Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

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

Test material

Constituent 1
Chemical structure
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)
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 w
ere 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: 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.
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%.
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, 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).
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.
Duration of test:
Males: 29 days
Females: 50-56 days (43-52 days for females that failed to deliver healthy offspring
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
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.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: At least twice daily (early in the morning and close to the end of the working day).
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: 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 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.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups were determined on post natal day 1 and daily thereafter. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective tables.
Body weights: Live pups were weighed on post natal day 1, 4, 7 and 13.
Sex: Sex was determined for all pups on post natal day 1 and 4.
Anogenital distance: Anogenital distance (AGD) was measured for all live pups on post natal day 1. The AGD was normalized to the cube root of body weight.
Areola/nipple retention: On PND 13, all males in each litter were examined for the number of areola/nipples.
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 (Dunnett, 1955) (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. (Ref. 1)
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data. (Ref. 2)
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution. (Ref. 3)
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. (Ref. 4)
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 (score s) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
References:
Ref. 1 Dunnett C.W., A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
Ref. 2 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Ref. 3 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).
Ref. 4 Kruskal W.H. and Wallis W.A.. Use of ranks in one-criterion variance analysis. Journal of the American Statistical Association 47 (260): 583-621, December (1952).
Indices:
Reproductive indices; For each group, the following calculations were performed:
- Mating index: (Number of females mated/Number of females paired) x 100
- Fertility index: (Number of pregnant females/Number of females paired) x 100
- Conception index: (Number of pregnant females/Number of females mated) x 100
- Gestation index: (Number of females bearing live pups/Number of pregnant females) x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring indices:
- Percentage live males at First Litter Check: (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check: (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage of postnatal loss Days 0-4 of lactation: (Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check) x 100
- Viability index: (Number of live pups on Day 4 of lactation / Number of pups born alive) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
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
Description (incidence and severity):
Organ weights and organ to body weight ratios of treated animals were considered to be similar to those of control animals.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. At 100 mg/kg bw/day, black discoloration and enlargement of both sides of the inguinal lymph nodes were recorded for one male at necropsy. Microscopic examination revealed a marked chronic bilateral vascular inflammation of the inguinal area. This was considered to be an incidental rare finding, unrelated to treatment with the test item. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain and were not related to the treatment with the test item.
Neuropathological findings:
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.
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The number of implantation sites was not considered to be affected by treatment. Low numbers of implantation sites were noted for one female each in the control and high dose group (n=3 and n=2, respectively). In the absence of a dose related incidence, it was considered to be an incidental finding. One high dose female had 2 implantation sites only; no live offspring. No abnormalities were seen in the reproductive organs of either sex that could explain why this female failed to deliver healthy offspring. She had a histologic normal cycle and the spermatogenic staging profile was normal for the male she was mated. Furthermore, all remaining pregnant females in the high dose group had normal litter sizes and no abnormalities in pup development were noted. Therefore, this isolated incidence was considered a chance finding and not to be related to treatment. For three females (two in the low dose and one in the mid dose group) the number of pups born was slightly higher than the number of implantation sites. This was considered to be due to normal resorption of these areas as the enumeration was performed on day 14 of lactation.
The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
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.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Duration of gestation was not considered to be affected by treatment.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
A total of three females at 10 mg/kg bw/day, one female at 100 mg/kg/day and two females at 1000 mg/kg/day were not pregnant. In the absence of a
dose-related trend and any correlating histopathological findings, this was not considered to be related to treatment.
Details on maternal toxic effects:
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 part urition 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 (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No maternal toxicity observed up to and including highest dose tested.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were unaffected by treatment.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
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.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was not affected by treatment.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The number of live offspring on day 13 after littering compared to the number of live offspring on day 4 (after culling) was not affected by treatment.
One control pup was found missing on lactation day 7. No pups in the treated groups were found dead/missing between lactation days 5 and 13.
External malformations:
no effects observed
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.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
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.
Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment.
Treatment up to and including 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.
Serum T4 levels in male and female PND 13-15 pups were considered not to be affected by treatment. All values remained within the normal range of biological variation.
Details on embryotoxic / teratogenic effects:
There was no evidence of teratogenic effects based on the absence of relevant clinical signs and external macroscopic findings.

Effect levels (fetuses)

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 adverse effects seen up to and ncluding highest dose tested.

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

The number of pregnant females was 10, 7, 9 and 7 in the control, 10, 100 and 1000 mg/kg bw/day groups, respectively. Consequently, fertility and conception indices were lower than normally expected in the low and high dose group. There were no morphological findings in the reproductive organs of either sex that suggested a test-item relationship or which could explain the failure of pregnancy. Spermatogenic staging profiles were normal for

all males examined and non-pregnant females showed histologic normal cycles. Furthermore, as the incidence of non-pregnant females was within normal limits, in the absence of a dose-related incidence trend, and as litters of the remaining pregnant females were unaffected, the unexpected low number of pregnant females in the low and high dose groups was considered not to be related to treatment with the test item.

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.