Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
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:
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:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
(July 2016)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
(July 2000)
Deviations:
no
Qualifier:
according to
Guideline:
other: other guidance as listed under Principles of method if other than guideline
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

Reference
Name:
Unnamed
Type:
Constituent
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 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 oral 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.
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.
Frequency of treatment:
Once daily, 7 d/w
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.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
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.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

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.

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

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

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

- All remaining males:
Epididymides and Testes

HISTOPATHOLOGY: Yes
According to test guidelines
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.

Results and discussion

Results of examinations

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 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):
not examined
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. The statistically increased prothrombin time noted in males and females at 1000 mg/kg/day was not considered to be adverse as changes compared to the concurrent control group were slight and no corroborative findings, such as increased bleeding or hemorrhage, were observed.
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.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
All results relevant for developmental and reproduction parameters are (further) summarized in sections 7.8.1 and 7.8.2.

Effect levels

Key result
Dose descriptor:
NOAEL
Remarks:
Parental generation
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 observed up to and including the highest dose level tested.

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Analysis of dose preparations:

In the control group formulation, no test substance was detected. The concentrations analysed in the formulations of groups exposed to 10, 100 and 1000 mg/kg bw/day were in agreement with target concentrations (i.e. mean accuracies 97.8%, 97.8% and 96.7%, respectively).

The formulations of the low and high dose group were homogeneous (i.e. coefficient of variation ≤ 1.2%).

Applicant's summary and conclusion

Conclusions:
Based on a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test performed with BMS-589154-01 according to OECD//EC guidelines and GLP principles, the NOAEL was determined to be at least 1000 mg/kg bw/day, based on no adverse effects at the highest concentration tested.
Executive summary:

A combined 28d 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 during 13 -15 days of lactation (for 43-52 days). Formulation analysis showed that the formulations were prepared accurately and homogenously. No toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day). The statistically increased prothrombin time noted in males and females at 1000 mg/kg bw/day was not considered to be adverse as changes compared to the concurrent control group were slight and no corroborative findings, such as increased bleeding or hemorrhage, were observed. 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). Based on the absence of adverse effects at the highst dose tested, a No Observed Adverse Effect Level (NOAEL) for BMS-589154-01 of at least 1000 mg/kg bw/day was established.