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Genetic toxicity: in vitro

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in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
FAT 11178/H
FAT 11178/H
Details on test material:
- Name of test material (as cited in study report): FAT 11178/H
- Aggregate state at RT: solid
- Colour: black
- Batch Number: Op. 129
- Purity: 96.4%
- Stability of test article: pure: stable for years; in solvent: > 2 hours in water
- Storage Conditions: room temperature, light protected
- Expiration date: March, 1992


Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix
Test concentrations with justification for top dose:
10.0; 100.0; 333.3; 1000.0; and 5000.0 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: A. dest.
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.
Untreated negative controls:
Negative solvent / vehicle controls:
Positive controls:
W/o S-9: 10 µg/plate sodium azide (TA 1535, Ta 100), 50 µg/plate 4-NOPD (TA 1537, TA 1538, TA 98); w/ S-9: 10 µg/plate 2-AA (all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

DETERMINATION OF CYTOTOXICITY: Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.
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 as positive if either a significant dose-related 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 significant dose-related 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 test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent 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 results
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
To evaluate the toxicity of the test article a pre-study was performed with strains TA 98 and TA 100. The plates with the test article showed normal background growth up to 5000.0 µg/plate in strain TA 98 and TA 100, respectively. According to the dose selection criteria, the test article was tested at the following concentrations: 10.0; 100.0; 333.3; 1000.0; and 5000.0 µg/plate.

In both experiments no toxic effects, normally evidenced by a reduction in the number of revertants, occurred in the test groups with and without metabolic activation in all strains used. The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

Applicant's summary and conclusion

Interpretation of results (migrated information):

Under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test article is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

The study followed OECD guideline 471 (1981) and the principles of GLP. In the presence and absence of rat liver S-9 microsomal activation system and at doses of up to 5000 μg/plate, S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 did not show an increased number of revertant colonies. In addition, there was no indication of toxicity to bacteria. Therefore, no evidence of a mutagenic potential was associated with the test substance. The purity of the test item was 94.6%. A. dest. was used as a vehicle.