Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 03rd, 1989 to November 01st, 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to international guidelines and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Official Journal of the European Communities No L 251 137-139
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
Guidelines 52 FR 19080 (May 20, 1987; effective June 19, 1987), 798.5395.
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Stability: Ensured by sponsor

Test animals

Species:
hamster, Chinese
Strain:
other: Chinese hamster (Cricetulus griseus) random outbred strain
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain as cited inthe reported: Chinese hamster (Cricetulus griseus) random outbred strain
- Source: CIBA-GEIGY Tierfarm, Sisseln.
- Weights at test initiation:
- tolerability test: females 27-33 g, males 31-33 g
- mutagenicity test: females 23-33 g, males 26-33 g
- Days of acclimatization: at least 3 days
- Diet: standard diet NAFAG No.924, ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23°C
- Humidity (%): 58-75%
- Air changes (per hr): air-conditioned room, no further details
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Klucel 0.5%
Duration of treatment / exposure:
The animals were sacrificed after 16, 24 and 48 hours post treatment
Frequency of treatment:
Single administration by gavage
Post exposure period:
16, 24 and 48 hours (8 female and 8 male animals per sampling time point were sacrificed)
Doses / concentrations
Remarks:
Doses / Concentrations:
5000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
8
Control animals:
yes, concurrent vehicle
Positive control(s):
64 mg/kg bw Cyclophosphamide in 20 mL/kg Klucel 0.5% served for positive control substance. the animals were sacrificed after 24 hours following treatment.

Examinations

Tissues and cell types examined:
Bone marrow smears.
Details of tissue and slide preparation:
Following sacrifice of the animals, bone marrow was harvested from the shafts of both femurs with fetal calf serum. After centrifugation small drops of the sediment mixture were transferred on the end of a slide, spread out with the aid of a polished cover glass and the preparations were air-dried. Within 24 hours, the slides were stained with undiluted May-Grünwald solution for 3 minutes and then with May-Grünwald solution/water (1:1) for 2 minutes. After being rinsed in distilled water, the slides were left immersed in diluted Giemsa solution (16.6%), for 10 miniutes. After rinsing with distilled water and air-drying, the slides were cleared in Xylene and mounted.
The slides of five animals from each sex showing the best differentiation between mature and polychromatic erythrocytes were selected for scoring. The slides of five female and five male animals each of the negative control group and of the dosage group sacrificed at 16, 24 and 48 hours post-treatment were examined. From the animals of the positive control group which were sacrificed 24 hours after application, the slides of five female and five male animals were scored. 1000 polychromatic erythrocytes per animal each were scored for the incidence of micronuclei. To determine the mitotic activity of the red compartment, the ratio of polychromatic to normochromatic erythrocytes was calculated for each animal by counting a total of 1000 erythrocytes. A low proportion of polychromatic erythrocytes was indicative for a mitosis inhibiting activity of the test substance.
Evaluation criteria:
A test substance is considered to be active in this test system if a statistically significant increase in the number of polychromatic erythrocytes with micronuclei in comparison with the negative control occurs at any sampling time.
Statistics:
Chi-squared test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Remarks:
There was no significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with 5000 mg/kg bw of the test item as compared with the negative control animals at all sampling times.
Toxicity:
yes
Remarks:
one case of mortality (male) was reported in the treatment group of 48 hours.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Tolerability test:
In the tolerability test the maximum dose of 5000 mg/kg bw caused no death in a group of four animals. Therefore, this dose was taken as the highest in the mutagenicity test.
Mutagenicity test:
The mutagenicity test was performed with the dosage of 5000 mg/kg bw.
One male animal died in the treatment group of 48 hours. The animals were treated once with 5000 mg/kg bw test material and bone marrow was harvested 16, 24 and 48 hours thereafter. There was no significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the dose of 5000 mg/kg bw of test material as compared with the negative control animals at all three sampling times.
By contrast, the positive control (cyclophosphamide, 64 mg/kg bw, sampling time 24 hours) yielded a marked increase of the percentage of micronucleated cells. Here the mean percentage of polychromatic erythrocytes with micronuclei was 2.0. In comparison with the negative control (0.03 %) this value is highly significant (p <0.05).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test item was tested in an in vivo micronucleus test (MNT) according to the OECD TG 474 (1983) using Chinese hamster. No evidence of mutagenic effects was obtained in Chinese hamsters treatedby gavage with 5000 mg/kg bw test material.
Executive summary:

The test article was tested in an in vivo micronucleus test (MNT) according to the OECD TG 474 (1983) using Chinese hamster. The test item was administered by single gavage at a dose of 5000 mg/kg bw, which had been selected based on a preliminary tolerability test. The treated animals were sacrificed 16, 24 and 48 hours thereafter. From the bone marrow smears were made and evaluation of any mutagenic effect of the test item on polychromatic erythrocytes as manifested by micronuclei was done.

At all three sampling times, no statistically significant increase in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals could be evidenced. The respective positive control with cyclophosphamide (64 mg/kg bw) yielded an average of 2.0% polychromatic erythrocytes with micronuclei. This was significantly different from the negative vehicle controls (0.03%). It is concluded that no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test article in the MNT.