Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
mutant histidine gene
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 the liver of Aroclor 1254 induced male Sprague Dawley rats.
Test concentrations with justification for top dose:
plate incorporation assay: 0, 50, 160, 500, 1600, 5000 µg/plate with and without S9 mix
preincubation assay: 0, 50, 160, 500, 1600, 5000 µg/tube with and without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item formed clear colourless solutions in DMSO.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
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.
Remarks:
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; independent repeat as preincubation testing (preincubation for 20 min. at 37 °C);
Testing for each strain and dose with and without S9 mix was performed in triplicate, which is also valid for solvent and positive controls.

DETERMINATION OF CYTOTOXICITY
- 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
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
None of the five strains used 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 and was confirmed by the results of the preincubation trials.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Substance precipitation occurred in the plate incorporation test at the dose of 5000 µg per plate and in the preincubation assay at the dose of 5000 µg per plate with S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY: There was no indication of a bacteriotoxic effect of the test item at doses of up to and including 5000 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Executive summary:

A bacterial reverse mutation test (Ames) according to OECD TG 471 was conducted with the following tester strains: Salmonella typhimurium TA 1535, TA 100, TA, 1537, TA 98, and TA 102. In this test the test substance non-mutagenic without and with S9 mix in the initially performed plate incorporation as well as in the preincubation modification, performed as independent repeat.