Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 February 2016 - 28 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Organisation of Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: other guidance as listed under Principles of method if other than guideline
Deviations:
no
Principles of method if other than guideline:
In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 2016);
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)
GLP compliance:
yes (incl. certificate)
Remarks:
d.d. 3 November 2015
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Appearance: White crystalline solid
Storage conditions: At room temperature protected from light
Specific details on test material used for the study:
Test substance handling: Amber glassware was used or container was wrapped in aluminum-foil

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Female animals: Charles River Deutschland, Sulzfeld, Germany. Male animals: Charles River Laboratories USA, Kingston.
- Age at study initiation:
Males: approximately 10-12 weeks.
Females: approximately 10-12 and 12-14 weeks for the pretest and main study, respectively.
- Weight at study initiation: 268-301 gr (males) or 201-231 gr (females)
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

ENVIRONMENTAL CONDITIONS (conditions set to maintain)
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 Februari 2016 to 28 June 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
Method of formulation: Formulations (w/w) were prepared daily and used within 5 hours after preparat ion. The formulations were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

Storage conditions of formulations: At room temperature protected from light.

Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.

Dose volume: 4 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The samples of the control group and 40 mg/kg bw/day treatment group were taken in duplicate from the middle position of the container and immediately stored on dry ice. Samples of the 4 mg/kg bw/day and 400 mg/kg bw/day treatment groups were taken in duplicate from the top, middle and bottom position of the container and immediately stored on dry ice. The samples were stored at a target temperature ≤-70°C and all samples were analyzed for BMS-587172-01.

The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Stability of formulations in the vehicle propylene glycol for at least 5 hours at room temperature protected from light (concentration range 1-200 mg/mL) was determined as part of the analytical method development and validation study.
Details on mating procedure:
- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After 14 days of unsuccessful pairing the female who had not shown evidence of mating was separated from her male.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to termination. Females that delivered were exposed for 51-64 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were exposed for 43 days (1 female) or 53 days (1 female). Two females (400 mg/kg bw/day dose level) were not dosed on one occasion as they were littering at the time of dosing.
Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Once daily for 7 d/w.
Duration of test:
Males: 29 days
Females: 51-64 days
Doses / concentrationsopen allclose all
Dose / conc.:
4 mg/kg bw/day (actual dose received)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Doses for the main study were selected based on the results from a previous single dose oral toxicity study in Sprague-Dawley rats and an oral dose range finding (DRF) study (10-17 days) in Wistar Han rats conducted with BMS-587172-01. In the single-dose oral toxicity study, no clinical signs of toxicity or macroscopic abnormalities were observed at the limit dose of 2000 mg/kg. In the dose range finding study, 17 daily oral doses of 200 and 400 mg/kg/day (n=3/female/group) were well tolerated and not associated with clear clinical evidence of toxicity. Because of a lack of toxicity observed at these doses, an additional dose
of 1000 mg/kg/day was added for 10 daily doses. At 1000 mg/kg/day, all animals exhibited clinical signs which included hunched posture, piloerection, increased salivation and rales. In addition, toxicity studies conducted with structurally similar compounds, BMS-587319-03 and BMS-512148-01, resulted in clinical toxicity at doses ≥ 200 mg/kg/day. Based on these data, the high dose of 400 mg/kg/day of BMS-587172-01 was selected for this study because it was anticipated to induce some evidence of toxicity, but within acceptable limits. The low dose of 4 mg/kg/day was selected because it was anticipated to be a no effect level, and the intermediate dose of 40 mg/kg/day was selected because it allowed for a 10 fold spacing between dose levels.

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.

Identification of pups:
On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

Culling:
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups. Selective elimination of pups, e.g. based upon body weight or anogenital distance, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.

On Day 2 of the mating period, a few females which had their mating confirmed were accidentally dosed based on their body weights from the previous day instead of their most recent body weights (day 0 post-coitum). Due to this error, 5 females across the test item-treated groups received a slightly higher dose. This deviation was not considered to have affected the study outcome as it occurred on a single day and the difference in applied dose volume was relative small (max. 1.25 % higher).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: At least twice daily (early in the morning and close to the end of the working day) for viability and mortality.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule: At least once daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals, at 1 hour (± 30 minutes) after dosing (i.e. on the peak period of anticipated effects after dosing). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT:
Yes
- Time schedule for examinations:Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1, 4, 7 and 13.

FOOD CONSUMPTION:
Yes
Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1, 4, 7 and 13 of lactation.

FOOD EFFICIENCY:
Yes
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data.

WATER CONSUMPTION :
Yes
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

GENERAL REPRODUCTION DATA
- Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.
- Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS:
No

GROSS PATHOLOGY
All animals were fasted overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.

The number of former implantation sites and corpora lutea were recorded for all paired females.

- Selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis: According to test guidelines
- All remaining animals and females which failed to deliver: According to test guidelines

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines

HISTOPATHOLOGY
According to test guidelines

Inadvertently, a few tissues were not available for histopathology. Reasons for missing a few tissues included that these tissues were not discernable at necropsy or trimming, or were erroneously not collected at necropsy. Missing tissues are listed in raw data and pathology report.
Evaluation: Sufficient data was available for adequate histopathological evaluation.

FUNCTIONAL OBSERVATIONS:
The following functional observation tests will be performed on each individual animal of the selected 5 animals/sex/group:
- hearing ability, pupillary reflex (L/R), static righting reflex;
- fore- and hind-limb grip strength (recorded as the mean of three measurements);
- locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system, total movements and ambulations are reported).
The selected females were tested once during the last week of lactation (e.g. PND 6-13).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities, anogenital distance and areola/nipple retention.
- Mortality: The numbers of live and dead pups on day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1,4 ,7 and 13 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation.
- Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- Areola/nipple retention: On PND 13, all males in each litter were examined for the number of areola/nipples.

GROSS EXAMINATION OF DEAD PUPS: Yes
If possible, defects or cause of death were evaluated.

SACRIFICE
Pups, younger than 7 days were killed by decapitation.
On PND 4 (at culling), from 2 pups per litter blood samples (0.4 mL in total) were collected by decapitation between 7.00 and 10.30 a.m.. Blood samples from the 2 pups per litter were collected into one serum tube.
On PND 13-15, from 2 pups per litter (if possible from one male and one female) blood samples were collected at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane, between 7.00 and 10.30 a.m., followed by exsanguination.
All remaining pups (PND 7-15) were sacrificed.

GROSS NECROPSY
All pups were sexed by both external as well as internal examination. Descriptions of all abnor malities were recorded. Particular attention was paid to the external reproductive genitals to exam ine signs of altered development. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. At terminal sacrifice (PND 13-15), the thyroid from 1 male and 1 female pup per litter was preserved in 10% buffered formalin.

THYROID HORMONE ANALYSIS:
- PND13-15 pups
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination between 7.00 and 10.30 a.m..
- How many animals: From 2 pups per litter (if possible from one male and one female)
- Parameters checked were: According to test guidelines
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 1; many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 3) was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test (Ref. 4) was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Indices:
Reproductive indices; For each group, the following calculations were performed:
- Mating index: (Number of females mated/Number of females paired) x 100
- Fertility index: (Number of pregnant females/Number of females paired) x 100
- Conception index: (Number of pregnant females/Number of females mated) x 100
- Gestation index: (Number of females bearing live pups/Number of pregnant females) x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring indices:
- Percentage live males at First Litter Check: (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check: (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage of postnatal loss Days 0-4 of lactation: (Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check) x 100
- Post-implantation survival index (%): (Total number of offspring born/Total number of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% when the number of offspring exceeded the number of implantation sites recorded.
- Live birth index (%): (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
- Viability index (%): (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100
- Lactation index (%): (Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling)) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Incidental findings that were noted included rales, scabbing, wounds and alopecia. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. As they were observed in the absence of a treatment related distribution and/or were seen for individual animals only, they were not considered to be test item related.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a trend towards higher food consumption (absolute) of females in all test item treated groups when compared to control animals. However, no clear dose relation could be established. Changes reached statistical significance for females in all dose levels during almost the entire post-coitum period, and for 400 mg/kg bw/day females during lactation days 4-7.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
There was a trend towards higher relative food consumption of females in all test item treated groups when compared to control animals. However, no clear dose relation could be established. Changes reached statistical significance for females in all dose levels during almost the entire post-coitum period.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Statistical significant higher urea levels were recorded for females at 400 mg/kg/day (15.1 mmol/L) when compared to their concurrent controls and the mean value was at the higher end of the historical range. In the absence of any correlating changes on organ level, these changes were not considered as adverse.
Any remaining statistically significant changes were considered to be chance findings as no dose related trend could be established and/or values remained within the range considered normal for rats of this age and strain. Furthermore, no correlating changes on organ level were noted.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The live birth index, viability index and lactation index were considered not to be affected by treatment.
Two control pups and one and three pups in respectively the 40 and 400 mg/kg/day groups were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
In the 40 mg/kg/day group, three pups within one litter were found dead on lactation days 2 ,4 and 13, respectively. No other pups were found dead/missing in the period between first litter check on day 1 and lactation day 13.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of premature birth. No deficiencies in maternal care were observed.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No toxicity was observed up to and including the highest dose level tested.

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The live birth index was considered not to be affected by treatment.
Two control pups and one and three pups in respectively the 40 and 400 mg/kg bw/day groups were found dead at first litter check. No toxicological relevance was attributed to these dead pups since
the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The viability index and lactation index were considered not to be affected by treatment.
In the 40 mg/kg bw/day group, three pups within one litter were found dead on lactation days 2 ,4 and 13, respectively. No other pups were found dead/missing in the period between first litter check on day 1 and lactation day 13.
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment up to 400 mg/kg bw/day. The median anogenital distance of male pups at 4 mg/kg bw/day was statistically significantly lower when compared to the concurrent control group but this decrease occurred without a dose related trend.
Treatment up to 400 mg/kg bw/day had no effect on areola/nipple retention. Nipples were not detected for any of the examined male pups on PND 13.

Serum T4 levels in male and female pups on PND 13-15 were considered not to be affected by treatment up to 400 mg/kg bw/day. The mean value for the 40 mg/kg bw/day group was statistically significantly higher when compared to the concurrent control group but this increase occurred without a dose related trend.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicity was observed up to and including the highest dose level tested.

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In an oral OECD 422 screening study, the NOAEL parental and NOAEL for reproduction were derived to be at least 400 mg/kg bw/day based on the absence of adverse effects up to and including the highest dose level tested.