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:
08 Aug - 14 Dec 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
other: HsdWin: NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: about 5 weeks
- Weight at study initiation: 22.0-29.7 g (males); 19.4-25.3 g (females)
- Assigned to test groups randomly: yes, by numbered index cards
- Fasting period before study: overnight
- Housing: five mice of the same sex were caged together in Makrolon-cage type III
- Diet: Altromin 1324 (Altromin International, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 65-75%
- Air changes (per hr): 8
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 Aug To: 14 Dec 2000

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: tap water
Duration of treatment / exposure:
not applicable
Frequency of treatment:
single application
Post exposure period:
24 h (all dose groups including control group) and 48 h (additional 1500 mg/kg bw and control group)
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000 and 1500 mg/kg bw (5, 10 and 15% w/w)
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: single application by gavage
- Doses / concentrations: 30 mg/kg bw (0.3% w/w)
- Vehicle: tap water

Examinations

Tissues and cell types examined:
Tissue: bone marrow
Cell type: bone marrow cells
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
Cell suspension was transferred to a clean, dry slide, a smear prpared, and the slide left to air-dry. The slides were stained manually according to the technique of Pappenheim with May-Grünwald and Giemsa solutions.

METHOD OF ANALYSIS:
The slides were examined under light microscope. At high magnification a total of at least 2000 erythrocytes per animal were examined. Each erythrocyte scored was classified as polychromatic or mature and at least 2000 poly-chromatic erythrocytes were examined for the presence or absence of micronuclei. The frequencies of micronucleated cells per 2000 polychromatic erythrocytes were then calculated together with the ratio of polychromatic to normochromatic cells.

Evaluation criteria:
A test substance is classified as mutagen if it induces a significant increase in the number of micronucleated PCEs for at least one time point. A test substance producing no significant increase in the number of micronucleated PCEs at any time point is considered as non-genotoxic in this system.
Statistics:
Mean number of NCEs and PCEs and the mean number of micronucleated PCEs were calculated for each sex and for both sexes combined together with the standard deviation. Assay data analysis was performed using the Mann-Whitney-U-test (P > 0.05).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
no depression in bone marrow proliferation
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The mean occurence of micronucleated PCEs was 0.17% and 0.06% 24 h and 48 h after application, respectively. These values are in the range of the historical negative control (0.06% - 0.24% micronucleated PCEs). The positive control animals had an increase in 4.13% indicating a genotoxic effect. In the test substance groups the frequency of detected micronuclei was in the range of the historical negative control in all doses and at any preparation time after application of the test substance. In the female animals 24h after application, there was a very slight dose-dependent increase in the frequency of micronuclei (500 mg/kg bw: 0.01%, 1000 mg/kg bw: 0.12%, 1500 mg/kg bw: 0.15%) in comparison to the corresponding negative controls (0.09% PCEs with micronuclei). However, statistical analysis of the data revealed no significant increase of the micronuclei ratio per 2000 PCEs. The observed very slight increase, which is still in the range of the historical negative controls, was considered to be of no biological relevance and does not indicate a dose-dependent treatment-related effect.

- Ratio of PCE/NCE (for Micronucleus assay): The ratio of PCE to NCE for the test substance groups were from 0.8 to 1.0 and are not different to the respective vehicle control groups.

- Statistical evaluation (Mann-Whitney U-Test, p > 0.05): Dose levels of 500, 1000 and 1500 mg/kg bw caused no statistical significant enhancement (in a test for p < 0.05) in the frequency of micronuclei compared to the negative control at any preparation interval. Because only one male animal survived until terminal sacrifice, statistical evaluation of the male dose group 1500 mg/kg bw 48 h p. a. was not possible. Significant increases over the negative controls were seen in positive control group animals



Any other information on results incl. tables

Application of 1500 mg/kg bw of the test substance resulted in toxic symptoms like sedation and excitation with tremors in male and female animals 24 h p.a.. In the high dose group 1 male was found dead 24 h after application and 3 males and 2 females were found dead 48 h after application. Animals of the high dose group showed body weight reduction of up to 15.5%. All other animals survived to scheduled termination. In the low and middle dose groups body weight gain was positive but in comparison to the negative control animals, the body weight gain was reduced.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this micronucleus assay, there was no evidence of an aneugenic or clastogenic effect of the test substance leading to micronucleus formation in polychromatic erythrocytes of treated mice 24 h or 48 h after oral administration up to 1500 mg/kg bw.