2-Cyclopentene-1-carboxylic acid, 5-(dimethylphenylsilyl)-2-(hydroxymethyl)-, (1R,2R)-2-amino-1,3-dihydroxy-1-(4-nitrophenyl)propyl ester, (1R,5S)-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 April 2005 - 26 March 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in 2006 according to OECD Method 407 and in accordance with GLP. Study material is well characterized.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

445-910-2

EC Name:

-

Cas Number:

1388152-30-4

Molecular formula:

Hill formula: C24 H32 N2 O7 Si CAS formula: C15 H20 O3 Si . C9 H12 N2 O4

IUPAC Name:

(1R,2R)-1,3-Dihydroxy-1-(4-nitrophenyl)propyl-2-ammonium (1R, 5S)-5-(dimethylphenylsilyl)-2-(hydroxymethyl)cyclopent-2-ene-1-carboxylate

Constituent 2

Reference substance name:

(1R,2R)-1,3-Dihydroxy-1-(4-nitrophenyl)propyl-2-ammonium (1R, 5S)-5-(dimethylphenylsilyl)-2-(hydroxymethyl)cyclopent-2-ene-1-carboxylate

IUPAC Name:

(1R,2R)-1,3-Dihydroxy-1-(4-nitrophenyl)propyl-2-ammonium (1R, 5S)-5-(dimethylphenylsilyl)-2-(hydroxymethyl)cyclopent-2-ene-1-carboxylate

Details on test material:

Test material is a white solid which was received at testing laboratory on 19 April 2005 and 1 July 2005 and stored at room temperature..

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals were doubly housed in elevated, stainless steel, wire mesh cages during the first week of the acclimation period and individually housed thereafter. At the start of the study the rats were in the weight range of 197-233 grams (males) and 158 -186 grams (females) and individual weights were within 20% of the mean weight for each sex. The rats were four to six weeks old and acclimatized for 2 weeks prior to initiation of the study. Free access to mains drinking water and food was allowed throughout the study. The temperature and relative humidity were set to achieve limits of 18 to 26 C and 30 to 70% respectively. The rate of air exchange was at least 15 changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours of darkness.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The appropriate amounts of BMS-296796-02 were weighed and corn oil slowly added to the desrired final volume. The mixtures were homogenized for approximately 3 minutes until they appeared homogenous and then stirred with a stirbar/stirplate until a uniform suspension was obtained. The appropriate aliquots needed daily were dispensed while the remaining aliquots were refrigerated until needed. Fresh solutions were made weekly.
The test and control articles were administered once daily by oral intubation using a flexible tube (catheter) fitted onto a syringe of appropriate size.
The test article was administered once daily, 7 days per week, for 28 days unitl necropsy. The dose levels of 1000 and 500 and 100 mg/kg/day were selected for the 28-day Main Study based on no test article effects seen at these same dose levels after dosing in the 7-day Range-Finding Study .

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses to determine homogeneity, stability, and concentration of the test article in vehicle under the conditions of this study were performed by the Testing Facility. Prior to initiation of the study, batches of low-concentration and high-concentration dose mixtures were prepared. Three samples each from the top, middle and bottom portion of each mixture were taken for analysis. Duplicate samples of the low- and high-concentration dose mixtures were assayed 4, 7, and 14 days after preparation. All dose levels were assayed weekly (2 samples per concentration).

Duration of treatment / exposure:

29 days

Frequency of treatment:

The test article was administered once daily, 7 days per week, for 28 days unitl necropsy

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
other: oral gavage

Doses / concentrations 2

Remarks:

Doses / Concentrations:
500 mg/kg/day
Basis:
other: oral gavage

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
other: oral gavage

No. of animals per sex per dose:

Range-Finding study: 3/sex/group for 3 dose levels and a control level for a total of 3 males and 3 females.
Main study: 3 test article-treated groups of 5 animals each and a negative control group for a total of 40 rats (20 males and females)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels of 1000 and 500 and 100 mg/kg/day were selected for the 28-day Main Study based on no test article effects seen at these same dose levels after dosing in the 7-day Range-Finding Study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily. Observations were made concurrent with dosing or at one of the viability checks.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice pretest and weekly
Observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as evaluations of respiration and palpation for tissue masses.

BODY WEIGHT: Yes
- Time schedule for examinations: twice pretest, weekly during treatment and terminally (after fasting).
Terminal, fasted body weights were obtained just prior to necropsy.

FOOD CONSUMPTION
- Feed consumption was measured (weighed) (4-7 daylweek), beginning one week prior to treatment. The average of the current and previous weight was used for all intervals.

HAEMATOLOGY: Yes (Blood obtained via the orbital sinus (retrobulbar venous plexus))
- Time schedule for collection of blood: at study termination
- Anaesthetic used for blood collection: Yes: under light isoflurane anesthesia
- Animals fasted: Yes
- How many animals:all animals (5 animals/sex/group)

CLINICAL CHEMISTRY: Yes Blood for clinical chemistry studies was collected (approximately 1.0rnL) into tubes with no anticoagulant, allowed to clot, and centrifuged to obtain serum.
- Time schedule for collection of blood:at study termination
- Animals fasted: Yes
- How many animals: all animals

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and Dose groups that were examined: all animals pretest and during Week 4. Testing was staggered over two sessions with approximately equal numbers of animals per sex per dose group in each session.
- Battery of functions tested:
-functional observational battery included home cage and open field evaluations, reflex assessments, grip strength, landing foot splay, proprioception, air righting reflex, body temperature and motor activity.

OTHER: Environmental conditions were measured and recorded twice daily, just prior to the first evaluation and just after the last evaluation,. Noise level was maintained within a level of 55 to 65 decibels.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table I)
Necropsy was performed on 5 animals/sex/group at termination of study. Exsanguination following carbon dioxide inhalation Complete macroscopic examinations were performed and organ weights obtained for all animals at the scheduled sacrifice interval.
HISTOPATHOLOGY: Yes (see table I for list of tissues analyzed)
Slides of the indicated tissues were prepared and examined microscopically for all animals in Groups 1 and 4. Additionally, target organs were examined in Groups 2 and 3. Any abnormalities not noted during macroscopic examinations which were seen during histology processing were recorded.

Statistics:

The following parameters were analyzed statistically: body weight body weight change from interval to interval feed consumption hematology coagulation clinical chemistry organ weights motor activity counts forelimb and hindlimb grip strength measurements landing foot splay measurements body temperature (rectal)
Mean values of all dose groups were compared to the mean value for the control group at each time interval.
Evaluation of equality of group means was made by the appropriate statistical method, followed by a multiple comparison test if needed.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

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

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
All animals survived until the termination of the study. No test article-related clinical signs were observed during the study.

BODY WEIGHT AND WEIGHT GAIN
At the end of the study, absolute body weights in the 500 and 1000 mg/kg/day males were lower than controls (-6% and -8%, respectively). A reduction in body weight gain became apparent in the 3'd and 4th weeks of dosing.

FOOD CONSUMPTION/EFFICIENCY
There were no apparent differences in feed consumption between groups of animals.

HAEMATOLOGY
Minor but statistically significant decreases in mean corpuscular hemoglobin concentration (MCHC) was noted in males at all dose levels. Coagulation: There were no differences between groups of animals.

CLINICAL CHEMISTRY
The most consistent and statistically significant changes following the test article administration were decreases in glucose (range: -19% to -34%, relative to controls) and slight increases in phosphorus in the 500 and 1000 mg/kg/day animals (both sexes) and negligible decreases in chloride in the 2 100 mg/kg/day females and 2 500 mg/kg/day males. Statistically significant, slight and not dose-related increases in potassium (females) and blood urea nitrogen (males) were noted at all doses.

NEUROBEHAVIOUR
There were no apparent differences in motor activityand functional observational battery and between groups of animals.

ORGAN WEIGHTS
Test article-related organ weight changes included increases in kidney and liver weights (see text-table below for details). Statistically significant differences were identified only with the liver-to-body-weight ratios at 500 (both sexes) and 1000 mg/kg/day (males). Kidney weight changes correlated with serum electrolytes variations and increased liver weight correlated with hepatocellular hypertrophy in only 1 female dosed with 1000 mg/kg/day

GROSS PATHOLOGY
There were no test article-related macroscopic findings. Findings occurred with comparable incidence and severity in the test article-treated and control animals, or occurred sporadically. These incidental findings were not considered to be test article- related.

HISTOPATHOLOGY: NON-NEOPLASTIC: All protocol tissues from the control and the 1000 mg/kg/day animals were examined. The liver, spleen and stomach were identified as potential target tissues and were also evaluated in the 100 and 500 mg/kg/day animals.
Liver: minimal hepatocellular hypertrophy was observed in only 1 female treated with 1000 mg/kg/day.
Spleen: minimal to moderate increases in extramedullary hematopoiesis (Em), predominantly erythropoiesis, was observed in 215 males per group treated with 100, 500
Huntingdon Life Sciences Study No. 05-2889 BMS-296796-02 Nonclinical Study Report
and 1000 mg/kg/day. In 1 male in the 1000 mg/kg/day group this correlated with decreased red blood cell counts and increased reticulocyte counts in the peripheral blood. Increases in EMH and reticulocytes occur as a regenerative response to decreased circulating red blood cells.
Stomach: the limiting ridge between the glandular and non-glandular mucosa was characterized by minimal to slight epithelial cell hyperplasia and hyperkeratosis in 115 males and 415 females dosed with 1000 mg/kg/day.
Other microscopic findings were seen sporadically or were at a similar incidence in control and test article treated groups. They were typical of those seen in rats of this age and strain and are considered incidental and unrelated to treatment.

Effect levels

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The test article dosing mixtures were analyzed for stability, homogeneity and concentration. These assays demonstrated dosing mixture stability for up to 14 days, that the mixtures were homogeneous and that the dosing mixtures were close in concentration to their intended values.

Applicant's summary and conclusion

Conclusions:

The principal findings following the oral administration of BMS-296796-02 to rats for 28 days included increased kidney weight with serum electrolyte variations at 2100 mg/kg/day, lower absolute body weight (males only), decrease in serum glucose and increased liver weight at 2500 mg/kg/day

Histopathologically, the findings were characterized by splenic extramedullary hematopoiesis in males at all doses, gastric epithelial cell hyperplasia and hyperkeratosis at 1000 mg/kg/day (both sexes) and hepatocellular hypertrophy in a single 1000 mg/kg/day female.

Under the conditions of this study, the no-observed-adverse-effect-level (NOAEL) for repeated dosing was 1000 mg/kg/day.

Executive summary:

The study is considered valid and assigned a reliability rating of 1. The principal findings following the oral administration of BMS 296796 -02 to rats for 28 days: low dose effects include: changes to red blood cell parameters, electrolyte disturbances, decreased body weihgr, decreased serum glucose, changes in clinical pathology parameters, increased organ weights included the kidney and liver. Microscopic changes were observed in the following organs: spleen, liver and stomach. Under the conditions of this study, the no-observed-arverse-effect-level (NOAEL) for repeated dosing was 1000 mg/kg/day.

Under the conditions of this study, the NOAEL for repeated dosing oral was 1000 mg/kg/day.

This finding is well above the classification criteria for serious damage to health by prolonged exposures. Therefore, the substance is not classified under the EU regulations based upon this 28 day repeated dose study.