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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 16 male and 16 female Crl:CD®(SD}BR rats were fed for 90 days with diets that contained 0, 100, 1000 or 5000 ppm of the test substance to assess its subchronic toxicity. At these dietary levels, male rats received an average of 0, 7.3, 71, and 365 mg/kg/day of the test substance in the 0, 100, 1000 and 5000 ppm groups, respectively. For female rats, the average daily dose was 0, 9.5, 88, and 453 mg/kg/day in the 0, 100, 1000, and 5000 ppm groups, respectively.

After 90 days on test, six rats per group were used to conduct a one-generation reproduction study. Male and female rats continued to receive their respective treatment group’s diet throughout mating, gestation, and lactation.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Purity: 96%

Test animals

Species:
rat
Strain:
other: Crl:CD®(SD)BR
Sex:
male/female

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: Certified Purina® Laboratory Chow #5002
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Certified Purina® Laboratory Chow #5002
- Storage temperature of food: Refrigerated (temperature not stated in the report)
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 15 days
- Proof of pregnancy: Copulation plug
- After successful mating each pregnant female was caged (how): After the 15-day mating period, each female rat was housed individually in polypropylene cages with bed-o'cobs® cage bedding
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Approximately 50-gram samples were taken during the study from each treatment level (excluding control diet) and analyzed for the concentration, homogeneity, or stability of the test substance in the diet. Homogeneity was determined by collecting a sample from the top, middle, and bottom of the mixing vessel. Stability was determined by collecting four samples from the middle of the mixing vessel. One of the four samples was immediately frozen, the second was refrigerated for ten days, the third was stored at room temperature for 24 hours, and the fourth was stored at room temperature for ten days. Homogeneity and stability samples were taken at the start of the study. Homogeneity samples were also taken after one week on test when mixers were changed from the Stephan to the Hobart high-speed mixer. Samples to verify the concentration of the test substance in the diet were taken at the end of the 90-day feeding portion of the study.

Diet samples were analyzed for the test substance by reversed-phase liquid chromatography. The test substance was ultrasonically extracted (~ 1 hour) from diet samples (10.0 g) into methylene chloride. The 0 ppm sample was extracted with 50 mL of the solvent; 100, 1000 and 5000 ppm diet samples were extracted with 100 mL of methylene chloride. Extracts (~ 5 mL) were filtered through 0.45 µm filters. Filtered
extracts were then diluted 4:10 (100 ppm), 1:25 (1000 ppm) or 1:100 (5000 ppm). Aliquots (1.50 mL) of the filtered extract (0 ppm) or diluted extracts (100, 1000, 5000 ppm) were evaporated to dryness under nitrogen. The resulting residues were ultrasonically resuspended in acetonitrile (1.50 mL, 5-10 min).

Recovery efficiencies were determined at the 5000 ppm level by adding 50.0 mg of the test substance directly to control diet (10.0 g) and at the 100 ppm level by addition of a test substance stock solution (1.00 mL, 1000 µg/mL) to control diet (10.0 g).

The reference test compound (96% purity) was used to make calibration standards (2.0, 4.0, 6.0 µg/mL) for the creation of the standard curve. Quantitation was performed using linear regression analysis.

The dietary concentration of INA-7881 was determined by duplicate injection with a Hewlett Packard 1090a liquid
chromatograph.
Duration of treatment / exposure:
1 Generation
Frequency of treatment:
Daily
Details on study schedule:
Six designated rats in each test group were used to initiate a one-generation, one-litter reproduction substudy at the end of the 90-day feeding study. Throughout the reproduction substudy, all rats received their respective group's diet. Each female rat was housed with a randomly selected male rat from the same test group for a period of fifteen days. During the 15-day mating period, each female rat was checked daily for the presence of a copulation plug. After the 15-day mating period, each female rat was housed individually in polypropylene cages with bed-o'cobs® cage bedding. Six days later, female rats were examined at least twice daily for the birth of pups.
Doses / concentrationsopen allclose all
Dose / conc.:
100 ppm
Dose / conc.:
1 000 ppm
Dose / conc.:
5 000 ppm
No. of animals per sex per dose:
6 males and 6 females
Control animals:
yes, plain diet
Positive control:
No

Examinations

Parental animals: Observations and examinations:
The male and female parent rats were sacrificed and discarded without pathological evaluation.
Litter observations:
Live and dead pups in each litter were counted as soon as possible after delivery was completed. Four days after birth:
• live pups were counted,
• the litter was weighed,
• ten pups, with an equal number of male and female pups where possible, were selected on the basis of their being representative of the
general health of their litter. Extra pups were sacrificed and discarded without pathological evaluation.
• litters of less than ten pups were not reduced, and
• reduced litters were weighed.

Twelve days after birth, live pups were counted. Twenty-one days after birth (weaning), each pup was sexed, weighed, sacrificed and discarded without pathological examination.
Postmortem examinations (parental animals):
The male and female parent rats were sacrificed and discarded without pathological evaluation.
Postmortem examinations (offspring):
Twenty-one days after birth (weaning), each pup was sexed, weighed, sacrificed and discarded without pathological examination.
Statistics:
The Fisher's Exact, Kruskal-Wallis, and/or Mann-Whitney U tests were used to evaluate measures of reproduction and lactation performance. Statistically significant differences were judged at the p < 0.05 probability level.
Reproductive indices:
Indices of reproduction were calculated on a per litter basis for female rats bearing litters with at least one live pup. The percentage was calculated for each litter and the average percentage for each group is reported. The following formulas were used:

Fertility Index (%) = Number females bearing litters/Number of females mated x 100

Gestation Index (%) = Number of females bearing litters with at least one live pup/Number of females bearing litters x 100
Offspring viability indices:
Number of Pups Born Alive (%) = Total number of pups born alive/Total number of pups born x 100

0-4 Day Viability Index (%) = Total number of pups alive at 4 days postnatal (prior to litter reduction)/Total number of pups born alive x 100

1-4 Day Viability Index (%) = Total number of pups alive at 4 days postnatal (prior to litter reduction)/Total number of pups 1 day postnatal x 100

Lactation Index (%) = Total number of pups alive at weaning (21 days postnatal)/Total number of pups alive after litter reduction (4 days postnatal) x 100

Litter Survival (%) = Number of litters at weaning/Number of litters delivered x 100

Average # of Pups/litter = Total number of pups born alive/Total number of litters delivered

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weight of dams in the high-dose group was greater than the control group (320 g in the control group and 336.6 g in the high-dose group). Although these changes were statistically significant, they were considered not to be biologically relevant or compound related.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
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:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Concentration 0 100 ppm 1000 ppm 5000 ppm

Fertility Index 100% 83.3% 83.3% 83.3%
Gestation Index 100% 100% 100% 100%
% Pups born alive/Litter 100% 95% 100% 95.4%
0-4 Day Viability Index/Litter 98.3% 92.7% 91.3% 100%
1-4 Day Viability Index/Litter 100% 98.5% 97.6% 100%
Lactation Index/Litter 100% 100% 98.0% 100%
Litter survival 100% 100% 100% 100%

Mean number of pups/litter at:

Concentration 0 100 ppm 1000 ppm 5000 ppm

Birth (Total) 9.5 13.2 14.6 12.8
Birth (Alive) 9.5 12.6 14.6 12.2
24 Hours 9.3 12.0 13.6 12.2
Day 4 9.3 11.8 13.2 12.2
Day 4 after reduction 7.8 9.4 10.0 8.4
Day 12 7.8 9.4 10.0 8.4
Day 21 7.8 9.4 9.8 7.4

Number of males 4.5 5.2 5.8 4.4
Number of females 3.3 4.2 4.0 4.0

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No compound-related effects were noted in the one-generation reproduction study. Therefore, the no-observable-effect level (NOEL) was 5000 ppm., the highest dose tested.

Applicant's summary and conclusion

Conclusions:
No compound-related effects were noted in the one-generation reproduction study. Therefore, the no-observable-effect level (NOEL) was 5000 ppm, the highest dose tested.
Executive summary:

Groups of 16 male and 16 female Crl:CD®(SD)BR rats were fed for 90 days with diets that contained 0, 100, 1000 or 5000 ppm of the test substance to assess its subchronic toxicity. At these dietary levels, male rats received an average of 0, 7.3, 71, and 365 mg/kg/day of the test substance in the 0, 100, 1000 and 5000 ppm groups, respectively. For female rats, the average daily dose was 0, 9.5, 88, and 453 mg/kg/day in the 0, 100, 1000, and 5000 ppm groups, respectively. After 90 days on test, six rats per group were used to conduct a one-generation reproduction study. Male and female rats continued to receive their respective treatment group’s diet throughout mating, gestation, and lactation.

No compound-related effects were noted. Therefore, the no-observable-effect level (NOEL) was 5000 ppm, the highest dose tested.