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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:
The experimental phase of the study was performed between 08 September 2005 and 24 October 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Meets the criteria for classification as reliable without restriction according to Klimisch et al (1997)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

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
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: FSM-003B (S196439)
Description: Black powder
Batch number: CE-501-2
Date Received: 23 August 2005
Storage conditions: room temperature in the dark under nitrogen over silica gel

Method

Target gene:
Histidine for Salmonella typhimurium
Tryptophan for Escherichia coli
Species / strain
Species / strain:
other: TA1535, TA1537, TA98, TA100 and Escherichia coli WP2uvrA-
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone /beta-naphthoflavone induced rat liver S9.
Test concentrations with justification for top dose:
Preliminary Toxicity Test
0, 0.15, 0.5, 1.5, 5, 15, 50, 500, 1500 and 5000 µg/plate


For Mutation Tests 1 and 2

Concentration range in the main test (with metabolic activation): 50, 150, 500, 1500 and 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50, 150, 500, 1500 and 5000 µg/plate

Formulated concentrations were adjusted to allow for the stated purity of the test material and therefore test concentrations relate to the concentration of active ingredient.
Vehicle:
Vehicle(s)/solvent(s) used: Dimethyl sulphoxide
Justification for choice of solvent/vehicle: The test item was fully soluble in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in house.
Controlsopen allclose all
Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Solvent controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rate of TA1535
Solvent controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rate of TA1537
Solvent controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rate of TA98
Solvent controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rate of WP2uvrA-
Solvent controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix
Negative controls:
yes
Remarks:
Spontaneous Mutation Rates of TA100
Solvent controls:
yes
Remarks:
Dimethyl Sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
With S9 mix
Negative controls:
yes
Remarks:
Spontaneous Mutation rate of TA1535
Solvent controls:
yes
Remarks:
Dimethyl Sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
With S9 mix
Negative controls:
yes
Remarks:
Spontaneous Mutation Rate of WP2uvrA-
Solvent controls:
yes
Remarks:
Dimethyl Sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
With S9 mix
Negative controls:
yes
Remarks:
Spontaneous Mutation Rate of TA98
Solvent controls:
yes
Remarks:
Dimethyl Sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix
Negative controls:
yes
Remarks:
Spontaneous Mutation Rate of TA1537
Solvent controls:
yes
Remarks:
Dimethyl Sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
With S9 mix
Details on test system and conditions:
Salmonella typhimurium strains TA1535, TA1537, TA98. TA100 and Escherichia 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). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as in the first experiment, fresh cultures of the bacterial strains and fresh test material formulations.
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS ( Kirkland D J (Ed) (1989) Statistical Evaluation of Mutagenicity TestData. UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III, Cambridge University Press.) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response. A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
None used

Results and discussion

Test results
Species / strain:
other: TA1535, TA1537, TA98, TA100 and Escherichia coli WP2uvrA-
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Remarks:
(> 5000 µg/plate)
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
Observations:
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate. A blue colour was observed at and above 50 ug/plate, this did not prevent scoring of the revertant colonies. 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 significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

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

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

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test item using the 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).

Results

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of test material, either with or without metabolic activation.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All positive controls responded as expected and confirmed that the test system was responding as expected.

Conclusion

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