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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:
Between 29 October 1998 and 13 november 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Commission Directive 92/69/EEC, B14 (Ames test).
Principles of method if other than guideline:
The study was conducted according to Safepharm Standard Method 700.04 and was designed to assess the mutagenic potential of the test material using a bacterial / microsome test system. The study was based on the in vitro technique described by Ames and his co-workers and Garner et al, in which mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium and tryptophan auxotrophs of Escherichia coli to various concentrations of the test material. The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EC and US EPA (TSCA) guidelines.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
histidine synthesising gene
Species / strainopen allclose all
Species / strain / cell type:
bacteria, other: Salmonella typhimurium: TA1535, TA1537, TA98 and TA100. Escherichia coli WP2uvrA-
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced, rat-liver S9.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 50 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50 ... 5000 µg/plate
Vehicle / solvent:
Solvent: Dimethyl sulphoxide
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
All of the strains were stored at -196C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limitedl lot number 201417 1/03) and incubated at 37 C for approximately 10 hours.

Rationale for test conditions:
Not discussed
Evaluation criteria:
The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible dose-related and statistically (Dunnett's method of linear regression(5)) significant increase in the revertant count in at least one strain of bacteria. If a greater than twofold increase in revertant count is observed in two experiments then this is taken as evidence of a positive response.
Statistics:
Dunnett's method of linear regression(5) is used to determine statistical significance in the increase in the revertant count.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA100, WP2uvrA-
Metabolic activation:
not applicable
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(> 5000 µg/plate)
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
(>5000 μg/plate)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(> 5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
(> 5000 µg/plate)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(> 5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(> 5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
not applicable
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:
not applicable
Positive controls validity:
valid
Additional information on results:
Observations:
Solvent control plates gave counts of revertant colonies within the normal range.

All positive control chemicals gave increases in revertants, either with or without the metabolising system as appropriate, within expected ranges. No statistically significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of the substance, either with or without metabolic activation. The substance was found to be non-mutagenic under the conditions of this test.
Remarks on result:
other: other: preliminary test
Remarks:
To test cytotoxicity

Any other information on results incl. tables

Please refer to attached pdf document for detailed results.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

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

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Eschericia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EC and USA EPA (TSCA) guidelines. The dose range was determined in a preliminary toxcity assay and was 50 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.

Validity of test

The vehicle (dimethyl sulphoxide) control plates gave 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.

Results and conclusions

The test material caused no visible reduction in the growth of the bacterial lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose of 5000 μg/plate.

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