L-Valine, N-(methoxycarbonyl)-3-methyl-

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for general toxicity was 500 mg/kg/day in the male and female rats.  The reproductive NOAEL was 500 mg/kg/day as there were no test article-related changes in estrous cycling in the female and mating and fertility in the males or females.  The NOAEL for viability and growth in the offspring was also 500 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 October 2015 - 26 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Please see "Principles of method if other than guideline" field

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 14 Oct 2015, Peanut oil, NF, batch (lot) no. 2EA0347 with a purity of 100%, stored at ambient conditions was used as the control article instead of Peanut oil, NF, batch (lot) no.
27A54j77 with a purity of 98%, stored in a freezer set to maintain -80ºC. This deviation did not impact the outcome of the study because the peanut oil used for the study was consistent throughout the study and was amended on the day of discovery.
• On 14 to 21 Oct 2015, the bulk test article was stored in a controlled temperature area set to maintain 21°C and protected from light instead of in a freezer set to maintain -80ºC prior to
the protocol being amended to revise the storage conditions. This deviation did not impact the outcome of the study because the appropriate storage conditions were used for the bulk test material in the interim.
• On 27 Oct 2015 and 20 Nov 2015, the time the dose formulations were transferred to the study room was not properly documented; therefore, it could not be confirmed that the dose
formulations were mixed for 30 minutes prior to use. This deviation did not impact the outcome of the study because it was presumed that the formulations were allowed to mix for
an adequate amount of time prior to usage.
• On Day 15 of Study (DS 15, 11 Nov 2015), female rats were dosed for 15 days prior to being placed into cohabitation instead of 14 days. This deviation did not impact the outcome of the study because the additional day of dose administration prior to cohabitation was not detrimental to the study.
• On 13 Nov 2015, during the dose administration the Argus Automated Data Collection and Management System malfunctioned and resulted in the loss of the predose clinical observation and/or dose volume for male rats 3835, 3836 and 3837 (Group 2). This deviation did not impact the outcome of the study because it was determined that a sufficient
amount of test article preparation was used to verify that the animals were dosed.

Husbandry
• On 17 to 19 Oct 2015, 08, 09 Nov 2015, and 09 Dec 2015, the relative humidity was out of range (as low as 24.0%) for no more than 51 hours in duration during the study. This
deviation did not impact the outcome of the study because the variations from the target humidity were not considered to be detrimental to the animals.

In-life Observations, Measurements, and Evaluations
• From 12 Oct 2015 through 04 Nov 2015, during the acclimation period, female rats did not have a body weight recorded at least once weekly. In addition male rats not assigned to
study (no. 21 and 32) did not have body weights or clinical observations recorded after Day 9 through 29. This deviation did not impact the outcome of the study because sufficient evaluation of these female rats was performed during the acclimation period.
• On 27 Oct 2015 through 04 Nov 2015, during the acclimation period, female rats 17, 22, 28, 37, 42 and 45 did not have clinical observations recorded between Day 21 and 29. This deviation did not impact the outcome of the study because sufficient evaluation of these female rats was performed during the acclimation period.
• On 11 Nov 2015, the first two animals in Group 2 were not mated in consecutive order. The animals were switched and a footnote to the data was added, however no reason for the switch was provided. This deviation did not impact the outcome of the study because the animals were mated and the study design was completed.
• On Day 21 of gestation (DG 21, 09 Dec 2015), a food left or fed value was not recorded for female 3866 (Group 1). This deviation did not impact the outcome of the study because sufficient data were available to evaluate this parameter.

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-214702-01 treatment of Crl:CD(SD) male and female rats before cohabitation, through mating, implantation, gestation and lactation. The design of this study is based on the study objective, the overall product development strategy for the test article, and OECD Guideline.

The test system was selected because: 1) it is a standard species accepted for use in fertility and early embryonic development studies; 2) this species and strain has been demonstrated to be sensitive to reproductive toxicants; and 3) historical data and experience exist at the Testing Facility. The number of animals chosen for this study was the smallest number considered necessary to provide the minimum number of pregnancies recommended by the applicable guidelines. The two additional male rats and six additional female rats were ordered to ensure a sufficient number of healthy rats were available for randomization onto study.

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

Charles River Inc., Raleigh, NC, United States

Sex:

male/female

Details on test animals or test system and environmental conditions:

Eighty eight (42 male and 46 female) rats were received.

The body weight range for the male rats was 298 g to 340 g on the day after arrival and was 316 g to 348 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 207 g to 235 g on the day after arrival and was 219 g to 294 g at randomization and study assignment. The female rats were approximately 66 days of age upon arrival at the Testing Facility.

The study room was 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 maintained at 66°F to 77°F (19°C to 25°C); relative humidity was targeted at 30% to 70%. An automatically controlled 12-hour light:12-hour dark fluorescent light cycle was maintained. Each dark period began at 1900 hours (± 30 minutes).

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.

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. All water was from a local source and passed through 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. 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 were documented in the study records and reviewed by the Study Director. None of the medical examinations had an adverse impact on the integrity of the study data or on the interpretation of the study results.

Route of administration:

oral: gavage

Vehicle:

peanut oil

Remarks:

Batch No: 2EA0347, 2EH0290 and 2EJ0376. Expiration date 31 Jan 2016, 31 Jul 2016 and 30 Sep 2016 respectively

Details on exposure:

P Generation
Before initiation of dose administration, males were selected for study on the basis of physical condition and body weights recorded during acclimation. Females were selected for study on the basis of physical condition, body weights recorded during the acclimation period. The rats were assigned to dose groups, by sex, based on computer-generated (weight-ordered) randomization procedures. No rats were replaced during this study.

F1 Generation
Litters were not culled during the lactation period because random selection of pups for culling could have resulted in potential biases in pup viabilities and body weight gains during this period.

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 7 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 DG 0 and assigned to individual housing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples to be analyzed were transferred on the day of preparation (on cold packs, protected from light) and stored in a refrigerator set to maintain 4ºC, protected from light, until analyzed. Analyses were performed by HPLC using a validated analytical procedure (MBAA00).

Duplicate (1 mL) sets of top, middle, and bottom samples (duplicate middle only for Groups 1 and 3) were collected and transferred to the analytical laboratory; triplicate sets of top, middle, and bottom samples (triplicate middle only for Groups 1 and 3) were taken in the same manner and stored in a refrigerator set to maintain 4°C, protected from light at the Testing Facility as backup samples. On days where only concentration analysis was required, the formulations were sampled from the middle. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 20% of theoretical concentration. For homogeneity, the criteria for acceptability were a relative standard deviation (RSD) of concentrations of ≤ 5% for each group. After acceptance of the analytical results, backup samples were discarded. One sample (10 mL) from Groups 2 through 4 was taken on the first day of preparation for determination of density.

Stability analyses performed previously in conjunction with Charles River Laboratories Study No. 20080535 demonstrated that the test article is stable in the control article when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Charles River Laboratories Study No.20080535.

Frequency of treatment:

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 DL 3 (rats that delivered a litter) or DG 24 (rats that did not deliver a litter; see Appendix 2, Deviations). Any dam in the process of parturition was not given the test and/or control article until the following work day. Such events were noted in the raw data.
F1 generation pups were not directly given the test article but may have been exposed in utero during gestation or via maternal milk during the lactation period.

Dose / conc.:

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

Remarks:

Control article.
Concentration: 0 mg/ml
Dose volume 10 ml/kg

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Concentration: 5mg/ml
Dose volume: 10 ml/kg

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Concentration: 15 mg/ml
Dose volume: 10 ml/kg

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Concentration: 50 mg/ml
Dose volume: 10 ml/kg

No. of animals per sex per dose:

10

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 rats and a 28-day repeat dose study in Sprague-Dawley rats.

In the acute oral toxicity study with a 14 day recovery period, animals were treated with 2000 mg/kg (n=3/sex). No clinical signs or mortality were observed. No abnormalities were observed at necropsy.

In the 28-day repeat dose study, animals were treated with 0, 15, 150, 1000 mg/kg/day (n=5/sex/group) of BMS-214702-01 in arachis oil BP. At doses ≥150 mg/kg/day, an increased incidence/severity of agglomeration of secretion and goblet cell hyperplasia in stomach was observed and considered to be a non-degenerative adaptive change. At 1000 mg/kg/day increased salivation was observed (day 6 onward). One male was euthanatized in extremis on day 25 due to a deterioration of condition (labored respiration developed on day 21; abdominal distension, dehydration developing on day 24; hunched posture, piloerection, ptosis, tiptoe gait on day 25). In the euthanatized male, small spleen, gaseous distension of intestines and stomach, and sloughing of non-glandular gastric epithelium was observed at necropsy. Dark liver was observed at necropsy in all males. The NOEL for this study was determined to be 150 mg/kg/day.

For this study, doses of 50, 150, and 500 mg/kg were selected for this reproduction and developmental toxicity study. The high dose of 500 mg/kg was expected to result in toxicity without causing mortality or severe effects. A low dose of 50 mg/kg was selected based on the NOEL from the 28 day study and is expected to result in a NOAEL. A mid-dose of 150 mg/kg was selected to evaluate the dose-response relationship of any potential BMS-214702-01-related adverse effects.

Positive control:

Not used

Parental animals: Observations and examinations:

The in-life procedures, observations, and measurements listed below were performed for all rats.
The rats were assessed for viability at least twice daily during the study.
The rats were observed for general appearance daily during the acclimation period, dose period and prior to scheduled euthanasia.
Postdose observations were recorded between 1 to 2 hours following dose administration.
Maternal observations were recorded daily during the postpartum period.
Body weights were recorded twice (males) or three times (females) during acclimation, daily during the dose period and on the day of scheduled euthanasia.

For the male rats, food consumption values were recorded once weekly during the dose period and on the day of euthanasia (food left weight). For the female rats, food consumption values were recorded once weekly during the dose period, DGs 0, 7, 10, 14, 18 and 21 and DLs 0 and 4. During cohabitation, when two rats occupied the same cage, replenishment of the food was documented, but individual values were not recorded.

Within each dose group, consecutive order was used to assign rats to cohabitation (i.e., pairing), one male per one female. The cohabitation period consisted of 7 days. Females with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be at DG 0 and assigned to individual housing.

Natural delivery observations (adverse clinical signs observed, duration of gestation [DG 0 to the time the first pup was observed], litter size, and pup viability at birth) were 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.

Sperm parameters (parental animals):

Not recorded

Litter observations:

The in-life procedures, observations, and measurements listed below were performed for all F1 generation litters, with the litter as the unit of measure.
Litters were observed for dead pups twice daily and the pups in each litter were counted once daily.
Clinical observations were recorded daily.
Body weights were recorded on Days 0 (birth) and 4 postpartum.

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 61, 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 DG 25.
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 identified in Text Table 7 (see attached pdf) 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 identified in Text Table 7 (see attached pdf) 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. 3821 and female no. 3870) 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.
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 4 were preserved in Bouin's solution for possible future evaluation.

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. 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.

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.

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 Comparisons 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.

Reproductive indices:

The following natural delivery/reproductive parameters were reported:
• Duration of Gestation: The duration of gestation was calculated from DG 0 to the day the first pup was observed.
• Fertility Index: Percentage of matings that resulted in pregnancies
• Gestation Index: Percentage of pregnancies that resulted in birth of live litters
• Number of offspring per litter: Live and dead pups
• Number of implantation sites and corpora lutea.

Offspring viability indices:

• General condition of dam and litter during the postpartum period.
• Viability Indices: Percentage of pups born that survived 4 days
• Lactation Index: Percentage of pups that survived 4 days

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All clinical signs that were observed in male and female were considered unrelated to the test article because: 1) the observations were not dose-dependent; 2) the observations occurred in only one or two rats in a dose group; and/or 3) the clinical observations were transient and did not persist.

These clinical signs for male rats included urine-stained abdominal fur; sparse hair coat on the limb(s) or head; chromodacryorrhea; rales; slight excess salivation; localized alopecia on the limb(s); piloerection; and a scab on the head.

The clinical signs for female rats included sparse hair coat on the neck or limb(s); urine-stained abdominal fur; a scab on the left axilla; localized alopecia on the neck; rales; chromorhinorrhea; hunched posture; decreased motor activity; ataxia; coldness to the touch; and hyperpnea.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two male rats (one in the control group and one in the 500 mg/kg/day dose group) were found dead prior to scheduled euthanasia. The death in the control group was related to a technician error. There were no adverse clinical signs prior to it being found dead and body weight and food consumption were comparable to other rats in the group. The single death in the 500 mg/kg/day dose group was not considered related to the test article as all other rats in this group survived to scheduled euthanasia, and there were no test article related adverse observations in this rat prior to it being found dead. All tissues appeared normal at necropsy. All other male rats survived until scheduled euthanasia.

One female rat (animal no. 3874) in the 50 mg/kg/day dose group was found dead on DS 22. There were no adverse clinical signs observed in this female prior to it being found dead. Body weight and food consumption values for this rat were generally comparable with the other rats in this dose group. All tissues appeared normal for the slight degree of autolysis at necropsy. This death was not considered to be test article related because it was not dose dependent. All other female rats survived until scheduled euthanasia.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gains in male rats were unaffected by doses of the test article up to and including 500 mg/kg/day. Body weight gains were 99%, 97% and 96% of the control group in the 50, 150 and 500 mg/kg/day dose groups, respectively, for the interval of DSs 1 to 61. There was a statistically significant increase (p≤ 0.05) in body weight gain observed in the male rats in the 500 mg/kg/day dose group on DSs 57 to 61 in comparison with the control group value. This increase was not considered to be test article related because it was a single occurrence and the increase did not impact the overall body weight gain for the study period.

Mean body weights and body weight changes values in the female rats were unaffected by administration of the test article at dose levels up to and including 500 mg/kg/day during the premating, gestation and lactation dose periods.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption in male and female rats was unaffected by doses of the test article up to and including 500 mg/kg/day. Food consumption values in male rats were 102%, 99% and 98% of the control group in the 50, 150 and 500 mg/kg/day dose groups, respectively, for the interval of DSs 1 to 61.

Food efficiency:

not examined

Description (incidence and severity):

Not examined

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

not specified

Description (incidence and severity):

Not specified

Ophthalmological findings:

not specified

Description (incidence and severity):

Not specified

Haematological findings:

not examined

Description (incidence and severity):

Not examined

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not examined

Urinalysis findings:

not examined

Description (incidence and severity):

Not examined

Behaviour (functional findings):

not specified

Description (incidence and severity):

Not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All rats appeared normal at necropsy.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

All rats appeared normal at necropsy.

Other effects:

no effects observed

Description (incidence and severity):

No other effects were observed in the study.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The predose and precohabitation estrous cycling observations [mean estrous stages per 14 days, rats with six or more consecutive days in diestrus and rats with six or more consecutive days of estrus] were unaffected by doses of the test article as high as 500 mg/kg/day.

Reproductive function: sperm measures:

not examined

Description (incidence and severity):

Not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

In male and female rats, 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 500 mg/kg/day.

Pregnancy occurred in 9, 9, 10 and 10 mated females in the 0 (Control Article), 50, 150 and 500 mg/kg/day dose groups, respectively, and all of the respective pregnant dams delivered litters.

In the 150 and 500 mg/kg/day dose groups, there was a statistically significant decrease (p≤ 0.05 or p≤ 0.01) in the mean pup weight per litter observed on PND 4 in comparison with the control group value. This decrease was not considered to be test article related because it was not dose dependent.

All other indexes were comparable among all dose groups.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: general toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

Description (incidence and severity):

From birth to day 4 postpartum no clinical observations in the F1 generation pups were attributable to maternal doses of the test article as high as 500 mg/kg/day. All 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 one or two litters in a particular dose group; and/or 3) the observations were sporadic and did not persist. These clinical signs included coldness to the touch; purple discoloration on the whole body or back; pale appearance of the whole body; swollen neck; portion of the tail missing; third and fourth digits missing from the left hindpaw; umbilical hernia; a laceration on the lower midline; and pups observed to be not nesting or not nursing.

Dermal irritation (if dermal study):

no effects observed

Description (incidence and severity):

No effects were observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

All pups that were stillborn appeared normal at necropsy. There were a few found dead pups that were observed with no milk present in the stomach at the time of necropsy; however, this did not occur in a dose dependent manner.

Body weight and weight changes:

not examined

Description (incidence and severity):

Not examined

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

Not examined

Food efficiency:

not examined

Description (incidence and severity):

Not examined

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

not examined

Description (incidence and severity):

Not examined

Ophthalmological findings:

not examined

Description (incidence and severity):

Not examined

Haematological findings:

not examined

Description (incidence and severity):

Not examined

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not examined

Urinalysis findings:

not examined

Description (incidence and severity):

Not examined

Sexual maturation:

not examined

Description (incidence and severity):

Not examined

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

Not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One pup that was euthanized on PND 4 was observed with slight dilation of the left pelvis of the kidney. This was not considered to be test article related because it was a single occurrence. All other pups appeared normal at the time of necropsy.

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no test article-related microscopic pathologic observations apparent in the epididymides, seminal vesicle glands, prostate glands, ovaries and testes.

Other effects:

no effects observed

Description (incidence and severity):

No other effects were observed.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not examined

Developmental immunotoxicity:

not examined

Description (incidence and severity):

Not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

Key result

Reproductive effects observed:

no

Conclusions:

Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for general toxicity was 500 mg/kg/day in the male and female rats.

The reproductive NOAEL was 500 mg/kg/day as there were no test article-related changes in estrous cycling in the female and mating and fertility in the males or females. The NOAEL for
viability and growth in the offspring was also 500 mg/kg/day.

Executive summary:

The objective of this study was to test for toxic effects/disturbances resulting from BMS-214702-01 treatment of Crl:CD(SD) male and female rats before cohabitation, through mating, implantation, gestation and lactation. The study design was as follows:

Group No.	Test material	Dose level (mg/kg)	Concentration (mg/ml)	Dose volume (ml/kg)	No of male rats	No of female rats
1	Control article	0	0	10	10	10
2	BMS-214702 -01	50	5	10	10	10
3	BMS-214702 -01	150	15	10	10	10
4	BMS-214702 -01	500	50	10	10	10

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 15 days prior to cohabitation and continued through Day 3 postpartum (DL 3; rats that delivered a litter) or Day 24 of Presumed Gestation (DG 24; rats that did not deliver a litter). Any dam in the process of parturition was not given the test and/or control article until the following work day.  Such events were noted in the raw data.

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 article 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, two male rats (one in the control group and one in the 500 mg/kg/day dose group) were found dead prior to scheduled euthanasia.  The death in the control group was related to a technician error.  The single death in the 500 mg/kg/day dose group was not considered related to the test article as all other rats in this group survived to scheduled euthanasia, and there were no test article related adverse observations in this rat prior to it being found dead.  One female rat in the 50 mg/kg/day dose group was found dead prior to scheduled euthanasia.  This death was not considered to be test article related because it was not dose dependent.  All additional male and female rats survived until scheduled euthanasia.

There were no test article-related clinical signs observed in the male or female rats during the study.  Body weights and food consumption were unaffected by the test article during the dose period in the males and during the pre-mating, gestation and lactation periods in the females at doses up to and including 500 mg/kg/day.

The precohabitation estrous cycling observations [mean estrous stages per 14 days, rats with six or more consecutive days in diestrus and rats with six or more consecutive days of estrus] were unaffected by doses of the test article as high as 500 mg/kg/day.  All mating and fertility parameters in the males and females were unaffected by doses of the test article as high as 500 mg/kg/day.

There were no test article-related necropsy observations in the male or female rats.  There were no toxicologically relevant differences in the organ weights collected in the males or females.

Pregnancy occurred in 9, 9, 10 and 10 mated females in the 0 (Control Article), 50, 150 and 500 mg/kg/day dose groups, respectively, and all of the respective pregnant dams delivered litters.

No clinical observations in the F1 generation pups were attributable to maternal doses of the test article as high as 500 mg/kg/day.  There were no test article-related necropsy observations in the F1 generation pups.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Based on the results of the reproductive/developmental toxicity screening study, a NOAEL of 500 mg/kg bw/day for viability and growth of the first generation of offspring (F1) was determined. F1 generation pups were not directly given the test article, but may have been exposed in utero during gestation or via maternal milk during the lactation period. No clinical observations in the F1 generation pups were attributable to maternal doses of the test article as high as 500 mg/kg bw/day. There were no test article-related necropsy observations in the F1 generation pups.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat