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Genetic toxicity: in vivo

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in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 - 26 Sept 1990
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
no version/adoption date specified
according to guideline
EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)
Version / remarks:
19 Sept 1984
GLP compliance:
Type of assay:
mammalian germ cell cytogenetic assay

Test material

Constituent 1
Reference substance name:
EC Number:
Cas Number:

Test animals

other: BOR:NMRI
Details on species / strain selection:
substrain SPF (Han.)
Details on test animals or test system and environmental conditions:
- Source: Frima Winkelmann, Versuchtierzucht, Gartenstr. 30, Borchen, Germany
- Age at study initiation: adult, about 3 months
- Weight at study initiation: males 22.3 - 25.0 g / females 20.7 - 24.3 g
- Assigned to test groups randomly: yes
- Housing: collective housing
- Cage type: Macrolon type II / max. 5 animals/cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

- Temperature (°C): 20 +/- 2
- Humidity (%): 55 +/- 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): 30 ml/kg bw
Details on exposure:
In a preliminare dose-range finding experiment, doses of 15,000, 10,000, 5,000 and 2,000 mg/kg bw were administered orally in a single application to 2 male and 2 female mice. A dose of 15.000 mg/kg was considered to be near the maximal tolerated dose. Based on the results of the dose-range fmding test, the test article was administered in a dose of 15,000 mg/kg bw in the main test. Negative control animals received only the vehicle (corn oil), whilst positive control animals were treated with Cyclophosphamide.

In each case a single oral administration in a volume of 30 ml/kg body weight (10 ml;kg for the positive control) was made. 5 male and 5 female animals were used in each group.prepared in corn oil.

PREPARATION OF DOSING SOLUTIONS: The test solution was prepared by suspending an appropriate amount of the prewarmed test article (70°C) at a volume of 50% in corn oil.
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
24 h, 48 h, and 72 h
Doses / concentrations
Dose / conc.:
15 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Neg. control (24 h): 5m, 5f
Neg. control (48 h): 5m, 5f
Neg. control (72 h): 5m, 5f
Pos. control (24 h): 5m, 5f
Treatment (24 h): 5m, 5f
Treatment (48 h): 5m, 5f
Treatment (72 h): 5m, 5f

a total of 35m and 35f
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (trade name: Endoxan)
- Route of administration: oal
- Doses / concentrations: 40 mg/kg bw
- Batch no.: 078487
- Vehicle for positive control: aqua bidest.


Tissues and cell types examined:
Monochromatic and polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: On the basis of the dose-range finding results, a dose of 15,000 mg/kg body weight was considered to be near the MTD (maximal tolerated dose) and was therefore chosen for the main study.

TREATMENT AND SAMPLING TIMES: 24 h, 48 h, and 72 h

After scrifice, femora were removed and the bone marrow was suspended in feta! calf serum. Samples were centrifuged at 1600 x g and subsequently decanted. One drop of each suspension was then smeared on a slide by means of a second slide. Two preparations were made from each animal, dried, fixed in absolute methanol (99%) for 5 min and then allowed to dry in air. Slides were stained with a May-Grunwald and Giemsa solution.

The cells were examined under a microscope at thousandfold magnification. A total of 1000 polychromatic erythrocytes were examined on each slide and the number of micronucleated cells in each sample was recorded. The ratio of polychromatic erythrocytes to normochromatic (mature) erythrocytes was calculated for a sample of 1000 cells.
Evaluation criteria:
Increase in the incidence of micronucleated polychromatic erythrocytes in any sex or at any time point. Percentage of polychromatic erythrocytes. Cell counts are based on a total of 1000 cells per animal. Historical control data of the laboratory indicate that an incidence of up to 8 micronucleated cells per 1000 polychromatic erythrocytes may be considered to be within normal limits.
In each test group and the corresponding negative control group, the number of polychromatic erythrocytes with micronuclei, the number of polychromatic erythrocytes, the number of normochromatic erythrocytes and the ratio of poly- to normochromatic erythrocytes were analysed statistically with the t-test for independent samples. Mean values of the negative and positive control groups were compared with the Mann-Whimey U-Test.

Results and discussion

Test results
Dose tested was near MTD (max. tolerable dose), causing reduced activity and piloerection
Vehicle controls validity:
Negative controls validity:
not examined
Positive controls validity:
Additional information on results:
Clinical findings in all animals treated with a single dose of 15,000 mg/kg bw of the test article were piloerection and reduced activity. No animals died in the main study.

The number of polychromatic erythrocytes with micronuclei was significantly increased 24 h post-injection in the positive control animals. This result confirms the high sensitivity of this particular strain of mice.

In relation to the untreated controls, the number of polychromatic erythrocytes with micronuclei was not increased in any test group (24, 48, 72 h p.a.).

In none of the parameters investigated, a significant difference was found between female animals treated with the test article and negative control animals. The number of polychromatic erythrocytes without micronuclei in group males (48 h p.a.) as well as the ratio of polychromatic to normochromatic erythrocytes was significantly increased. The same parameters were decreased in group males (72 h p.a.). These effects might be explained by a stimulation of the formation of polychromatic erythrocytes in male mice after 48h by the test article resulting in a subsequent increase of normochromatic erythrocytes, which arise from polychromatic erythrocytes, after 72h.

In conclusion, the test article is considered to be non-mutagenic under the experimental conditions of this study.

Any other information on results incl. tables

Please refer to pdf document attached for detailed tables.

Applicant's summary and conclusion

Interpretation of results: