JAUNE REACTIF 181

Genetic toxicity: in vivo

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:

03 April 1990 to 04 July 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:
BG 3247/TV 6
- Expiration date of the lot/batch:
March 1995

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
room temperature
- Stability under storage conditions:
Pure: stable; In vehicle: stable in water, methanol, DMSO, DMF, acetone >72 hours

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal which has been used for many years 'as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test

Sex:

male/female

Details on test animals or test system 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

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

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 40400/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 40400/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:

FAT 40400/A did not induce micronuclei in bone marrow cells of the mouse.

Executive summary:

A study was performed to investigate the potential of FAT 40400/A to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD guideline 474. The test article was formulated in carboxymethylcellulose suspension (1 %). This suspending agent was used as negative control. The volume administered orally was 20 ml/kg b.w. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The 5000 mg/kg b.w. dose level of the test article was investigated at 24 h, 48 h, and 72 h preparation interval. In a pre-experiment this dose level was estimated to be the maximum attainable dose.

After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects. In comparison to 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. An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FAT 40'400/A is considered to be non-mutagenic in this micronucleus assay.