1H-Imidazolium, 3-ethyl-1-methyl-, C11-13-branched alkylbenzenesulfonates

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

It has been concluded that EMI-DBS does not induce reverse mutation in the bacterial strains investigated, and is not capable of inducing chromosome aberration in cultured CHO-K1 cells.

Link to relevant study records

Reference

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:

other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 471.

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

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Based on the results of preliminary range-finding test, the main test was performed on 5 dose levels with and without metabolic activation, as follows:

In the absence of S9 mix: TA100, TA1537 = 0, 156, 313, 625, 1250 and 2500 µg/plate and TA98, TA1535, WP2uvrA = 0, 313, 625, 1250, 2500, 5000 µg/plate
In the presence of S9 mix: All strains = 0, 313, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2); 2-aminoanthracene

Remarks:

The control substances were recommended by the relevant OECD and NIER test guidelines

Details on test system and experimental conditions:

Supplier of bacterial strains: Molecular Technology Inc.
Storage method: Kept in freezer
Storage temperature: -80°C (deep freezer DF9007)
Composition: Bacterial strain = 0.8 ml, DMSO = 0.07 ml

Pre-cultivation conditions
Nutrient broth: Name - No. 2, Manufacturer - Oxoid, Lot No. - 588285
Cultivation time: 10 hours (stop incubation in the early stationary phase)
Shaking incubator: Model - SHKE-5000-1CE, Manufacturer - Barnstead International USA
Incubation method: Shaking type - rotation (RPM: 180/min), Rotation diameter - 2.54 cm
Culture vessel: Type - Erlenmeyer flask, Capacity - 50 ml
Volume of culture medium: 15 ml
Volume of inoculum: 30 µl

Medium: Top Agarr; Minimum glucose agar plate; Vogel-Bonner medium E(10x)

Evaluation criteria:

The result was judged as "positive" if there was a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.

Statistics:

The statistical method for analysis of the results was not applied in this study.

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

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

Regardless of the metabolic activation system, the number of revertant colony did not show an increase compared with the number of revertant colony of the negative control group.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Cytotoxicity was observed in 2500 µg/plate dose levels (TA100, TA1537 strains) and in the 5000 µg/plate dose levels (TA98, TA1535 strains) in the absence of metabolic activation system. In the presence of metabolic activation system, cytotoxicity was observed in the 5000 µg/plate dose levels (TA98, TA100 and TA1537 strains). When compared to the negative control, significant increases in the number of revertant colonies were not observed in the 5 strains regardless metabolic activation system.

The sterility of the solvent and the S9 mix were certified by the sterility test. It was counted that the number of revertant colonies of positive control groups and negative control groups were within or close to the range of the historical data. Therefore this test was performed properly.

Conclusions:

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

The EMI-DBS was found to not induce reverse mutation under the conditions of the study.

Executive summary:

The genetic toxicity in vitro of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 471. The mutagenic potential of EMI-DBS in bacteria was evaluated using the bacterial reverse mutation study. The study was performed using the preincubation method using four histidine-requiring strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537, and one tryptophan-requiring strain of Escherichia coli WP2uvrA in the presence and absence of metabolic activation. Based on the results of preliminary range-finding test, the main test was performed at the following 5 dose levels:

- in the absence of metabolic activation system (S9 mix (-)): TA100, TA1537 = 0, 156, 313, 625, 1250, 2500 µg/plae and TA98, TA1535, WP2uvrA = 0, 313, 625, 1250, 2500, 5000 µg/plate

- in the presence of metabolic activation (S9 mix (+)): 0, 313, 625, 1250, 2500, 5000 µg/plate.

There was no increase in the number of revertant colonies seen in TA98, TA100, TA1535, TA1537 and WP2uvrA strains when compared with the negative control groups in the presence and absence of metabolic activation system. Based on these findings, it has been concluded that EMI-DBS does not induce reverse mutation in the bacterial strains investigated in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

A study was conducted on the registered substance to determine the in vitro genetic toxicity of the substance. The genetic toxicity in vitro of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 471. The mutagenic potential of EMI-DBS in bacteria was evaluated using the bacterial reverse mutation study. The study was performed using the preincubation method using four histidine-requiring strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537, and one tryptophan-requiring strain of Escherichia coli WP2uvrA in the presence and absence of metabolic activation. Based on the results of preliminary range-finding test, the main test was performed at the following 5 dose levels:

- in the absence of metabolic activation system (S9 mix (-)): TA100, TA1537 = 0, 156, 313, 625, 1250, 2500 µg/plae and TA98, TA1535, WP2uvrA = 0, 313, 625, 1250, 2500, 5000 µg/plate

- in the presence of metabolic activation (S9 mix (+)): 0, 313, 625, 1250, 2500, 5000 µg/plate.

There was no increase in the number of revertant colonies seen in TA98, TA100, TA1535, TA1537 and WP2uvrA strains when compared with the negative control groups in the presence and absence of metabolic activation system. Based on these findings, it has been concluded that EMI-DBS does not induce reverse mutation in the bacterial strains investigated in this study.

Further, a study was conducted in accordance with the OECD Guideline for Testing of Chemicals 473. The study was performed to assess the ability of EMI-DBS to induce chromosomal aberration in Chinese Hamster Ovary (CHO-K1) cells with and without metabolic activation (S9 mix). The test substance was dissolved in DMSO. In order to assess the cytotoxicity of the test substance in cultures CHO-K1 cells, Relative Cell Count (RCC) was calculated for all cultures treated with the test substance at the 8 dose levels (0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10 mM). On the basis of this preliminary test, the following treatment times and concentrations were selected for the main study: 24 hours continuous treatment (without S9 mix) = 0.039, 0.078 and 0.156 mM; 6 hours treatment and 18 hours recovery (without S9 mix) = 0.039, 0.078 and 0.156 mM; 6 hours treatment and 18 hours recovery (with S9 mix) = 0.078, 0.156 and 0.313 mM. In the absence of S9 mix and with 24 hours continuous treatment at all dose levels, the test substance caused no statistically significant chromosomal aberration when compared with the negative control group. And also in the group of 6 hours treatment and 18 hour recovery, the test substance caused no statistically significant increase at all dose levels in the number of cells with chromosome aberration. In the presence of S9 mix (6 hours treatment and 18 hours recovery) the test substance caused no statistically significant increase at all dose levels when compared with the negative control group. In the presence and absence of S9 mix, the test substance caused no statistically significant increase in the number of cells with polyploidy and endoreduplication, when compared with the negative control group. Based on the above results, it is concluded that the test substance EMI-DBS is not capable of inducing chromosome aberration in cultured CHO-K1 cells under the conditions of this study.

Justification for selection of genetic toxicity endpoint

This study was conduced in a GLP accredited laboratory using OECD Testing Guideline 471.

Justification for classification or non-classification

No reverse mutation or induction of chromosome aberration was observed in the OECD 471 and OECD 473 in vitro genetic toxicity studies conducted on the substance.