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:
29 February 2016 - 28 June 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 (incl. certificate)
Remarks:
d.d. 3 November 2015
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Appearance: White crystalline solid
Storage conditions: At room temperature protected from light
Specific details on test material used for the study:
Test substance handling: Amber glassware was used or container was wrapped in aluminum-foil

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Female animals: Charles River Deutschland, Sulzfeld, Germany. Male animals: Charles River Laboratories USA, Kingston.
- Age at study initiation:
Males: approximately 10-12 weeks.
Females: approximately 10-12 and 12-14 weeks for the pretest and main study, respectively.
- Weight at study initiation: 268-301 gr (males) or 201-231 gr (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).
- Water: Free access to tap water.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

ENVIRONMENTAL CONDITIONS (conditions set to maintain)
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 Februari 2016 to 28 June 2016

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item.
Test item was administired via oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
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.

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: 4 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:
The samples of the control group and 40 mg/kg bw/day treatment group were taken in duplicate from the middle position of the container and immediately stored on dry ice. Samples of the 4 mg/kg bw/day and 400 mg/kg bw/day treatment groups were taken in duplicate from the top, middle and bottom position of the container and immediately stored on dry ice. The samples were stored at a target temperature ≤-70°C and all samples were analyzed for BMS-587172-01.

The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Stability of formulations in the vehicle propylene glycol for at least 5 hours at room temperature protected from light (concentration range 1-200 mg/mL) was determined as part of the analytical method development and validation study.
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 termination. Females that delivered were exposed for 51-64 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 days (1 female) or 53 days (1 female). Two females (400 mg/kg bw/day) were not dosed on one occasion as they were littering at the time of dosing.
Frequency of treatment:
Once daily, 7 d/w
Doses / concentrationsopen allclose all
Dose / conc.:
4 mg/kg bw/day (actual dose received)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
400 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 single dose oral toxicity study in Sprague-Dawley rats and an oral dose range finding (DRF) study (10-17 days) in Wistar Han rats conducted with BMS-587172-01. In the single-dose oral toxicity study, no clinical signs of toxicity or macroscopic abnormalities were observed at the limit dose of 2000 mg/kg bw/day. In the dose range finding study, 17 daily oral doses of 200 and 400 mg/kg bw/day (n=3 females/group) were well tolerated and not associated with clear clinical evidence of toxicity. Because of a lack of toxicity observed at these doses, an additional dose of 1000 mg/kg bw/day was added for 10 daily doses. At 1000 mg/kg bw/day, all animals exhibited clinical signs which included hunched posture, piloerection, increased salivation and rales. In addition, toxicity studies conducted with structurally similar compounds, BMS-587319-03 and BMS-512148-01, resulted in clinical toxicity at doses ≥ 200 mg/kg bw/day. Based on these data, the high dose of 400 mg/kg bw/day of BMS-587172-01 was selected for this study because it was anticipated to induce some evidence of toxicity, but within acceptable limits. The low dose of 4 mg/kg bw/day was selected because it was anticipated to be a no effect level, and the intermediate dose of 40 mg/kg bw/day was selected because it allowed for a 10 fold spacing between dose levels.

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination, organ weights and histopathology (see also respective paragraphs).

On day 2 of the mating period, a few females which had their mating confirmed were accidentally dosed based on their body weights from the previous day instead of their most recent body weights (Day 0 post-coitum). Due to this error, 5 females across the test item-treated groups received a slightly higher dose. This deviation was not considered to have affected the study outcome as it occurred on a single day during a total treatment period of 51-64 days and the difference in applied dose volume was relative small (max. 1.25 % higher).
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) for viability and mortality.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule: At least once daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals, at 1 hour (± 30 minutes) after dosing (i.e. on the peak period of anticipated effects 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.

FUNCTIONAL OBSERVATIONS:
The following functional observation tests will be performed on each individual animal of the selected 5 animals/sex/group:
- hearing ability, pupillary reflex (L/R), static righting reflex;
- fore- and hind-limb grip strength (recorded as the mean of three measurements);
- locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system, total movements and ambulations are reported).
The selected males were tested once during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13).

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

FOOD CONSUMPTION:
Yes
- Weekly, for males and females. Food consumption was not recorded during the mating period 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 on Days 1, 4, 7 and 13 of lactation.

FOOD EFFICIENCY:
Yes.
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data.

WATER CONSUMPTION : Yes.
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 between 7.00 and 10.30 a.m..
- Anaesthetic used for blood collection: Yes (iso-flurane)
- 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

THYROID HORMONE ANALYSIS:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination between 7.00 and 10.30 a.m..
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: All males
- Parameters checked were: According to test guidelines

URINALYSIS: No
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:
Cowper’s glands, epididymides, glans penis, levator ani plus bulbocavernosus muscle complex (LABC), testes and thyroid.



HISTOPATHOLOGY: Yes
According to test guidelines

Inadvertently, a few tissues were not available for histopathology. Reasons for missing a few tissues included that these tissues were not discernable at necropsy or trimming, or were erroneously not collected at necropsy. Missing tissues are listed in raw data and pathology report.
Evaluation: Sufficient data was available for adequate histopathological evaluation.
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 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 Fisher Exact-test (Fisher, 1950) was applied to frequency data. (Ref. 2)
- 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 (scores) in the summary tables. No statistical analysis was performed on histopathology findings.

Details on reproductive and developmental parameters are included in section 7.8.1 and 7.8.2.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental findings that were noted included rales, scabbing, wounds and alopecia. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. As they were observed in the absence of a treatment related distribution and/or were seen for individual animals only, they were not considered to be test item related.
Mortality:
no mortality observed
Description (incidence):
One high dose male was euthanized on Day 9 of the pre-mating period due to problems with his hindleg, most likely caused by an accident. Clinical signs of this male included abnormal posture, abnormal gait (at a slight degree), and swelling of his left hindleg (at a slight to moderate degree) from pre-mating Day 5 onwards. The slight body weight loss (1%) recorded from premating Days 1 to 8 was probably related to a reduced food intake caused by his impaired mobility. Necropsy findings included thickened left hindleg and isolated reddish foci on the stomach mucosa.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a trend towards higher food consumption (absolute) of both males and females in all test item treated groups when compared to control animals. However, no clear dose relation could be established. Changes reached statistical significance for females in all dose levels during almost the entire post-coitum period, and for 400 mg/kg bw/day females during lactation Days 4-7.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
There was a trend towards higher food relative to body weight for both males and females in all test item treated groups when compared to control animals. However, no clear dose relation could be established. Changes reached statistical significance for females in all dose levels during almost the entire post-coitum period.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Statistical significant higher urea levels were recorded for males at 40 and 400 mg/kg bw/day (12.2 and 12.3 mmol/L, respectively) and females at 400 mg/kg bw/day (15.1 mmol/L) when compared to their concurrent controls. In males (40 and 400 mg/kg bw/day), values were above the available historical range, but no dose related trend could be established. In females (400 mg/kg bw/day), mean value was at the higher end of the historical range. In the absence of any correlating changes on organ level, these changes were not considered as adverse.
Lower glucose concentration was noted in all test item treated groups, reaching statistical significance for males, but not females. No dose dependency could be established.
Any remaining statistically significant changes were considered to be chance findings as no dose related trend could be established and/or values remained within the range considered normal for rats of this age and strain. Furthermore, no correlating changes on organ level were noted.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There was a test item-related increase in absolute and relative kidney weights in the males of all test item-treated groups without any correlating pathomorphologic alterations.
Any other differences, including those that reached statistical significance were considered not to be treatment-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There was a higher incidence in the follicular cell hypertrophy in the thyroid glands of males treated at 4 and 400 mg/kg bw/day. Based on the minimal degree, which is a common background finding in male rats, and the absence of a dose-response effect it was considered not to be related to the test item.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Serum levels of T4 in F0-males were considered not to be affected by treatment.

Effect levels

Key result
Dose descriptor:
NOAEL
Remarks:
Parental generation
Effect level:
>= 400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicity was observed up to the highest dose level tested.

Target system / organ toxicity

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 mean accuracies for the 4, 40 and 400 mg/kg bw/day dose levels were 99.5%, 96.2% and 97.2%, respectively. The coefficient of variantion for the formulations of the 4 and 400 mg/kg bw/day dose levels were 4.0% and 0.9%, respectively.

Applicant's summary and conclusion

Conclusions:
In an oral OECD422 screening study with rats, the NOAEL was determined to be at least 400 mg/kg bw/day, based on the absence of adverse effects up to and including the highest dose level tested.