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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Disperse Red 184

Method

Target gene:
chromosomal aberrations in cultured mammalian cells
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Source of cells: cell bank of "Genetic Toxicology", HMR Germany
Test organism: cell line V79 of Chinese hamster lung fibroblasts
Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
The test compound was suspended in DMSO and tested at the following concentrations:

without S9-mix:
20 h: 3.16#, 10.0#, 31.6, 100.0, 316.0 and1000.0* µg/mL
28 h: 31.6#, 100.0#, 316.0 and 1000.0* µg/mL

with S9-mix:
20 h: 31.6#, 100.0, 316.0 and 1000.0 µg/mL
28 h: 316.0# and 1000.0 µg/mL

* not evaluated because of high toxicity
# not used because higher concentrations were evaluated
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Evaluation criteria:
Analysis of metaphases
The slides were coded and 25 - 100 metaphases per experimental group and cell culture were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed. Only metaphases with 22 ±2 chromosomes are included in the analysis. The metaphases were examined for the following aberrations: chromatid gap, chromosome gap, chromatid break, chromosome break, minute, double minute, chromatid deletion, chromosome deletion, chromatid exchanges including intrachanges, chromosome exchanges including intrachanges, dicentrics, pulverization and ring formation. Furthermore the incidence of polyploid metaphases was determined in 1000 cells of each cell culture.
Additionally a mitotic index was determined by counting the number of cells undergoing mitosis in a total of 1000 cells. The mitotic index is given in per cent.
After the metaphases had been evaluated, the code was broken. The values for the control group were compared with the results from the dose groups and the positive control at each sampling time.

Criteria for a valid assay
The assay was considered valid if the following criteria are met:
the solvent control data were within the laboratory's normal control range for the spontaneous mutant frequency
the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range

Criteria for a positive response
The evaluation of the results was performed as follows:
The test compound is classified as mutagenic if it induces a statistically significant increase in the aberration rate (without gaps) with one or more of the concentrations tested as compared with the solvent controls.
The test compound is classified as mutagenic if there is a concentration-related increase in the aberration rate (without gaps).
The test compound is classified as non-mutagenic if the tests are negative both with and without metabolic activation.
Statistics:
Statistics
The Biometry of the results was performed with a one-sided Fisher - Exact test.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)

Applicant's summary and conclusion

Conclusions:
There was an enhancement of the aberration rates inclusive and exclusive gaps 20 h and 28 h after the start of the treatment with the concentration of 316 µg/mL (relative mitotic index 19.4% and 44%, respectively) without S9-mix. In addition, an increased number of cells with break events was found in these groups. In the presence of S9-mix no relevant reproducible enhancement of metaphases over the range of the solvent control was found with any of the concentrations used.

As the increase in aberration rates were only observed at 316 µg/mL without metabolic activation and in the presence of cytotoxicity and visible test substance precipitation, the relevance and reliability of this result is questionable.
Executive summary:

In this study the potential of Disperse Red 184 to induce chromosome aberrations was investigated in V 79 cells of the Chinese hamster lung in vitro. For each experiment two cell cultures were used.

Disperse Red 184 was suspended in DMSO. Evaluation of the solubility of that suspension in cell culture medium showed that 1000 µg/mL was the highest practicable concentration and produced precipitate. Accordingly, a preliminary toxicity study was carried out using a maximum concentration of 1000 µg/mL and a range of lower dose levels down to 10 µg/mL.

Following treatment in the absence of S9 metabolic activation, severe toxicity was observed at 500 µg/mL and above. Survival declined in a dose-related manner reaching 29.2 % of the solvent control value at the highest dose level, 1000 µg/mL.

In the presence of metabolic activation (S9-mix) there was only a slight indication of toxicity up to the limit of solubility.

Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test compound solutions did not have any effect on these parameters.

Hence, the test compound was suspended in DMSO at the following concentrations:

without S9-mix:

20 h: 3.16#, 10.0#, 31.6, 100.0, 316.0 and 1000.0* µg/mL

28 h: 31.6#, 100.0#, 316.0 and 1000.0* µg/mL

with S9-mix:

20 h: 31.6#, 100.0, 316.0 and 1000.0 µg/mL

28 h: 316.0# and 1000.0 µg/mL

* not evaluated because of high toxicity

# not used because higher concentrations were evaluated

The highest concentrations produced no relevant lowering of the mitotic index in the presence of metabolic activation and a distinct lowering of the mitotic index in the absence of metabolic activation. Microscopic visible precipitation of the test compound was observed at 10 µg/mL and above in the absence of S9-mix and at 100 µg/mL and above in the presence of S9-mix.

After treatment with the test compound there was no relevant increase in the number of polyploid cells as compared with the solvent controls.

There was an enhancement of the aberration rates inclusive and exclusive gaps 20 h and 28 h after the start of the treatment with the concentration of 316 µg/mL (relative mitotic index 19.4% and 44%, respectively) without S9-mix. In addition, an increased number of cells with break events was found in these groups. In the presence of S9-mix no relevant reproducible enhancement of metaphases over the range of the solvent control was found with any of the concentrations used.

The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

As the increase in aberration rates were only observed at 316 µg/mL without metabolic activation and in the presence of cytotoxicity and visible test substance precipitation, the relevance and reliability of this result is questionable.