Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Result determined using OECD test guideline 474. Negative result - no genotoxicity or toxicity.
Link to relevant study records
Reference
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:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable non-guideline study with QAU statement. Limited details on procedure and results are provided.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
limited details reported
Principles of method if other than guideline:
The animals were treated once by gavage with the highest applicable dose of 5000 mg/kg and sacrificed 16, 24 and 48 hours thereafter. From the bone marrow smears were made and the number of micronucleated polychromatic erythrocytes was analyzed.
GLP compliance:
no
Remarks:
but QAU statement available
Type of assay:
micronucleus assay
Species:
hamster, Chinese
Strain:
other: random outbred strain
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CIBA-GEIGY Tierfarm, Sisseln
- Age at study initiation: females 6-10 weeks, males 4-9 weeks
- Weight at study initiation: females 22-26 g, males 27-33 g (tolerability test), females 24-34 g, males 24-35 g (mutagenicity test)
- Housing: Individually
- Diet (e.g. ad libitum): NAFAG No. 924
- Water: ad libitum
- Acclimation period: 4-5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 53-58
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
CMC 0.5%
Frequency of treatment:
once
Post exposure period:
16, 24 and 48 hours
Remarks:
Doses / Concentrations:
5000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
Tolerability test: 2 females and 2 males.
Mutagenicity test: 24 females and 24 males in the treatment group and in the negative control group. 8 females and 8 males in the positive control group.
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (ENDOXAN)
- Route of administration: oral, gavage
- Doses / concentrations: 64 mg/kg in 20 ml/kg vehicle, sacrificed at 24 hours.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
5000 mg/kg was determined as the highest applicable dose in a preliminary tolerability test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
a) test substance
5000 mg/kg in 20 ml/kg vehicle, sacrificed at 16 hours.
5000 mg/kg in 20 ml/kg vehicle, sacrificed at 24 hours.
5000 mg/kg in 20 ml/kg vehicle, sacrificed at 48 hours.
b) Negative control:
20 ml/kg vehicle, sacrificed at 16 hours.
20 ml/kg vehicle, sacrificed at 24 hours.
20 ml/kg vehicle, sacrificed at 48 hours.
c) Cyclophosphamide:
64 mg/kg in 20 ml/kg vehicle (positive control), sacrificed at 24 hours.

DETAILS OF SLIDE PREPARATION:
Bone marrow is harvested from the shafts of both femurs with fetal calf serum. After centrifugation small drops of the sediment mixture are transferred on the end of a slide, spread out with the aid of a polished cover glass and the preparations are air-dried. Within 24 hours, the slides are stained in undiluted May-Grünwald solution for 3 min then in May-Grünwald solution/water 1/1 for 2 min. After being rinsed in distilled water, the slides are left immersed in diluted Giemsa solution (16.6%), for 10 min. After rinsing with distilled water and air-drying, the slides are cleared in Xylene and mounted.

METHOD OF ANALYSIS:
Prior to analysis the slides are coded. Thereafter the quality of staining is evaluated. The slides of five animals from each sex showing the best differentiation between mature and polychromatic erythrocytes are selected for later 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 are examined. From the animals of the positive control group which are sacrificed 24 hours after application, the slides of five female and five male animals are scored. 1000 polychromatic erythrocytes per animal each are scored for the incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes is determined for each animal by counting a total of 1000 erythrocytes.
Statistics:
The significance of difference is assessed by X2-test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
There was no significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the dose of 5000 mg/kg of the test substance as compared with the negative control animals. By contrast, the positive control (cyclophosphamide, 64 mg/kg) yielded a marked increase of the percentage of micronucleated cells. Here the mean percentage of polychromatic erythrocytes with micronuclei was 3.16. In comparison with the negative control (0.04) this value is highly significant (p <0.05).
Conclusions:
Interpretation of results (migrated information): negative
During the described mutagenicity test and under the experimental conditions reported, the test item did not induce chromosomal aberrations in bone marrow cells after oral administration to Chinese hamsters.
Executive summary:

In this experiment the animals were treated with the test substance once by gavage with the highest applicable dose of 5000 mg/kg and sacrificed 16, 24 and 48 hours thereafter. From the bone marrow smears were made. The bone marrow smears from the animals treated with the dose of 5000 mg/kg showed no statistically significant increase (p >0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at all three sampling times. The respective "positive control" experiments with cyclophosphamide (64 mg/kg) yielded an average of 3.16% polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.04%) treated with the vehicle (CMC 0.5%) alone. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test substance.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Data are available from three Ames tests in vitro, two Chromosome aberrations tests in vitro, two gene mutation assays in vitro and two micronucleus tests in vivo.

All in-vitro and in-vivo tests performed have produced the same negative result, with and without metabolic activation.


Justification for selection of genetic toxicity endpoint
Result determined using OECD test guideline 474.

Justification for classification or non-classification

Based on the data available the substance does not trigger any of the requirements for classification.

The registered substance is therefore not classified.