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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
Study period:
2006/03/10-2006/07/19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was conducted according to Good Laboratory Practice (GLP) and followed the OECD test guideline 471 (Bacterial Reverse Mutation Test).
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
This test substance shows spontaneous degradation in water and thus the post degradation products have been tested in this assay. Therefore, this assay was performed with a multi-constituent test material of the degradation products formed in aqueous environment.

Method

Target gene:
His Operon / Trp Operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
- Type and identity of media: Vogel-Bonner HIS deficient MGA medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: mutations in the His operon
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
- Type and identity of media: Vogel-Bonner HIS deficient MGA medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: mutations in the His operon
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
- Type and identity of media: Vogel-Bonner HIS deficient MGA medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: mutations in the His operon
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: Vogel-Bonner HIS deficient MGA medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: mutations in the His operon
Species / strain / cell type:
E. coli WP2
Details on mammalian cell type (if applicable):
- Type and identity of media: Vogel-Bonner TRP deficient MGA medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: mutations in the Trp operon
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)
Test concentrations with justification for top dose:
15, 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Positive controls:
yes
Positive control substance:
9-aminoacridine
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Details on test system and experimental conditions:
The dose range of the test material was 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test was performed by mixing 0.1 mL of bacterial culture, 0.1 mL of test material formulation, 0.5 mL of phosphate buffer or S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar. Ten concentrations of the test material (single dose) and a vehicle control (sterile distilled water) were tested. In addition, 0.1 mL of the maximum concentration of the test material and 2 mL of molten, trace histidine or tryptophane supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. The plates were incubated for a nominal 48 hours at 37°C after an initial overnight equilibration period and then assessed for revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.
Evaluation criteria:
NA
Statistics:
NA

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
NA

Any other information on results incl. tables

NA

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without S9

The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction.

The method has been designed to comply with the requirements of the Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries. The method also complies with the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC, and the USA, EPA (TSCA) OPPTS harmonised guidelines (870.5100, Aug 1998).

This test substance shows spontaneous degradation in water and thus the post degradation products have been tested in this assay. Therefore, this assay was performed with a multi-constituent test material of the degradation products formed in aqueous environment.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia soli strain WP2uvrA were treated with the test material using the Ames plate incorporation method at six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity test and was 15 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Results.

The vehicle (sterile distilled water) and untreated control plates produced counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused a visible reduction in the growth of the bacterial background lawn at the maximum recommended dose level (5000 µg/plate), in the presence of S9 only, to the majority of bacterial strains. The sensitivity of the bacterial tester strains to the toxicity of the test material varied slightly between strain type, exposure with S9 and Experiment number. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix.

No toxicologically 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.

A small, statistically significant increase in revertant colony frequency was observed in bacterial strain TA 1535, without S9 only, in Experiment 1 at 15 µg/plate. This response was considered not to be toxicologically significant because the response was non-reproducible, the mean count was only 1.60 times the concurrent vehicle control value and the revertant counts at 15 µg/plate were within the acceptable range for the tester strain.

Conclusion.

The test material was considered to be non-mutagenic under the conditions of this test.