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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
The Ames test (in vitro) showed mutagenic effects in several tester strains in the presence of metabolic activation, whereas the micronucleus test (in vivo) showed no mutagenic effects.
Link to relevant study records
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed GLP and OECD guideline study
according to
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
Only a single dose was tested in the main study. This test dose was accurately determined in a dose range finding study in order to find the maximum dose producing distinct toxicity but no lethality.
GLP compliance:
Type of assay:
micronucleus assay
Details on test animals and environmental conditions:
- Strain specifics: Hsd
- Source: Harlan Winkelmann GmbH, Gartenstr. 27, 33178 Borchen, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 174 -188 g g, mean 181.5 g, females: 131 - 145 g, mean 138.1 g
- Housing: in groups of five in Macrolon type 4 cages in fully air-conditioned room, softwood granulate
- Diet (e.g. ad libitum): rat/mice diet ssniff R/M-H (V 1524) , ad libitum
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: 5 days

- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
- Vehicle(s)/solvent(s) used: Tylose H4000 G4 PHA, 0.5 %
- Concentration of test material in vehicle: 20% (w/v)
- Amount of vehicle (if gavage or dermal): 10 ml/kg body weight
- Purity: Oleum sesame Ph. Eur. III, Fa. Pharm. Fabrik GmbH, Frankfurt/Main, Germany
Details on exposure:
The test compound was suspended in the vehicle and dosed twice orally at 2000 mg per kg bw at an interval of 24h to male and female mice, upon the results of the previously conducted dose range finding assay
Duration of treatment / exposure:
treatment: twice orally at 200 mg per kg bw (interval 24h)
exposure: animals were killed 24 h after second administration
Frequency of treatment:
Twice at an interval of 24 h
Doses / Concentrations:
2000 mg/kg bw
nominal conc.
No. of animals per sex per dose:
5 males and 5 females (10 animals) per dose group
Control animals:
yes, concurrent vehicle
Positive control(s):
40 mg/kg body weight cyclophosphamide (Endoxan (R), 5 males and 5 females); dissolved in distilled water 0.4 % (w/v)
Tissues and cell types examined:
polychromatic and normochromatic erythrocytes from the femoral bone marrow of each animal
Evaluation criteria:
2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded. In addition, the ratio of polychromatic to 200 normochromatic erythrocytes was determined. Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test item, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes; comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase) .
The results of the treatment groups (test substance) in the micronucleus test at each dose and killing time were compared with corresponding control values.The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided). All statistical results are based on a 95% level of significance. Due to a change in species from mouse to rat, there are no historical data for comparison available.
Wilcoxon (paired, one-sided, increase) and Wilcoxon (paired, two sided).
All statistical results are based on a 95% level of significance.
Vehicle controls validity:
Negative controls validity:
not examined
Positive controls validity:
Additional information on results:
All animals survived after application of 2000 mg per kg bw. The following signs of toxicity were observed: reduced spontaneous activity.

The dissection of the animals revealed no test substance related macroscopic findings.
Interpretation of results (migrated information): negative
Oral administration of the test item did not lead to a substantial increase of micronucleated polychromatic erythocytes and was not mutagenic in the in vivo micronucleus test
Executive summary:

The test item was tested in the micronucleus test according to OECD 474. The substance was suspended in Tylose H4000 G4 PHA, 0.5 %, and dosed twice (intervall 24 h) orally at 2000 mg per kg bodyweight to male and female rats, upon the results of the previously conducted dose range finding assay. According to the test procedure the animals were killed 24 h after administration.

The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with the test item and was statistically not different from the control values.

EndoxanRwas used as positive control substance and was administered orally at a dose of 40 mg per kg bodyweight.

EndoxanR induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity af the system.

The results indicate that, under the conditions of the present study, the test item is not mutagenic in the micronucleus test.

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

Additional information

Additional information from genetic toxicity in vivo:

Justification for selection of genetic toxicity endpoint
micronucleus test in vivo

Justification for classification or non-classification

Available genetic toxicity reports (Ames tests, Micronucleus in vivo) presented in this IUCLID are not sufficient to classify the substance anisbase as mutagenic according to 67/548/EEC, (EC) No 1272/2008 respectively.

Results of a Micronucleus test in vivo show no clastogenic effects for this substance.