JAUNE REACTIF 181

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 August 1990 to 19 November 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 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Revised Chemical Substance Law (1987) according to the notification of December 9, 1986 by EA, Environmental Agency (No. 700); MHW, Ministry of Health and Welfare (No. 1039) and MITI, Ministry of International Trade and Industry (No. 1014), Japan.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

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
Lot/batch No.: BG 3247/TV 6
Stability: Pure: stable for 5 years, In solvent: > 72 hours in water, methanol, acetone, DMSO, and DMF
Storage condition of test material: room temperature
Expiration date of the lot/batch: 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: 5 years; In solvent: in water, methanol, acetone, DMSO and DMF > 72 hours
- Stability under test conditions:

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

Evaluated test concentrations: with and without S9 mix: 7 hours: 5.0 mg/mL, 18 hours: 0.5, 4.0, 5.0 mg/mL, 28 hours: 5.0 mg/mL

Vehicle / solvent:

culture medium without fetal calf serum

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation: Two days old logarithmically growing stock cultures more than 50 % confluent were trypsinised and a single cell suspension was prepared. The cells were seeded into Quadriperm dishes ( Heraeus, D-6450 Hanau, F.R.G. ) which contained microscopic slides (2 chambers per dish and test group). In each chamber 5E4 - 1E5 cells were seeded with regard to preparation time. The medium was MEM + 10 % FCS.
- Exposure: After 48 hours (7 hours, 28 hours preparation interval) and 55 hours (18 hours preparation interval) the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 20 µL/mL S9 mix. After 4 hours this medium was replaced with normal medium after rinsing twice with 'saline G'.
- Fixation: 5, 15.5 and 25.5 hours after the start of the treatment colcemid was added (0.2 µg/mL culture medium) to the cultures. 2 hours (7 hours
interval) or 2.5 hours later, (18 hours and 28 hours interval) the cells were treated on the slides in the chambers with hypotonic solution (0.4 % KCl) for 20 minutes at 37 °C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 absolute methanol + glacial acetic acid. All two slides per group were prepared. After fixation the cells were stained with Giemsa (Merck, D-6100 Darmstadt, F.R.G.).

NUMBER OF CELLS EVALUATED:
- At least 100 well spread metaphases per slide were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome number of 22 ± 1 were included in the analysis.

DETERMINATION OF CYTOTOXICITY
- To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points. A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

Statistics:

chi-square test

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Additional information on genotoxicity:
Both, in the absence and presence of S9 mix the test article did not increase significantly the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (0.5 % - 4.0 %) were in or near to the range of the control values : 0.0 % - 2.0 %. At fixation interval 18 hours in the absence of S9 mix in one sample treated with 4.0 mg/mL the aberration rate (4.0 %) was slightly increased as compared to the control value (2.0 %). This effect was observed only in one of the duplicate cultures. As this value falls within our historical data range: 0.0 % - 4.0 % the observed effect is not regarded as biologically relevant. Also, the occurrence of two exchanges is restricted to this sample and do not influence in this case the evaluation of the test article. Therefore, a dose-depended increase in the exchange rate (1 % with 4.0 mg; 1.5 % with 5.0 mg/mL) can not be evaluated unequivocally. In addition, the exchange rate found in the sample treated with 5.0 mg/mL is near to our historical exchange control range. 0.0 % - 1.0 %.

- Additional information on cytotoxicity:
In the pre-experiment on toxicity (colony forming ability) in the presence of S9 mix after treatment with 5.0 mg/mL the colony forming ability was distinctly reduced; in the absence of S9 mix only a slight reduction was observed. Higher concentrations were not applied. Also, in the main experiment the mitotic index was reduced in the high dose range (1.0 - 5.0 mg/mL) at fixation intervals 7 and 28 hours both in the presence and absence of S9 mix. At fixation interval 18 hours, only in the presence of S9 mix some indications of toxicity were observed (using the mitotic index as indicator).

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

FAT 40400/A is considered to be not clastogenic in this chromosomal aberration test.

Executive summary:

In a GLP-compliant chromosome aberration test, tested according to OECD guideline 473, chinese hamster lung fibroblast (V79) were exposed to the test substance with and without metabolic activation.

The test article FAT 40400/A was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro. Preparation of chromosomes was done 7 h (high dose), 18 h (low, medium and high dose), and 28 h (high dose) after start of treatment with the test article. The treatment interval was 4 h. In each experimental group two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations.

The following dose levels were evaluated:

without S9 mix:

7 h: 5.0 mg/ml

18 h: 0.5; 4.0; 5.0 mg/ml

28 h: 5.0 mg/ml

with S9 mix:

7 h: 5.0 mg/ml

18 h: 0.5; 4.0; 5.0 mg/ml

28 h : 5.0 mg/ml

 

The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response. Treatment with 5.0 mg/ml reduced distinctly the plating efficiency of the V79 cells in the presence of S9 mix; in the absence of S9 mix only a slight reduction was observed. Also, toxic effects were observed in the main experiment using the mitotic index as indicator for toxicity. Higher concentrations than 5.0 mg/ml were not applied. There was no relevant increase in cells with structural aberrations after treatment with the test article at any fixation interval either without or with metabolic activation by S9 mix. Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, FAT 40400/A is considered to be non-clastigenic in this chromosomal aberration test.