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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
of 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
of 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: semi-solid, wax-like (at 20°C)
Details on test material:
Name of the test material (as cited in the Study): WS400102

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: essential amino acid requiring strains
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from male Wistar rats treated with phenobarbital and beta-naphthoflavone for enzyme induction.
Test concentrations with justification for top dose:
Range-finding test: 0; 10; 31.6; 100; 316; 1000; 2500 and 5000 μg/plate
Experiment 1: 0; 1.581; 5; 15.81; 50; 158.1; 500; 1581 and 5000 μg/plate
Experiment 2: 0; 1.581; 5; 15.81; 50; 158.1; 500; 1581 and 5000 μg/plate
Complementary to Experiment 2: 0; 0.1581; 0.5; 1.581; 5; 15.81; 50; 158.1 and 500 μg/plate
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Justification for choice of solvent/vehicle:
In a solubility trial, DMSO and acetone were suitable vehicles for exposure to the test substance up to the maximum guideline recommended test substance concentration of 5000 μg/plate, whereas in distilled water the test material was insoluble. DMSO was selected as vehicle, because of its better biocompatibility with the test system than acetone.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
without and with S9 mix
Negative solvent / vehicle controls:
yes
Remarks:
without and with S9 mix
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine; sodium azide; 9-aminoacridine; methyl-methanesulfonate.
Remarks:
Positive control substances for tests without metabolic activation (S9 mix). All of them are well established reference mutagens.
Untreated negative controls:
yes
Remarks:
without and with S9 mix
Negative solvent / vehicle controls:
yes
Remarks:
without and with S9 mix
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene; Activity of S9 was additionally tested with Benzo[a]pyrene
Remarks:
Positive control substances for tests with metabolic activation (S9 mix). All of them are well established reference mutagens.
Details on test system and experimental conditions:
Range-finding test & Experiment 1: Standard Plate Incorporation Tests
Experiment 2 & Complementary to Experiment 2: Pre-incubation Test

All tests, except the Complementary to Experiment 2, were performed without and with metabolic activation (S9 mix).
The Complementary to Experiment 2, was performed without metabolic activation.
Proportion of S9 fraction in the S9 mix was 10% v/v and of S9 fraction in the final medium ca. 2% v/v in all tests with metabolic activation.

The following positive controls were used to check mutability of the bacteria and activity of the S9 mix:

Without metabolic activation (S9 mix):

4-nitro-1,2-phenylene-diamine: - 4 μg/plate, dissolved in DMSO: - strain: TA 98

Sodium azide: - 2 μg/plate, dissolved in distilled water: - strains: TA 100, TA 1535

9-Aminoacridine: - 50 μg/plate, dissolved in DMSO: - strain: TA 1537

Methyl-methanesulfonate: - 2 μL/plate, dissolved in distilled water: - strain: WP2 uvrA


With metabolic activation (S9 mix):

2-Aminoanthracene: - 2 μg/plate, dissolved in DMSO: - strains: TA 1535, TA 1537, TA 98, TA 100

2-Aminoanthracene: - 50 μg/plate, dissolved in DMSO: - strain: WP2 uvrA

In addition, adeqaute biological activity of the S9 batch used in the present study was confirmed by use of two mutagens, Aminoanthracene and Benzo(a)pyrene, that require metabolic activation by microsomal enzymes.

Evaluation criteria:
The test substance is considered to exhibit mutagenic activity in this assay if the following criteria are met:
- a dose–related increase in the number of revertants and/or;
- a reproducible, biologically relevant positive response for at least one of the dose groups in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if in at least one of the five tested strains the number of reversion was more than twice higher than the reversion rate of the vehicle (solvent) control.

A test substance is considered non-mutagenic in this test if:
Neither a dose-related increase in the number of revertants nor a reproducible, biologically relevant positive response is evident in any of the dose groups, with or without metabolic activation.

The study was considered valid if:
- the number of revertant colonies of the vehicle (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.
Statistics:
The data were not statistically analysed. The study result was unequivocal.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
but confined to 5000 µg/plate (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
but confined to 5000 µg/plate (+S9) and 2500 & 5000 µg/plate (-S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Confined to Experiment 2 to 500, 1581 & 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
reduced background lawn down to 158.1 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Viability checked by a plating experiment in each test was satisfactory.
Remarks on result:
other: tables of results attached as background material

Any other information on results incl. tables

At a test material concentration of 5000 µg/plate, precipitate was observed in the Range-finding test in both bacterial strains (TA98 and TA100) with metabolic activation, and in Experiment 2 in all tested strains (S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2) with and without metabolic activation.

Applicant's summary and conclusion

Conclusions:
negative without and with metabolic activation (S9 mix)