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:
24 May - 3 June 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Scientifically acceptable; Guideline conform; GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982
Report Date:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
The animals were treated twice at interval of 24hours at dose levels of 50, 500 and 5000 mg/kg bw. Bone marrow cells were collected 6 hours after the second treatment.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Pharma Forschung Toxikologie, Hattersheim, Germany
- Age at study initiation:7 to 12 weeks
- Weight at study initiation: 27 - 38 g (males), 22 - 31 g (females)
- Housing: groups of five animals in Makrolon type-3 cages with wire mesh tops and softwood bedding
- Diet (e.g. ad libitum): pelleted Altromin diet 1324 ad libitum
- Water (e.g. ad libitum): Community tap-water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 27°C
- Humidity (%): 42 - 65%

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: sesame oil
- Amount of vehicle (if gavage or dermal): 25 mg/ kg bw
Details on exposure:
A pre study revealed that 5000 mg/kg bw/d was the maximum tolerated dose.

Treatment:
Five males and five females were assigned to each test group. Animals were treated orally twice (one application per day) with either three doses of the test substance, the positive control (Cyclophosphamide) or the vehicle control (deinised water). Six hours after the last application the bone marrow of the animals was prepared.

Preparation of the animals:
The animals were killed and the femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1000 rpm for 5 min and the supernatant was discarded. A small drop of re-suspended cell pellet was spread on a slide. The smear was air-dried and stained with May-Grünwald/Giemsa.

Bone marrow smears were scored microscopically for micronucleated polychrmoatic erythrocytes. To analyse target organ cytotoxicity the ratio between polychrmoatic and normochromatic erythrocytes was determined. 2000 polychromatic erythrocytes per animal were analysed for appearance of micronuclei. The count of micronuclei containing normocytes was determined.
Duration of treatment / exposure:
The animals were treated twice at interval of 24hours.
Frequency of treatment:
twice
Post exposure period:
6 hours after second treatment
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 500, 5000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide, 100 mg/kg bw

Examinations

Tissues and cell types examined:
2000 immature erythrocytes per animal are examined for the incidence of micronucleated immature erythrocytes.
1000 mature erythrocytes per animal are examined for the incidence of micronucleated mature erythrocytes.
The proportion of immature among total (immature + mature) erythrocytes was determined.
Details of tissue and slide preparation:
Staining Methods: May-Gruenwald
Statistics:
Performed by computer programm "Diamant" (Code: SG-PA Nr. 4453) programmed by the Department of Praktische Methamatik of the Hoechst AG, Frankfurt, Germany.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
For all treated animals the incidences of micronucleated immature erythrocytes were comparable to control values. Also the incidence of micronucleated mature erythrocytes were not changed. The ratios of immature erythrocytes to total erythrocytes were not affected by the treatment.

Any other information on results incl. tables

Animals treated with up to 5000 mg/kg bw/d (2 applications, one daily) did not show any clinical sign. All animals survived until the study termination.

The number of micronuclei containing polychromatic erythrocytes and normocytes were not influenced by the treatment with Hostanox SE 10. The ratio polychromatic erythrocytes/normocytes did not differ to the ratio obtained from the negative control animals.

Cyclophosphamide (positive control) showed a significant increase of induced mirconucleus frequency.

In conclusion, it can be stated that during the study the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The oral application of Hostanox SE 10 to mice did not induce increased micronucleated polychromatic erythrocytes in bone marrow.
Executive summary:

The test substance was investigated for its micronuclei formation potential in bone marrow in mice. Each 5 males and 5 females were treated by gavage at daily doses of 0, 50, 500 and 5000 mg/kg bw for two days. No treatment related clinical effect was observed. The ratios of polychromatic erythrocytes to normochromatic erythrocyte were not affected by the treatment. No increase of micronuclei formation was observed for treated animals.