1H-Pyrazolo[3,4-c]pyridine-3-carboxylic acid, 4,5,6,7-tetrahydro-1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-, ethyl ester

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Sprague-Dawley CD® rats (10 animals/sex/group) were dosed by oral gavage once daily with 0 (1% w/v Methylcellulose), 10, 100 or 1000 mg/kg/day of BMS-589152-01. The male animals were dosed during the pre-cohabitation, cohabitation and post-mating periods (approximately 35 days) and then euthanized and necropsied. The female animals were dosed during the precohabitation and cohabitation periods and during gestation and lactation (through Lactation Day [LD] 13, approximately 70 days) and then euthanized and necropsied. The dose volume was 5 mL/kg/day for all dose groups. Parameters evaluated during the study for the F0 animals were: viability, clinical observations, body weights, food consumption, estrous cycling, mating and fertility, parturition and littering, functional assessments, clinical pathology (at termination) and thyroid hormone analysis (at termination), organ weights, macroscopic observations and microscopic pathology. Parameters evaluated for the F1 pups were: viability, clinical observations, sex, body weights, ano-genital distance, nipple retention, thyroid hormone analysis (at Postnatal Days [PND} 4 and 13), thyroid weights, macroscopic observations and microscopic pathology (thyroids).

An additional 5 animals/sex in Groups 1 and 4 were dosed by oral gavage once daily for 35 days with 0 (1% w/v Methylcellulose) or 1000 mg/kg/day BMS-589152-01. At the end of the treatment period, these animals were held for a 14 day treatment-free recovery period and then euthanized and necropsied. Parameters evaluated during the study were: viability, clinical observations, body weights, food consumption, clinical pathology (hematology and clinical chemistry at end of recovery; coagulation at end of dosing and end of recovery), organ weights, macroscopic and microscopic observations.

The study was performed in compliance with OECD 422 guideline for testing of chemicals adopted 28.07.15: Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test. There was a BMS-589152-01-related effect on the Viability index (%) for Day 4 at 1000 mg/kg/day (-9.6%). There were four unscheduled P0 decedents (i.e., one male and two females at 1000 mg/kg/day and one female at 10 mg/kg/day) over the course of this study. Due to the absence and/or nature of the clinical signs and pathology findings, the causes of these deaths could not be unequivocally attributed to treatment with BMS-589152-01. There were no clinical signs associated with the treatment of BMS-589152-01 in males and females that survived until scheduled termination. There were no body weight or food consumption differences, organ weight, clinical pathology or thyroid hormone differences, macroscopic or microscopic changes attributed to BMS-589152-01.

There were no BMS-589152-01-related adverse effects on estrous cycling, mating and fertility indices, delivery and litter size or on litter observations. There were no BMS-589152-01-related effects on pup sex, mean body weights, anogenital distance, nipple retention, functional assessments, or macroscopic changes attributed to BMS-589152-01. With all findings considered, the male and female systemic NOAEL was 1000 mg/kg/day and the reproductive NOAEL was 100 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:

The study was conducted between 16 May 2016 and 06 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The purpose of this study was to assess possible effects associated with repeat exposures to the test item over a relatively limited period of time as well as to assess any potential effects on reproductive performance, in male and female rats when the test chemical is administered by oral gavage. The study was expected to permit detection of effects on gonadal function, mating behavior, conception, development of the conceptus, parturition and pup survival to PND 13. However, it did not provide complete information on all aspects of reproduction and development and did not allow for definite claims of no reproductive/developmental effects.

Specific details on test material used for the study:

Description: Off-white to yellow solid
Storage conditions: Room temperature and protected from light (amber jars)
Batch/Lot number: AAG8999N/15600T0001
Purity: 100.1%
Retest date: 27 December 2017

Species:

rat

Strain:

other: Albino Rats (Outbred) VAF/Plus CD (Sprague-Dawley derived) [Crl:CD (SD)BR]

Details on species / strain selection:

The OECD 422 Test Guideline is designed for use with the rat. Exposure studies in a rodent species are required by the chemical regulatory agencies. In addition, a historical data base is available for comparative evaluation.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Supplier: Charles River Laboratories, Raleigh, North Carolina 27610
Age of the animals at start of treatment: Approximately 9 to 10 weeks
Weight range at start of treatment Males: 308 g to 402 g
Females: 193 g to 252 g

Pretest Period
The pretest period was approximately 2 weeks; all animals were checked for viability twice daily. For males, pretest procedures, other than routine husbandry care and identification procedures, were not performed until after the animals were stabilized for 8 days. Prior to assignment to study, all male animals were examined to ascertain suitability for study.
For females, routine husbandry care, identification procedures and vaginal lavage for estrous evaluation commenced on the day after arrival. All other procedures were not performed until after the female animals were stabilized for at least 5 days. Prior to assignment to study, all female animals were examined to ascertain suitability for study.

Animal Care and Husbandry
Facilities Management/Animal Husbandry
Currently acceptable practices of good animal husbandry were followed e.g., Guide for the Care and Use of Laboratory Animals; National Academies Press, 2011. Envigo, East Millstone, New Jersey is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

Veterinary Care
Animals were monitored by the technical staff for any conditions requiring possible veterinary care.

Environmental Control
Light/dark cycle: A twelve hour light/dark cycle was provided and controlled via an automatic timer.
Temperature and relative humidity: Monitored and maintained within the range of 19 to 25 °C and 30 to 70%.
There were no deviations from these ranges.

Animal Housing and Environmental Enrichment
Cages:Polycarbonate cages with a stainless steel mesh lid.
Number of animals per cage: From arrival until one day prior to treatment, animals were pair or group housed (2 or 3 rats of the same sex per cage, respectively) in solid bottom cages with cellulose-based contact bedding.
From the initiation of treatment (pre-cohabitation), due to the odd number of animals per offset (n=5), one main study F0 male was individually housed to avoid invalidating food consumption data. The remaining F0 males for each offset were pair-housed [same sex and treatment group per cage (except during the cohabitation phase)]. All F0 males were housed in suspended, solid bottom cages with cellulose-based contact bedding.
From the initiation of treatment (pre-cohabitation phase), the main study F0 animals were pair housed [same sex and treatment group per cage] in suspended, solid bottom cages with cellulose-based contact bedding. During the cohabitation period, one main study male and one main study female from the same group were co-housed (1:1 in the male’s cage) until evidence of mating was seen or 14 consecutive days of cohabitation had elapsed. Per the OECD 422 guideline, the main study F0 females were individually housed during presumed gestation and housed with her litter after delivery.
From the initiation of treatment, due to the odd number of animals in the recovery group, one male and one female from each dose group were individually housed to avoid invalidating food consumption data. All remaining recovery animals were pair-housed with an animal of the same sex and dose group.
Bedding Teklad 7070C Certified Diamond Dry Cellulose Bedding
Envigo, Madison, Wisconsin
Provided to each cage throughout the study and changed at appropriate intervals each week.
Analytical results of the bedding, provided by the manufacturer, are maintained on file at the Testing Facility. There were no known contaminants in the bedding that were expected to interfere with the objectives of this study.
Environmental enrichment Provided to each cage throughout the study and replaced when necessary.

Feed
Diet: Teklad Global 16% Protein Rodent Diet (Certified), 2016C, Envigo, Madison, Wisconsin
Availability: Without restriction (removed overnight before blood sampling for clinical pathology). Fresh feed was presented weekly.
Analysis: Analysis of each feed lot used during this study was performed by the manufacturer. Results were provided to the Testing Facility and are maintained on file at the Testing Facility. There were no known contaminants in the feed that were expected to interfere with the results of this study.

Water
Supply: Potable water from the public supply (New Jersey-American Water Company, Cherry Hill, New Jersey) via an automated watering system.
Availability: Without restriction.
Analysis: Water analyses are conducted by New Jersey-American Water Company, Cherry Hill, New Jersey (Raritan-Millstone Plant) to ensure that water meets standards specified under the EPA Federal Safe Drinking Water Act Regulations (40 CFR Part 141). In addition, water samples are collected biannually from representative rooms in the Testing Facility; chemical and microbiological water analyses are conducted on these samples by a subcontract laboratory. Results of all water analyses are maintained on file at the Testing Facility. There were no known contaminants in the water which were expected to interfere with the results of this study.

Identification of animals
Upon arrival of the first 5 animals/sex/group, each animal was identified by tail mark on the day of arrival and implanted 3 days later with a BMDS IMI-1000 Implantable Radio Frequency Transponder (microchip) programmed with a unique number. Upon arrival of the second 5 animals/sex/group, each animal was implanted with a transponder (microchip) programmed with a unique number. This number was cross referenced with an animal number assigned by the Testing Facility; this number plus the study number comprised a unique identification number for each animal. In addition, each cage was provided with a cage card that was color-coded for dose level identification and contained study number and facility-assigned animal number information.
Each pup within a litter was assigned an identification number on PND 1 that was tattooed on the ventral surfaces of the paws prior to the first body weight. This number, the dam number, and the study number comprised the unique animal number for each pup and was included on the cage cards.

Route of administration:

oral: gavage

Vehicle:

other: 1% w/v Methylcellulose (USP) in distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
BMS-589152-01 was prepared for administration by mixing with the control item (vehicle) to achieve the desired concentrations.
The test chemical was used as supplied when calculating quantities to be used during dose preparation. Fresh formulations were prepared once weekly and stored refrigerated (2 – 8 °C) when not in use.

Volume dose : 5 mL/kg body weight.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Control (Group 1): Vehicle at the same volume dose as the treated groups.

Formulation: Dose formulations were stirred for at least 30 minutes prior to dosing and throughout the dosing period.
A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.

Details on mating procedure:

There was a pre-cohabitation period of 2 weeks. During the cohabitation period, one main study male and one main study female from the same group were cohoused (1:1 in the male’s cage) until evidence of mating was seen
or 14 consecutive days of cohabitation had elapsed. Female rats were observed each morning for the presence of a vaginal plug or sperm in the vaginal smear. If not mated, the stage of the estrous cycle was recorded. The day on which evidence of mating was observed was defined as Day 0 of presumed gestation (GD 0). Once mated, the female rat was removed from the mating cage and housed individually until delivery.
Per the OECD 422 guideline, the main study F0 females were individually housed during presumed gestation and housed with her litter after delivery.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of Dose Formulations
Homogeneity
Three samples each from the top, middle and bottom portion of the formulations for the low- and high-concentrations prepared for the first week were taken for analysis.
Stability
Stability under storage conditions used in this study was determined from the dose formulations prepared using the proposed preparation procedure at 24 hour (room temperature) and 9 days ± 1 day (refrigerated) from preparation. Stability was performed under the method validation study TT51SK.
Dose Concentration
Four samples (direct sampling and volume as per analytical method) were taken from the middle region of each formulation (including the control) prepared for the first, third and last week of the study. Two samples were analyzed for dose confirmation analysis and two samples were retained refrigerated (2 to 8°C). Retained samples were discarded after valid analytical results were obtained.
Sample Storage Conditions
Dose formulation samples were maintained refrigerated (2 to 8 °C) until analysis.
Method of Analysis
Concentration of BMS-589152-01 in formation, including controls, was determined by HPLC-UV (FIA Method No FIA 013-16). Analyses were performed by the Department of Formulation and Inhalation Analysis at the Testing Facility.

Duration of treatment / exposure:

The duration of exposure (males – pre-cohabitation up to 2 weeks and continuing until the day prior to necropsy [a minimum of 35 consecutive days]; Females - 2 weeks pre-cohabitation, cohabitation up to 3 weeks, 22 days of gestation and continuing until LD 13 [approximately 70 consecutive days]) was intended to achieve adequate exposure over the dosing period, to mimic potential occupational exposure. Recovery males and females were treated for 35 consecutive days followed by a 14-day test chemical-free recovery phase per OECD 422 guideline recommendation.

Frequency of treatment:

daily

Details on study schedule:

Pretest period: 2 weeks
Pre-cohabitation period (treatment intiated): 2 weeks
Co-habitation period: Up to 14 days
The main study F0 males were dosed once daily.
The main study F0 females were dosed for 2 weeks precohabitation, up to 2 weeks during cohabitation and continuing until LD 13, approximately 60 days.
The recovery study male and female animals were treated for 35 consecutive days followed by a 14-day test chemical-free recovery period.

Scheduled Necropsy
Necropsy was performed on up to 10 main study F0 males/group after males had been treated for 5 weeks. Necropsy schedules were established to ensure that approximately equal numbers of males from each group were examined at similar times of the day.
Necropsy was performed on up to 10 main study F0 females/group.
Female rats who failed to deliver a litter were euthanized on GD 26. Female rats with total litter loss (all pups died prior to scheduled sacrifice) were euthanized on that day. For apparently non-pregnant F0 females, the numbers of uterine implantation sites were checked after staining with ammonium sulfide [modification of the Salewski staining technique (Salewski, 1964)].
Necropsy was performed on up to 5 recovery study animals/sex/Groups 1 and 4 after animals had been treated for 35 days followed by a 14-day recovery period.
Selected F1 pups (2 females/litter) for Thyroid Hormone Blood Collection) were euthanized on PND 4 and discarded without further evaluation.
Surviving F1 pups were euthanized on PND 13.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses for this study were selected based on the results from a previous oral single dose and 14 day dose range-finding studies (BMS Study No. BMY 657/024062/AC and Envigo Study No. NF79LD) conducted in rats with BMS-589152-01. In the single-dose oral toxicity study, no clinical signs of toxicity or macroscopic abnormalities occurred at the limit dose of 2000 mg/kg. In the dose range finding study, 14 daily oral doses of 100, 500 or 1000 mg/kg/day (n=6 females and 6 males / group) were well tolerated and not associated with clear clinical evidence of toxicity. In addition, toxicity studies conducted with a structurally similar compound, BMS-562247-01 revealed increased prothrombin time (PT) and activated partial thromboplastin time (aPTT) at doses as low as 25 mg/kg/day, although similar changes did not occur with BMS-589152-01. Based on these data, the limit dose of 1000 mg/kg of BMS-589152-01 was selected as the high dose for this study because it is anticipated to induce some evidence of toxicity without mortality. The low dose of 10 mg/kg was selected because it is anticipated to be a no effect level, and the intermediate dose of 100 was selected because it allows for a 10 fold spacing between dose levels.

- Rationale for animal assignment (if not random): More animals than required for the study were purchased and stabilized. Animals considered suitable for study were distributed into 2 groups (Groups 1 and 4) of 15 animals per sex and 2 groups (Groups 2 and 3) of 10 animals per sex by a computerized random sort program so that body weight means for each group were comparable. Only females with regular estrous cycles (4 to 5 days of cyclicity) were included for randomization to study groups. Individual weights of animals placed on test were within ±20% of the mean weight. Information as to the disposition of all animals not utilized in the study is maintained in the study file.

- Rationale for selecting satellite groups: To determine reversibility of potential effects of the test chemical, a satellite group of recovery animals for Groups 1 and 4 (the highest group administered) was included in this study. Per the OECD 422 guideline, 5 animals per sex for Groups 1 and 4 were sufficient to assess for reversibility, persistence or delayed occurrence of systemic toxicologic effects, following a 14 day test chemical-free phase.

- Number of animals: The number of animals in this study was considered the minimum necessary to allow for meaningful interpretation of the data, as required by the OECD 422 guideline. Eight (8) pregnancies per group were considered the minimum acceptable number for screening for reproductive and developmental toxicities. The group size of 10 males and 10 females used in this study, retaining a one male to one female pairing ratio, with expected pregnancy rates of 80-90%, was anticipated to provide at least 8-10 pregnancies per group for evaluation. Females were evaluated pre-exposure for estrous cyclicity and animals that fail to exhibit typical 4-5 day cycles were not included in the study; therefore, extra females (n=3 per group) were recommended by OECD in order to yield at least 10 regularly cycling females per group. Three test chemical-treated groups was considered the minimum number of groups necessary to provide a range of effects.

Parental animals: Observations and examinations:

Daily Observations
Animals were observed in their cages at least twice daily for mortality and general condition. Animals in extremely poor health or in a possible moribund condition were identified for further monitoring and possible euthanasia.

Clinical Signs
During the treatment period, animals were observed in their cages for signs of toxic or pharmacologic effects at least once daily, 3 ± 1 hours post dose.

Physical Examination
Main study F0 male rats were observed approximately 1 week prior to initiation of dosing and once weekly during the treatment period until termination. Main study F0 female rats were observed approximately one week prior to initiation of dosing,
once weekly during the pre-cohabitation period and daily during cohabitation. Once mating was confirmed, animals were observed on GDs 0, 7, 14 and 20 and LDs 1, 4, 7 and 13. Maternal behavior was observed daily. Recovery study male and female rats were observed once approximately one week prior to the initiation of dosing and once weekly during the treatment and recovery periods until termination. Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as an evaluation of respiration.

Body Weight
Non-fasted body weights of the main study F0 male rats were recorded approximately one week prior to the initiation of dosing and once weekly during the pre-cohabitation and cohabitation periods. Terminal, fasted body weights were recorded just prior to necropsy. Non-fasted body weights of the main study F0 female rats were recorded from the initiation of dosing and weekly during pre-cohabitation and cohabitation phases until mating. Mated female rats were weighed on GDs 0, 7, 14 and 20 and female rats that delivered litters were weighed on LDs 1, 4, 7 and 13. Terminal, fasted body weights were recorded just prior to necropsy. Non-fasted body weights of the recovery study rats were recorded approximately one week prior to initiation of dosing and once weekly during the treatment and recovery periods. Terminal fasted body weights were recorded just prior to necropsy.

Food Consumption
Food consumption for the main study F0 male and female rats was measured (weighed) during the week prior to the initiation of dosing and weekly during the pre-cohabitation phase. Food consumption was not measured during the cohabitation phase when main study male rats were co-housed with main study female rats. Food consumption for the main study male rats was measured weekly post-cohabitation phase. Food consumption for the mated main study female rats was measured on GDs 0-7, 7-14 and 14-20 and for the females that delivered litters on LDs 1-7 and 7-13. Food consumption for the recovery study male and female rats was measured during the week prior to initiation of dosing, on the day dosing initiated and weekly throughout the treatment and recovery periods.

Parturition and Lactation
On GD 18, several days prior to expected parturition, examination for signs of parturition was performed 3 times daily (morning, mid-day, and afternoon). Whenever possible a female that was in the process of delivery was not disturbed. Dosing and other activities (e.g., detailed physical observations and body weights) were not performed during parturition. The day on which parturition was observed as completed was defined as LD 1 (dams) = PND 1 (pups).

Functional Assessments – F0 Animals
Sensory Reactivity
Sensory reactivity evaluations (visual approach, audition assessment, pinna reflex, proprioception, pain perception, pupil response and air righting) were performed according to the Testing Facility’s SOPs once prior to dosing early in Week 5 for the first five main study F0 males in each group and on LD 8 ± 1 for five main study F0 females in each group.

Grip Strength
Forelimb and hindlimb grip strength evaluations were performed using a Chatillon Digital Force Gauge (Model E-DFE-010) and accompanying Columbus Instruments Grip Strength Platform according to the Testing Facility’s SOPs once early in Week 5 for the first five main study F0 males in each group and on LD 8 ± 1 for five main study F0 females in each group.

Locomotor Activity
Spontaneous exploratory activity was measured once in early Week 5 for the first five main study F0 males in each group and on LD 8 ± 1 for five main study F0 females in each group. Each animal was placed in a separate shoebox cage in an automated Motor Monitor System (Kinder Scientific). Activity was monitored during a 60-minute session composed of 12 5-minute intervals. The total number of horizontal and vertical photo-beam breaks that occurred during each of the 5-minute intervals was recorded.

Open Field
Evaluations for abnormalities of posture (post) or gait and for any abnormal behavior or vocalization were performed according to the Testing Facility SOPs once in early Week 5 for the second five main study F0 males in each group and once on LD 8 ± 1 the second five main study F0 females in each group.

Functional assessments and Scoring
Evaluations were performed according to the Testing Facility’s Standard Operating Procedures that include defined scales. Behaviors not adequately described by the defined scales were further characterized by descriptive comments. Testing proceeded from the least to the most interactive with the subject.

Clinical Pathology – F0 Adults
Blood for evaluation hematology, coagulation and clinical chemistry parameters was obtained at termination from anesthetized (isoflurane) main study F0 animals (up to 5 animals/sex/group) via the aorta as a terminal procedure (animals were not allowed to recover from anesthesia). Animals were fasted overnight prior to blood collection. Blood for evaluation of coagulation parameters was obtained from unanesthetized recovery study animals (up to 5 animals/sex/group) via the jugular vein at termination of dosing. Blood for evaluation of hematology, coagulation and clinical chemistry parameters was obtained at terminations of recovery from anesthetized (isoflurane) recovery study animals up to 5 animals/sex/group) via the aorta as a terminal procedure (animals were not allowed to recover from anesthesia). Animals were fasted overnight prior to each blood collection interval. For the terminal collections, blood was obtained from anesthetized (isoflurane) F0 adult animals (the first up to 5 animals/sex/group at termination and from the remaining up to 5 animals/sex/group), via the aorta, as a terminal procedure (animals were not allowed to recover from anesthesia and necropsied). Animals were fasted overnight prior to blood collection.

Clinical Chemistry
Blood samples (approximately 1.0 mL) were collected into tubes with no anticoagulant, allowed to clot, centrifuged to obtain serum and analyzed for the following using a Siemens ADVIA 1800 Chemistry Analyzer:
Aspartate aminotransferase (AST) (Kinetic - Modified Bergmeyer)
Alanine aminotransferase (ALT) (Kinetic - Modified Bergmeyer)
Alkaline phosphatase (ALKP) (Kinetic – Tietz AMP Buffer)
Blood urea nitrogen (BUN) (Enzymatic – Roch-Ramek with Urease)
Creatinine (CREAT) (Jaffe Picric Acid in Alkaline Medium)
Glucose (GLU) (Glucose Hexokinase II Method)
Cholesterol (CHOL) (Enzymatic esterase/oxidase – Trinder Endpoint)
Triglycerides (TRIG) (Fossati Three Step Enzymatic – Trinder Endpoint)
Total protein (TP) (Biuret Technique)
Albumin (ALB) (Bromocresol Green Method)
Total bilirubin (TBILI) (Oxidation with Vandate)
Sodium (NA+) (Ion Selective Electrode)
Potassium (K+) (Ion Selective Electrode)
Chloride (Cl-) (Ion Selective Electrode)
Calcium (Ca++) (Michaylova & Ilkova, Arsenazo III)
Inorganic phosphorus (PHOS) (Phosphomolybdate - UV Method)

Hematology
Blood samples (approximately 0.25 mL) were collected into tubes containing K2EDTA anticoagulant and analyzed for the following using a Siemens ADVIA 120 Hematology Analyzer:
Hemoglobin (HGB)
Hematocrit (HCT)
Red blood cell count (RBC)
Platelet count (PLT)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Red cell distribution width (RDW)
Total white blood cell count (WBC)
Reticulocyte count (RETIC)
Differential white blood cell count
Neutrophils (ANEU)
Lymphocytes (ALYM)
Eosinophils (AEOS)
Basophils (ABASO)
Monocytes (AMONO)
Large unstained cells (ALUC)
A peripheral blood smear was prepared for each animal at each blood collection interval and was available for confirmation of automated results and/or other evaluations deemed necessary by the Clinical Pathologist.

Coagulation
Blood samples (approximately 1.0 mL) were collected into tubes containing sodium citrate anticoagulant and analyzed for the following using a Diagnostica Stago Products STA Compact mechanical clot detection system:
Prothrombin time (PT)
Activated partial thromboplastin time (APTT)

Oestrous cyclicity (parental animals):

During the pre-cohabitation and cohabitation phases, vaginal smears were obtained daily from each main study F0 female and the stage of estrous was determined. In addition, a vaginal smear was collected from each main study F0 female on the day of scheduled termination and evaluated for stage of estrous cycle.

Sperm parameters (parental animals):

During the microscopic examination of the testes, special emphasis was placed on the stage of spermatogenesis and the interstitial testicular cell structure. Any cell- or stage-specificity of testicular finds was noted.

Litter observations:

Litters were observed as soon as possible after parturition completion for the number of live and dead pups, runts and pup abnormalities and the sex of each pup was determined. Thereafter, litters were observed once daily and had viability checks performed twice daily. All pups in the litter were uniquely identified by tattoo after parturition completion. The full number of pups remained in each litter, the litters were not culled. The presence of dead pups was recorded and these were removed from the litter as found and necropsied.

Physical Examinations
Each pup was given a physical examination (including observation for any abnormal behavior) on PNDs 1, 4, 7 and 13.

Body Weight and Sexing
Individual pup body weights data were recorded on PNDs 1, 4, 7 and 13. The sex of each pup was identified on PND 1 and verified on PNDs 4 and 7.

Anogenital Distance and Nipple Retention
Anogenital distance was recorded on PND 4 for all F1 pups. Nipple retention was assessed for all F1 male pups on PND 12 or 13.

Postmortem examinations (parental animals):

Adult males and females were euthanized by exsanguination following isoflurane inhalation.

Macroscopic postmortem examinations were performed on all main study F0 rats, including unscheduled decedents. Postmortem examinations included examination of the external surface, all orifices, cranial cavity, nasal cavity (external examination), neck and its associated tissues and organs, thoracic, abdominal and pelvic cavities and their associated tissues and organs, and external surfaces of the brain. Special attention was paid to the organs of the reproductive system. The number of implantation sites or scars and corpora lutea were recorded for reach female rat. Corresponding tissues from several control animals were saved for comparative purposes when gross lesions were found. Macroscopic postmortem examinations were performed on recovery study rats. Postmortem examinations included examination of the external surface, all orifices, cranial cavity, nasal cavity (external examination), neck and its associated tissues and organs, thoracic, abdominal and pelvic cavities and their associated tissues and organs, and external surfaces of the brain. Special attention was paid to the organs of the reproductive system. The number of implantation sites or scars and corpora lutea were recorded for each female rat.

Organ Weights
Organs indicated in Table 1 (see section 'any other information on materials and methods') were weighed for the first 5 main study F0 animals/sex/group. The organs listed in Text Table 2 were weighed for the second 5 main study F0 animals/sex/group at the scheduled necropsy interval. Prior to weighing, the organs were carefully dissected and properly trimmed to remove adipose and other contiguous tissues in a uniform manner. Organs were weighed as soon as possible after dissection in order to avoid drying. Paired reproductive organs were weighed separately.

Postmortem examinations (offspring):

Pups were euthanized using an intraperitoneal injection of sodium pentobarbital.

Macroscopic post-mortem examinations (external only) were performed on all surviving F1 pups on PND 13. Postmortem examinations included examination of external surface, all orifices, cranial cavity, nasal cavity (external examination), neck and its associated tissues and organs, thoracic, abdominal and pelvic cavities and their associated tissues and organs, and external surfaces of the brain. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. Gross abnormalities, unusual observations and/or the absence of milk in the stomach were noted and then the carcasses were discarded. F1 pups that died during the study were given a macroscopic postmortem examination and gross lesions were saved and preserved in 10% neutral buffered formalin. Particular attention was paid to the external reproduction genitals which were examined for signs of altered development. Unusual observations and the absence of milk in the stomach were noted and the carcasses were discarded.

Organ Weights
The thyroid gland was weighed for 1 male and 1 female F1 pup/litter at the scheduled necropsy interval.

Statistics:

Statistical analysis was applied where there was an indication of possible meaningful differences. All statistical analyses were performed using the individual animal (or litter) as the basic experimental unit. For litter findings, the litter was the treated experimental unit and the basis for statistical analysis and biological significance were assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
Significant differences between control and treated groups were expressed at the 5% (p<0.05), 1% (p<0.01) or the 0.1% (p<0.001) level. The key to annotations used on the tables that contain statistical results is given below.
* p<0.05
** p<0.01
*** p<0.001

Reproductive indices:

Pre-implantation Loss (%):
Number of corpora lutea - Number of implantation sites/Number of corpora lutea x 100
Post-implantation Loss (%):
([Number of implantations – Number of live-born pups] / Number of implantations) x 100
Post-implantation Survival Index (%):
Number of live-born pups/Number of implantation sites x 100
Sex Ratio (% Males)
Number of male pups/number of total pups x 100
Female Mating Index (%):
Number of females with confirmed mating (sperm and/or vaginal plug) + Number of pregnant females without evidence of mating (no sperm or vaginal plug)/Number of females placed with males x 100
Female Fertility Index (%):
Number pregnant/Number copulated x 100
Male Mating Index (%):
Number of males with confirmed mating with a female or pregnancy for females without evidence of mating/Number of males placed with females x 100
Male Fertility Index (%):
Number of males mating and impregnating a female + Number of males with a pregnant female without evidence of mating/Number of males with confirmed mating + number of males with a pregnant female without evidence of mating x 100

Offspring viability indices:

Viability Index (%):
PND 4: Number of pups alive on PND 4/Number of live pups on PND 1 x 100
PND 7 and PND 13: Number of pups alive on PND 7 or PND 13/(Number of pups alive on PND 4 – pups selected for thyroid hormone analysis) x 100
Live Birth Index (%):
Total number of live-born pups/total number of pups born x 100

Clinical signs:

no effects observed

Description (incidence and severity):

Main Study
There were no clinical signs associated with treatment with BMS-589152-01 in males and females that survived until scheduled termination. Any clinical signs noted were considered common to this laboratory animal species and/or occurred sporadically, and thus were not attributed to treatment with BMS-589152-01.

Recovery Phase
There were no clinical signs associated with treatment with BMS-589152-01 in males and females that survived until scheduled termination. Any clinical signs noted were considered common to this laboratory animal species and/or occurred sporadically, thus not attributed to treatment with BMS-589152-01.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male and three females were electively euthanized over the course of this study. Due to the absence and/or nature of the clinical signs and pathology findings, these deaths were not considered to be related to treatment with BMS-589152-01. Animal No. 4101 was euthanized on Day 4 of the dosing phase due to respiratory distress. The macroscopic evaluation revealed discolored red lungs which correlated to minimal hemorrhage histologically, and a discolored liver which did not have a microscopic correlation. Animal Nos. 2538 and 4578 failed to deliver litters by GD 25 so were electively euthanized on GD 26 where non-pregnancy status was confirmed. The only macroscopic observation was implantation scars for Animal No. 4578, whereas Animal No. 2538 was considered to be within normal limits.
Animal No. 4571 experienced a total litter loss and was electively euthanized on LD3.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Main Study
Males: There were no BMS-589152-01-related effects on the mean body weights or body weight change of males during the pre-cohabitation, cohabitation or post-cohabitation phases of the study. Any differences from the control were not considered to be BMS-589152-01-related due to their direction, small magnitude, infrequent occurrence and/or lack of relation to dose.

Females: There were no BMS-589152-01-related effects on the mean body weights of females during the pre-cohabitation, cohabitation, gestation or lactation phases of the study. Any differences from the controls were not considered to be BMS-589152-01-related due to their direction, small magnitude, infrequent occurrence and/or lack of relation to dose.

Recovery Phase
There were no BMS-589152-01-related effects on the mean body weights or body weight change in treated males and females. Any differences from controls were not considered to be BMS-589152-01-related due to their direction, small magnitude, infrequent occurrence and/or lack of relation to dose.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Main Study
Males: There were no BMS-589152-01-related effects on food consumption for the males during the pre-cohabitation or post-cohabitation phases of the study.
Females: There were no BMS-589152-01-related effects on food consumption for the females during the pre-cohabitation, gestation or lactation phases of the study.

Recovery Phase
There were no BMS-589152-01-related effects on food consumption for treated males and females. Any differences from the respective controls, statistically significant or otherwise, were considered to be BMS-589152-01-related due to their direction, small magnitude, infrequent occurrence and/or lack of relation to dose.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

Main Study
There were no BMS-589152-01-related effects on hematology. Any differences from controls were not considered to be BMS-589152-01-related due to their direction, small magnitude, infrequent occurrence and/or lack of relation to dose.

Recovery Phase
There were no BMS-589152-01-related effects on hematology. Any differences from the controls were not considered to be BMS-589152-01-related due to their direction, small magnitude, infrequent occurrence and/or lack of relation to dose.

Coagulation
Main Study
There were no BMS-589152-01-related effects on coagulation. Any differences from controls were not considered to be BMS-589152-01-related due to their direction, small magnitude, infrequent occurrence and/or lack of relation to dose.

Recovery Phase
There were no BMS-589152-01-related effects on coagulation. Any differences from controls were not considered to be BMS-589152-01-related due to their direction, small magnitude, infrequent occurrence and/or lack of relation to dose.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Main Study
There were no BMS-589152-01-related effects on clinical chemistry. Any differences from the controls were not considered to be BMS-589152-01-related due to their direction, small magnitude, infrequent occurrence and/or lack of relation to dose.

Recovery Phase
There were no BMS-589152-01-related effects on clinical chemistry. Any differences from the controls were not considered to be BMS-589152-01-related due to their direction, small magnitude, infrequent occurrence and/or lack of relation to dose.

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional Observational Battery
Main Study
There were no BMS-589152-01-related effects on the Functional Observational Battery including sensory reactivity.
Grip Strength
There were no BMS-589152-01-related adverse effects on grip strength.
Motor Activity
There were no BMS-589152-01-related effects on horizontal or vertical (rearing) movements in either sex. Any changes were considered small in magnitude, sporadic in nature, and all animals habituated to their environment.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Main Study
There were no BMS-589152-01-related microscopic observations. All microscopic findings were considered to be unrelated to test chemical based on low incidence, distribution/severity, and/or consistency with background pathology of rats.

Recovery Phase
Target organs were not identified in the terminal phase of this study. Therefore, tissues from recovery rats were not examined, except for the single euthanized moribund male.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

There were no thyroid hormone differences attributed to BMS-589152-01

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no BMS-589152-01-related effects on estrous cycling. The number of cycles (regular and irregular), as well as mean cycle length, were within individual control range values.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No treatment associated effects to the testes were reported

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no BMS-589152-01-related effects on mating and fertility. Any differences from controls were not considered to be BMS-589152-01-related due to their direction, small magnitude, infrequent occurrence and/or lack of relation to dose. There were 10, 10, 10 and 10 females that mated in the 0, 10, 100 and 1000 mg/kg/day groups. Of these 10, 9, 10 and 9 females in the respective groups were pregnant. All pregnant animals delivered live litters. The number of animals mating, number of dams conceiving, number of days to mating, and gestation length, and number of live litters were comparable among groups. The male and female mating, fertility and gestation indices were comparable among groups.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: At dose levels of 10, 100 or 1000 mg/kg bw/day the substance was well tolerated and no treatment related adverse effects were reported in F0 adults

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: viability of offspring

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no BMS-589152-01-related clinical observations

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There was a BMS-589152-01-related effect on the Viability index (%) for Day 4 at 1000 mg/kg/day (-9.6%).
There were no test item related effects on viability indices for Days 7 and 14, post-implantation survival or live birth indices (%).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no BMS-589152-01-related effects on pup sex or absolute mean body weights.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no BMS-589152-01-related macroscopic observations in terminal necropsy rats. All macroscopic findings occurred sporadically or at similar incidence and severity in control and test chemical-treated groups and were considered incidental.

Histopathological findings:

not examined

Description (incidence and severity):

Microscopic pathology was only carried out on the thyroid, no effects were reported

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Delivery and Litter Size
Main Study
There was no effect on the number of implantation sites, gestation length, total litter size, or live litter size on PNDs 1, 4, 7, and 13. One litter at 1000 mg/kg/day had total litter loss, although this was of low incidence and once removed, mean values were comparable to the control range.

Litter Observations
Main Study
There were no BMS-589152-01-related litter observations.

Anogenital Distance and Nipple Retention
Main Study
There were no BMS-589152-01-related effects on anogenital distance or nipple retention in male or female pups.

Thyroid Hormone
Main Study
There was a decrease of -18.2%, -12.6% and -12.4% in levels of thyroxin (T4) in female pups at 10, 100 and 1000 mg/kg/day, respectively. However, this effect showed no dose relationship and so it cannot be unequivocally attributed to the treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Analysis of preliminary mixes confirmed that the preparation procedure used for this study produced homogeneous mixtures. Analyses confirmed that dose suspensions of appropriate concentration were administered.

Mean Analytical Concentrations

	Group 1	Group 2	Group 3	Group 4
Interval	(0.0 mg/mL)	(2.0 mg/mL)	(20.0 mg/mL)	(200.0 mg/mL)
Week 1	ND	114.5^1	116.1^1	104.3
Week 1 (Back-up)	-	105.8	81.2^1	-
Week 3	ND	99.1	100.6	100.7
Week 9	ND	101.7	101.5	107.4

¹Result did not meet acceptance criteria.

Conclusions:

There was a BMS-589152-01-related effect on the Viability index (%) for Day 4 at 1000 mg/kg/day (-9.6%). Additionally, there was a decrease of -18.2%, -12.6% and -12.4% in levels of thyroxin (T4) in female pups at 10, 100 and 1000 mg/kg/day, respectively. However, this effect showed no dose relationship and so it cannot be unequivocally attributed to the treatment.
There were four unscheduled P0 decedents (i.e., one male and two females at 1000 mg/kg/day and one female at 10 mg/kg/day) over the course of this study. Due to the absence and/or nature of the clinical signs and pathology findings, the causes of these deaths could not be unequivocally attributed to treatment with BMS-589152-01.
There were no clinical signs associated with treatment with BMS-589152-01 in males and females that survived until scheduled termination. There were no body weight or food consumption differences, organ weight, clinical pathology or thyroid hormone differences, macroscopic or microscopic changes attributed to BMS-589152-01.
There were no BMS-589152-01-related adverse effects on estrous cycling, mating and fertility indices, delivery and litter size or on litter observations. There were no BMS-589152-01-related effects on pup sex, mean body weights, anogenital distance, nipple retention, functional assessments, or macroscopic changes attributed to BMS-589152-01.
With all findings considered, the male and female systemic NOAEL was 1000 mg/kg/day and the reproductive NOAEL was 100 mg/kg/day.

Executive summary:

Sprague-Dawley CD® rats (10 animals/sex/group) were dosed by oral gavage once daily with 0 (1% w/v Methylcellulose), 10, 100 or 1000 mg/kg/day of BMS-589152-01. The male animals were dosed during the pre-cohabitation, cohabitation and post-mating periods (approximately 35 days) and then euthanized and necropsied. The female animals were dosed during the precohabitation and cohabitation periods and during gestation and lactation (through Lactation Day [LD] 13, approximately 70 days) and then euthanized and necropsied. The dose volume was 5 mL/kg/day for all dose groups. Parameters evaluated during the study for the F0 animals were: viability, clinical observations, body weights, food consumption, estrous cycling, mating and fertility, parturition and littering, functional assessments, clinical pathology (at termination) and thyroid hormone analysis (at termination), organ weights, macroscopic observations and microscopic pathology. Parameters evaluated for the F1 pups were: viability, clinical observations, sex, body weights, ano-genital distance, nipple retention, thyroid hormone analysis (at Postnatal Days [PND} 4 and 13), thyroid weights, macroscopic observations and microscopic pathology (thyroids).

An additional 5 animals/sex in Groups 1 and 4 were dosed by oral gavage once daily for 35 days with 0 (1% w/v Methylcellulose) or 1000 mg/kg/day BMS-589152-01. At the end of the treatment period, these animals were held for a 14 day treatment-free recovery period and then euthanized and necropsied. Parameters evaluated during the study were: viability, clinical observations, body weights, food consumption, clinical pathology (hematology and clinical chemistry at end of recovery; coagulation at end of dosing and end of recovery), organ weights, macroscopic and microscopic observations.

The study was performed in compliance with OECD 422 guideline for testing of chemicals adopted 28.07.15: Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test. There was a BMS-589152-01-related effect on the Viability index (%) for Day 4 at 1000 mg/kg/day (-9.6%). There were four unscheduled P0 decedents (i.e., one male and two females at 1000 mg/kg/day and one female at 10 mg/kg/day) over the course of this study. Due to the absence and/or nature of the clinical signs and pathology findings, the causes of these deaths could not be unequivocally attributed to treatment with BMS-589152-01. There were no clinical signs associated with the treatment of BMS-589152-01 in males and females that survived until scheduled termination. There were no body weight or food consumption differences, organ weight, clinical pathology or thyroid hormone differences, macroscopic or microscopic changes attributed to BMS-589152-01.

There were no BMS-589152-01-related adverse effects on estrous cycling, mating and fertility indices, delivery and litter size or on litter observations. There were no BMS-589152-01-related effects on pup sex, mean body weights, anogenital distance, nipple retention, functional assessments, or macroscopic changes attributed to BMS-589152-01. With all findings considered, the male and female systemic NOAEL was 1000 mg/kg/day and the reproductive NOAEL was 100 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available (further information necessary)

Effects on developmental toxicity

Description of key information

See summary under effects on fertility

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed