6-chloro-2-(6-chloro-4-methyl-3-oxobenzo[b]thien-2(3H)-ylidene)-4-methylbenzo[b]thiophene-3(2H)-one

Genetic toxicity: in vitro

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed in compliance with GLP and OECD Test guideline 471

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

6-chloro-2-(6-chloro-4-methyl-3-oxobenzo[b]thien-2(3H)-ylidene)-4-methylbenzo[b]thiophene-3(2H)-one

EC Number:

219-163-6

EC Name:

6-chloro-2-(6-chloro-4-methyl-3-oxobenzo[b]thien-2(3H)-ylidene)-4-methylbenzo[b]thiophene-3(2H)-one

Cas Number:

2379-74-0

Molecular formula:

C18H10Cl2O2S2

IUPAC Name:

6-chloro-2-[(2E)-6-chloro-4-methyl-3-oxo-2,3-dihydro-1-benzothiophen-2-ylidene]-4-methyl-2,3-dihydro-1-benzothiophen-3-one

Test material form:

solid: nanoform

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (phenobarbital/ß-naphthoflavone-induced rat liver S9)

Test concentrations with justification for top dose:

Experiment I (plate incorporation): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate both with and without metabolic activation
Experiment II (pre-incubation): 33, 100, 333, 1000, 2500 and 5000 µg/plate both with and without metabolic activation

Vehicle / solvent:

DMSO, purity >99% (Merck, Darmstadt, Germany). The solvent was chosen because of its solubility properties and its relative non-toxicity to bacteria

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: sodium azide (TA 1535, TA 100), 4-nitro-o-phenylene-diamine (TA 1537, TA 98), methyl methane sulfonate (WP2 uvrA), With metabolic activation: 2-aminoanthracene (TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
plate incorporation (experiment I); preincubation (experiment II). Since experiment I gave negative results, experiment II was performed as a
preincubation assay.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Each concentration, including the controls, was tested in triplicate.

DETERMINATION OF CYTOTOXICITY: To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Not mandatory according to OECD guideline 471

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Pre-experiment was reported as experiment I because the criterion (evaluable plates (>0 colonies) at five concentrations or more in all strains are used) was met.

COMPARISON WITH HISTORICAL CONTROL DATA:
yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in both experiments.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. A minor reduction observed in strain TA 1537 at 2500 µg/plate without S9 mix in experiment II is judged to be based on biologically irrelevant fluctuations and does not represent a true toxic effect.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).

There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The test item did not induce gene mutations by frameshifts or base-pair substitutions in the genome of the strains used when tested in a bacterial reverse mutation assay (Ames test, test strains: Salmonella typhimurium TA 98, TA 100, TA 1535, TA1537; E. coli WP2 uvrA) with and without metabolic activation (rat liver S9 and hamster liver S9) at concentrations up to 5000 µg/plate. Since the test item did not induce gene mutations the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of test item to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.