Dodecylbenzenesulphonic acid, compound with isopropylamine (1:1)

Genetic toxicity: in vitro

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:

2018

Report date:

2018

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dodecylbenzenesulphonic acid, compound with isopropylamine (1:1)

EC Number:

247-556-2

EC Name:

Dodecylbenzenesulphonic acid, compound with isopropylamine (1:1)

Cas Number:

26264-05-1

Molecular formula:

C18H30O3S.C3H9N

IUPAC Name:

sodium dodecylbenzenesulfonate

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium TA98, TA100, TA1535 and TA1537. Escherichia coli WP2 uvrA.

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from the livers of rats treated with Aroclor.

Test concentrations with justification for top dose:

Dose range finder/first mutation assay: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (TA100 and WP2uvrA) with and without metabolic activation. Followed by testing at 5.4, 17, 52, 164, 512 and 1600 µg/plate (TA1535, TA1537 and TA98).
Experiment 2: 50, 100, 200, 400, 800 and 1600 µg/plate in the absence of S9 mix. WP2 uvrA was also tested at 2500 and 5000 µg/plate.
Experiment 2: 78, 156, 313, 625, 1250 and 2500 µg/plate in the presence of S9 mix. WP2 uvrA was also tested at 5000 µg/plate.
Experiment 3: 12.5, 25 and 50 µg/plate (TA1537 without S9 mix only).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: standard as per OECD guidelines

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 (TA1537 without metabolic activation) and 2-aminoanthracene for all strains with metabolic activation

Details on test system and experimental conditions:

Test substance preparation: No correction was made for the purity/composition of the test item. A solubility test was performed based on visual assessment. The test item formed a clear colorless solution in ethanol. In the second experiment, the stock solution was treated with ultrasonic waves until the test item had completely dissolved. Test item concentrations were used within 4 hours after preparation. Any residual volumes were discarded.

Mutation assay: At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain. The dose-range finding study with the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation experiment. In the second part of this experiment, the test item was tested both in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In two follow-up experiments with additional parameters, the test item was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in ethanol and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Cell counting: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Rationale for test conditions:

Standard as per OECD guidelines

Evaluation criteria:

In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
 
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

Not performed

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose-range Finding Test/First Mutation Experiment:
The test substance was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose-range finding test, the following dose-range was selected for the first mutation experiment with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 5.4, 17, 52, 164, 512 and 1600 μg/plate. Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.
Toxicity: To determine the toxicity of the test substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. Cytotoxicity as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.
Mutagenicity: No increase in the number of revertants was observed upon treatment with Rhodacal VS1 under all conditions tested.

Second Mutation Experiment
To obtain more information about the possible mutagenicity of the test substance, a second mutation experiment was performed in the absence and presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test item was tested at a concentration range of 50 to 1600 µg/plate and 78 to 2500 µg/plate in the absence and presence of 10% (v/v) S9-mix, respectively in the tester strains TA1535, TA1537, TA98 and TA100 and at a concentration range of 156 to 5000 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strain WP2uvrA.
Precipitate: Precipitation of the test substance on the plates was not observed at the start of the incubation period. At the end of the incubation period the test substance precipitated at a concentration range of 800 µg/plate and upwards in tester strain TA98 in the absence of S9-mix. In the presence of S9-mix the test substance precipitate was observed in tester strains TA1535, TA1537, TA98 and TA100.
Toxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.
Mutagenicity: In the second mutation assay, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

Third Mutation Experiment
Due to the cytotoxicity in the second experiment, only three analyzable dose levels were left for the determination of the mutagenicity of the test item in tester strain TA1537 in the absence of S9-mix. Therefore a third experiment was performed with concentrations of 12.5, 25 and 50 µg/plate to complete the data of the second experiment.
Precipitate: Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.
Toxicity: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Mutagenicity: In the third mutation assay, no increase in the number of revertants was observed upon treatment with Rhodacal VS1 under all conditions tested.

All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in three experiments. The negative control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA100 (presence of S9-mix) in the second experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 2 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.