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:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed GLP and OECD guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
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:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- 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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- 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
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
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)

Examinations

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.
Statistics:
Wilcoxon (paired, one-sided, increase) and Wilcoxon (paired, two sided).
All statistical results are based on a 95% level of significance.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
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.

Applicant's summary and conclusion

Conclusions:
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.