1H-Pyrazolo[3,4-c]pyridine-3-carboxylic acid, 4,5,6,7-tetrahydro-1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxo-1-piperidinyl)phenyl]-, ethyl ester

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 March 2016 - 12 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(July 2016)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

(July 2000)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other:

Principles of method if other than guideline:

In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 2016);
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000);
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008);
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008);
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-1H,4H,5H,6H,7H-pyrazolo[3,4-c]pyridine-3-carboxylate

EC Number:

700-890-7

Cas Number:

503614-91-3

Molecular formula:

C27 H28 N4 O5

IUPAC Name:

ethyl 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-1H,4H,5H,6H,7H-pyrazolo[3,4-c]pyridine-3-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

Appearance: White to off-white powder
Storage conditions: At room temperature protected from light

Specific details on test material used for the study:

Purity/composition correction factor: No correction factor required;
Test substance handling: amber glassware used or container wrapped in aluminumfoil.

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation (start treatment F0): Approximately 9-10 weeks (males), approximately 10-12 weeks (females).
- Weight at study initiation: 255-284g (males) or 204-237g (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), except maximum 24 hours before sacrifice.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 15 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 22 March 2016 to 12 July 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity: 1.036

Details on exposure:

Method of formulation: Formulations (w/w) were prepared daily and used within 5 hours after preparation. The formulations were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

Appearance of test item formulations: White, cloudy solutions.

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

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

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

- M/F ratio per cage: 1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In the main study, analyses were conducted on a single occasion during the treatment phase, according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). 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%.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to neropsy. Females were exposed for 50-56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were exposed for 43-52 days.

Pups were not treated directly, but were potentially exposed to the test substance in utero, through lactational transfer and from exposure to maternal urine/faeces.

Frequency of treatment:

Once daily for 7 d/w.

Details on study schedule:

- Age at mating of the mated animals in the study: Approximately 13 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Doses for the main study were selected based on the results from a previous oral single dose study (BMS-589154-01: Acute Oral Toxicity Study in the Rat (Study BMY 667/024060/AC), Bristol-Myers Squibb Company; 2003) and a 10 day dose range finding study (BMS-589154-01: Dose Range Finder (Gavage) Toxicity Study in the Rat (Study NF79LD), Bristol-Myers Squibb Company; 2016) conducted in rats with BMS-589154-01.
In the dose range finding study, 6 females (3 per group) were exposed for 10 days to dose levels 500 and 1000 mg/kg bw/day. Mortality was checked at least twice daily.
Detailed clinical observations were made in all animals at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing.
Body weights were measured on days 1, 3, 5, 7 and 10 food consumption was measured on days 1-5 and 5-10. Blood samples were collected at the end of the treatment period to determine prothrombin time and activated partial thromboplastin time. All animals were subjected to an external, thoracic and abdominal examination on day 11 (scheduled necropsy) or sooner (decedents). No organs were fixed. Animals were deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy.

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

Positive control:

no

Examinations

Parental animals: Observations and examinations:

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

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were conducted at least immediately after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded.

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

FOOD CONSUMPTION:
Yes. Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on post natal days 1, 4, 7 and 13.

FOOD EFFICIENCY:
Yes.

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

OPHTHALMOSCOPIC EXAMINATION: No

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

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

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Yes
- Time schedule for examinations: The selected males were tested during week 4 of treatment and the selected females were tested once during the last week of lactation.
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength (mean of three measurements per animal) and locomotor activity(recording period: 1-hour under normal laboratory light conditions). Total movements and ambulations were reported.

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.
On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.

Sperm parameters (parental animals):

Slides of the testes were prepared for histopathological staging of spermatogenesis (5 males of the control and high dose group and all males suspected to be infertile).

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on days 1, 4, 7 and 13 of lactation.
- Sex: Sex was determined for all pups on days 1 and 4 of lactation (anogenital distance was measured for all live pups on post natal day 1. The anogenital distance was normalized to the cube root of body weight). On post natal day 13, all males in each litter were examined for the number of areola/nipples.

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

Postmortem examinations (parental animals):

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

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

- Selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis: According to test guidelines

- All remaining animals and females which failed to deliver: According to test guidelines

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

- All remaining males:
Epididymides and Testes

HISTOPATHOLOGY
According to test guidelines

Postmortem examinations (offspring):

SACRIFICE
Pups, younger than 7 days were killed by decapitation.
All remaining pups were sacrificed using Euthasol® 20% (AST Farma B.V., Oudewater, The Netherlands) by intraperitoneal (ip) injection.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
No.

Statistics:

The following statistical methods were used to analyse 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 Fisher Exact-test was applied to frequency data.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- 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. Individual values, means and standard deviations may have been rounded off before printing.

Reproductive indices:

For each group, the following calculations were performed:
- Mating index: (Number of females mated/Number of females paired) x 100
- Fertility index: (Number of pregnant females/Number of females paired) x 100
- Conception index: (Number of pregnant females/Number of females mated) x 100
- Gestation index: (Number of females bearing live pups/Number of pregnant females) x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring 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 Days 0-4 of lactation: (Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check) x 100
- Viability index: (Number of live pups on Day 4 of lactation / Number of pups born alive) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were observed during the observation period. Incidental findings that were noted included scabs, alopecia, scales, focal erythema, rales and slight salivation. These findings occurred within the range of background findings to be expected for rats of this age and strain which were housed and treated under the conditions in this study. At the incidence observed, these were not considered to be signs of toxicological relevance.

Mortality:

no mortality observed

Description (incidence):

No mortality of parental rats occurred during the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in body weights and body weight gain were noted up to 1000 mg/kg bw/day. In the absence of a dose-related trend, the statistically significantly higher body weight gain at 100 mg/kg bw/day on day 4 and at 10 mg/kg bw/day on day 13 of lactation, as compared to the concurrent control group, was considered not to be related to treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption or relative food consumption were noted. In the absence of a dose-related trend, the statistically significantly higher food consumption at 100 mg/kg bw/day on lactation days 1-4 (absolute) and on lactation days 1-13 (relative to body weight) was considered not to be related to treatment.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Prothrombin time was statistically significantly increased in males and females at 1000 mg/kg bw/day (17.7 and 19.5 for respectively control and high dose males; 15.7 and 17.6 for respectively control and high dose females). All remaining haematology parameters were unaffected by treatment up to 1000 mg/kg bw/day in both sexes.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry parameters were considered not to have been affected by treatment. When compared to the concurrent control group, a statistically significantly lower group
mean value of sodium concentration was recorded in females at 10 mg/kg bw/day (138.3 and 135.5 mmol/L for respectively control and low dose females). No toxicological significance was attached to this finding, as it occurred in the absence of a treatment-related distribution and the value remained within the range considered normal for rats of this age and strain. Serum levels of T4 in F0 males were not considered to be affected by treatment. All values remained within the normal range of biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed. The statistically significantly lower grip strength of the hind limb in females at 10 mg/kg bw/day was considered not to be treatment-related due to lack of dose dependency. No effects on the forelimbs and no effects on other functional observations or clinical signs were seen in any of the sexes. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.
There were 3/10 couples treated at 10 mg/kg bw/day, 1/10 couples treated at 100 mg/kg bw/day and 4/10 couples treated at 1000 mg/kg bw/day that failed to deliver healthy pups. Histopathology did not reveal any changes in the reproductive organs which could account for the lack of
offspring. Spermatogenic profiles were normal for all males examined.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females had a regular 4- or 5-day estrous cycle during the pretest and treatment period until mating occurred.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Spermatogenic profiles were normal for all males examined.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Precoital time was not considered to be affected by treatment. Number of implantation sites was not considered to be affected by treatment. There were 3/10 couples treated at 10 mg/kg bw/day, 1/10 couples treated at 100 mg/kg bw/day and 4/10 couples treated at 1000 mg/kg bw/day that failed to deliver healthy pups (10/10 for controls delivered healthy pups). At 1000 mg/kg bw/day, one of the 4 coupkes without pups did not show evidence of mating. The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment. Sex ratio of pups was not affected by treatment.

Details on results (P0)

No toxicologically relevant findings for parturition were noted among the pregnant females. One female in the high dose group was noted with a slightly prolonged parturition which lasted for two days. Clinical observations did not indicate any difficulties with giving birth. As she had a completely normal litter with 11 healthy pups, and no signs of difficult or prolonged parturition were noted among the other pregnant females, no toxicological significance was attached to the slightly prolonged parturition of this single female. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects seen up to and including the highest dose tested.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment. Incidental clinical symptoms of pups consisted of lean appearance, and blue discolouration, scabbing and wounds on different parts of the body. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment. The number of live offspring on day 1 after littering compared to the total number of offspring born was not affected by treatment. One pup in the control group was found dead at first litter check. The number of live offspring on day 4 before culling compared to the number of offspring on day 1 was not affected by treatment. No pups were found dead/missing between lactation days 2 and 4.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were unaffected by treatment.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female post natal day 13-15 pups were considered not to be affected by treatment. All values remained within the normal range of biological variation.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment. Treatment up to 1000 mg/kg bw/day had no effect on areola/nipple retention. Nipples were not observed in any of the examined male pups at post natal day 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment. The only macroscopic finding in this study was scabbing on the head noted for one pup in the 10 mg/kg bw/day group. Due to its isolated occurrence in the low dose group it was considered to be a chance finding. No other macroscopic findings of pups were noted.

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no substance-related effects observed up to and including the highest dose tested.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

As also one high dose female had implantations only (no live offspring), this resulted in a relatively low number of females with live offspring (n=6) in the high dose group. It should be noted that the number of not-mated (n=1), non-pregnant (n=2) females and females with implantation sites only (n=1) in the high dose group were within the normal range of biological variation. Moreover, different mechanisms/processes are involved in mating, conception, implantation and/or early prenatal development. Therefore, the relatively low number of females with live offspring in the high dose group was considered not to be test item related but rather a chance finding. The OECD guideline mentions that 8 pregnant females per group normally is the minimum

acceptable number of pregnant females per group. However, in the present study sufficient information could be obtained from the available litters in all groups for a thorough evaluation of possible treatment-related developmental effects.

Applicant's summary and conclusion

Conclusions:

In an oral 28-day repeated dose toxicity study combined with reproduction/developmental toxicity screening performed according to OECD/EC guidelines and GLP principles, the parental, reproduction and developmental NOAEL of BMS-589154-01 was concluded to exceed 1000 mg/kg bw/day.

Executive summary:

A combined 28-day repeated dose study with screening for reproductive and/or developmental effects was performed according to OECD/EC guidelines and GLP principles. BMS-589154-01 was administered by daily oral gavage to male and female rats at dose levels of 10, 100 and 1000 mg/kg bw/ day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 13 days of lactation (for 50-56 days). Females which failed to deliver healthy offspring were exposed for 43-52 days. Formulation analysis showed that the formulations were prepared accurately and homogenously. No toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No toxicologically relevant changes were noted in any of the parental parameters investigated in this study (i.e. viability/mortality, clinical appearance, functional observations, body weight, food consumption, haematology, clinical biochemistry, serum concentration of the thyroid hormone T4 (males only), macroscopy and organ weights). There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item. There were 3/10 couples treated at 10 mg/kg bw/day, 1/10 couples treated at 100 mg/kg bw/day and 4/10 couples treated at 1000 mg/kg bw/day that failed to deliver healthy pups. One high dose animal did not show evidence of mating.It should be noted that the number of not-mated (n=1), non-pregnant (n=2) females and females with implantation sites only (n=1) in the high dose group were within the normal range of biological variation. Moreover, different mechanisms/processes are involved in mating, conception, implantation and/or early prenatal development. Therefore, the relatively low number of females with live offspring in the high dose group was considered not to be test item related but rather a chance finding. The OECD guideline mentions that 8 pregnant females per group normally is the minimum acceptable number of pregnant females per group. However, in the present study sufficient information could be obtained from the available litters in all groups for a thorough evaluation of possible treatment-related developmental effects.Histopathology did not reveal any changes in the reproductive organs which could account for the lack of offspring. Spermatogenic profiles were normal for all males examined. Mating index, fertility and conception index were unaffected by treatment. Precoital time, the number of implantation sites, gestation index and duration of gestation were not considered to be affected by treatment. No toxicologically relevant findings for parturition were noted among the pregnant females. No treatment-related changes toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance (post natal day 1), areola/nipple retention (post natal day 13 males), T4 thyroid hormone levels (post natal day 13-15) and macroscopy.

Based on absence of adverse effects seen at the highest dose tested, the parental, reproduction and developmental NOAEL of BMS-589154-01 was concluded to exceed 1000 mg/kg bw/day.