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Genetic toxicity in vitro

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Reference
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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions: only 4 strains tested
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
, no E.coli WP2 or S. typhimurium TA 102 tested
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix from liver homogenates of Aroclor 1254 induced male rats
Test concentrations with justification for top dose:
initial testing (plate incorporation; with and without S9 mix): 19.2-12000 µg/plate
repeat (plate incorporation; with and without S9 mix): 19-9600 µg/plate;
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Na-azide (NaN3, only TA 1535), nitrofurantoin (NF, only TA 100), 4-nitro-1,2-phenylene diamine (4-NPDA, TA 1537 and TA 98), 2-aminoanthracene (2-AA, all strains)
Remarks:
The positive controls NaN3, NF, and 4-NPDA were used without S9 mix; the positive control 2-AA was used with S9 mix.
Details on test system and experimental conditions:
Strains were obtained from Prof. B. Ames in 1982 and maintained properly.

METHOD OF APPLICATION: According to Ames et al. (Proc. nat. Acad. Sci. 70, 2281-2285, 1973; Mutation Res. 31, 347-364, 1975).
Plate incorporation for initial testing and independent repeat; for each strain and dose four plates were used in parallel, which was also valid for solvent and positive controls.

DETERMINATION OF CYTOTOXICITY
- 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. This increase should be about twice that of negative controls.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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:
Evidence of mutagenic activity was not found. Neither a dose-related doubling of the mutant count nor a biologically relevant increase in the same, in comparison with the negative controls, was observed.
The positive controls had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Substance precipitation occurred at and above 1200 µg/plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Doses up to and including 19.2 µg/plate did not cause any bacteriotoxic effects. The total bacteria counts remained unchanged. No inhibition of growth was observed. At higher doses the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a very limited extent up to 12000 µg/plate for evaluation purposes.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Executive summary:

A bacterial reverse mutation assay according to the protocol of Ames et al. (Proc. nat. Acad. Sci. 70, 2281-2285, 1973; Mutation Res. 31, 347-364, 1975) was conducted for the evaluation of point mutagenic effects. In this assay 4 strains of Salmonella typhimurium were used (TA 1535, TA 100, TA 1537, and TA 98). The initial plate incorporation test used test substance doses of 19.2-12000 µg/plate. Due to bacteriotoxic effects doses for independent repeat were lowered to a maximum of 9600 µg/plate.

Substance precipitation occurred at and above 1200 µg/plate. Doses up to and including 19.2 µg/plate did not cause any bacteriotoxic effect. The total bacteria counts remained unchanged and thus no inhibition of growth was observed. At higher doses the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a very limited extent for evaluation purposes.

Evidence of mutagenic activity was not found. Neither a dose-related doubling of the mutant count nor a biologically relevant increase in the same, in comparison with the negative controls, was observed. The positive controls had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

For the assessment of genetic toxicity two Ames tests are available for the substance. Both were conducted comparable to the protocol of OECD TG 471, but have acceptable restrictions.

The first Ames test was carried out with the 5 tester strains S. Typhimurium TA 100, TA1535, TA98, TA1537, and E. coli WP2uvrA-. Doses up to 5000 µg/plate with and without S9 mix were employed in a range finding test and, due to bacteriotoxic effects, up to 1000 µg/plate in the mutagenicity test.

The test substance did not cause a doubling or more of the number of revertant colonies compared to the solvent control, whereas the positive controls increased the number of revertant colonies by more than twice, and thus indicated that the test was carried out appropriately.

The second Ames test used only 4 S. Typhimurium strains (TA 1535, TA 100, TA 1537, and TA 98), but was conducted more accurate compared to the first (e.g. additionally independent repeat of mutagenicity test, four replicate plates for each strain and dose). The initial plate incorporation test here used doses of 19.2-12000 µg/plate. Due to bacteriotoxic effects doses for independent repeat were lowered to a maximum of 9600 µg/plate.

Substance precipitation occurred at and above 1200 µg/plate. Doses up to and including 19.2 µg/plate did not cause any bacteriotoxic effect. The total bacteria count remained unchanged and thus no inhibition of growth was observed. At higher doses the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a very limited extent for evaluation purposes.

Evidence of mutagenic activity was not found. Neither a dose-related doubling of the mutant count nor a biologically relevant increase in the same, in comparison with the negative controls, was observed. The positive controls had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Overall, the two negative Ames tests led to the conclusion that the substance is non-mutagenic in bacteria.


Justification for selection of genetic toxicity endpoint
The study with the more conscientiously conduction is selected. Nevertheless, both Ames-tests are relevant for the assessment of genetic toxicity.

Justification for classification or non-classification

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