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Toxicological information

Genetic toxicity: in vitro

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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:
1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Pre-test on cell growth inhibition with 4h and 24h treatment was performed in order to determine the toxicity of the test article and also in the evaluations of results. These deviations had no detrimental impact on the outcome of the study.
Principles of method if other than guideline:
None
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Solid
Details on test material:
None

Method

Target gene:
None
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimal essential Medium; SEROMED; D-12247 Berlin)
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix liver microsomal fraction
Test concentrations with justification for top dose:
- Test concentrations in the pre-test on toxicity (with and without S9 mix, exposure time 4 hours and 24 hours):
39.1, 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0, 5000.0 µg/ml.

- Main experiment (Without S9-mix):
40, 80, 120, 160 µg/ml.

- Main experiment (With S9-mix):
30, 60, 90, 120 µg/ml.
Vehicle / solvent:
On the day of the experiment (immediately before treatment), the test article was dissolved in deionised water.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
MES (Without metabolic activation); CPA (with metabolic activation)
Details on test system and experimental conditions:
DURATION
- Exposure duration: 18 hours without S9 mix; 4 hours with S9 mix.

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

DETERMINATION OF TOTOXICITY
- In the pre-test the toxicity of the test article was examined using the determination of the cell number. Cell numbers of two cultures (10 coordinate defined fields per culture) were determined for each experimental group.



Evaluation criteria:
A test article is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- no significant increase of the number of structural chromosome aberrations are observed.

A test article is classified as mutagenic if:
- the number of induced structural chromosome aberrations are not in the range of our historical control data (0.0 - 4.0 % aberrant cells
exclusive gaps).
- either a concentration-related or a significant increase of the number of structural chromosome aberrations are observed.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the Fisher's-exact-test. Evaluation was performed only for cells carrying aberrations exclusive gaps.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
-In the pre-test, no precipitation of the test article in culture medium was observed neither in the presence nor in the absence of S9 mix. No influence of the test article on the pH value or osmolarity was observed (solvent control: 280 mOsm, pH 7.3 versus 284 mOsm and pH 7.3 at 5000 ug/ml).

- In the main experiment in the presence and absence of S9 mix no reduced mitotic indices (81.1 % and 95.4 % of control) were observed. In the absence of S9 mix the cell number was not reduced after treatment with 120 ug/ml (85 % of control) whereas the cell number in the presence of S9 mix was reduced to 55 % of control. However, at the next higher concentration in the absence of S9 mix (160 ug/ml) cytogenetic evaluation was not possible, since no mitotic cells were observed.

- In the absence and presence of S9 mix, neither a significant nor a statistically relevant increase in the number of cells carrying structural chromosome aberrations was observed. In the absence and in the presence of S9 mix, the aberration rates of the cells after treatment with the test article (1.0 % and 3.5 %) were near the range of the solvent control values (1.0% - 2.0%, see table 7) and within the range of our historical control data:
0.0 % - 4.0 %.

EMS (600 ug/ml) and CPA (0.71 ug/ml) were used as positive controls and showed distinct increases in cells with structural chromosome
aberrations.

Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article FAT 40061/E did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line).
Executive summary:

An in vitro screening assay was performed to assess the potential of FAT 40061/E to induce structural chromosome aberrations. Evaluation of cytogenetic damage induced in V79 cells (cell line from the lung of the Chinese Hamster) was performed in the absence and the presence of metabolic activation. Cultures of the highest evaluable concentration were investigated after 18 hours.

The experiment was performed according OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) and follows GLP methodology.

In the absence and the presence of S9 mix the highest possible test article concentrations were evaluated for cytogenetic damage. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

- Test concentrations in the pre-test on toxicity (with and without S9 mix, exposure time 4 hours and 24 hours): 39.1, 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0, 5000.0µg/ml.

 

- Main experiment (Without S9-mix): 40, 80, 120, 160µg/ml.

- Main experiment (With S9-mix): 30, 60, 90, 120µg/ml.

The highest concentration used in a pre-test on toxicity (5000 ng/ml) was chosen with regard to the current OECD-Guideline for in vitro mammalian cytogenetic tests. Dose selection of the cytogenetic experiments was performed considering the toxicity data of the pre-test. In this study no influence of the test article on the pH value or osmolality was observed.

In the main experiment, reduced cell numbers were observed in the presence of 120 ng/ml S 9 mix after treatment. In the absence of S9 mix, at the evaluated concentration, neither reduced mitotic indices nor reduced cell numbers were observed.

Neither a significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test article.

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) 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 chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

Therefore, FAT 40061/E is considered to be non-mutagenic in this chromosome aberration test.