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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2015
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
See principals of method section for summary
Principles of method if other than guideline:
All deviations that occurred during the study have been authorized/acknowledged by the Study Director, assessed for impact, and documented in the study records. All protocol deviations are listed below.
None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Formulations and Dosing
• On 21 Oct 2015, there was no documentation present to confirm that the Sponsor had reviewed the formulation instructions prior to preparation. This deviation did not impact the outcome of the study because the Sponsor’s Representative was on site for the initial preparation and the procedure was reviewed as it was in progress.
• On 27 Oct 2015 and 09 Nov 2015, it could not be conclusively determined if the aliquots were allowed to mix for at least 30 minutes prior to use and the time the aliquots were transferred to the dose room was not recorded. This deviation did not impact the outcome of the study because it was presumed that the aliquots were allowed to mix for a sufficient amount of time prior to usage on study.
• On 19 Nov 2015, two dose volumes were recorded for male 4726 in Group 3. This deviation did not impact the outcome of the study because it was presumed that the rat was dosed only
once and this was a data entry issue.
• On 16 Dec 2015, the dosing details data were missing from males 4739 and 4740 in Group 4. It was presumed that these males both received a dose volume of 4.9 mL. This deviation did not impact the outcome of the study because it is presumed that the appropriate dose volume was administered to the animal based on the fact that a body weight was recorded and the knowledge of the standard practicesemployed at the Testing Facility.
• On 28 Dec 2015, the samples that were transferred to the analytical department were not documented as being transferred refrigerated on cold packs or protected from light. This deviation did not impact the outcome of the study because it was presumed that the samples were handled in an appropriate manner during transferral.

Husbandry
• On 17-19, 26, 28 Oct 2015, 26, 27 Nov 2015, and 09 Dec 2015, the temperature was out of range (as high as 81.0°F) for no more than 9 hours during the study and the relative humidity was out of range (as low as 18.3% and as high as 71.2%) for no more than 50 hours during the study. This deviation did not impact the outcome of the study because the variations from the target temperature and humidity were not considered to be detrimental to the animals.
• On 06 Nov 2015, water was not available ad libitum for approximately 80 minutes for females 4759 and 4760 in Group 2. The vet staff was notified and the animals were examined; however, the Study Director was inadvertently not notified of this occurrence. This deviation did not impact the outcome of the study because the amount of time without water was minimal and the issue was addressed immediately once it was discovered

In-life Observations, Measurements, and Evaluations
• On 04 Nov 2015, a postdose clinical observation was not recorded for male 4726 in Group 3. This deviation did not impact the outcome of the study because sufficient data were available
to evaluate this parameter.
• On 08 Nov 2015, postdose clinical observations were performed 1 or 6 minutes early for females 4770 in Group 3 and 4771 and 4772 in Group 4. This deviation did not impact the
outcome of the study because the evaluations were performed and the variation from the targeted time was minimal.
• On 15 Nov 2015, postdose clinical observations were not recorded for all of the female rats. This deviation did not impact the outcome of the study because sufficient data were available
to evaluate this parameter.
• On 02-15 Dec 2015, female rat 4777 (Group 4, 1000 mg/kg/day) did not receive food consumption values at least weekly during this period. This deviation did not impact the outcome of the study because this female had no confirmed date of mating; therefore, the values for this female would not have been included in tabulation or statistical analyses.
• On 15 Dec 2015, animal 4773 (Group 4, 1000 mg/kg/day) did not receive a clinical observation or body weight. This deviation did not impact the outcome of the study because
sufficient data were available to evaluate these parameters.

Postmortem and Pathology
• On 09 Dec 2015, fetal body weights were recorded, gross examinations were performed and the fetuses were retained for a female (4723) that was euthanized on GD 20 due to adverse clinical observations. This is above what was required the protocol for dams that were found dead or euthanized prior to scheduled euthanasia. This deviation did not impact the outcome of the study because the additional observations were not detrimental to the study.
• On 14 Dec 2015, one male pup was found dead on PND 4 and was noted as being saved in 10% neutral buffered formalin from litter 4747 in Group 1. This pup was not noted with any
gross lesions, and therefore, was inadvertently retained. This deviation did not impact the outcome of the study because the retention of this pup was not detrimental to the study.
• On 16 Dec 2015, two pups were inadvertently not retained in Bouin's solution from litter 4775 in Group 4. It was presumed that the sex of the found dead pups were one male and one female and the two missing pups were both female after reviewing the in-life transaction record. It was also presumed that the found dead pups were partially cannibalized since the
litter observation sheet stated that the heads appeared normal. This deviation did not impact the outcome of the study because sufficient data were available to evaluate this parameter.
• On 25 Dec 2015, there was no directive to process the epididymides into 10% neutral buffered formalin from Bouin's solution. In addition, there was no documentation of when the testes and epididymides were rinsed and transferred into 10% neutral buffered formalin. This deviation did not impact the outcome of the study because it was presumed that the tissues were rinsed and processed correctly.

Other
• On 02 Mar 2016, no inferential statistical analyses were to be performed on necropsy observations; however, a Fisher’s exact evaluation was performed on the F1 generation pup necropsy data. This deviation did not impact the outcome of the study because the additional statistical analyses evaluation was not detrimental to the study.
• On 22 Oct 2015, there was no Individual Scientist assigned in the protocol to oversee the analysis of the bulk test article at Bristol-Myers Squibb Company and a GLP-compliant stability report was not generated to provide the results of this analysis. This deviation did not impact the results of this study because the analysis was being performed to verify stability during the course of the study and the analysis was performed by the Sponsor under acceptable conditions.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
The objective of this study was to test for toxic effects/disturbances resulting from BMS-217947-01 treatment of Crl:CD(SD) male and female rats before cohabitation, through mating, implantation, gestation and lactation.

Test material

Constituent 1
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Crl:CD(SD) Sprague Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Eighty eight (42 male and 46 female) Crl:CD(SD) rats were received from Charles River Inc., Raleigh, NC, United States. The body weight range for the male rats was 287 g to 332 g on the day after arrival and was 336 g to 396 g at randomization and study assignment. The male rats were approximately 71 days of age upon arrival at the Testing Facility.
The body weight range for the female rats was 197 g to 240 g on the day after arrival and was 241 g to 288 g at randomization and study assignment. The female rats were approximately 66 days of age upon arrival at the Testing Facility.
Husbandry
All cage sizes and housing conditions were in compliance with the Guide for the Care and Use of Laboratory Animals.

Housing
Animals were co-housed in solid-bottomed cages by dose group (no more than 2 per cage/sex) until cohabitation. During the cohabitation period, one male rat and one female rat were pair
housed in the male rat’s solid-bottomed cage. After cohabitation, female rats were individually housed until delivery or scheduled euthanasia. Male rats were returned to the same sex pair that
was established prior to the mating period. Each dam and delivered litter was housed in a common nesting box.

The study rooms were maintained under conditions of positive airflow relative to a hallway and independently supplied with a minimum of 10 changes per hour of 100% fresh air that had been
passed through 99.97% HEPA filters. Room temperature and humidity were monitored constantly throughout the study. Room temperature was targeted at 66°F to 77°F (19°C to 25°C); relative humidity was targeted at 30% to 70% (see Appendix 2, Deviations). An automatically controlled 12-hour light:12-hour dark fluorescent light cycle was maintained. Each dark period began at 1900 hours(± 30 minutes).

Nesting Material
Nesting material (Bed-o'Cobs®) was provided. Nesting material was changed as often as necessary to keep the animals dry and clean. The nesting material was analyzed for environmental contaminants and results of the analysis are on file at the Testing Facility. It was considered that there were no known contaminants in the nesting material that would interfere with the objectives of the study.

Food
Rats were given Certified Rodent Diet® #5002 (PMI® Nutrition International) available ad libitum from individual feeders. The food was analyzed for environmental contaminants and results of the analysis are on file at the Testing Facility. It was considered that there were no known contaminants in the food that would interfere with the objectives of the study.

Water
Water was available ad libitum from individual bottles attached to the cages and/or from an automatic watering access system (see Appendix 2, Deviations). All water was from a local source and passedthrough a reverse osmosis membrane before use. Chlorine was added to the processed water as a bacteriostat; processed water was expected to contain no more than 1.2 ppm chlorine at the time of analysis. Periodic analysis of the water was performed, and results of these analyses are on file at the Testing Facility. It was considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
For psychological enrichment, rats were provided with a chewing object and Crink L Nest™. It was considered that there were no known contaminants in the enrichment devices that would interfere with the objectives of the study.

Veterinary Care
Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations, food supplementation, and therapeutic treatments were documented in the study records and reviewed by the Study Director. Food supplementation included moistened meal and therapeutic treatments included daily bedding changes, and use of a warming pad and/or Crink L Nest™. None of the medical examinations, food supplementation or therapeutic treatments had an adverse impact on the integrity of the study data or on the interpretation of the study results.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
P generation male rats were administered the test and/or control article for 28 days prior to cohabitation, throughout cohabitation and continued until the day prior to euthanasia. P generation female rats were administered the test and/or control article for 14 days prior to cohabitation and continued through Lactation Day 3 (LD 3; rats that delivered a litter) or Gestation Day 24 (GD 24; rats that did not deliver a litter). The female not mated after completion of the 14-day cohabitation period was considered to be GD 0 on the last day of cohabitation and continued to be administered the test article through the postpartum period until LD 3.
Details on mating procedure:
Within each dose group, consecutive order was used to assign P generation rats to cohabitation, one male rat per female rat. The cohabitation period consisted of 14 days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be at GD 0 and assigned to individual housing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis for all concentrations at the first and last preperations, Homogeneity wa assessed for the first preperations in groups 2 and 4. The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations was averaged and utilized as the concentration results.

Samples to be analyzed will be submitted within 1 week of preparation. All samples to be analyzed will be transferred (on cold/ice packs, protected from light) to the analytical laboratory at the Testing Facility.
Any residual/retained analytical samples (and test article used in analysis) will be discarded before issue of the Final Report.
Duration of treatment / exposure:
P generation male rats were administered the test and/or control article for 28 days prior to cohabitation, throughout cohabitation and continued until the day prior to euthanasia.

P generation female rats were administered the test and/or control article for 14 days prior to cohabitation and continued through Lactation Day 3 (LD 3; rats that delivered a litter) or Day 24 of Gestation Day (GD 24; rats that did not deliver a litter). The female not mated after completion of the 14-day cohabitation period was considered to be GD 0 on the last day of cohabitation and continued to be administered the test article through the postpartum period until LD 3.

F1 generation pups were not directly given the test substance but may have been exposed in utero during gestation or via maternal milk during the lactation period.
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control article - Peanut oil
Concentration: 0mg/kg
Dose Volume 10mg/kg
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Concentration:1 mg/L
Dose Volume: 10 mL
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Concentration:10 mg/L
Dose Volume: 10 mL
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Concentration:100 mg/L
Dose Volume: 10 mL
No. of animals per sex per dose:
10 M/F per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The oral (gavage) route was selected for use because: 1) in comparison with the dietary route, the exact dose could be accurately administered; and 2) it is one of the proposed routes for clinical use. Doses for this study were selected based on the results from a previous acute oral toxicity study in Sprague-Dawley CD rats and a 28-day repeat dose study in Sprague-Dawley CD rats.
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
The in-life procedures, observations and measurements listed below were performed for all rats:
Viability checks were conducted at least twice daily
Clinical Observations:
-General appearances were examined twice (males) or 4 times (females) during acclimation, daily before administration during dose period and prior to scheduled euthanasia
-Postdose observations were recorded 1 to 2 hours following dose administration
-Maternal observations were recorded daily during the postpartum period.
-Body weights were recorded twice (males) or 4 times (females) during acclimation, daily during the dose period and on the day of scheduled euthanasia
-Food consumption values for the male rats were recorded once weekly during the dose period and on the day of euthanasia. For the female rats, food consumption values were recorded once weekly during the dose period, GDs 0, 7, 10, 14,
18 and 21 and LDs 0 and 4 (food left value). During cohabitation, when 2 rats occupied the same cage, replenishment of the food was documented, but individual values were not recorded
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage for 14 consecutive days before initiation of dose administration, and for 14 consecutive days after onset of dosing beginning with the day after the first dose administration and then until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period.
Litter observations:
The in-life procedures, observations and measurements listed below were performed:
Viability checks for dead pups was conducted twice daily and the pups in each litter were counted once daily
Clinical oberservations were recorded daily
Body weights were recorded daily on day 0 and 4 portpartum
Postmortem examinations (parental animals):
The rats that were euthanatized before scheduled termination were examined for the cause of condition as soon as possible after the observation was made. A gross necropsy was performed. The rats were examined for gross lesions.
On DS 63, surviving male rats were euthanatized, and a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed.
After completion of the 4-day postpartum period, surviving P generation female rats were euthanatized and examined for gross lesions. The P generation female rat that did not deliver a litter was euthanatized on GD 25. Dams with no surviving pups were euthanatized after the last pup was found dead or missing, presumed cannibalized.
The reproductive tract was dissected from the abdominal cavity. The number and distribution of implantation sites and corpora lutea was recorded. The uterus of the apparently nonpregnant rat was examined while being pressed between glass plates to confirm the absence of implantation sites. The uterus and ovaries of the apparently nonpregnant rat was retained in 10% neutral buffered formalin.

A gross necropsy of the thoracic, abdominal, and pelvic viscera were for all rats. Images were generated for illustration of or consultation on gross observations. Generation of such images was documented. Images and associated documentation were retained and were archived.

The organs were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for the rats found dead. Organ to body weight ratios (using the terminal body weight obtained at necropsy) were calculated. Paired organs were weighed together.

Representative samples of the tissues were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated. A table of random units was used to select 1 male and 1 female P generation rat from the control group (male no. 4702 and female no. 4742) from which all tissues collected at necropsy were retained in order to provide control tissues for any possible histopathological evaluations.

Tissues were shipped (ambient conditions) to the Charles River Laboratories, Inc. facility located in Frederick, MD, for processing. Tissues from the rats that were found dead, all animals in Groups 1 and 4, and all animals with gross lesions were trimmed, embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

Pathological evaluations were performed by a board-certified veterinary pathologist. Pathological examinations were performed on the rats that were found dead, all rats in Groups 1 and 4, and all rats with gross lesions.
Postmortem examinations (offspring):
Pups were euthanatized by an intraperitoneal injection of sodium pentobarbital (390 mg/mL).
The pup that died before examination of the litter for pup viability was evaluated for vital status at birth. The lungs were removed and immersed in water. The lungs that sank from the pup that died before examination was identified as stillborn. Pups with lungs that float were identified as liveborn and to have died shortly after birth.
The pups that died were examined for gross lesions and the cause of death on the day the observation was made. Pups found on PNDs 1 to 3 were preserved in Bouin's solution for possible future evaluation. A pup found on PND 4 was preserved in neutral buffered 10% formalin.

All surviving pups were euthanatized on PND 4 and examined for gross lesions. Gross lesions were preserved in neutral buffered 10% formalin for possible future evaluation. Necropsy included a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly.
Statistics:
Descriptive statistics including number, mean, percentages and/or standard deviation were reported as appropriate. Litter values were used where appropriate. Clinical and necropsy observations data were summarized but no inferential statistical analysis were performed
Statistically significant pair-wise comparison probabilities were reported as either p≤ 0.05 or p≤ 0.01, unless otherwise noted below.

Proportional data were analyzed using Fisher's Exact Test, as appropriate. Continuous data were analyzed using Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e., Bartlett’s Test was not significant (p> 0.001)]. If the Analysis of Variance was significant (p≤ 0.05), Dunnett’s Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett’s Test was significant (p≤ 0.001)], the Kruskal-Wallis Test was used. In cases where the Kruskal-Wallis Test was statistically significant (p≤ 0.05), Dunn’s Method of Multiple Comparisons10 was used to identify the statistical significance of the individual groups. Count data, such as the number of estrous cycles, were analyzed using the Kruskal-Wallis Test, and in the event of a significant result (p≤ 0.05), Dunn's Test was used to compare the groups given the test article with the control group.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Males:
In the 1000 mg/kg/day dose group, there was an increase in the total number of males observed with excess salivation as well as the number of males observed with slight or moderate excess salivation in comparison with the control group values. All other clinical signs that were observed were considered unrelated to the test article because: 1) the observations were not dose-dependent; 2) the observations occurred in only 1 or 2 rats in a dose group; and/or 3) the clinical observations were transient and did not persist. In the 1000 mg/kg/day dose group, there was an increase in the total number of females observed with excess salivation during the premating, gestation and lactation periods in comparison with the control group values. There was also an increase in the number of females in the 1000 mg/kg/day dose group observed with urine-stained abdominal fur during the premating period in comparison with the control group value.

Females:
In the 1000 mg/kg/day dose group, there was an increase in the total number of females observed with excess salivation during the premating, gestation and lactation periods in comparison with the control group values. There was also an increase in the number of females in the 1000 mg/kg/day dose group observed with urine-stained abdominal fur during the premating period in comparison with the control group value.
All other clinical signs that were observed were considered unrelated to the test article because: 1) the observations were not dose-dependent; 2) the observations occurred in only 1 or 2 rats in a dose group; and/or 3) the clinical signs did not persist. These clinical signs included ungroomed coat; a scab on the back or nose; hunched posture; mild dehydration (based on skin turgor); a laceration and ulceration on the back; decreased motor activity; chromorhinorrhea;
chromodacryorrhea; ptosis; sparse hair coat on the back, limb(s) or underside; twitches; splayed limb(s); low carriage; vocalization to touch; swollen head and nose; and whole body tremors.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male (animal no. 4727) in the 100 mg/kg/day dose group was euthanized on day 28 of study (DS 28) due to adverse clinical observations. This rat was observed with decreased motor activity, limited use of the left forelimb, hunched posture and rales on DS 28 prior to euthanasia. A perforation was observed in the esophagus of the rat at the time of necropsy; all other tissues appeared normal. This death was considered to be the result of an intubation error and was not considered to be test article related.
One female (animal no. 4763) in the 100 mg/kg/day dose group was euthanized on day 20 of presumed gestation (GD 20) due to adverse clinical observations. This female was observed
with a scab on the nose; decreased motor activity; low carriage; vocalization to touch; and a swollen head and nose on GD 20 prior to euthanasia. Body weight and food consumption values for this rat were generally comparable with the other rats in this dose group. All tissues appeared normal at necropsy.
The litter consisted of 13 fetuses in utero. All fetuses appeared normal at gross external examination. This death was not considered to be test article related because it was not dose dependent. All other females survived until scheduled euthanasia.
All other rats survived until scheduled euthanasia
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males:
In the 1000 mg/kg/day dose group, there was a statistically significant decrease (p≤ 0.01) in body weight gain observed in the males during the overall dose period (DSs 1 to 63) and at DSs 1 to 8, 1 to 28 and 61 to 63 in comparison with the control group values. There was also a statistically significant decrease (p≤ 0.05 or p≤ 0.01) in the mean body weight for the males in the 1000 mg/kg/day dose group at all intervals beginning on DS 8 and continuing through the remainder of the dose period in comparison with the control group values.
Body weights and body weight gains were unaffected by doses of the test article up to and including 100 mg/kg/day. During the study period (DSs 1 to 63), the mean body weight gain in the male rats was 101%, 93%, and 68% of the control group in the 10, 100 and 1000 mg/kg/day dose groups, respectively

Females:
Mean body weights and body weight changes were unaffected by administration of the test article at dose levels up to and including 1000 mg/kg/day during the premating, gestation and lactation dose periods. In the 10 and 100 mg/kg/day dose groups, there was a statistically significant decrease (p≤ 0.05 or p≤ 0.01) in body weight gain observed in the females during the overall premating period (DSs 1 to 15) in comparison with the control group value. These decreases were not considered to be test article related because they were not dose dependent. There was a statistically significant increase (p≤ 0.05) in body weightgain observed at 1000 mg/kg/day on DSs 8 to 15 during the premating period in comparison with the control group value. This increase was not considered to be test article related because it did not impact the overall body weight change for this dose group during the premating period (DSs 1 to 15). There was a statistically significant increase (p≤ 0.01) in body weight gain observed at 1000 mg/kg/day during the overall lactation
period (LDs 0 to 4) as well as on LDs 0 to 1 in comparison with the control group value. There was also a statistically significant increase (p≤ 0.01) in the mean body weight observed on LD 3 in the 1000 mg/kg/day dose group in comparison with the control group value. These increases were not considered to be test article related because a decrease rather than an increase would be considered to be toxicologically relevant.
Food consumption and compound intake (if feeding study):
no effects observed
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:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
The predose estrous cycling observations [mean estrous stages per 14 days, rats with 6 or more consecutive days in diestrus and rats with 6 or more consecutive days of estrus] were unaffected by doses of the test article as high as 1000 mg/kg/day.
In the 1000 mg/kg/day dose group, there was a statistically significant decrease (p≤ 0.01) in the mean estrous stages per 14 days and a statistically significant increase (p≤ 0.05) in the number of rats with 6 or more consecutive days in diestrus during the precohabitation period in comparison with the control group values.
All mating and fertility parameters [numbers of days in cohabitation, rats that mated, fertility index (number of pregnancies per number of rats that mated), rats with confirmed mating dates during the first week of cohabitation and number of pregnancies per number of rats in cohabitation] were unaffected by doses of the test article as high as 1000 mg/kg/day.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
All mating and fertility parameters [numbers of days in cohabitation, rats that mated, fertility index (number of pregnancies per number of rats that mated), rats with confirmed mating dates
during the first week of cohabitation and number of pregnancies per number of rats in cohabitation] were unaffected by doses of the test article up to and including 1000 mg/kg/day.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic/general toxicity
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There was an increase in the number of pups in the 1000 mg/kg/day dose group that were observed with coldness to the touch in comparison with the control group value.
All other clinical signs that were observed were considered unrelated to the test article because: 1) the observations occurred in only 1 litter in a particular dose group; and/or 2) the observations were sporadic and did not persist. These clinical signs included a purple neck, back, nose and/or base of tail; mild dehydration; a missing portion of the tail, right hindlimb and/or right hindlimb digits; and a scab on the head.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Two dams in the 1000 mg/kg/day dose group were observed with all pups dying between PNDs 0 to 3. The death of these litters was considered to be due to lack of maternal care as the dams were observed with adverse clinical signs after parturition. In the 1000 mg/kg/day dose group, there was a statistically significant increase (p≤ 0.01) in the number of pups that were found dead or presumed cannibalized on PNDs 1 to 4 in comparison with the control group
value. There was also a statistically significant decrease (p≤ 0.01) in the viability/lactation index (average number of pups on PND 4 per number of liveborn pups on PND 0) in the 1000 mg/kg/day dose group in comparison with the control group value. There was a statistically significant decrease (p≤ 0.05) in the number of surviving pups per litter observed in the 1000 mg/kg/day dose group in comparison with the control group value.
In the dose groups up to and including 1000 mg/kg/day, values for the numbers of dams delivering litters; duration of gestation; averages for corpora lutea and implantation sites per delivered litter; dams with stillborn pups; dams with no liveborn pups; the gestation index (number of dams with 1 or more liveborn pups/number of pregnant rats); and the number of dams with all pups dying days 3 to 4 postpartum were comparable among the dose groups.
The values for total pups delivered; liveborn pups; stillborn pups; and the percentage of male pups per number of pups sexed per litter were comparable among the 4 dose groups during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The live litter size at weighing and the pup weights per litter were also comparable among the 4 dose groups. There was a statistically significant increase (p≤ 0.05) in the mean pup weight per litter observed on PND 0 in the 10 mg/kg/day dose group in comparison with the control group value. This increase was not considered to be test article related because it was not dose dependent.
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
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test article related findings apparent in the pups that were found dead prior toscheduled euthanasia. All pups that were euthanized on PND 4 appeared normal at the time of necropsy.
Histopathological findings:
not examined
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for general toxicity was 100 mg/kg/day in the male and female rats. There were adverse clinical signs observed in both sexes at 1000 mg/kg/day and decreased body weight gain observed in the males at 1000 mg/kg/day. The reproductive NOAEL was 1000 mg/kg/day as there were no test article-related changes in mating and fertility in the males or females. There was a decrease in the mean estrous stages per 14 days and an increase in the number of rats with 6 or more consecutive days in diestrus at 1000 mg/kg/day during the precohabitation period. This was not considered to be an adverse finding because this did not impact the mating and fertility of the females at 1000 mg/kg/day.
The NOAEL for viability and growth in the offspring was 1000 mg/kg/day. The decrease in pup viability observed in the 1000 mg/kg/day dose group was attributed to lack of maternal care as the dams were observed with adverse clinical signs shortly after parturition.
Executive summary:

The objective of this study was to test for toxic effects/disturbances resulting from BMS-217947-01 treatment of Crl:CD(SD) male and female rats before cohabitation, through mating, implantation, gestation and lactation. BMS-217947-01 was administered at 0, 10, 100 and 1000 mg/kg. P generation male rats were administered the test and/or control article for 28 days prior to cohabitation, throughout cohabitation and continued until the day prior to euthanasia. P generation female rats were administered the test and/or control article for 14 days prior to cohabitation and continued through Lactation Day 3 (LD 3; rats that delivered a litter) or Gestation Day 24 (GD 24; rats that did not deliver a litter). The female not mated after completion of the 14-day cohabitation period was considered to be GD 0 on the last day of cohabitation and continued to be administered the test article through the postpartum period until LD 3. Doses were adjusted based on the most recently recorded body weight and administered at approximately the same time each day. F1 generation pups were not directly given the test substance but may have been exposed in utero during gestation or via maternal milk during the lactation period. The following parameters and end points were evaluated in this study: viability, clinical signs, food consumption, body weights, body weight changes, reproductive capacity, maternal behavior, natural delivery observations, gross necropsy observations, organ weights, and histology and pathological evaluations. During the study, one male and one female in the 100 mg/kg/day dose group were euthanized due to adverse clinical observations. The death in the male was the result of an intubation error and the death in the female was not considered to be test article related because it was not dose dependent. All additional male and female rats survived until scheduled euthanasia. In the 1000 mg/kg/day dose group, there was an increase in the number of males and females observed with excess salivation during the dose period. There was also an increase in the number of females in the 1000 mg/kg/day dose group observed with urine-stained abdominal fur during the premating period. In the males at 1000 mg/kg/day, there was a statistically significant decrease in body weight gain observed during the overall dose period and at Days 1 to 8 of Study (DSs 1 to 8), 1 to 28 and 61 to 63. There was also a statistically significant decrease in the mean body weight for the males Page 14 Final Report Testing Facility Study No. 20080537 Unaudited/Audited Draft/Interim/Final Report Page 15 Sponsor Reference No. XX Testing Facility Study No. XX at 1000 mg/kg/day at all intervals beginning on DS 8 and continuing through the remainder of the dose period. In the females at 1000 mg/kg/day, there was a statistically significant decrease in the mean estrous stages per 14 days and a statistically significant increase in the number of rats with 6 or more consecutive days in diestrus during the precohabitation period. All mating and fertility parameters in the males and females were unaffected by doses of the test article as high as 1000 mg/kg/day. There were no test article-related necropsy observations in the males or females. There was a statistically significant decrease in terminal body weight observed in the males at 1000 mg/kg/day. There was a statistically significant increase in the mean liver weights and the liver weight to terminal body weight ratios observed in the males and females at 1000 mg/kg/day. Pregnancy occurred in 10, 9, 10 and 10 mated females in the 0 (Control Article), 10, 100 and 1000 mg/kg/day dose groups, respectively. All of the respective pregnant dams delivered litters except for the female in the 100 mg/kg/day dose group that was euthanized due to agonal clinical observations. Two dams in the 1000 mg/kg/day dose group were observed with all pups dying between PNDs 0 to 3. The death of these litters was considered to be due to lack of maternal care as the dams were observed with adverse clinical signs after parturition. In the 1000 mg/kg/day dose group, there was a statistically significant increase in the number of pups that were found dead or presumed cannibalized on PNDs 1 to 4. There was a statistically significant decrease in the viability/lactation index (average number of pups on PND 4 per number of liveborn pups on PND 0) in the 1000 mg/kg/day dose group. There was also a statistically significant decrease in the number of surviving pups per litter observed in the 1000 mg/kg/day dose group. No clinical observations in the F1 generation pups were attributable to maternal doses of the test article as high as 1000 mg/kg/day. There were no test article-related necropsy observations in the F1 generation pups.