implantation sites, resorptions and live fetuses, was not adversely affected by the treatment. The treatment did not affect embryonal development as measured by weight and crown-rump length of the fetuses. Only two fetuses, each one from a different litter, were found with multiple malformations. Both were from litters exposed to the intermediate level (100 ppm) of the test substance. They were considered as spontaneous congenital malformations of genetic origin and are seen in this strain of rat at about the same frequency as in this study. These defects were not treatment related.

Under the conditions of this test, the test substance was not embryotoxic or teratogenic when fed to the rats.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

Rat (diet): Developmental toxicity study, 100 ppm (9.4 mg/kg bw/day) based on decreased body weight and food consumption in dams at 400 ppm.
Foetal: >400 ppm; no adverse effect observed


Rabbit (gavage): Developmental toxicity study, 6 mg/kg bw/day based on decreased maternal body weight, mortality, and clinical signs of cholinesterase inhibition at 16 mg/kg bw/day.
Foetal: >16 mg/kg bw/d; no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The teratogenic potential of the test substance when fed to pregnant rats during the period of fetal organogenesis was conducted. 25 pregnant females were assigned to each group. During the treatment period (days 6 through 15 of gestation) the diets containing 50, 100 and 400 ppm of the test substance were administered along with a concurrent control diet group. All rats were sacrificed by chloroform inhalation on the day 21 of gestation. At the time of sacrifice, the abdominal wall of the female was opened and both ovaries and uterus were removed and inspected. The uterus was then opened, and the fetuses removed and examined. About one half of the fetuses from each litter were preserved for subsequent examination to detect skeletal abnormalities. The remaining fetuses were examined for visceral and neural anomalies. In the group fed 400 ppm to pregnant rats resulted in decreased food consumption and body weight gains. Body weight gains were significantly lower. Only a slight effect on food consumption was noted at the intermediate level (100 ppm). With the exception of one female at the intermediate level with liver fibrosis, no clinical signs of toxicity, changes in behavior, or gross pathological changes in organs or tissues were observed in treated rats. The outcome of pregnancy measured by the number of implantation sites, resorptions and live fetuses, was not adversely affected by the treatment. The treatment did not affect embryonal development as measured by weight and crown-rump length of the fetuses. Only two fetuses, each one from a different litter, were found with multiple malformations. Both were from litters exposed to the intermediate level (100 ppm) of the test substance. They were considered as spontaneous congenital malformations of genetic origin and are seen in this strain of rat at about the same frequency as in this study. These defects were not treatment related. Under the conditions of this test, the test substance was not embryotoxic or teratogenic when fed to the rats.

A developmental study in rabbits was also conducted. Solutions of the test substance in reverse osmosis processed deionized water were administered by gavage on days 7 through 19 of presumed gestation to artificially-inseminated rabbits which had been randomly assigned to each of four dosage groups (20 rabbits/group). The test substance as administered to rabbits at dosages of 0 (vehicle), 2, 6 and 16 mg/kg/day (calculated as the active ingredient) at a dosage volume of 5 mL/kg/day. Agent-related deaths occurred for one rabbit in the 6 mg/kg/day dosage group and seven (P =0.003) rabbits in the 16 mg/kg/ day dosage group. These deaths generally followed observed evidence of central nervous system (CNS) stimulation. One low dosage group rabbit aborted on day 27 of gestation, and one middle dosage group rabbit delivered on day 29 of gestation. These occurrences were considered to be spontaneous. CNS stimulation occurred for high dosage group rabbits and was considered to be an effect of the test agent. Dosage-dependent increases (P =0.047 to P <0.0001) in incidence of tremors, hyperactivity, body jerks, excess salivation, aggressive behavior (stamping of hind feet), hyperpnea, and convulsions were observed for high dosage group rabbits, as compared with control. Dosage-dependent increased incidences were also observed for ataxia, impaired or lost righting reflex, self-inflicted or spontaneous lesions, dyspnea, hyper sensitivity, pin-point pupils and vocalization. These incidences were not significantly different (P >0.05) from those observed for control rabbits. Postmortem observations included a perforated stomach and evidence of excess salivation in the middle dosage group rabbit. Excess salivation was also observed for high dosage group rabbits, and the incidence was significantly increased (P =0.008), as compared with the control group. As compared with the control group, the incidence of anorexia was significantly decreased (P =0.009) for rabbits in the high dosage group. This observation was considered to be a rebound in feed consumption after the dosage period. During the last part of gestation, spontaneous anorexia did occur among the control, low and middle dosage group rabbits. A small, dosage-related increase (P <0.05) in maternal body weight gain occurred between days 0 and 29 of gestation; however, only the gain of the high dosage group differed significantly from the control value. This difference was due to the decreased incidence of anorexia of the dams of the high dosage group during the latter portion of gestation. A significant decrease (P =0.024) in maternal body weight was observed among the dams in the 2 mg/kg/day dosage group on days 0 to 7, as compared with that for the control group. This decrease was not test substance-related, since it occurred before the dosage period. The incidence of pregnancy and number of corpora lutea, implantations and resorptions per dam were similar for all dosage groups. A dosage-related increase in average fetal body weight occurred; the difference from the control value was statistically significant only for the middle (P =0.025) and high (P =0.003) dosage groups. This increase in fetal body weight was not considered to be an adverse effect, since it was not observed to be due to the presence of edema. It probably was due to increased feed consumption of the dams during the latter part of gestation. Administration of 2, 6 and 16 mg/kg/day dosages of the test substance to does did not result in adverse effects on embryo/ fetal viability or fetal sex ratio. Similarly, no gross external, soft tissue or skeletal malformations, developmental variations or variations due to retarded development observed for fetuses were attributed to administration of the test substance to the does. On the basis of these data, administration of aqueous solutions of the test substance to pregnant rabbits at dosages of 2, 6 and 16 mg/kg/day on days 7 through 19 of gestation was neither teratogenic nor embryo-fetal toxic, even in the presence of maternal toxicity. The maternal toxicity was expressed as death and CNS stimulation.

Toxicity to reproduction: other studies

Description of key information

Study Type	Species	Findings	Guideline	Reliability
3-Generation Feeding study	Rat	No adverse effects on reproduction or development	no guideline followed	2

Additional information

The test substance was fed to male and female rats for approximately three months at levels of 0, 0.005 and 0.01% of a nutritionally complete diet. After completion of the feeding period, a reproduction study was conducted with 10 male and 20 female rats within each group in which F1A and F1B litters were cast. 10 male and 20 female weanling rats were selected from the F1B litter in each group and continued on their respective diets for three months, at which time they were bred within groups to produce F2A and F2B litters. This same procedure was followed with the F2B litter to produce the F3A and F3B litters. 10 male and 10 female weanling rats of the F3B litter from the control and each test group were subjected to a histopathological evaluation following gross necropsy. The indices and litter data obtained for the test groups were comparable with those for the control group. The average weaning weights of the second (F2) and third (F3) generation pups fed 0.01% the test substance were slightly lower than those of the controls, but the difference was statistically significant only with the F2B litter; these differences were not considered related to administration of the test substance. A postweaning growing phase was conducted with female pups from an additional third generation litter (F3C) . The test litters were fed the test substance for nine weeks and received only control feed from Week 10 through Week 10. Body weight gains from Week 0 through Week 9 for the litter fed 0.01% the test substance were significantly lower than those for the control litter; however, the decreased growth in the 0.01% test litter was not considered related to administration of the test substance, but was attributed to reduced food Intake; food consumption through the 14-week growing phase for the 0.01% test Litter was significantly lower than that for the control litter; and body weight gains in the test litter remained lower than the control after the test substance was withdrawn, and the pups were fed the control diet only. No test substance-related gross or histopathologic changes were observed in any of the tissues examined from F3B weanling rats. Organ/body weight and brain/body weight ratios were determined. The organ weight values for weanlings in the test groups showed no test substance-related differences from control values. The test substance was fed to rats at dietary levels of 0.005 and 0.01% for three generations without any adverse effect upon reproduction and without any gross or histologic evidence of test substance-related congenital anomalies.

Justification for classification or non-classification

There is no evidence to suggest that the test substance causes adverse effects on reproduction or development of the offspring. Therefore, the test substance is not classified for reproductive or developmental toxicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.