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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): FAT 40'224C
- Description: red powder

Test animals

Species:
mouse
Strain:
other: non-consanguinous OF-1 albino mice originating from an SPF colony
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: IFFA-CREDO, L'Arbresle, France
- Weight at study initiation: 25g
- Housing: Animals were housed 5 of the same sex per cage in Makrolon type III cages.
- Diet (e.g. ad libitum): Aliment Rats-Souris Charles River, produced by U.A.R., Villemoisson/Orge, France, ad libitum
- Water (e.g. ad libitum): ad libitum
- Quarantaine period: 1 week at Battelle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Vehicle: distilled water
The test substance was dissolved in distilled water to obtain a stock solution of 250 mg/mL.
Details on exposure:
Intragastric intubation: using 0.5 mL of a solution at 250 mg/mL per 25 g body weight.
Frequency of treatment:
single treatment
Post exposure period:
20, 44, 68 hours
Doses / concentrations
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Thio-TEPA (N, N', N"-triethylenethiophosphoramide), reference N° 509-227, made by Lederle Laboratories Ltd.
- Doses / concentrations: 20 mg/kg

Examinations

Tissues and cell types examined:
normochromatic and polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
First a preliminary range finding test was carried out by treating six groups of three female mice each at concentrations of 5500, 5000, 4000, 3000, 2000 and 1000 mg/kg body weight. As no death occurred within 72 hours, the concentration of 5000 mg/kg was chosen for the main-test.

TREATMENT AND SAMPLING TIMES:
1. Negative control (distilled water) 0.5 ml - sacrificed at 44 hours
2. Positive control (Thio-TEPA): 20 mg/kg - sacrificed at 44 hours
3. FAT 40'224/C 5000 mg/kg - Sacrificed at 20 hours
4. FAT 40'224/C 5000 mg/kg - Sacrificed at 44 hours
5. FAT 40'224/C 5000 mg/kg - Sacrificed at 68 hours

DETAILS OF SLIDE PREPARATION:
After sacrifice of the animals the femurs were taken and broken open at one end. Bone marrow cells were suspended in foetal calf serum using a small syringe, and the cells were centrifuged at 120 x g for 5 minutes. The supernatant was removed with a Pasteur pipette, cells were spread on a microscope slide and the smears allowed to dry in air. The following day smears were stained with Giemsa (1:6 in water), dried and mounted with a coverslip.

METHOD OF ANALYSIS:
Two types of erythrocytes were observed in the bone marrow smears: normochromatic (mature red blood cells about to pass into the blood stream) and polychromatic (immature red blood cells). The latter are stained blue by Giemsa for around 24 h after the expulsion of the erythroblast nucleus: the staining is probably due to traces of RNA remaining in these cells. The proportion of polychromatic erythrocytes containing one or more micronuclei was compared with the total number of polychromatic erythrocytes, and statistical comparisons were made between these ratios for the different groups. A minimum of 500 polychromatic erythrocytes were counted per smear (two smears per animal). In each smear an evaluation was made of the number of nucleated cells and the two types of erythrocytes (normochromatic and polychromatic) were counted up to a total of 2000 erythrocytes per animal. This was done in order to gain information on the mode of action of the test compound in bone marrow cells, and also to identify possible artifacts. All slides were given coded labels and were microscopically analysed without knowledge of their treatment groups.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
At low magnification of the microscope no noticeable differences in bone marrow nucleated cells were observed between animals treated with FAT 40'224/C and negative controls. In the positive control group (Thio-TEPA) decreased numbers of nucleated bone marrow cells were noted.

There was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in animals exposed to 5000 mg/kg of FAT 40'224/C compared to negative control animals. In animals treated with Thio-TEPA there was a statistically significant increased number of micronucleated cells.
The ratio of polychromatic to normochromatic erythrocytes was markedly decreased in mice treated with Thio-TEPA. There was no statistically significant difference in this ratio between animals treated with FAT 40'224/C and the negative controls.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test substance is not considered to be mutagenic in the micronucleus test.
Executive summary:

In a GLP-compliant micronucleus test, tested according to OECD guideline 474, 5 non-consanguinous OF-1 albino mice originating from an SPF colony per sex per treatment group were treated once by oral gavage with the test substance 5000 mg/kg bw dissolved in distilled water followed by a 20, 44, 68 hour exposure period. In a preliminary range finding study the concentration of 5000 mg/kg bw was chosen for the main-test. A positive control (Thio-TEPA) administered at a concentration of 20 mg/kg bw showed pronounced evidence of mutagenicity 44 h after administration.

No mutagenic effect was observed in bone marrow smears taken 20, 44 and 68 h after administration of the test substance.