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other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.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:
1990
Report Date:
1990

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)
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: FAT 40'400/A
Batch No: BG 3247/TV 6
Stability: Pure: stable; In solvent: > 72 hours in water, methanol, acetone, DMSO, and DMF
Storage: room temperature
Expiration date: March 1995

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf, CH-4414 Füllinsdorf/Basel, Switzerland
- Age at start of acclimatization: minimum 10 weeks
- Weight at study initiation: approximately 30 g
- Fasting period before study: 18 hours
- Housing: Individually in Makrolon type-1 cages with wire mesh top (EHRET GmbH, D-7830 Emmendingen) with granulated soft wood bedding (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
- Diet: pelleted standard diet (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
- Water: tap water, ad libitum (Gemeindewerke, D-6101 Roßdorf, F.R.G.)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: carboxymethylcellulose-suspension (1%)
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals.
- Concentration of test material in vehicle: 5000 mg/kg bw
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw
Frequency of treatment:
Single treatment
Post exposure period:
24, 48, and 72 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
5000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide;
- Route of administration: orally
- Doses: 40 mg/kg bw
- Volume administrated: 10 mL/kg bw
- Vehicle: physiological saline

Examinations

Tissues and cell types examined:
Normochromatic and polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A preliminary study on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity study. The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on survival within 72 hours.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 mL syringe. The cell suspension was centrifuged at 1,500 rpm for 5 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grunwald (Merck, D-6100 Darmstadt F.R.G.)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg F.R.G.). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1,000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.
Evaluation criteria:
A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points. A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
In vivo, but no cytotoxicity was observed.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg b.w., FAT 40'400/A suspended in 1% CMC. The volume administered was 20 mL/kg b.w. One male animal expressed eyelid closure and one male animal showed a reduction of spontaneous activity.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with FAT 40'400/A were in the same range as compared to the negative control groups.
- Ratio of PCE/NCE: The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that FAT 40'400/A had no cytotoxic properties.
- 40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronucleus frequency.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test substance did not induce micronuclei in bone marrow cells of the mouse.
Executive summary:

In a GLP-compliant erythrocyte micronucleus test, tested according to OECD guideline 474, 6 NMRI mice per sex were treated once by oral gavage with the test substance (5000 mg/kg bw) dissolved in 1% CMC followed by a 24, 48 and 72 hours post exposure period. In a pre-experiment 5000 mg/kg bw was estimated to be the maximum tolerated dose. The finding that the mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the negative controls, indicated that the test substance had no cytotoxic properties. In comparison with the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article and therefore it could be concluded that the test substance did not induce micronuclei in this study.