Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2015 - September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is performed according to OECD/EC guidelines and in compliance with GLP principles.
Reason / purpose:
reference to same study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 1996
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, July 2000.
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 1995.
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral)) May 2008.
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 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
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
other: Crl:WI)Han
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 10-12 weeks.
- Weight at study initiation: males: 301-337 grams; females: 202-244 grams
- Fasting period before study: no
- Housing: Pre-cohabitation: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages. Cohabitation: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages. Post-cohabitation: Males were housed in their home cage (Macrolon plastic cages, with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages. Lactation: Pups were kept with the dam until termination in Macrolon plastic cages. During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied. During locomotor activity monitoring, animals were housed individually in a polycarbonate cage without cage-enrichment, bedding material, food and water.
- 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 treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 – 21.3
- Humidity (%): 41 - 73
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 July 2015 To: 22 September 2015
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
specific gravity 1.036
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and 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 substance.
Dose volume was 5 mL/kg bw/day. Actual dose volumes were calculated according to the latest body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe. Based on information provided by the sponsor.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: a maximum of 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post-coitum
- After successful mating each pregnant female was caged: Females were individually housed in Macrolon plastic cages. Pups were kept with the dam until termination in Macrolon plastic cages.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (31 July 2015; Study Day 3), according to a validated method (Project 509278). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 29-33 days, i.e. 2 weeks prior to cohabitation, during cohabitation and up to the day prior to scheduled necropsy.
Females were exposed for 40-55 days, i.e. during 2 weeks prior to cohabitation, during cohabitation, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Pups were not dosed directly but could have potentially be exposed to the test substance in utero, via maternal milk and/or from exposure to maternal urine/feces.
Frequency of treatment:
Once daily for 7 days per week.
Details on study schedule:
A maximum of 14 days will be allowed for mating, after which females who have not shown evidence of mating will be separated from their males. In case less than 9 females per group have shown evidence of mating, each non-mated female may be re-mated once with a proven male of the same group for a maximum of 7 days (if possible).
Remarks:
Doses / Concentrations:
1.5, 15, 150 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of a 10-day dose range finding study.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from treatment onwards up to the day prior to necropsy. 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 dosing and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight, with a maximum of 24 hours)
- How many animals: 5 animals/sex/group
- Parameters examined: according to guidelines

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: Yes (overnight, with a maximum of 24 hours)
- How many animals: 5 animals/sex/group
- Parameters examined: according to guidelines

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected at the end of the cohabitation period (i.e. overnight prior to scheduled necropsy of males)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: During the sampling period animals were deprived of food but water was available.
- Parameters examined: according to guidelines

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period.
- Dose groups that were examined: 5 animals/sex/group
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity.
Estrous cyclicity (parental animals):
no
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight, sperm morphology.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Following completion of the cohabitation period (a minimum of 28 days of dose administration). All surviving animals up to Day 29-33
- Maternal animals: All surviving animals up to Day 40-55 (at least 4 days of lactation)
Females which delivered Lactation Days 5-7.
Females which failed to deliver: Post-coitum Day 26 or approximately 21 days after the last day of the cohabitation period.
Females with total litter loss: Within 24 hours of litter loss.

GROSS NECROPSY
Gross necropsy according to guidelines.

HISTOPATHOLOGY / ORGAN WEIGHTS
According to guidelines.
Postmortem examinations (offspring):
SACRIFICE
Pups surviving to planned termination were euthanized by decapitation on Days 5-7 of lactation.
All pups were sexed externally and descriptions of all external abnormalities were recorded. The
stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk.
If possible, defects or cause of death were evaluated.
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 (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 (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test 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.
Reproductive indices:
Precoital time: Number of days between start of mating and confirmation of mating

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 viability 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: Number of dead pups before planned necropsy/ Number of live pups at First Litter Check) x 100

Viability index: (Number of live pups before planned necropsy/ Number of pups born alive) x 100
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined
Reproductive function: estrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
No mortality occurred during the study period. No toxicologically relevant clinical signs of toxicity were noted during the observation period. Salivation seen after dosing among all animals at 150 mg/kg bw/day was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
No toxicologically relevant changes in body weight and body weight gain were noted. Males at 150 mg/kg showed a statistically significant reduced body weight gain during the mating period. Since the mean weight gain difference to control males over the treatment period was only 4%, food intake remained normal and general health condition was normal, this was considered not to represent a toxicologically meaningful difference.
No changes in food consumption before or after correction for body weight were noted that were considered to be related to treatment. Any variations noted in food intake did not show a dose-related trend and/or were not consistently seen as treatment-duration increased.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS);
no data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS):
no test-item related effects observed

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected. Mean precoital time appeared higher at 15 and 150 mg/kg bw/day, but precoital time for individual rats remained within the expected range for rats of this age and strain, and the control mean was lower than expected (1.7 vs 2.7 days expected based on background data). Overall, variations in precoital time did not show an apparent dose-related trend, and as such this was considered unrelated to treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS):
Statisticaly significant higher liver weights were observed in males at 150 mg/kg bw/day (relative to body weights) and females (absolute and relative to body weights); mean relative weight was approximately 17 and 37% higher than the control mean for males and females, respectively.
Statistically significant lower prostate gland weight (absolute and relative) was observed at 15 and 150 mg/kg bw/day (no clear dose-related trend); mean relative weight was approximately 23 and 19% lower than the control mean at 15 and 150 mg/kg bw/day, respectively.
Statistically significant lower thymus weight was observed in high dose females (absolute and relative); mean relative weight was approximately 29% lower than the control mean.
Statistically significant higher uterus weight was observed at 15 mg/kg bw/day (relative) and 150 mg/kg bw/day (absolute and relative); mean relative weight was approximately 41 and 40% higher than the control mean at 15 and 150 mg/kg bw/day, respectively.
The remaining organ weight differences observed, including those that reached statistical significance (increased relative weight of male kidneys), were considered related to the slightly lower on terminal body weight and had no histopathological correlates. These variations were therefore considered unrelated to the administration of BMS-587319-03.

GROSS PATHOLOGY (PARENTAL ANIMALS):
There were no test-item related macroscopic findings.

HISTOPATHOLOGY (PARENTAL ANIMALS):
Test item-related microscopic findings were present in the thyroid gland of both sexes and trachea, liver, spleen and adrenal glands of females:
Thyroid glands: Hypertrophy of follicular cells was recorded at a slightly increased severity in males at 150 mg/kg bw/day and at an increased incidence and severity in females at 150 mg/kg bw/day. The incidences and severities recorded for the remaining dose groups are within background pathology for rats of this age and strain.
Trachea: Hypertrophy of the ciliated epithelium was recorded at a minimal or slight degree in 4/5 females at 150 mg/kg bw/day.
Liver: Hepatocellular hypertrophy was recorded at a minimal or slight degree in 2/5 females at 150 mg/kg bw/day.
Spleen: An increased incidence and/or severity of extramedullary hematopoiesis (up to slight at 1.5 mg/kg bw/day, up to moderate at 15 and 150 mg/kg bw/day) was present in females starting at 1.5 mg/kg bw/day.
Adrenal glands: An increased incidence and severity (up to slight degree) of hypertrophy of the zona fasciculata was recorded in females at 150 mg/kg bw/day.
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed on reproductive parameters investigated in this study up to and including 150 mg/kg bw/day, the highest dose tested.
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING):
No test-substance related effects observed.

CLINICAL SIGNS (OFFSPRING):
Incidental clinical symptoms consisted of blue spots on the head, snout and left eye for a single surviving pup. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

BODY WEIGHT (OFFSPRING):
Body weights of pups were considered to have been unaffected by treatment.

GROSS PATHOLOGY (OFFSPRING):
Incidental macroscopic findings of pups that were found dead included absence of milk in the stomach. The nature and incidence of this finding remained within the range considered normal for pups of this age, and was therefore considered to be of no toxicological relevance.
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed on developmental parameters investigated in this study up to and including 150 mg/kg bw/day, the highest dose tested.
Reproductive effects observed:
not specified
Conclusions:
No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites, spermatogenic profiling, and histopathological examination of reproductive organs). Based on these results, the NOAEL for reproduction toxicity in this study is at least 150 mg/kg bw/day, the highest dose tested.
Executive summary:

BMS-587319-03 was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 1.5, 15 and 150 mg/kg bw/day. Males were exposed for 2 weeks prior to cohabitation, during cohabitation, and up to termination (for 29-33 days). The females were exposed for 2 weeks prior to cohabitation, during cohabitation, during post-coitum, and at least 4 days of lactation (for 40-55 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Treatment up to 150 mg/kg bw/day was well-tolerated; no mortality occurred, no treatment-related changes during functional observation tests were seen, and no toxicologically relevant clinical signs or changes in body weights and food intake were noted. Test-substance related effects were observed in high dose animals and included changes in haematological parameters (Hb, RDW, counts of neutrophils, lymphocytes, reticulocytes), clinical biochemical parameters (prot, chol, Ca, Cl, ALAT), urinalysis (volume, glu, Na, pH), organ weight of liver, prostate gland, uterus, thymus (f), and microscopic findings (hypertrophy in cells of thyroid, trachea, liver, adrenals, haematopoiesis spleen). Based on the effects obeserved in high dose animals, the NOAEL for parental (systemic) toxicity for BMS-587319 -03 in this study is 15 mg/kg bw/day.

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites, spermatogenic profiling, and histopathological examination of reproductive organs). Based on these results, the NOAEL for reproduction toxicity in this study is at least 150 mg/kg bw/day, the highest dose tested.

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy). Based on these results, the NOAEL for developmental toxicity in this study is at least 150 mg/kg bw/day, the highest dose tested.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites, spermatogenic profiling, and histopathological examination of reproductive organs). Based on these results, the NOAEL for reproduction toxicity in this study is at least 150 mg/kg bw/day, the highest dose tested.

(parent, females) NOAEL = 150 mg/kg ; (parent, males) NOAEL = 150 mg/kg

Maternal effects include: liver effects. Paternal effects include: liver effects. No effects were found on mating or fertility.


Short description of key information:
One key study; oral (daily) Study of Fertility and Early Embryonic Development (rat).

Justification for selection of Effect on fertility via oral route:
One key study; oral (daily) Study of Fertility and Early Embryonic Development (rat).

Effects on developmental toxicity

Description of key information
One key study; oral (daily) Study of Fertility and Early Embryonic Development (rat)..
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2015 - September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is performed according to OECD/EC guidelines and in compliance with GLP principles.
Reason / purpose:
reference to same study
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) March 1996.
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, July 2000.
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral)) May 2008.
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 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
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
other: Crl:WI)Han
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 10-12 weeks.
- Weight at study initiation: males: 301-337 grams; females: 202-244 grams
- Fasting period before study: no
- Housing: Pre-cohabitation: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages. Cohabitation: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages. Post-cohabitation: Males were housed in their home cage (Macrolon plastic cages, with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages. Lactation: Pups were kept with the dam until termination in Macrolon plastic cages. During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied. During locomotor activity monitoring, animals were housed individually in a polycarbonate cage without cage-enrichment, bedding material, food and water.
- 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 treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 – 21.3
- Humidity (%): 41 - 73
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 July 2015 To: 22 September 2015
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
specific gravity 1.036
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and 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 substance.
Dose volume was 5 mL/kg bw/day. Actual dose volumes were calculated according to the latest body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe. Based on information provided by the sponsor.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (31 July 2015; Study Day 3), according to a validated method (Project 509278). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: a maximum of 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post-coitum
- After successful mating each pregnant female was caged: Females were individually housed in Macrolon plastic cages. Pups were kept with the dam until termination in Macrolon plastic cages.

A maximum of 14 days will be allowed for mating, after which females who have not shown evidence of mating will be separated from their males. In case less than 9 females per group have shown evidence of mating, each non-mated female may be re-mated once with a proven male of the same group for a maximum of 7 days (if possible).
Duration of treatment / exposure:
Males were exposed for 29-33 days, i.e. 2 weeks prior to cohabitation, during cohabitation and up to the day prior to scheduled necropsy.
Females were exposed for 40-55 days, i.e. during 2 weeks prior to cohabitation, during cohabitation, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Pups were not dosed directly but could have potentially be exposed to the test substance in utero, via maternal milk and/or from exposure to maternal urine/feces.
Frequency of treatment:
Once daily for 7 days per week.
Duration of test:
From two weeks prior to cohabitation up to at least 4 days of lactation.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of a 10-day dose range finding study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from treatment onwards up to the day prior to necropsy. 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 dosing and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

OTHER: haematology, clinical chemistry, urinalysis, FOB, gross pathology, organ weights, histopathology
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: Yes
- Number of late resorptions: Yes
Fetal examinations:
gestation index and duration, parturition, maternal care and early postnatal pup development (Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio, clinical signs, body weight and macroscopy)
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 (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 (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test 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.
Indices:
Precoital time: Number of days between start of mating and confirmation of mating
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
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: Number of dead pups before planned necropsy/ Number of live pups at First Litter Check) x 100
Viability index: (Number of live pups before planned necropsy/ Number of pups born alive) x 100
Details on maternal toxic effects:
Maternal toxic effects:yes
Details on maternal toxic effects:
In high dose females statistically significant changes compared to controles included higher relative neutrophil counts, lower realtive lymphocyte counts, higher relative reticulocyte count, higher red cell distribution with, and lower haemoglobin concentration.
In high dose animals statistically significant changes compared to controls included higher total protein (not statistically significant for females), higher cholesterol (not statistically significant for males), higher calcium, higher ALAT in females, lower chloride in females.
In high dose animals statistically significant changes compared to controls included higher urinary volume, higher glucose, higher sodium excretion in males, higher urinary pH in females. The lower calcium concentration in females at 1.5, 15 and 150 mg/kg bw/day showed no dose-related trend. Also, when corrected for urinary volume, calcium excretion was not statistically different to controls at 1.5 and 150 mg/kg bw/day. Also, the lower potassium concentration in males at 150 mg/kg bw/day, was not statistically different to controls when corrected for urinary volume. These variations were therefore considered not toxicologically relevant.
Statisticaly significant higher liver weights were observed in males at 150 mg/kg bw/day (relative to body weights) and females (absolute and relative to body weights); mean relative weight was approximately 17 and 37% higher than the control mean for males and females, respectively.
Statistically significant lower prostate gland weight (absolute and relative) was observed at 15 and 150 mg/kg bw/day (no clear dose-related trend); mean relative weight was approximately 23 and 19% lower than the control mean at 15 and 150 mg/kg bw/day, respectively.
Statistically significant lower thymus weight was observed in high dose females (absolute and relative); mean relative weight was approximately 29% lower than the control mean.
Statistically significant higher uterus weight was observed at 15 mg/kg bw/day (relative) and 150 mg/kg bw/day (absolute and relative); mean relative weight was approximately 41 and 40% higher than the control mean at 15 and 150 mg/kg bw/day, respectively.
Test item-related microscopic findings were present in the thyroid gland of both sexes and trachea, liver, spleen and adrenal glands of females:
Thyroid glands: Hypertrophy of follicular cells was recorded at a slightly increased severity in males at 150 mg/kg bw/day and at an increased incidence and severity in females at 150 mg/kg bw/day. The incidences and severities recorded for the remaining dose groups are within background pathology for rats of this age and strain.
Trachea: Hypertrophy of the ciliated epithelium was recorded at a minimal or slight degree in 4/5 females at 150 mg/kg bw/day.
Liver: Hepatocellular hypertrophy was recorded at a minimal or slight degree in 2/5 females at 150 mg/kg bw/day.
Spleen: An increased incidence and/or severity of extramedullary hematopoiesis (up to slight at 1.5 mg/kg bw/day, up to moderate at 15 and 150 mg/kg bw/day) was present in females starting at 1.5 mg/kg bw/day.
Adrenal glands: An increased incidence and severity (up to slight degree) of hypertrophy of the zona fasciculata was recorded in females at 150 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING):
No test-substance related effects observed. Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment.

CLINICAL SIGNS (OFFSPRING):
Incidental clinical symptoms consisted of blue spots on the head, snout and left eye for a single surviving pup. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

BODY WEIGHT (OFFSPRING):
Body weights of pups were considered to have been unaffected by treatment.

GROSS PATHOLOGY (OFFSPRING):
Incidental macroscopic findings of pups that were found dead included absence of milk in the stomach. The nature and incidence of this finding remained within the range considered normal for pups of this age, and was therefore considered to be of no toxicological relevance.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed on developmental parameters investigated in this study up to and including 150 mg/kg bw/day, the highest dose tested. Fetal
Abnormalities:
not specified
Developmental effects observed:
not specified

Accuracy of the preparations was between 100% and 103%. The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

Homogeneity coefficient of variation was 0.27 -0.75%. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Analysis of Group 2 formulations after storage yielded a relative difference of 0.6%. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours.

Conclusions:
No treatment-related changes were noted in any of the developmental parameters investigated in this study (maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy). Based on these results, the NOAEL for developmental toxicity in this study is at least 150 mg/kg bw/day, the highest dose tested.
Executive summary:

BMS-587319-03 was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 1.5, 15 and 150 mg/kg bw/day. Males were exposed for 2 weeks prior to cohabitation, during cohabitation, and up to termination (for 29-33 days). The females were exposed for 2 weeks prior to cohabitation, during cohabitation, during post-coitum, and at least 4 days of lactation (for 40-55 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Treatment up to 150 mg/kg bw/day was well-tolerated; no mortality occurred, no treatment-related changes during functional observation tests were seen, and no toxicologically relevant clinical signs or changes in body weights and food intake were noted. Test-substance related effects were observed in high dose animals and included changes in haematological parameters (Hb, RDW, counts of neutrophils, lymphocytes, reticulocytes), clinical biochemical parameters (prot, chol, Ca, Cl, ALAT), urinalysis (volume, glu, Na, pH), organ weight of liver, prostate gland, uterus, thymus (f), and microscopic findings (hypertrophy in cells of thyroid, trachea, liver, adrenals, haematopoiesis spleen). Based on the effects obeserved in high dose animals, the NOAEL for parental (systemic) toxicity for BMS-587319 -03 in this study is 15 mg/kg bw/day.

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites, spermatogenic profiling, and histopathological examination of reproductive organs). Based on these results, the NOAEL for reproduction toxicity in this study is at least 150 mg/kg bw/day, the highest dose tested.

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy). Based on these results, the NOAEL for developmental toxicity in this study is at least 150 mg/kg bw/day, the highest dose tested.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

BMS-587319-03 was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 1.5, 15 and 150 mg/kg bw/day. Males were exposed for 2 weeks prior to cohabitation, during cohabitation, and up to termination (for 29-33 days). The females were exposed for 2 weeks prior to cohabitation, during cohabitation, during post-coitum, and at least 4 days of lactation (for 40-55 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Treatment up to 150 mg/kg bw/day was well-tolerated; no mortality occurred, no treatment-related changes during functional observation tests were seen, and no toxicologically relevant clinical signs or changes in body weights and food intake were noted. Test-substance related effects were observed in high dose animals and included changes in haematological parameters (Hb, RDW, counts of neutrophils, lymphocytes, reticulocytes), clinical biochemical parameters (prot, chol, Ca, Cl, ALAT), urinalysis (volume, glu, Na, pH), organ weight of liver, prostate gland, uterus, thymus (f), and microscopic findings (hypertrophy in cells of thyroid, trachea, liver, adrenals, haematopoiesis spleen). Based on the effects obeserved in high dose animals, the NOAEL for parental (systemic) toxicity for BMS-587319 -03 in this study is 15 mg/kg bw/day.

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites, spermatogenic profiling, and histopathological examination of reproductive organs). Based on these results, the NOAEL for reproduction toxicity in this study is at least 150 mg/kg bw/day, the highest dose tested.

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy). Based on these results, the NOAEL for developmental toxicity in this study is at least 150 mg/kg bw/day, the highest dose tested.


Justification for selection of Effect on developmental toxicity: via oral route:
One key study; oral (daily) Study of Fertility and Early Embryonic Development (rat)..

Justification for classification or non-classification

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites, spermatogenic profiling, and histopathological examination of reproductive organs). Based on these results, the NOAEL for reproduction toxicity in this study is at least 150 mg/kg bw/day, the highest dose tested.

No treatment-related changes were noted in any of the developmental parameters investigated in this study (maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy). Based on these results, the NOAEL for developmental toxicity in this study is at least 150 mg/kg bw/day, the highest dose tested. There is no justification for classification for either fertility or developmental effects based upon the observations made during the study.