2-(6-methoxybenzofuran-2-yl)-1,3-dimethyl-5-(methylsulphonyl)1H-benzimidazolium acetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April from 02 to 18, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fluorescent Brightener 363

IUPAC Name:

Fluorescent Brightener 363

Method

Target gene:

histidine auxotrophs and tryptophan auxotrophs

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:

rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Since no relevant toxic effects were observed with 5000 µg/plate in the pre-experiment/experiment I, it was chosen as maximal concentration.

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO treated

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD) for strains TA 1537, TA98 without S9; 2-aminoanthracene (2-AA) as positive control for all the used strains with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for experiment I; preincubation for experiment II

Pre-Experiment for Toxicity
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. The experimental conditions in this pre-experiment were the same as for the experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, if the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Dose Selection
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I. Since no relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades. The following concentrations of the active ingredient were tested in experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate.

Experimental Performance
For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 µl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µl Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µl Overlay agar
In the pre-incubation assay 100 µl test solution, 500 µl S9 mix/ S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

Acceptability of the Assay
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

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:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: 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

Vehicle 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

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The plates incubated with the test item showed normal background growth up to 5000 µg/plate. Only in experiment II in the absence of metabolic activation, reduced background growth was observed at 5000 µg/plate in strains used.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. Minor reductions in the number of revertants was observed in the absence of metabolic activation in strain TA 1537 at 3 µg/plate in experiment I and in strain TA 100 at 5000 µg/plate in experiment II.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 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:

The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test item was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. In the Pre-Experiment/Experiment I, the test item was tested at the concentrations of 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate, while in the experiment II it was tested at 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. Only in experiment II in the absence of metabolic activation, reduced background growth was observed at 5000 µg/plate in strains used. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. Minor reductions in the number of revertants was observed in the absence of metabolic activation in strain TA 1537 at 3 µg/plate in experiment I and in strain TA 100 at 5000 µg/plate in experiment II. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 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.

Conclusion

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.