Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

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

Test material

Test material form:
solid: particulate/powder
Specific details on test material used for the study:
- Stability under test conditions: A stability test in the solvent, did not reveal significant degradation of the active ingredient.


Target gene:
histidine locus
Species / strain
Species / strain / cell type:
other: TA 1535, TA 100, TA 1537, TA 98, TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix from rat liver homogenates with Aroclor 1254 for enzyme induction
Test concentrations with justification for top dose:
Initial test (plate incorporation): 0, 50, 158, 500, 1581, 5000 µg/plate with and without S9-mix.
Independent repeat (preincubation method): 0, 50, 158, 500, 1581, 5000 µg/tube (with and without S9-mix).
Vehicle / solvent:
Ethylene glycol dimethylether
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: Na-azide (only TA 1535), nitrofurantoin (only TA 100), 4-nitro-1,2-phenylene diamine (TA 1537 and TA 98), mitomycin C (only TA 102 in plate incorporation assay), cumene hydroperoxide (only TA 102 in preincubation assay), 2-aminoanthracene.
The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C and cumene hydroperoxide were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for initial testing; as preincubation method for independent repeat (preincubation for 20 min. at 37 °C).
Testings for each strain and dose with and without S9 mix was performed in triplicate, which is also valid for solvent and positive controls.

- gross appraisal of background growth
- mutant count: A toxic effect was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls.
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 100, TA 1537, TA 98, TA 102
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
Untreated negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
None of the five strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix

- Precipitation: At 1581 μg per plate, the substance started to precipitate, so that for TA 1537 the dose 5000 μg per plate could not be used for assessment purpose.

ADDITIONAL INFORMATION ON CYTOTOXICITY: There was no indication of a bacteriotoxic effect at assessable doses of up to and including 5000 μg per plate and up to and including 1581 μg per tube.
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Executive summary:

The substance was investigated in a bacterial reverse mutation (Ames) test according to OECD TG 471 on Salmonella typhimurium strains TA 1535, TA 100, TA, 1537, TA 98, and TA 102 with and without S9 mix. The initial testing used doses up to and including 5000 µg/plate. The independent repeat was performed as preincubation for 20 minutes at 37 °C in the same dose range.

Substance precipitation occurred at 1581 µg/plate and above. Assessable doses up to and including 5000 µg per plate did not cause any bacteriotoxic effect. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed, therefore the test substance was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification.