Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990-10-02 to 1990-12-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-condform study under GLP without deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Version / remarks:
First Addendum to the OECD Guideline for Testing of Chemicals, Section 4, No. 474, adopted May 26, 1983, "Micronucleus Test".
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: The vehicle was chosen to its nontoxicity for the animals.
- Concentration of test material in vehicle: 20 mg/ml
- Amount of vehicle (if gavage or dermal): 20 ml/kg b.w.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was dissolved in aqua dest..

Duration of treatment / exposure:
24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis.
Frequency of treatment:
single application
Post exposure period:
not applicable
Doses / concentrations
Remarks:
Doses / Concentrations:
400 mg/ kg bw
Basis:
actual ingested
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): none provided
- Route of administration: orally
- Doses / concentrations: 30 mg/kg b.w., volume 10 ml/kg b.w.

Examinations

Tissues and cell types examined:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the bone marrow was flushed out. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
400 mg/kg was determined by a preliminary study to be the maximum tolerated dose.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.

DETAILS OF SLIDE PREPARATION:
The cell suspension was centrifuged at 1,500 rpm for 5 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald / Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with l00x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides.

OTHER:
Evaluation criteria:
A test article is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes at for at least one of the test points. A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at anyone of the test points is considered non-mutagenic in this system.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Remarks:
dose level 400 mg/kg bw
Toxicity:
no effects
Remarks:
dose level 400 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 400 to 5000 mg/kg bw
- Solubility: no data
- Clinical signs of toxicity in test animals: At 5000 mg/kg bw reduction of spontaneous activity, abdominal position, eyelid closure, apathy. All animals died within 6 hours after treatment. at 400 mg/kg bw reduction of spontaneous reaction: two males and two females eyelid closure: one male and one female
- Evidence of cytotoxicity in tissue analyzed: not reported
- Rationale for exposure: acording to the astudy report, the first appearance of micronuclei in PCEs is at least 10-12 hours after a clastogenic exposure.
- Harvest times: 24, 48, and 72 h after treatment, respectively
- High dose with and without activation: 5000 mg/kg bw was the highest dose applied
- Other:

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): the registered substance had no cytotoxic properties.
- Induction of micronuclei (for Micronucleus assay): The substance had no cytotoxic properties.
- Ratio of PCE/NCE (for Micronucleus assay): No apparent cytotoxic effect (PCE/NCE ratio)
- Appropriateness of dose levels and route: Both dose level and route of exposure are considered to be appropriate
- Statistical evaluation: Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
During the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the substance is considered to be non-mutagenic in this micronucleus assay.