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Toxicological information

Genetic toxicity: in vitro

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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:
2004
Report Date:
2004

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Method

Target gene:
histidine locus of S. typhimurium
tryptophan locus of E. coli
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: uvrB-deletion, rfa-mutation
Species / strain / cell type:
E. coli WP2
Additional strain / cell type characteristics:
other: uvrA-deletion
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from the liver of phenobarbitone/ beta-naphthoflavone induced rats
Test concentrations with justification for top dose:
preliminary toxicity test: 0.15-5000 µg/plate
mutation test 1 (dose finding; with and without metabolic activation): 1.5-5000 µg/plate
mutation test 2 (main test), without metabolic activation: [TA100] 1.5-1500 µg/plate; [TA1535 and TA1537] 5-5000 µg/plate; [WP2uvrA- and TA98] 15-5000 µg/plate; with metabolic activation: [WP2uvrA-, TA98, TA1535, and TA1537] 15-5000 µg/plate; [TA100] 5-5000 µg/plate
Vehicle / solvent:
DMSO (dried prior to use with molecular sieves (sodium alumino-silicate, nominal pore diameter 4 x 10exp-4 µm)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG; for WP2uvrA-, TA100, and TA1535), 9-Aminoacridine (9AA; for TA1537), 4-Nitroquinoline-1-oxide (4NQO; for TA98), 2-Aminoanthracene (2AA; for TA100, TA1535, TA1537, and WP2uvrA-), Benzo(a)pyrene (BP; for TA98)
Remarks:
The positive controls ENNG, 9AA, and 4NQO were used without S9 mix; the positive controls 2AA and BP with S9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION for dose finding and main test: in agar (plate incorporation) in triplicate; plates were incubated at 37 °C for approx. 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity was preliminarily assessed based on relative total growth and gross appraisal of the background growth.
Evaluation criteria:
The test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnett's method of linear regression (Kirkland D J, Statistical Evaluation of Mutagenicity Test Data, UKEMS Subcomittee on Guidelines for Mutagenicity Testing, Report - Part III, Cambridge University Press, 1989).

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, E. coli WP2uvrA-
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:
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Result of preliminary toxicity test: The test material was non-toxic to the background lawns of the strains of bacteria used (TA100 and WP2uvrA-), however, a substantial decrease in the frequency of revertant colonies was observed at 1500 and 5000 µg/plate, without and with metabolic activation respectively.

Results of mutation tests: The test material caused a visible reduction in the growth of the bacterial background lawn to all of the Salmonella tester strains initially at 500 and 1500 µg/plate, without and with metabolic activation respectively. A substantial decrease in the frequency of revertant colonies was also observed in tester strain TA100 at 150 and 500 µg/plate, without and with metabolic activation, respectively. No toxicity was observed to tester strain WP2uvrA-. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on strain type and presence or absence of metabolic activation.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: was observed at and above 1500 µg/plate with an opaque film noted at 5000 µg/plate, this did not prevent the scoring of revertant colonies.
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 assay (Ames) according to OECD TG 471 was conducted for the evaluation of point mutagenic effects. In this assay 4 histidine auxotrophic mutants of Salmonella typhimurium (TA 1535, TA 1537, TA 100, TA 98) and 1 tryptophan auxotrophic mutant of Escherichia coli (WP2uvrA-) were used. The doses ranged between 1.5 and 5000 µg/plate, based on a preliminary toxicity assay and depending on the strain type and presence or absence of metabolic activation. The main mutation test was conducted independently from a dose-finding mutation test using similar doses.

In the main test the test substance caused a visible reduction in the growth of the bacterial background lawn to all of the Salmonella tester strains initially at 500 and 1500 µg/plate, with the exception of TA100 showing already a substantial decrease in the frequency of revertant colonies at 150 µg/plate, all without and with metabolic activation respectively. No toxicity was observed to tester strain WP2uvrA-.

The vehicle (DMSO) control plates gave counts of revertant colonies in the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation, thus the test substance was considered to be non-mutagenic in the bacterial reverse mutation assay.