Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Specific details on test material used for the study:
- Stability under test conditions: The stability of the substance in the formulation was analytically verified for at least 24 hours.
- The substance in the test item is dissolved in approx. 30 % solvent. Test concentrations in the Ames-test were adjusted to the substance content (approx. 70 %).
Target gene:
histidine locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix from rat liver induced with phenobarbital/beta-naphthoflavone.
Test concentrations with justification for top dose:
Experiment I (plate incorporation), with and without S9-mix: 10, 25, 50, 160, 500, 1600, and 5000 µg/plate for each tester strain
Experiment II (pre-incubation method), with and without S9-mix: 10, 25, 50, 160, 500, 1600, and 5000 µg/plate for each tester strain
Vehicle / solvent:
EGDE (Ethylene glycol dimethylether)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
EGDE
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
cumene hydroperoxide
mitomycin C
other: 4-Nitro-1,2-phenylene diamine (used for TA1537), Anthracene-2-amine (used for all strains).
Remarks:
The positive control Anthracene-2-amine was only used with S9 mix, the others were used without S9-mix..
Details on test system and experimental conditions:
METHOD OF APPLICATION: Initially the plate incorporation method was used. As independent repeat the preincubation modification (20 min. at 37°C) was performed.
For each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
The toxicity of the substance was assessed by a gross appraisal of background growth on the plates for mutant determination. Moreover, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the solvent 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 TA1535, TA100, TA1537 and TA98 this increase should be about twice that of solvent controls. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Species / strain:
S. typhimurium TA 1535
Remarks:
plate incorporation trial
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Remarks:
plate incorporation trial
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Remarks:
plate incorporation trial
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Remarks:
plate incorporation trial
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Remarks:
plate incorporation trial
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Remarks:
preincubation trial
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/tube
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Remarks:
preincubation trial
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Remarks:
preincubation trial
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/tube for trials without S9-mix, no cytotoxicity for trials with S9-mix, but tested up to limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Remarks:
preincubation trial
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Remarks:
preincubation trial
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1600 µg/tube for trials without S9-mix, no cytotoxicity for trials with S9-mix, but tested up to limit concentrations
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 solvent controls in the plate incorporation test. This applied both to the tests with and without S9 mix and was confirmed by the results of the preincubation trials. The positive controls increased mutant counts to well over those of the solvent controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Substance precipitation occurred at 5000 µg per plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Doses of up to and including 5000 µg per plate did not produce bacteriotoxic effects in the plate incorporation method. The preincubation method showed the test item to produce strain-specific bacteriotoxic effects at the dose of 5000 µg per plate.
Executive summary:

A bacterial reverse mutation test (Ames test) was conducted according to OECD TG 471 to investigate the potential of the test substance to induce gene mutations. The assay was performed in two independent experiments, the initial plate incorporation and the subsequent preincubation test, both with and without S9 mix and the following tester strains: Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98, and TA 102. The doses applied were up to and including 5000 µg/plate (the content of the substance in the test item, which is approx. 70 % in approx. 30 % solvent, was considered).

Substance precipitation occurred at 5000 µg per plate. None of the five strains used showed a dose-related and biologically relevant increase in mutant counts over those of the solvent controls in the plate incorporation test. This applied both to the tests with and without S9 mix and was confirmed by the results of the preincubation trials.

The positive controls increased mutant counts to well over those of the solvent controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

In conclusion, the test substance was considered to be non-mutagenic in the Ames test with and without S9 mix.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A bacterial reverse mutation test (Ames test) was conducted according to OECD TG 471 to investigate the potential of the test substance to induce gene mutations. The assay was performed in two independent experiments, the initial plate incorporation and the subsequent preincubation test, both with and without S9 mix and the following tester strains: Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98, and TA 102. The doses applied were up to and including 5000 µg/plate (the content of the substance in the test item, which is approx. 70 % in 30 % solvent, was considered).

Substance precipitation occurred at 5000 µg per plate. None of the five strains used showed a dose-related and biologically relevant increase in mutant counts over those of the solvent controls in the plate incorporation test. This applied both to the tests with and without S9 mix and was confirmed by the results of the preincubation trials.

The positive controls increased mutant counts to well over those of the solvent controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

In conclusion, the test substance was considered to be non-mutagenic in the Ames test with and without S9 mix.

Justification for classification or non-classification

According to Regulation (EC) No 1272/2008, Annex I, no classification is warranted for genetic toxicity.