Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP and Guideline study but study period or date of report are not indicated in the study record.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratory Japan Inc. (CRJ), Atsugi Shiiku Center
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 27.0 - 32.3 g
- Assigned to test groups randomly: yes
- Housing: individually
- Diet (e.g. ad libitum): solid diet (CE-2, CLEA Japan), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24.5
- Humidity (%): 46 - 69
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: olive oil
- Justification for choice of vehicle: test substance is soluble in oil but not in water.
- Concentration of test material in vehicle: 25.0, 50.0, and 100 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg/d
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed and dissolved in olive oil in order to prepare a stock solution with a maximum concentration. The solution was thaen further diluted. After preparation it was stored in the dark in the fridge and used within 48 hours.
Duration of treatment / exposure:
48 hours
Frequency of treatment:
24 hour-interval
Post exposure period:
24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
250, 500, and 1000 mg/kg/day
Basis:
nominal conc.
Nominal concentrations were confirmed by analytical measurement. A recovery rate of between 103% was detected.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg (10 mL/kg/day)

Examinations

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on a preliminary toxicity test in both sexes it was conducted that male mice are more sensitive. Maximum tolerable doses of 1000 mg/kg bw/d for males and 2000 mg/kg bw/d for females was determined. Therefore, the main micronucleus test was conducted uisng male mice and a maximum dose of 1000 mg/kg bw/d.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Bone marrow smears for observations of micronuclei were prepared about 24 hours after the final administration.

DETAILS OF SLIDE PREPARATION: Each mouse was euthanized by cervical dislocation, the femurs on both sides were harvested, both the ends were cut, and the bone marrow cells were washed in a centrifuge tube with 0.6 mL fetal bovine serum (Lot No.: 1248874, Life Technologies) and centrifuged at 200 × g for 5 minutes to remove the supernatant. After the deposit was pipetted, 3 bone marrow smears were prepared per individual from the bone marrow cell suspension. The bone marrow smears were dried naturally and then fixed in methanol for 5 minutes. They were stored at room temperature.

METHOD OF ANALYSIS: Each bone marrow smear was stained with a few drops of a 40 μg/mL Acridine Orange (AO) solution dissolved in Sorensen’s 1/15mol/L phosphate buffer immediately before observation, covered with a cover glass, and observed under a blue excitation fluorescence microscope equipped with a 510 nm absorption filter with use of a 100-power objective lens and a 10-power ocular lens.
Evaluation criteria:
The test was considered valid if the negative and positive control fell within the variation range (average value +/- 3* standard deviation) of the historical control data.
Statistics:
The Fisher’s exact test (one-tailed test) was conducted in the negative control group, respective test article treated groups, and the positive control group. In the exact test, the Bonferroni correction was used in order to detect multiplicity. For dose (logarithms) dependence of micronucleus emergence frequencies, the Cochran-Armitage test for trend (one-tailed test) was conducted. In any of the tests, the significance level was 5 % and 1 %.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
up to the highest dose group of 1000 mg/kg bw/d.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 250 - 2000 mg/kg bw/d
- Solubility: The test substance was completely soluble in olive oil.
- Clinical signs of toxicity in test animals: only in males decrease in locomotor activity, abdominal postures, piloerection.
- Evidence of cytotoxicity in tissue analyzed: No
- Rationale for exposure: clinical signs of the male animals at 2000 mg/kg bw/d.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No statistically significant increase in micronuclei was observed in any of the dose groups.
- Ratio of PCE/NCE: As for the percentage of polychromatic erythrocytes in erythrocytes, no statistically significant differences were recognized between the negative control group and the other groups, and there were no antiproliferative activities of bone marrow cells resulting from administration of the test substance.
- Appropriateness of dose levels and route: Since 1000 mg/kg/day (in vivo maximum tolerable dose) was set as the highest dose in the present test, the test results are considered to be highly reliable to evaluate micronucleus inducibility.
- Statistical evaluation: The negative and positive controls fell well within the variation range of the historical control data.

Applicant's summary and conclusion