Benzene, poly(propene) derivatives

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

5 July 2007 to 10 August 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Sponsor's identification: AL305B
Description: clear, colourless, slightly viscous liquid
Purity: 100%
Batch number: TSO7003
Storage conditions: room temperature, in the dark

Method

Target gene:

S. typhimurium: Hisidine locus.
E. coli: Tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/β-naphthoflavone induced rat liver homogenate metabolising system (S9).

Test concentrations with justification for top dose:

15 to 5000 microgram/plate

Vehicle / solvent:

Tetrahydrofuran

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: 4-Nitroquinoline-1-oxide, 2-Aminoanthracene and Benzo(a)pyrene, N-ethyl-N'-nitro-N-nitrosoguanidine.

Details on test system and experimental conditions:

PRELIMINARY TOXICITY TEST
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. 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 (TA100 or WP2uvrA-), 0.025 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 (tetrahydrofuran) were tested. In addition, 0.025 mL of the maximum concentration of the test material ad 2 mL of molten, trace histidine or tryptophan 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 ºC 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.

RANGE-FINDING TEST
Six concentrations of the test material (15, 50, 150, 500, 1500 and 5000 µG/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten trace histidine or tryptophan supplemented, top agar, 0.025 mL of the test material formulation and vehicle or 0.1 mL of positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of VogeI-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix. The plates were incubated for a nominal 48 hours at 37 ºC after an initial: overnight equilibration period and the frequency of revertant colonies assessed using a Domino colony counter.

MAIN TEST
The second experiment was performed using methodology as described for the range-finding test using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (15 to 5000 µg/plate).

Evaluation criteria:

The test material w as considered positive in this test if the following criteria were met:

The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria. If a greater than two or threefold increase in revertant count (depending on tester strain type) is observed in two experiments then this is taken as evidence of no positive response.

Statistics:

Not stated

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

On the day of each experiment, the tester strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments were shown to be sterile. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the mutation test.

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 µg/plate. An oily precipitate was observed, with the aid of a microscope, at 500 µg/plate becoming visible by eye from 1500 µg/plate. This observation did not prevent the scoring of revertant colonies.

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. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was shown to be satisfactory.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Detailed results are presented in Tables 1-6 (attached).

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

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).

 

Methods

Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA 100 and Escherichia coli 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 (tetrahydrofuran) 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 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 5000µg/plate. An oily precipitate was observed, with the aid of a microscope, at 500µg/plate, becoming visible by eye from1500 µg/plate. This observation did not prevent the scoring of revertant colonies.

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.

 

Conclusion

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