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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name: FAT 92'504/A
Terasil blau GRN Isomerengemisch roh feucht
- Batch-No.: 1/95
Aggregate State at RT: solid
Colour: dark blue
Analysis: cf. data in the sponsor's files
Purity: 46 % (active ingredient)
Stability: Pure: stable until April, 1999
Storage: room temperature
Expiration Date: April 1999

On the day of experiment, the test article was dissolved in DMSO. The solvent was chosen because of its solubility properties and its relative non toxicity to the bacteria.
The test article did precipitate in the overlay agar at concentrations of 2500 µg/plate and above.

Method

Target gene:
Histidine auxotrophs
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Microsomal Fraction S9 Mix from rats
Test concentrations with justification for top dose:
33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate (active ingredient)
The highest dose of 5000 µg/plate was chosen on the basis of a pre-toxicity experiment, where no toxic effects were observed.
Vehicle / solvent:
- distilled water for TA 1535 and TA 100 without metabolic activation,
- DMSO for TA 1537, TA 98 without metabolic activation,
- DMSO for all strains with metabolic activation.
Controls
Untreated negative controls:
yes
Remarks:
Concurrent untreated and solvent controls were performed
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: Without S9: For TA 1535 and TA 100: Sodium azide and for TA 1535 and TA 98:4-nitro-o-phenylene-diamine, With S9: 2-aminoanthracene for all the strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation);
Evaluation criteria:
The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.

A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
A test article is considered mutagenic if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No toxic effects, evident as a reduction in the number of revertants, occurred in any of the test groups with and without metabolic activation up to the highest concentration.
The plates incubated with the test article showed normal background growth at all concentrations with and without metabolic activation.

Substantial, dose dependent increases in revertant colony numbers were observed in strains TA 98 and TA 1537 following treatment with FAT 92504/A; Terasil blau GRN Isomerengemisch roh feucht in the absence and presence of metabolic activation (S9 mix) in both experiments. TA 100 showed minor increases of colony numbers in the first experiment with and without metabolic activation but this effect could not be reproduced in the second experiment.

The enhancement factor of three was exceeded at concentrations of 333 µg/plate and above in the first and 1000 µg/plate and above in the second experiment in the absence of metabolic activation using strain TA 98. With metabolic activation the threshold of three was exceeded at a concentration of 1000 µg/plate and above in both experiments. In strain TA 1537 this threshold was reached or exceeded at 333 µg/plate and above in the presence or absence of metabolic activation in the first experiment and at 333 µg/plate or higher in the absence of metabolic activation and at 33 µg/plate and above with metabolic activation in experiment II.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
FAT 92504/A was found to induce gene mutations by frameshift with the strains Salmonella typhimurium TA 98 and TA 1537.
Executive summary:

This study was performed to investigate the potential of FAT 92504/A (mixture of Disperse Blue 054 and Disperse Blue 077) to induce gene mutations according to the plate incorporation test (experiments I and II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The assay was performed in two independent experiments, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate (active ingredient). No toxic effects, evident as a reduction in the number of revertants, occurred in any of the test groups with and without metabolic activation up to the highest concentration. The plates incubated with the test article showed normal background growth at all concentrations with and without metabolic activation. Substantial, dose dependent increases in revertant colony numbers were observed in strains TA 98 and TA 1537 following treatment with FAT 92'504/A; Terasil blau GRN Isomerengemisch roh feucht in the absence and presence of metabolic activation (S9 mix) in both experiments. TA 100 showed minor increases of colony numbers in the first experiment with and without metabolic activation but this effect could not be reproduced in the second experiment. The enhancement factor of three was exceeded at concentrations of 333 µg/plate and above in the first and 1000 µg/plate and above in the second experiment in the absence of metabolic activation using strain TA 98. With metabolic activation the threshold of three wasexceeded at a concentration of 1000 µg/plate and above in both experiments. In strain TA 1537 this threshold was reached or exceeded at 333 µg/plate and above in the presence or absence of metabolic activation in the first experiment and at 333 µg/plate or higher in the absence of metabolic activation and at 33 µg/plate and above with metabolic activation in experiment II. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article (FAT 92504/A) did induce gene mutations by frameshifts in the genome of the strains TA 98 and TA 1537.