Chlorothalonil

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Effects on fertility

Description of key information

- NOAEL for parental systemic toxicity is <500 ppm (equal to 36.1 and 40.9 mg/kg bw for males and females, respectively) based on the decreased body weight in the F1 animals and the results of the macroscopic and microscopic examination in both males and females; EPA OPP 83 -4, Lucas and Benz, 1990

- NOAEL for reproductive toxicity is > 3000 ppm (equal to 225 and 255 mg/kg bw for males and females, respectively), based on no adverse effects in the highest examined dose group (3000 ppm); EPA OPP 83 -4, Lucas and Benz, 1990

- NOAEL for developmental toxicity is 500 ppm (equal to 36.1 and 40.9 mg/kg bw for males and females, respectively) based on the decreased pup body weight; EPA OPP 83 -4, Lucas and Benz, 1990

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Sep 1988 to 22 Dec 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD-VAF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: (P) x 27 days old upon receipt and 40 days old at the initiation of the test material administration;
- Housing: The rats were housed two per suspended stainless steel cage upon receipt and for seven days thereafter in order to aid in their acclimation to the automatic watering. After randomization and for the remainder of the study, the animals were housed individually except during mating and lactation. A solid stainless steel bottom vas placed in the cages of the females from Day 18 of gestation through lactation. Ground corncob bedding vas provided during the period when the cage bottom vas in place.
- Diet: rodent chow ad libitum during the acclimation period. This rodent chow vas also used in the preparation of the diets for the study. Fresh diets were provided as often as necessary to ensure an adequate food supply, but routinely on a weekly basis. Feeders designed to minimize soiling and scattering were used and any incidence of excessive spillage vas noted. The feeders were sanitized weekly.
- Water: fresh potable water was available ad libitum by means of an automatic water system during both the acclimation and study periods

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24
- Humidity (%): 40 - 60
- Air changes: 11 or more fresh air changes per hour
- Photoperiod: alternating 12 hour light/dark cycle

IN-LIFE DATES: From: 20 Sep 1988 To: 22 Dec 1989

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
For each dietary level, appropriate amounts of the test material were mixed fresh weekly with a small amount of basal diet in a mortar and pestle in order to make a premix. The premix was then blended with the remainder of the basal diet for a 10 minute period in a Marion ribbon blender. The prepared diets were stored at room temperature in the dark. Diets were prepared at a constant concentration (ppm)

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: two weeks
- Proof of pregnancy: a copulation plug in the vagina and/or the presence of sperm in a vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: A solid stainless steel bottom vas placed in the cages of the females from Day 18 of gestation through lactation. Ground corncob bedding vas provided during the period when the cage bottom was in place.
- Females that did not exhibit evidence of mating were cohabited with the same male each night until evidence of mating was observed or until the two week mating period vas completed. In some instances, there were more females than males in a group due to the death of one or more males. When this occurred, selected males that had been sucessfully mated the first night were placed with the females that lacked mates on the second day of mating.
- All animals were mated during the second mating period regardless of whether they had delivered a litter following the first mating period. Different pairs of rats were mated for the second mating periods. During mating for the F1 generation animals care was taken to avoid brother-sister matings.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity
Prior to the initiation of the in life phase of the study, diets at concentrations of 500 ppm and 3000 ppm were prepared using the same methods of mixing as would be used subsequently in the study. Five samples at each concentration were assayed to evaluate homogeneity of mixing.

Stability
Prior to the initiation of the in life phase of the study, samples of diet at 500 ppm and 3000 ppm, which had been prepared for the homogeneity study were analyzed after sitting for 7 or 14 days under study room conditions.

Weekly Diet Samples
The concentration of the test in diets was measured weekly during the first 10 weeks of the study and biweekly for the remainder of the study.

Duration of treatment / exposure:

15 months

Frequency of treatment:

F0 parental animals were treated continuously throughout the 10 week growth period and also throughout the mating, gestation, lactation and rest phases of the study. All offspring and the F1 parents were exposed to the test material throughout their time on study.

Details on study schedule:

- F1 parental animals not mated until 14 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.

Dose / conc.:

500 ppm

Remarks:

P0 Group 2: 36.1 and 40.9 mg/kg bw for males and females, respectively

Dose / conc.:

1 500 ppm

Remarks:

P0 Group 3: 110 and 124 mg/kg bw for males and females respectively

Dose / conc.:

3 000 ppm

Remarks:

P0 Group 4: 225 and 255 mg/kg bw for males and females, respectively

No. of animals per sex per dose:

35 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

The parental, animals from each generation were treated continuously during the growth period prior to mating and then throughout the mating, gestation,
lactation and resting phases of the study. The growth period was ten weeks for the F0 animals and fourteen weeks for the F1 animals. Each generation of parental animals was mated twice to produce the F1a, F1b, F2a and F2b litters. The offspring was exposed to the test diets throughout the lactation periods. Thirty-five F1b males and females per group were selected to become the F1 generation after weaning.

Selection of Offspring for the F1 Generation
On Day 21 of lactation the size of each F1b litter was adjusted by random selection. For Group 1, one half of the litters was adjusted to yield three males and two females when possible and the other half of the litters was adjusted to yield two males and three females when possible. For the other groups (2, 3 and 4), each litter was adjusted to yield two males and two females when possible. The extra animals were selected from Group 1 because the number of litters available for selection necessitated having one additional animal in each litter to ensure sufficient animals for selection of the F1 generation.
When the number of male or female pups per litter was less than necessary to yield the selected number of pups, a partial adjustment was made; for example, a litter of six males and one female was adjusted to yield two male and one female pups. No adjustments were made for litters of two or less male and female pups.
After the last F1b litter was weaned (Day 21 of lactation), thirty-five male and thirty-five female pups were randomly selected from each group to produce the F1 generation. When possible at least one male and one female was selected from each litter.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Throughout the period of compound administration the animals were checked early in the morning and in the afternoon for overt signs of toxicity, moribundity and mortality. Prior to the initiation of treatment the animals were checked once a day.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Complete physical examinations were carried out weekly for all male animals throughout the study and for all females throughout the growth period prior to mating and during the two week resting period.

BODY WEIGHT:
- Time schedule for examinations: Individual body weights were recorded weekly from one week prior to initiation of treatment for the F0 animals and from the day of selection for the F1 animals. For males body weights were recorded each week until termination except during the mating periods when no weights were taken. Body weights for females were recorded during the growth periods and during the two week rest periods.
Body weights of females which showed positive signs of mating were recorded on Days 0, 7, 14 and 20 of gestation. Following parturition, body weights were recorded on Days 0, 7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Individual food consumption was measured weekly prior to mating and during the two week rest period. Food consumption was not recorded for males or females during the mating, gestation and lactation periods. Food was provided ad libitum during those periods.
Mean compound consumption (mg/kg/day) was calculated at weekly intervals prior to mating and during the two week rest period using the body weight, food consumption data and nominal dietary concentrations.

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Not examined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 ] offspring:
Date delivered, number, weight and sex of live and stillborn pups and any abnormalities observed in the offspring were recorded. Each litter was examined twice daily for survival and overt clinical signs of toxicity throughout the lactation period. Number and sex of live pups were recorded on Day 0, 1, 4 (pre- and post-cull), 7, 14 and 21 of lactation. Total litter weights for live pups were measured on Days 0, 4 (pre- and post-cull), 7 and 14 of lactation. Individual weights for live pups were measured on Day 21 of lactation.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
All surviving F0 and F1 adults were killed after the F1b or F2b litters were weaned.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY
- The following tissues were prepared for microscopic examination:
adrenal glands (2), bone (femur), bone marrow (sternum), brain, esophagus, eyes (2), gonads, testes with epididymides and seminal vesicles, ovaries with uterus and vagina, heart with aorta, intestines (duodenum, jejunum, ileum, cecum), colon, rectum, kidneys (2), liver (left and median lobe), lungs - fixed by inflation, pancreas, pituitary, prostate, salivary glands with cervical lymph nodes, skeletal muscle with peripheral nerve, skin with mammary tissue, spinal cord (cervical), spleen, stomach, thymus, thyroid and parathyroid glands, trachea, urinary bladder, all gross lesions

Postmortem examinations (offspring):

SACRIFICE
Those pups culled on Day 4 of lactation were killed on that day. All remaining offspring were killed on Day 21 of lactation, with the exception of the F1b offspring which were saved for selection to be F1 parents. Those F1b offspring not selected to be F1 parents were killed at the time of selection of F1 parents.

GROSS NECROPSY
All grossly abnormal offspring were necropsied and preserved in 10% neutral buffered formalin.

Statistics:

Contingency table analyses were conducted on gestation length, Day 4 pup viability indices and lactation indices instead of the proposed analyses. This course was chosen because for each set of data almost all of the observations were identical, so that contingency table analyses were more appropriate than those proposed. For the contingency table analysis the Hantel-Baenszel test for a trend in the proportions was conducted instead of the Armitage test for trend. The change was made because the computer programming for the Armitage test was not readily available and the analyses give similar results. Except for Bartlett's test and the test for lack-of-fit, the statistical analyses were reported at the 1% level in addition to the 5% level of significance.

Reproductive indices:

Male Indices:
Mating index = (Number of males producing females with positive signs of mating/Total number of males mated) x 100

Male fertility index = (Number of males producing pregnant females/Number of males producing females with positive signs of mating) x 100

Female Indices:
Mating Index = (Number of females showing positive signs of mating/Total number of females mated) x 100

Female Fertility Index = (Number of pregnant females/Number of females showing positive signs of mating) x 100

Offspring viability indices:

Sex Ratio Index = (Live born males/Total live born pups) x 100

Live Born Index = Number of pups live born/Total number of pups born) x 100

Stillborn Index = (Number of pups stillborn/Total number of pups born) x 100

Day 4 Pup Viability Index = (Number of pups alive Day 4 (pre-cull)/Total number of pups born alive) x100

Lactation Index = (Number of pups alive Day 21/Number of pups alive Day 4 (post-cull)) x 100

Litter Indices:

Live Born Index = (Number of litters with live pups Day 0/Total number of litters) x 100

Stillborn Index = (Number of litters with stillborn pups/Total number of litters) x 100

Day 4 Viability Index = (Number of litters with live pups Day 4 (pre-cull)/Number of litters with live pups Day 0) x 100

Lactation Index = (Number of litters with live pups Day 21/Number of litters with live pups day 4 (post-cull)) x 100

Viability Index = (Number of litters with live pups Day 21/Number of litters with live pups Day 0) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The only observation which could have been treatment related was an observation of loose stools in seven male rats from the high dose group. For six of the animals the observation of loose stools was noted only once and so was not considered a significant finding.There were no observation of clinical signs which were considered to be treatment related during gestation or lactation.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a clear treatment-related effect on body weight in both sexes.
For the high dose males the mean body weight was approximately 10% lower than the controls throughout the P0 study period. This represented 11 and 15% lower body weight gain by week 10 and 30 respectively. Both the body weight and the body weight gain showed a statistically significant difference for every week on study.
For the mid-dose male group, mean body weight gain was statistically significantly lower for Week 1 and for almost every week from Week 5 onwards. Mean body weights for the mid-dose group reached statistical significance after Week 18. At this time the difference from the control group was approximately 5%. Although the mean body weight and mean body weight gain of the low dose group were slightly lower than the control values throughout the study period, statistical significance vas not reached at any time for either parameter. The no observable effect level (NOEL) for body weight of the F0 males was considered to be 500 ppm.
For the females at the high dose level there were statistically significant differences in mean body weight and mean body weight gain throughout the growth and rest periods. The difference in body weight from the control group vas approximately 5% through to Week 10 and then increased to 7 or 8% during the rest period between the F1a and F1b littering. The body weight gain for the high dose was 13% lower than the controls by the end of the growth period. Although the mean body weight and mean body weight gains of the mid- and low dose groups for the F0 females were consistently lower than the control values, there were no statistically significant differences between groups. It can be concluded that 1500 ppm is the NOEL for weight effects on F0 females.

At Day 0 of gestation the mean body weight of the high dose group vas 7% lower than the control group and the mean body weight of the mid-dose group vas 5% lower than the control group. Both values were statistically significant when compared with the control group. The low dose group was comparable with the control group. During the gestation period the differences from the control group narrowed slightly so that the difference from control was reduced to 5% at the high dose and 3.5% at the mid-dose. On Day 20 of gestation only the high dose mean body weight was statistically lower than the control. The mean body weight of the low dose group was comparable with controls throughout gestation.

At Day 0 of lactation the mean body weight of the high dose group was 6.5% lower than the control group and the mean body weight of the mid-dose group was 5% lower than the control group. Both differences were statistically significant from control group. During lactation the differences between the groups disappeared and at Days 14 and 21 the mean body weights for all groups were comparable.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

For males and females the mean absolute and relative food consumptions were statistically reduced for Week 1 for the high dose groups. There were no significant differences in absolute food consumption after Week 1 for the high dose males and females which were considered treatment related.
Since absolute food consumption was similar to controls but body weight was lower than the controls, food consumption relative to body weight was higher for most study weeks for the high dose male and female F0 animals. Absolute and relative food consumption for the mid- and low dose levels for both sexes shoved sporadic incidences of statistically significant differences between groups but no consistent pattern which could be considered treatment related.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically, treatment-related effects were observed in the stomachs of male and female rats at all dose levels. These alterations to the stomach were characterized microscopically as squamous epithelial hyperplasia and hyperkeratosis.
Treatment-related effects were observed microscopically in the kidneys from male rats at all dose levels and from females at the mid- and high dose level. The effects increased in incidence and severity in a dose-related fashion and the findings were increased in incidence and severity in the males compared to the females.
Significant incidences of tubular epithelial hyperplasia and tubular hypertrophy were observed in both sexes and foci of clear cell hyperplasia and pigmentation were present in the kidney cortex of males as well as one Group 4 female.
In males the number of animals with cervical lymph nodes which were described as enlarged at necropsy was higher in the mid- and high dose groups than in the control group animals but most of the observations were estimated as only 2 to 5 times the normal size. Microscopically the enlarged lymph nodes showed slight to moderately severe lymphoid hyperplasia. Since the severity and incidence of the finding vas not related to dose and enlarged lymph nodes are a common finding, the increase was not considered treatment related.
No effects which were considered treatment related were observed in the primary or accessory sex organs for male or female rats. All of the findings in the sex organs were either seen in comparable severity and incidence in the controls and treated groups or were considered common in rats of this strain and age.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Two males in the high dose group had primary renal neoplasms, one a tubular adenoma and one a tubular carcinoma. The neoplasms were probably associated with the dietary administration of the test substance.

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The Day 0 mean litter size (total pups and liveborn pups) shoved no statistically significant difference between groups although the litter size and number of live offspring were slightly lower for the high dose group compared with the other groups.

The number of litters with stillborn pups in the treated groups were comparable and slightly lower than the number in the control groups. The mean index of stillborn pups was slightly higher in the control group than in the treated groups.

The mating and fertility indices showed no adverse effect of treatment at any dose level. Out of the 35 females mated from each group, 18, 23, 27 and 27 produced litters from the control, low, mid- and high dose groups, respectively. This represents a pregnancy rate of 51%, 66%, 77% and 77%, respectively. The pregnancy rates for all groups and especially the control group was lower than the pregnancy rate in the F1a
littering. The difference may have been due to the age or size of the females at the time of the F1b littering.

There were no significant differences between the mean number of days of gestation in the treated groups and the control group. The majority of animals delivered on Days 22 or 23 of gestation.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity (originally reported NOELs reevaluated to yield NOAELs, see study and endpoint summaries)

Effect level:

< 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

gross pathology

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity (originally reported NOELs reevaluated to yield NOAELs, see study and endpoint summaries)

Effect level:

>= 3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other: Not determinable due to absence of adverse effects

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 500 ppm

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Four male and two female F1 parental rats died while on study. The males were from each of the three treatment groups and the females were both from the mid-dose group. The deaths were not considered treatment related. There were no clinical observations during the F1 period which were considered to be treatment related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

On Week 0 of the F1 generation the mean body weights of the male and female control groups were 6% higher than the mean weights of the low dose groups. The F1 animals were selected from the F1b pups. At weaning of the F1b pups there was no significant difference between the control and the low dose group. Since the F1 animals were selected randomly from the F1b pups with no restrictions imposed on mean body weight, it is possible that the lower body weight at the beginning of the F1 generation for the low dose groups was due to a fortuitous
choice of heavier and lighter pups from the litters of the control and low dose groups. For males, the mean body weight of the high dose group was 9% lower than the control value at the beginning of the growth period and the difference, which was statistically significant each week, varied from 6 to 11% during the F1 period. In addition, the mean body weight gain of the high dose group was statistically significantly lower than the control group for almost every week. The difference over the entire 34 week period was 11%. The mean body weights and body weight gains of the mid-dose group males shoved no significant differences from the control values at any time during the F1 period but both the mean body weight and body weight gain were slightly lower than the means for the controls towards the end of the F1 period. Since the mid-dose level had a clear effect on body weight and body weight gain in the Fo generation which developed with time, it is possible that the differences in mean body weight and body weight gain in the F1 males, which were 5% and 6% respectively by Week 34, were, in fact, treatment related.
At Week 0 of the F1 growth period the mean body weight of the low dose group males was 6% lower than the control group. Because of the difference in the mean body weights at the beginning of the F1 period, it is considered more relevant to compare body weight gain data rather than body weight data to study the possibility of there being an effect on body weight for the low dose group males during the F1 growth period. There were no statistically significant differences between the mean body weight gains for the low dose group and the control group after Week 5. The differences up to Week 5 were not considered treatment related because the mid-dose group body weight data was comparable to the control data for Weeks 0 to 5. The low dose level, 500 ppm in the diet, is considered a no observable effect level (NOEL) for male body weight effects during the F1 period.
For the females the mean body weight and mean body weight gains for all treatment groups were lower than the·values for the control group for most of the F1 study period.
For approximately the first ten weeks of the F1 period, the mean values for all three dose groups were quite similar and the differences from the control group did not show a dose response. From Week 10 and Week 12 onwards for mean body weight and mean body weight gain respectively, the statistical analysis for trend showed a fit. At Weeks 24 and 25 there were no statistically significant differences in body weight gain between the controls and the low dose group. At the end of the F1 growth period, the percentage difference in mean body weight gain for the
high dose group and control group was similar in the F0 generation (14%) and the F1 generation (15%) which suggests the data for the high dose from the F1 females is valid.
The available information suggests that the difference in body weight data during the growth period for the low dose level was probably not related to treatment but that the lover body weight data at the high dose was treatment related.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

For males there vas no treatment-related effect on absolute food consumption during the F1 period. For several weeks the absolute food consumption was significantly lower in the low dose group but these differences were not considered to be treatment related since there were no consistent differences in the high or mid-dose groups.
Since the mean body weights of the high and mid-dose group males were lower than the control group but the mean absolute food consumption remained comparable with the control group, the food consumption relative to body weight was higher than for the control group and it is possible that utilization of food was affected by the test substance.
For the F1 females the mean absolute food consumption values for the treated groups, especially the low dose group, were frequently lower than the values for the control group but the data did not form a dose-response pattern. The differences, therefore, in absolute food consumption were not considered treatment related. The high dose F1 females showed an increase in relative food consumption compared with the controls almost every week food consumption was measured. The difference which was similar to that observed in the mid- and high dose male groups was considered to be treatment related. There were no statistical differences for the mid- or low dose groups.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related effects were observed in the stomach and kidney both by gross and microscopic pathology.
At necropsy, there were a few observations of raised areas or foci in the mucosa of the forestomach in the male and female rats which had received the test material, but the incidences did not increase in a treatment-related manner for either sex.
There was also a slight increase in the incidence of mottled kidneys in the mid- and high dose groups for both males and females relative to the control and low dose groups, but the observations were more common in the males. There was also a slight increase in the incidence of mottled kidneys in the mid- and high dose groups for both males and females relative to the control and low dose group.
There were two, one and three observations of small testes at necropsy in the low, mid- and high dose groups of male rats. The small testes correlated with the microscopic observations of spermatogenesis.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically, treatment-related effects were observed in the stomachs of male and female rats at all dose levels. The alterations to the stomach were characterized microscopically as squamous epithelial hyperplasia and hyperkeratosis. Both the incidence and severity of the microscopic lesions increased in a dose-related manner.
There was an increase in the number of observations made at necropsy in the kidney in the mid- and high dose level groups for both males and females, but the observations were more common in the males. The treatment-related observations were enlarged kidney, granular cortical surface and alteration of colour to pale or green.

Treatment-related effects were observed microscopically in the kidneys from male rats at all dose levels and from females at the mid- and high dose level. The effects, which increased in incidence and severity in a dose-related fashion, were more severe in the males. Tubular epithelial hyperplasia, tubular hypertrophy, foci of clear cell hyperplasia, pigmentation and karyomegaly were the treatment-related findings. In females only, a treatment-related increase in regenerative epithelium was also observed. It is likely that regenerative epithelium was not observed in the males because it was masked by the more severe lesions present in the kidneys of the male rats.
At the high dose level there was one rat with bilateral spermatogenesis, three rats with unilateral spermatogenesis and two with bilateral focal spermatogenesis. Since for most of the rats the finding was focal or unilateral, the incidence of spermatogenesis did not form a dose response and the incidence was considered normal by the pathologist, the spermatogenesis was not considered treatment related. There were no abnormal findings in female reproductive organs which were considered treatment related.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity (originally reported NOELs reevaluated to yield NOAELs, see study and endpoint summaries)

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity (originally reported NOELs reevaluated to yield NOAELs, see study and endpoint summaries)

Effect level:

> 3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3 000 ppm

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

F1a: The Day 0 mean litter size (total pups and liveborn pups) showed no statistically significant difference between groups although the litter size and number of live offspring were slightly lower for the high dose group compared with the other groups.

F1b: The Day 0 mean litter size (total pups and liveborn pups) showed no treatment-related differences. The number of litters with stillborn pups and the mean index of stillborn pups were slightly lower in the control group than in the treated groups. The slight difference were not considered treatment related since the control group from the F1b littering had less stillborns than the control groups in the other three litterings and the values for treated animals were within the range of control values.
No treatment-related effect on offspring viability was observed when the litter indices for the F1b reproduction were evaluated. All pregnant females delivered litters with live pups on Day 0 of lactation. There was one total litter loss from the high dose group but originally there was only one pup in the litter.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F1a: The only mean pup weight which was statistically different from the control values during the lactation period was the Day 21 weight of the high dose group which was 92.9% of the control value.

F1b: The mean pup weights on Day 0 were comparable and showed no dose-related reduction. On Day 4, prior to culling, the pup body weights of the mid-dose group were significantly lower than the controls but post-cull the difference was not significant. On Days 7, 14 and 21 of lactation, the mean pup weights for the mid- and high dose groups were significantly lower than the mean weight of the control group. When the pup weights were analyzed by analysis of covariance with litter size as the covariate, there were no statistical differences in mean pup weights between groups on Day 4 precull or Day 7. On Day 21 the differences between the control and the mid- and high doses were 7.7% and 14.1%, respectively. The mean pup weigh ts of the low dose group were comparable with the mean weights of the control group throughout the lactation period.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1a: Very few abnormal observations were made at the necropsies of the stillborn, spontaneous deaths or culled pups and the incidences do not suggest a treatment-related effect.
The only unusual finding in F1a pups was a single observation of a stillborn pup from the high dose group with an abnormal head which was described as having a small anterior portion with no eye lids, only one nostril apparent and no visible external opening for the oral cavity.
Dilation of the renal pelvis was the only observation from the necropsy of the pups terminated at 21 days of age which was higher in the treated groups. The percentage of litters with at least one finding of dilated renal pelvis was 24% in the control group, 34% in the low dose group, 26% in the mid-dose group and 38% in the high dose group. The percentage of pups with the finding followed the same pattern.
The only incidences of extremely dilated renal pelvises in F1a pups were from the mid- or high dose groups which might suggest a treatment-related effect; however, the data from the F1b, F2a and F2b litterings did not confirm this finding and the finding was not considered treatment related.
F1b: Very few abnormal observations were made at the F1b necropsies and the incidences do not suggest a treatment related effect

Histopathological findings:

no effects observed

Other effects:

not examined

Description (incidence and severity):

F1a: There were no significant differences between the groups for mean ratio of live born males to total live born pups
F1b: There were no significant differences between the groups for mean ratio of live born males to total live born pups.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity (originally reported NOELs reevaluated to yield NOAELs, see study and endpoint summaries)

Generation:

F1

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

F2a: The Day 0 mean litter size (total pups and liveborn pups) showed no statistically significant difference between groups although the litter size and number of live offspring were slightly lower for the treatment groups compared with the control group. The data did not fall into a dose response pattern, and the mean size of the litter for each group was within the range of the mean control values from the other litterings in the study. The difference was not considered to be treatment related.
The numbers of litters with stillborn pups in the treated groups were comparable and, for the high dose group, slightly lower than the number in the control group.
No treatment-related effect on offspring viability was observed when the litter indices for the F2a reproduction were evaluated. All pregnant females delivered litters with live pups on Day 0 of lactation except for one dam which died during delivery. There was only one total litter loss throughout the lactation period, which was from the low dose group.

F2b: The Day 0 mean litter size and the mean number of live offspring on Day 0 showed no treatment-related differences. The number of litters with stillborn pups in the treated groups were comparable and slightly lower than the number in the control group. The mean index of stillborn pups was slightly higher in the control group than in the treated groups.
No treatment-related effect on offspring viability was observed when the litter indices for the F2b reproduction were evaluated. All pregnant females delivered litters with live pups on Day 0 of lactation and there were no total litter losses throughout the lactation period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

F2a: When standard statistics were used, the only statistically significant difference in mean pup weight for the F2a pup was for the high dose group at 21 days, which had a mean pup weight 8.6% lower than for the control group. When litter size was used as a covariant in the statistical analysis, the low- dose group showed a significantly lower mean pup weight. Because the mid-dose group pup weight was comparable with the control group, the low dose difference was not considered treatment related.

F2b: The mean pup weights from Day 0 up until Day 14 showed no treatment-related reduction. On Day 21 the mean pup weights in the high and lov dose groups were lower than the controls and the differences were significant at the 0.01 level for the high dose and the 0.05 level for the low dose. The mean pup weights on Day 21 for the mid-dose vas close to that of the control group. The statistical test for trend on Day 21 was significant; however, there was a lack of fit because the mid-dose group had a value close to the control value;

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F2a: Very few abnormal observations were made at the necropsies of the stillborn, spontaneous deaths or culled pups and the incidences do not suggest a treatment-related effect. The only unusual finding in F2a pups was two male pups from a control litter which were joined from the thoracic region up to the head.
The necropsy of the pups terminated at 21 days of age showed a high background incidence of dilated renal pelvis that vas not treatment related.

F2b: No abnormal observations were made at the necropsies of the stillborn, spontaneous deaths or culled pups. The necropsy of the pups terminated at 21 days of age showed several incidences of dilation of the renal pelvis but there was no significant difference in incidence between groups.

Histopathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

F2a: There were no significant differences between the groups for mean ratio of live born males to total live born pups.
F2b: There were no significant differences between the groups for mean ratio of live born males to total live born pups.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity (originally reported NOELs reevaluated to yield NOAELs, see study and endpoint summaries)

Generation:

F2

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Diet Analysis
The pre-test evaluation of homogeneity and stability of the test substance in the diets demonstrated that a homogeneous dispersion of the test material in the feed was prepared and that the test material was adequately stable in the feed for 7 or 14 days under study room conditions. During the study the overall mean analyzed concentrations of chlorothalonil in the diets were 98%, 99% and 99% of the nominal concentrations for the 500 ppm, 1500 ppm and 3000 ppm
groups, respectively. The results indicated that the diets were prepared at the intended concentrations throughout the study

Table 1. Mean body weights and body weight gain (g) – selected timepoints

	Dietary concentration (ppm)
	F0 Males				F0 Females
Week	0	500	1500	3000	0	500	1500	3000
0	159.1	158.1	161.6	158.6	140.7	138.5	137.8	138.3
5	387.5	378.5	375.1	357.8*	231.9	228.8	225.6	218.4**
10	492.5	478.4	473.2	457.0**	273.7	268.8	262.3	253.3**
20	604.6	577.5	571.8*	540.8**	312.3	308.4	303.7	291.0**
30	650.2	621.9	614.4	577.1**	-	-	-	-
0-10	333.4	320.3	311.6*	298.3**	133.1	130.3	124.5	115.0**
	F1 Males				F1 Females
	0	500	1500	3000	0	500	1500	3000
0	126.4	118.5	125.5	115.3**	112.1	104.9	107.5	102.3**
5	394.5	373.5*	395.3	367.2**	244.9	223.3**	226.7**	223.3**
10	510.9	490.9	505.3	469.7**	294.9	268.0**	274.5**	261.9**
20/24	628.4	601.5	613.4	573.4**	343.8	322.9	323.9	308.3**
30	676.7	638.2*	649.7	608.4**	-	-	-	-
0-10	384.5	372.4	379.9	354.4**	182.9	163.1**	166.9*	159.5**

* Significantly different from control P < 0.05
** Significantly different from control P < 0.01

 

Table 2. Mean food consumption (g/rat/day) – selected timepoints

	Dietary concentration (ppm)
	F0 Males				F0 Females
Week	0	500	1500	3000	0	500	1500	3000
1	23.6	22.4	22.3*	19.3**	17.6	16.2**	16.6	14.5**
5	27.1	26.3	25.9	25.5*	18.7	18.2	17.9	17.9
10	27.9	27.1	27.2	27.0	19.4	18.5	18.7	18.5
Week 10 (g/kg/day)	56.6	56.2	57.2	59.2**	70.8	68.5	70.9	72.1
	F1 Males				F1 Females
	0	500	1500	3000	0	500	1500	3000
1	22.0	19.7*	22.1	21.2	18.3	17.5	17.7	18.2
5	30.4	29.2	31.3	29.5	22.0	20.3*	20.7	21.1
10	29.8	27.9*	30.5	28.4	20.4	19.7	19.9	19.4
Week 10 (g/kg/day)	58.1	56.9	60.0	60.4**	69.3	73.0	72.4	73.9**

* Significantly different from control P < 0.05
** Significantly different from control P < 0.01

 

Table 3. Reproductive Performance (selected parameters)

	Dietary concentration (ppm)
	F1a litter				F1b litter
Observation	0	500	1500	3000	0	500	1500	3000
Number mated	35	35	35	35	35	35	35	35
Number of matings	26	34	34	35	27	32	32	32
Number of pregnancies	25	29	31	32	18	23	27	27
Fertility index (%)	96	85	91	91	67	72	84	84
Mean gestation length (days)	22.1	22.3	22.1	22.0	22.5	22.3	22.0	22.1
Mean pups still born	0.4	0.1	0.2	0.2	0.1	0.1	0.4	0.1
Mean live litter size at birth	13.1	12.9	12.9	12.1	12.3	12.6	13.5	12.9
Mean live day 4 (pre-cull)	13.0	12.4	12.8	11.9	12.2	12.5	13.5	12.8
Mean live day 21	8.0	7.7	7.9	7.7	7.8	7.7	8.0	8.0
Live born index (%)	97.6	99.3	98.7	98.4	99.2	97.8	97.4	98.7
Viability index days 0-4 (%)	99.4	96.1	99.3	98.3	98.8	99.3	99.7	99.5
Lactation index days 4-21 (%)	100	99.6	100	100	100	100	100	96.3
	Dietary concentration (ppm)
	F2a litter				F2b litter
Observation	0	500	1500	3000	0	500	1500	3000
Number mated	35	35	34	35	35	34	33	34
Number of matings	29	29	28	27	31	30	22	27
Number of pregnancies	23	27	24	22	14	23	17	17
Fertility index (%)	79	93	86	81	45	77	77	63
Mean gestation length (days)	22.3	22.2	22.5	22.1	22.6	22.3	22.5	22.4
Mean pups still born	0.7	0.5	0.7	0.1	0.8	0.1	0.1	0.1
Mean live litter size at birth	13.2	12.4	12.4	12.7	12.2	13.0	12.2	13.1
Mean live day 4 (pre-cull)	13.0	12.2	12.7	12.4	12.0	12.9	12.2	13.1
Mean live day 21	8.0	7.8	7.7	7.8	7.7	7.9	7.6	7.8
Live born index (%)	95.0	96.3	94.2	99.1	94.1	99.0	99.1	99.2
Viability index days 0-4 (%)	98.5	94.6	98.2	97.4	97.5	99.7	100	99.6
Lactation index days 4-21 (%)	100	100	99.5	100	100	100	100	99.3

 

Table 4. Pup body weights (g)

	Dietary concentration (ppm)
	F1a				F1b
	0	500	1500	3000	0		500	1500	3000
Mean pup weight at birth	6.2	6.2	6.3	6.3	6.6	6.3		6.2	6.3
Mean pup weight day 7	15.4	15.7	15.5	15.5	17.3	16.9		16.0*	15.9*
Mean pup weight day 14	31.0	31.1	30.8	30.2	33.8	33.0		31.4*	29.5**
Mean pup weight day 21	49.6	49.2	47.9	46.1*	56.2	54.6		51.9*	48.3**
F2a	F2b
Mean pup weight at birth	6.3	6.1	6.3	6.3	6.3	6.1		6.5	6.2
Mean pup weight day 7	16.2	15.7	16.4	15.8	16.5	15.7		16.8	16.0
Mean pup weight day 14	32.5	31.0	33.2	31.1	32.8	31.2		32.2	31.2
Mean pup weight day 21	53.3	50.2	52.5	48.7**	55.0	50.4*		52.1	48.6**

* Significantly different from control P < 0.05
** Significantly different from control P < 0.01

Conclusions:

The NOAEL for parental toxicity is 500 ppm (equal to 22.6 mg/kg bw/day) based on the decreased body weight in the F1 animals and the results of the macroscopic and microscopic examination in both males and females (effects on kidneys and stomach). Given the decrease in body weight of the F1 pups (day statistically significant on days 7, 14 and 21), the NOAEL for developmental effects is established at 500 ppm (equal to 22.6 mg/kg bw/day). All reproductive parameters investigated showed no treatment-related effects except for pup weights at Day 21 which had a no observable effect level of 1500 ppm.

Executive summary:

In this two generation reproduction study (EPA OPP 83 -4, under GLP) with two litters per generation, the test substance was administered to Crl CD rats by dietary admixture at concentrations of 500, 1500 and 3000 ppm. There were 35 rats of each sex in the three groups. An additional group of 35 rats of each sex received untreated basal diet. The parental animals from each generation were treated continuously during the growth period prior to mating and then throughout the mating, gestation, lactation and resting phases of the study. The growth period was ten weeks for the F0 animals and Fourteen weeks for the F1 animals. Each generation of parental animals were mated twice to produce the F1a, F1b, F2a and F2b litters. The offspring were exposed to the test diets throughout the lactation periods. Thirty-five F1b males and females per group were selected to become the F1 generation after weaning. All animals were observed twice daily for overt signs of toxicity, moribundity and mortality. Complete physical examinations, body weights and Food consumption were recorded at regular intervals. All animals were necropsied and selected organs were preserved after completion of the F1b and F2b lactation periods. Histopathological examinations of F0 and F1 adults were conducted on reproductive organs as well as the target organs of test substance toxicity. All litters were examined daily for survival and overt clinical signs of toxicity throughout the lactation period. The date delivered, number, weight and sex of live and stillborn pups and any abnormalities observed in the offspring were recorded. On Day 4 of lactation the size of litters with nine or more pups was reduced to eight pups. The number, sex and weight of live pups were recorded at regular intervals during lactation. All surviving pups were sexed, weighed and necropsied at the end of each 21 day lactation period, except for selected F1b pups which continued as the F1 parental generation.

The study report describes the No Effect Levels (NOELs) for Parent, reproductive and developmental effects. Following assessment of the study data and the observed effects, the following No Adverse Effect Levels (NOAELs) are derived.

No mortalities or clinical signs of toxicity associated with administration of test substance were observed in the F0 or F1 animals. There was a treatment-related effect towards a lowered body weight in the F0 and F1 adults. The no observable adverse effect level for body weights was 500 ppm. The body weight effect was comparable in the F0 and F1 generations for both males and females. Increased relative Food consumption was observed in the groups that showed lower body weights. The anticipated treatment-related lesions in the kidneys and stomachs of adult animals were observed by gross and microscopic pathology. Chronic studies have shown that the no observable adverse effect levels for stomach and kidney effects were at lower treatment levels than the lowest dose achieved in this study. The effects were observed in the kidney at all dose levels in males and at 1500 and 3000 ppm in females. Stomach effects were observed at all dose levels in both sexes. The stomach and kidney effects were comparable in the F0 and F1 generations for both sexes.

Reproductive parameters in F0 and F1 animals, including mating and fertility indices and gestation length, were not affected by treatment of test substance. No gross malformations which were considered treatment related were observed in offspring in any of the groups throughout the study. No effects on the number of live and stillborn pups, pup sex ratio, pup survival and physical condition of the pups during lactation were observed. No findings which were considered treatment related were observed during necropsy of the pups. The only effect observed in pups in this study was lowered body weight compared to the control on Day 21 of lactation. The no observable adverse effect level for this effect was considered to be 1500 ppm. 

In summary, body weight, kidney and stomach effects were observed in parental animals for this reproduction study with test substance. The no adverse observable effect level (NOAEL; = NOEL) for body weight effects was 500 ppm but the stomach and kidney effects were observed at all dose levels. All reproductive parameters investigated showed no treatment-related effects except for pup weights at Day 21 which had a no observable adverse effect level of 1500 ppm. 

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

225 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

GLP compliant EPA OPP 83-4

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Two generation study

In this two generation reproduction study (EPA OPP 83 -4, under GLP) with two litters per generation, the test substance was administered to Crl CD rats by dietary admixture at concentrations of 500, 1500 and 3000 ppm. There were 35 rats of each sex in the three groups. An additional group of 35 rats of each sex received untreated basal diet. The parental animals from each generation were treated continuously during the growth period prior to mating and then throughout the mating, gestation, lactation and resting phases of the study. The growth period was ten weeks for the F0 animals and Fourteen weeks for the F1 animals. Each generation of parental animals were mated twice to produce the F1a, F1b, F2a and F2b litters. The offspring were exposed to the test diets throughout the lactation periods. Thirty-five F1b males and females per group were selected to become the F1 generation after weaning. All animals were observed twice daily for overt signs of toxicity, moribundity and mortality. Complete physical examinations, body weights and Food consumption were recorded at regular intervals. All animals were necropsied and selected organs were preserved after completion of the F1b and F2b lactation periods. Histopathological examinations of F0 and F1 adults were conducted on reproductive organs as well as the target organs of test substance toxicity. All litters were examined daily for survival and overt clinical signs of toxicity throughout the lactation period. The date delivered, number, weight and sex of live and stillborn pups and any abnormalities observed in the offspring were recorded. On Day 4 of lactation the size of litters with nine or more pups was reduced to eight pups. The number, sex and weight of live pups were recorded at regular intervals during lactation. All surviving pups were sexed, weighed and necropsied at the end of each 21 day lactation period, except for selected F1b pups which continued as the F1 parental generation.

The study report describes the No Effect Levels (NOELs) for Parent, reproductive and developmental effects. Following assessment of the study data and the observed effects, the following No Adverse Effect Levels (NOAELs) are derived.

No mortalities or clinical signs of toxicity associated with administration of test substance were observed in the F0 or F1 animals. There was a treatment-related effect towards a lowered body weight in the F0 and F1 adults. The no observable adverse effect level for body weights was 500 ppm. The body weight effect was comparable in the F0 and F1 generations for both males and females. Increased relative Food consumption was observed in the groups that showed lower body weights. The anticipated treatment-related lesions in the kidneys and stomachs of adult animals were observed by gross and microscopic pathology. Chronic studies have shown that the no observable adverse effect levels for stomach and kidney effects were at lower treatment levels than the lowest dose achieved in this study. The effects were observed in the kidney at all dose levels in males and at 1500 and 3000 ppm in females. Stomach effects were observed at all dose levels in both sexes. The stomach and kidney effects were comparable in the F0 and F1 generations for both sexes.

Reproductive parameters in F0 and F1 animals, including mating and fertility indices and gestation length, were not affected by treatment of test substance. No gross malformations which were considered treatment related were observed in offspring in any of the groups throughout the study. No effects on the number of live and stillborn pups, pup sex ratio, pup survival and physical condition of the pups during lactation were observed. No findings which were considered treatment related were observed during necropsy of the pups. The only effect observed in pups in this study was lowered body weight compared to the control on Day 21 of lactation. The no observable adverse effect level for this effect was considered to be 1500 ppm. 

In summary, body weight, kidney and stomach effects were observed in parental animals for this reproduction study with test substance. The no adverse observable effect level (NOAEL; = NOEL) for body weight effects was 500 ppm but the stomach and kidney effects were observed at all dose levels. All reproductive parameters investigated showed no treatment-related effects except for pup weights at Day 21 which had a no observable adverse effect level of 1500 ppm. 

 

Effects on developmental toxicity

Description of key information

- NOAEL for maternal toxicity is 10 mg/kg bw/day based on reduced food consumption and lowered body weight (gain) and for developmental toxicity 20 mg/kg bw/day based on the absence of adverse effects; EPA OPP 83 -3, Wilson 1988

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 Dec 1987 to 22 Jan 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Remarks:

[Hra: (NZW) SPF]

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 4 to 5 months (females), 7 to 9 months (males)
- Weight at study initiation: Females: 3869 g (mean), 3010 to 4779 g (range)
- Fasting period before study: Feed was withheld on the day of arrival.
- Housing: Animals were housed individually, except during mating, in stainless steel suspended cages with wire mesh floors and subcage floor pans in a separate room from other animals in the facility. A stainless steel feeder was affixed to the front of each cage. Feeders were cleaned at weekly intervals.
- Diet: Ad libitum, Certified High-Fiber Rabbit Chow
- Water: Ad libitum, tap water
- Acclimation period: 61 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64 to 77 (measured during test; 18 - 25°C)
- Humidity (%): 29 to 69 (measured during test)
- Air changes (per hr): 12.6 (measured during test)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 5 Dec 1987 To: 22 Jan 1988

Route of administration:

oral: gavage

Vehicle:

other:

Remarks:

0.5% (w/v) methylcellulose in water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose volumes were derived from Day 7 body weights and were adjusted during the remaining treatment period to the most recent body weights. Suspensions for each dose level were prepared daily and stirred prior to and during the dosing procedure.

VEHICLE
- Concentration in vehicle: 2.5, 5.0 and 10.0 mg/mL
- Amount of vehicle: 2 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to initiation of the study, one pre-test mix was prepared and analyzed for the 2.5 mg/mL and 10.0 mg/mL concentration levels (i.e., Groups II and IV , respectively). As during the dosing procedure, the suspensions were stirred continuously. Seven samples of each of the suspensions were collected by drawing up 5 mL into syringes and dispensing into tared 250 mL amber glass jars. The jars were then weighed to determine the net weight of each sample and the net weight of each sample was added to the label information. Five of the samples were frozen immediately after collection. The other two samples were maintained at room temperature for 24 hours and then frozen. A 100 mL sample of the vehicle was also collected and frozen immediately. Samples were shipped on dry ice for analysis. A duplicate set of seven samples of each suspension (Groups II and IV) and the vehicle were collected in the same manner and maintained frozen at the test facility.

Details on mating procedure:

Natural mating was used. Each female selected for mating was placed into the male 's cage. When coitus was observed, the female was removed and returned to her original cage. Following an interval of one to two hours , the female was placed into the cage of a different male. When coitus was observed with the second male, the female was considered mated and returned to her cage. The day on which coitus was observed with both males was designated Day 0 of gestation.

Duration of treatment / exposure:

From day 7 to day 19 of gestation

Frequency of treatment:

Once daily

Duration of test:

Until day 30 of gestation

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Remarks:

Group 2: Low dose

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Remarks:

Group 3: Mid dose

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Remarks:

Group 4: High dose

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: Twice daily
- Cage side observations checked included: Appearance, behaviour, signs of toxicity, moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Days 0, 7, 10, 13, 16, 19, 24 and 30 of gestation. On Days 7, 10, 13, 16 and 19 of gestation, the detailed physical exams were performed after all animals had been dosed for that day.

BODY WEIGHT:
- Time schedule for examinations: Body weights were recorded three times during the acclimation period. Body weights were also recorded on Days 0, 3, 7, 10, 13, 16, 19, 24 and 30 of gestation. Day 30 body weights are presented as actual and corrected (the actual Day 30 body weight minus the weight of the gravid uterus ).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day
- Time schedule: Food consumption was measured for Days 1, 3, 7, 10, 13, 16, 19, 24 and 29 of gestation.

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 30
- Gross postmortem examinations were performed on all mated rabbits including those dying spontaneously during the study. Only abnormal tissues were saved (10% neutral buffered formalin). Females showing signs of premature delivery (presence of fetuses/ pups at Day 26 of
gestation or later ) were killed via intravenous injection of sodium pentobarbital in the marginal ear vein on the day such evidence was observed. Reproductive systems were examined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter

Statistics:

Data were analyzed between control (Group I) and treated Groups II- IV.

Clinical signs:

no effects observed

Description (incidence and severity):

No adverse effect of treatment was evident from the physical examination data.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Mortality rates did not differ statistically between the control or treated groups. No mortality occurred in the control or low-dose group. In the control group, two females delivered prematurely and the remaining 18 females survived to scheduled sacrifice on Day 30 of gestation. In the low-dose group , one female was killed following premature delivery and the remaining 19 females survived to Day 30 gestation scheduled sacrifice. In the mid-dose group, one female died (mortality rate 3 5.0%), two females delivered prematurely and were sacrificed and the remaining 17 females survived to scheduled sacrifice ( Day 30 of gestation ). One female died on Day 10 of gestation after receiving three days of treatment. This female was pregnant as evidenced by 10 uterine implantation sites. At gross postmortem evaluation, this animal had fluid in the lungs and frothy fluid in the trachea, both findings are suggestive of a pulmonary oedema which may or may not have resulted from an intubation error. In the high-dose group, one female died (mortality rate 3 5.0% ), one female delivered prematurely and the remaining 18 females survived to scheduled sacrifice. One female died on Day 13 of gestation after receiving six days of treatment. This female was pregnant as evidenced by 13 uterine implantation sites, determined on the basis of postmortem findings.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights for each recorded interval during gestation were comparable between the control and each treated group. Mean body weight gains noted for the control and each treated group during the pre- and post-treatment intervals (Days 0-7 and 19-30 of gestation, respectively) were considered comparable. Over the entire treatment period (i.e. , Days 7-19), a comparable mean weight gain was seen in the control, low- and mid-dose groups. The high-dose group experienced a slight mean weight loss during the Day 7-19 treatment period and this difference from control was statistically significant. Likewise, the incidence of females losing weight over the Day 7-19 gestation interval was increased in the high-dose group; these incidences for the control, low-, mid- and high-dose groups were 10% (2/ 20), 10% (2/ 20), 20% (4/ 20) and 47.4% (9/19), respectively
A mean weight loss was noted for the control and each treated group during the Day 7-30 gestation interval using the Day 30 corrected body weights. These data were comparable between the control and each treated group. Thus, no adverse effect of treatment at the 5 or 10 mg/kg bw/day dose levels was evident from body weight data during gestation. At the 20 mg/kg bw/day dose level, a mean weight loss was experienced over the Day 7-19 treatment period and this difference from control data was statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption for the pre-treatment (Days 1 and 3), treatment (Days 7, 10, 13, 16 and 19) and post-treatment (Days 24 and 29) intervals were considered comparable between the control, low- and mid-dose groups. In the high-dose group, mean food consumption was comparable to control during the pre- and post-treatment intervals. During the treatment period, mean food consumption in the high-dose group was lower than control for each recording interval; however, only on Day 7 was this difference from control statistically significant. Differences in mean food consumption data for the high-dose group in comparison to control during the treatment period ranged from -8.7% at Day 10 to -35.0% at Day 17. Thus, no adverse effect of treatment on food consumption was evident at the 5 or 10 mg/kg bw/day dose levels. At the 20 mg/kg bw/day dose level, food consumption was reduced from control levels for each recording interval during the treatment period; however, only on Day 7 was this difference from control statistically significant.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

For all groups, numerous animals had discolored lungs (primarily red/ tan /brown foci /areas); these tended to be seen more frequently in Groups III and IV as compared to Group I. The incidences of females with discolored lungs for the control, low-, mid- and high-dose groups were 60% (12/ 20), 60% (12/ 20), 70% (14/ 20) and 80% (16/20), respectively. In both treated and control animals these discolorations were qualitatively similar; none were considered to be unique. In the opinion of the pathologist, these various discolorations of the lungs were not considered to be of toxicological significance with respect to the test article.
Other postmortem findings either occurred in the treated and control animals with comparable incidences and severities or they occurred sporadically. They were not considered to be related to the test article.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

No aborted pregnancies were noted during the study.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of corpora lutea and mean number of uterine implantations per pregnant female were comparable between the control and treated groups. The mean pre-implantation loss index was considered comparable between these same groups. A slight increase in the mean pre-implantation loss index was seen in the mid-dose group as compared to the control group; however, this difference from control was not statistically significant and in the absence of a similar increase in the high-dose group, was not considered indicative of a treatment-related effect.
No adverse effect of treatment was evident from uterine implantation data.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of resorption sites, the mean ratio of resorptions to implants and the incidence of females with at least one resorption site in utero comparable between the control and each treated group.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of live fetuses per pregnant female was comparable between the control and treated groups. The only dead fetus recovered in utero was from the litter of control.

Changes in pregnancy duration:

effects observed, non-treatment-related

Description (incidence and severity):

Premature delivery of a litter during the Day 26-30 gestation interval was seen for two control females (10.0%), one low-dose female (5.0%), two mid-dose females (10.0%) and one high-dose female (5.3%). These incidences were comparable between the control and treated groups and no adverse effect of treatment was indicated.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Pregnancy rates were comparable between the control and treated groups. The pregnancy rate was 100% in the control, low and mid-dose groups and 95% (19/ 20 ) in the high-dose group. A total of 72 females (18 control, 19 low-dose, 17 mid-dose and 18 high-dose females) survived to scheduled sacrifice (Day 30 of gestation). Only one of these females (high-dose female) did not have uterine implantations at sacrifice and no foci were visualized following uterine staining. This was the only non-pregnant animal on study.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean fetal weights distinguished by sex and as a composite for both sexes, were slightly lower than control in each of the treated groups but these differences from control data were not statistically significant. Mean fetal weight data for the treated groups were within the range of recent historical control data for this laboratory (Males: mean = 46.7 g; range = 40.0 to 50.4 g. Females: mean = 45.5 g; range = 39.6 to 48.1 g. Both: mean = 46.3 g; range = 40.5 to 49.6 g).
No adverse effect of treatment was evident from fetal weight data.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The mean number of male and female fetuses per litter and the ratio of male to female fetuses were comparable between the control and treated groups.
No adverse effect of treatment was evident from fetal sex distribution data.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidences of fetuses with external malformations for the control and low-dose groups were 0.6% (1/173 fetuses) and 0.6% (1/170 fetuses), respectively, No external malformations were seen in the 156 mid-dose fetuses (17 litters) or 158 high-dose fetuses (17 litters) evaluated. The following external malformations were seen during this study: distended abdomen in one fetus from the litter of control female; and the presence of a single external nare and absence of upper incisors in one fetus from the litter of low-dose female.
No external malformations were seen among the pups/fetuses recovered from females that delivered prematurely. A total of 39 pups were examined externally [14 control pups from two litters, six low-dose pups from a single litter and 19 mid-dose pups /fetuses from two litters].
No adverse effect of treatment was evident from the fetal external examination data.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidences of fetuses with skeletal malformations for the control, low-, mid- and high-dose groups were 5.2% (9/173 fetuses), 8.9% (15/169 fetuses), 5.8% (9/156 fetuses) and 3.8% (6/158 fetuses), respectively. The incidences of litters containing fetuses with skeletal malformations for these same groups were 44.4% (8/18 litters), 47.4% (9/19 litters), 52.9% (9/17 litters) and 29.4% (5/17 litters), respectively. The incidences of litters containing fetuses with skeletal malformations did not differ statistically between the control and treated groups and were considered comparable.
Fused sternebrae, a minor skeletal malformation, was the most common malformation seen during the study. The incidences of fetuses with fused sternebrae for the control, low-, mid- and high-dose groups were 2.3% (4/173), 3.6% (6/169), 0.6% (1/156) and 3.2% (5/158), respectively. The incidences of litters containing fetuses with fused sternebrae for the control, low-, mid- and high-dose groups were 22.2% (4/18), 10.5% (2/19), 5.9% (1/17) and 23.5% (4/17), respectively. The incidence of fused sternebrae both on a per fetus and per litter basis for this study was considered similar between the control and treated groups and within the range of recent historical control data.
Angulated arch (es) of the hyoid, also considered a minor skeletal malformation, was seen at low incidence in the control, low- and mid-dose groups; this malformation was not seen among the 158 high-dose fetuses. The incidences of fetuses with angulated arch (es) of the hyoids for the control, low- and mid-dose groups were 2.9% (5/173), 2.4% (4/169) and 1.9% (3/156), respectively. These incidences were similar between the control and treated groups and were within the range of recent historical control data.
Excluding those fetuses with only minor skeletal malformations as presented above, the incidences of fetuses with other skeletal malformations for the control, low- , mid- and high-dose groups were 1.2% (2/173), 3.0% (5/169), 3.2% (5/156) and 0.6% (1/158), respectively. Dissimilar malformations of the cranial bones (frontals , nasals) were seen in three fetuses (one fetus from each of the control, low- and mid-dose groups) that had either external or visceral malformations.
One fetus from the litter of control female had misshapen nasals and fused frontal bones; this fetus was noted during the visceral examination with defects of the aortic arch and a cardiac malformation. One fetus from the litter of low-dose female had fused frontal bones, nasals fused and misshapen and fused premaxillary bones; this fetus was noted externally with a single external nare. One fetus from the litter of mid-dose female had fused frontal bones and at visceral examination this fetus was noted with ectopic kidneys.
The only other malformation seen with some frequency during the study involved fused/branched ribs with or without vertebral involvement. Fused/branched ribs were seen in one control fetus (0.6%), one low-dose fetus (0.6%) and two mid-dose fetuses from different litters (1.3%). The incidence of litters containing fetuses with skeletal malformations was similar for all study groups (control and treated) ; most of these observations involved minor skeletal malformations (fused sternebrae , angulated arch of the hyoid) which were seen with similar incidence among the control and treated groups.
Other skeletal malformations seen during the study among the treated groups , occurred in low incidence, were dissimilar in nature and showed no dose-related trends. Thus, no adverse effect of treatment was indicated from skeletal malformation data.
No skeletal malformations were seen in the 39 pups/fetuses recovered from females that delivered prematurely (14 control , 6 low-dose and 19 mid-dose pups/fetuses were evaluated).
The incidences of fetuses with at least one ossification variation for the control, low-, mid- and high-dose groups were 64.2% (111/173), 52.7% (89/169), 66.0% (103/156) and 62.7% (99/158), respectively. The incidence of litters containing at least one fetus with an ossification variation was comparable between the control and treated groups; this was 94.4% for the control group and 100% for each of the treated groups. No adverse effect of treatment was evident from ossification variation data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidences of fetuses with visceral malformations for the control, low-, mid- and high-dose groups were 1.2% (2/173), 0.6% (1/170), 1.3% (2/156) and 1.3% (2/158), respectively. The incidences of litters containing fetuses with visceral malformations for the control, low-, mid- and high-dose groups were 11.1% (2/18), 5.3% (1/19), 11.8% (2/17) and 5.9% (1/17), respectively and these incidences were comparable between the control and each treated group.
In the control group, one fetus from the litter of female had persistent truncus arteriosus (a defect of the aortic and pulmonary arches) and interventricular septal defect. Ascites (fluid in the abdominal cavity) and an enlarged heart were seen in the one control fetus noted externally with a distended abdomen.
In the low-dose group, the only visceral malformation noted was slight distention of the lateral ventricles of the brain and this malformation was seen in one fetus from the litter of female. Distended renal pelvis (without a renal papillae present) was seen in one fetus from the litter of mid-dose female and two fetuses from the litter of high-dose female. The only other visceral malformation seen during the study was ectopic kidney seen in one fetus from the litter of mid-dose female. Several of these malformations have been noted at low incidence in historical control data for our laboratory.
No adverse effect of treatment was evident from the visceral malformation data.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Effect level:

>= 20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

effects observed, non-treatment-related

Key result

Developmental effects observed:

no

Table 1. Overview of study results

Dose (mg/kg bw/day)	0	5	10	20	dr
Maternal effects
Mortality	-	-	1a	1a	-
Clinical signs and	-	-	-	-	-
behaviour	-	-	-	-	-
Pregnant animals	20/20	20/20	20/20	20/20	-
Abortions	-	-	-	-	-
Early delivery	2	1	2	1	-
Females with viable foetuses at C-section	18	19	17	17	-
Body-weight gain
- day 7-10	6	8	-9	-26	dr
- day 10-13	24	3	22	8
- day 13-16	86	78	53	21	dr
- day 16-19	21	22	6	-9	dr
- day 19-30	94	38	75	111
- day 7-19	136	111	71	-8*	dr
Food consumption
- day 7-8	47	45	42	40*b
Pathology	No treatment related findings				-
Litter response
Live foetuses	No treatment related findings				-
Fetal weight	No treatment related findings				-
Post implantation loss	No treatment related findings				-
Sex ratio	No treatment related findings				-
Examination of foetuses
External observations	No treatment related findings				-
Skeletal findings	No treatment related findings				-
Visceral findings	No treatment related findings				-

dc/ic: statistically significantly decreased/increased compared to the controls

d/i: decreased/increased, but not statistically significantly compared to the controls

dr: dose-related

+/-: increase/decrease without statistical analysis

a: The female that died in the middle dose group had fluid in the lungs and trachea, both suggestive of pulmonary oedema. No indications on the cause of death of the other female were found.

b: The mean food intake in high dosed females during the treatment period differed from controls ranging from -8.7% at day 10 to -35.0% at day 17.

* p<0.05, ** p<0.01

 

Table 2. Reproductive performance parameters

Observation	Dose level (mg/kg bw/day)
	0 (control)	5	10	20
Animals Assigned (Mated)	20	20	20	20
Animals Pregnant	20	20	20	19
Animals Non-pregnant	0	0	0	1
Animals aborted	0	0	0	0
Premature births	2	1	2	1
Litters with viable foetuses	18	19	17	17
Litters with all resorptions	0	0	0	0
Mortality	0	0	1	1
Mean no corpora lutea/dam	11.0	10.1	11.2	10.0
Mean no implants/dam	10.2	9.5	9.5	9.9
Mean pre-implantation loss (%)	0.077	0.063	0.153	0.061
Number viable foetuses	172	170	156	158
Mean litter size	9.6	8.9	9.2	9.3
Mean number of males/litter	4.0	3.9	4.6	4.5
Mean number of females/litter	4.0	5.1	4.6	4.8
Dead foetuses	1	0	0	0
Number of resorptions	11	10	6	11
Mean number resorptions	0.6	0.5	0.4	0.6
Mean number resorptions/implants	0.054	0.0449	0.031	0.067
Litters with resorptions	7	2	4	10
Mean foetal weight – viable foetuses (g)	45.60	43.30	43.32	42.91
Mean male foetal weight (g)	46.88	43.19	43.34	43.09
Mean female foetal weight (g)	44.93	43.34	41.99	41.00
Ratio of viable foetuses (total males/total females)	1.0	0.8	1.0	1.0

 

 

 

Conclusions:

In conclusion, the NOAEL for maternal toxicity is 10 mg/kg bw/day based on reduced food consumption and lowered body weight (gain) and for developmental toxicity 20 mg/kg bw/day based on the absence of adverse effects.

Executive summary:

In this study (83-3 under GLP), the test substance was tested for its embryotoxic, fetotoxic, and teratogenic potential in rabbits. Test article was suspended in 0.5% (w/v) aqueous methyl cellulose vehicle and administered by gastric intubation as a single dose daily during the Day 7-19 gestation interval. Dose levels evaluated were 5, 10 and 20 mg/kg bw/day. Each test group contained 20 mated female New Zealand White rabbits. Included in the study was a vehicle-treated control group which also contained 20 mated females. All animals were observed twice daily (morning, afternoon) for obvious toxicological effects and mortality. Each animal was weighed and given a detailed physical examination on Days 0, 3, 7, 10, 13, 16, 19, 24 and 30 of gestation. Food consumption was recorded on Days 1 , 3, 7, 10, 13, 16, 19, 24 and 29 of gestation. Animals aborting or delivering prematurely were killed on the day such evidence was observed and given a gross postmortem evaluation. Intact pups/fetuses recovered from females that delivered prematurely (Days 26-30 of gestation) were evaluated for external malformations, eviscerated (gross evaluation of viscera) and evaluated for skeletal malformations following processing for Alizarin Red S staining of the ossified structures. Surviving females were killed on Day 30 of gestation and given a gross postmortem examination. The gravid uterus was removed, weighed and evaluated for the presence of live, dead and resorbed fetuses. Fetuses recovered in utero were weighed and given a gross external examination. Subsequently, each fetus was processed for visceral evaluation (fresh microdissection procedure ) and skeletal evaluation (Alizarin Red S stained specimens). The number of corpora lutea on each ovary was also recorded for each female.

No mortality occurred in the control or low-dose group. In the control group, two females delivered prematurely and the remaining 18 females survived to scheduled sacrifice on Day 30 of gestation. In the low-dose group (5 mg/kg/day), one female delivered prematurely and the remaining 19 females survived to Day 30 scheduled sacrifice. The pregnancy rate for the control and low-dose group was 100%. No maternal toxic, embryotoxic, fetotoxic or teratogenic effects were evident at the 5 mg/kg bw/day dose level. In the 10 mg/kg bw/day dose group, one female died on Day 10 of gestation after receiving three days of treatment. At gross postmortem examination, this female was noted with fluid in the lungs and frothy fluid in the trachea, both findings are suggestive of a pulmonary oedema which may or may not have resulted from an intubation error. Two mid-dose females delivered prematurely and the remaining 17 females in this group survived to Day 30 scheduled sacrifice. Pregnancy rate for the mid-dose group was 100%. The 10 mg/kg bw/day dose level was not considered to be maternally toxic, embryotoxic, fetotoxic or teratogenic. In the 20 mg/ kg/day dose group, one pregnant female died, one female delivered prematurely and the remaining 18 females survived to scheduled sacrifice. Seventeen of these eighteen high-dose females were pregnant as evidenced by the presence of uterine implantation sites (pregnancy rate = 95%). One high-dose female died on Day 13 of gestation after six days of treatment. The cause of death of this female could not be determined on the basis of postmortem findings. High-dose females experienced a mean weight loss during the treatment period (Days 7-19) in comparison to a mean weight gain in the control group over this same interval and these data differed statistically between these two groups. Mean food consumption for the high-dose group during the treatment period (Days 7, 10, 13, 16 and 19) was also lower than control data; however, only on Day 7 was this difference from control statistically significant. No other maternal toxicity was evident at the 20 mg/ kg/day dose level and this dose level was not considered to be embryotoxic, fetotoxic or teratogenic.

In conclusion, the NOAEL for maternal toxicity is 10 mg/kg bw/day based on reduced food consumption and lowered body weight (gain) and for developmental toxicity 20 mg/kg bw/day based on the absence of adverse effects.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subchronic

Species:

rabbit

Quality of whole database:

GLP compliant EPA OPP 83-3

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

Key: Developmental toxicity study - Rabbit

This study (83-3 under GLP), the test substance was tested for its embryotoxic, fetotoxic, and teratogenic potential in rabbits. Test article was suspended in 0.5% (w/v) aqueous methyl cellulose vehicle and administered by gastric intubation as a single dose daily during the Day 7-19 gestation interval. Dose levels evaluated were 5, 10 and 20 mg/kg bw/day. Each test group contained 20 mated female New Zealand White rabbits. Included in the study was a vehicle-treated control group which also contained 20 mated females. All animals were observed twice daily (morning, afternoon) for obvious toxicological effects and mortality. Each animal was weighed and given a detailed physical examination on Days 0, 3, 7, 10, 13, 16, 19, 24 and 30 of gestation. Food consumption was recorded on Days 1 , 3, 7, 10, 13, 16, 19, 24 and 29 of gestation. Animals aborting or delivering prematurely were killed on the day such evidence was observed and given a gross postmortem evaluation. Intact pups/fetuses recovered from females that delivered prematurely (Days 26-30 of gestation) were evaluated for external malformations, eviscerated (gross evaluation of viscera) and evaluated for skeletal malformations following processing for Alizarin Red S staining of the ossified structures. Surviving females were killed on Day 30 of gestation and given a gross postmortem examination. The gravid uterus was removed, weighed and evaluated for the presence of live, dead and resorbed fetuses. Fetuses recovered in utero were weighed and given a gross external examination. Subsequently, each fetus was processed for visceral evaluation (fresh microdissection procedure ) and skeletal evaluation (Alizarin Red S stained specimens). The number of corpora lutea on each ovary was also recorded for each female.

No mortality occurred in the control or low-dose group. In the control group, two females delivered prematurely and the remaining 18 females survived to scheduled sacrifice on Day 30 of gestation. In the low-dose group (5 mg/kg/day), one female delivered prematurely and the remaining 19 females survived to Day 30 scheduled sacrifice. The pregnancy rate for the control and low-dose group was 100%. No maternal toxic, embryotoxic, fetotoxic or teratogenic effects were evident at the 5 mg/kg bw/day dose level. In the 10 mg/kg bw/day dose group, one female died on Day 10 of gestation after receiving three days of treatment. At gross postmortem examination, this female was noted with fluid in the lungs and frothy fluid in the trachea, both findings are suggestive of a pulmonary oedema which may or may not have resulted from an intubation error. Two mid-dose females delivered prematurely and the remaining 17 females in this group survived to Day 30 scheduled sacrifice. Pregnancy rate for the mid-dose group was 100%. The 10 mg/kg bw/day dose level was not considered to be maternally toxic, embryotoxic, fetotoxic or teratogenic. In the 20 mg/ kg/day dose group, one pregnant female died, one female delivered prematurely and the remaining 18 females survived to scheduled sacrifice. Seventeen of these eighteen high-dose females were pregnant as evidenced by the presence of uterine implantation sites (pregnancy rate = 95%). One high-dose female died on Day 13 of gestation after six days of treatment. The cause of death of this female could not be determined on the basis of postmortem findings. High-dose females experienced a mean weight loss during the treatment period (Days 7-19) in comparison to a mean weight gain in the control group over this same interval and these data differed statistically between these two groups. Mean food consumption for the high-dose group during the treatment period (Days 7, 10, 13, 16 and 19) was also lower than control data; however, only on Day 7 was this difference from control statistically significant. No other maternal toxicity was evident at the 20 mg/ kg/day dose level and this dose level was not considered to be embryotoxic, fetotoxic or teratogenic.

In conclusion, the NOAEL for maternal toxicity is 10 mg/kg bw/day based on reduced food consumption and lowered body weight (gain) and for developmental toxicity 20 mg/kg bw/day based on the absence of adverse effects.

Supporting: Developmental toxicity study - Rat

Potential maternal, embryotoxic and teratogenic effects of the test substance were evaluated in this study in rats (EPA OPP 83-3, GLP). The test material was suspended in 0.5% aqueous methylcellulose and administered to three groups of 25 bred Sprague-Dawley COBS CD female rats as a single daily dose from days 6 through 15 of gestation. Dosage levels of 25, 100 and 400 mg/kg bw/day were selected from results of a previous study conducted in this laboratory. For comparative purposes, a concurrent control group also composed of 25 dams was dosed with the vehicle only on a comparable regimen. The route of administration was oral via gastric intubation. Throughout gestation all females were observed twice daily for toxicity, weighed at specified intervals and sacrificed on gestation day 20 for the scheduled Caesarean section. All fetuses were weighed, sexed and examined for external malformations. One-half of the fetuses were processed and examined skeletally after staining with Alizarin Red S. The remaining one-half of the fetuses were fixed in Bollin's solution and examined for visceral anomalies and developmental variations.

Three rats died in the 400 mg/kg/day group during the experimental period. These deaths were considered treatment-related. One mortality also occurred in the 100 mg/kg/day group but the cause of death was accidental as evidence of intubation error was observed at necropsy. Dose-related decreases in maternal body weight gain occurred during the test material administration period (gestation days 6-15) at all levels tested. Body weight gains remained reduced in all groups in the post-treatment period, gestation days 15-20. Food intake was decreased in dose-related fashion during the first six days of test material administration in all treatment groups when compared with the control group. Thereafter, food consumption was not adversely affected by treatment in the 25 and 100 mg/kg bw/day groups. Food intake remained reduced in the high dose group during the final three days of dosing but was significantly increased once treatment had concluded. The only sign of embryotoxicity observed in the study was an increase in early embryonic death in the 400 mg/kg/day group. No significant differences were noted in the number and types of fetal malformations or developmental variations when the treated groups were compared with the control group.

In conclusion, the maternal NOAEL was set on 100 mg/kg bw/day based on reduced body weights and food consumption and the  NOAEL for maternal developmental toxicity was set on 100 mg/kg bw/day based on an increased number of early embryonic death. The test substance was nonteratogenic at dosages of 400 mg/kg/day or less.

Justification for classification or non-classification

Based on the available information the substance is not classified as for reproductive toxicity in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.