Benzene, di-C10-14-alkyl derivs., sulfonated, sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Ames test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 14, 2016 to August 1, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

Benzene, di-C10-14-alkyl derivs., sulfonated, sodium salts

EC Number:

285-586-8

EC Name:

Benzene, di-C10-14-alkyl derivs., sulfonated, sodium salts

Cas Number:

85117-37-9

Molecular formula:

Molecular formula not applicable for this UVCB.

IUPAC Name:

Benzene, di-C10-14-alkyl derivs., sulfonated, sodium salts

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Benzene, di-C1 0-14-alkyl derivs., sulfonated, sodium salts
- ZS name: ALKYLBENZOLNACHLAUF, SULFONIERT, NA-SALZ
- ZS number: 1344
- Batch: SEALS 2011-104-5-8
- Appearance, colour: brown clear liquid

Method

Target gene:

Histidine and tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver S9-mix induced Aroclor 1254)

Test concentrations with justification for top dose:

From 1.7 to 5,000 µg/plate
In the dose range finding study, the test substance was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test substance did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants was observed in tester strain TA100 in the absence of S9-mix at the highest tested concentration. No toxicity was observed in tester strain WP2uvrA.

Vehicle / solvent:

Milli-Q water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene, ICR-191

Details on test system and experimental conditions:

- Dose range finding test
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and the WP2uvrA, both with and without S9-mix. Eight concentrations: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate. The highest concentration of the test substance used in the subsequent mutation assays was 5000 μg/plate. At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain in the absence and presence of S9-mix.
The first experiment was a direct plate assay and the second experiment was a pre-incubation assay.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
- The first experiment: direct plate assay
The above mentioned dose range finding study with two tester strains is reported as a part of the direct plate assay. In the second part of this experiment, the test substance was tested both in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. 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 (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in Milli-Q waterand either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of nonactivation 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.
- The second experiment: pre-incubation assay.
The test substance was tested both in the absence and presence of S9-mix in all tester strains. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were pre-incubated for 30 minutes by 70 rpm at 37°C, 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), 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in Milli-Q water. After the pre-incubation period the solutions were added to 3 ml molten top agar. 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.
- Colony counting
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test substance precipitate to interfere with automated colony counting were counted manually. Evidence of test substance 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:

- Recommended test system in international guidelines (e.g. OECD, EC)
- Dose range finding test

Evaluation criteria:

- Number of revertants
- Cytotoxicity
- Precipitation

Statistics:

No formal hypothesis testing was done. 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 substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
- A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

1rst test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the highest concentration tested

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

1rst test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the highest dose tested

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

1rst test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the highest dose tested

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

1rst test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

2nd test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

decrease in the number of revertants at 1600 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

2nd test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- First experiment: direct plate assay
The test substance was initially tested in the tester strains TA100 and WP2uvrA as a dose range finding test with 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 mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate. Precipitation of the test substance on the plates was observed at the start of the incubation period at the highest concentration of 5000 µg/plate. No precipitation was observed at the end of the incubation period. Toxicity (cytotoxicity) was measured by the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies and was observed in tester strains TA100, TA1537 and TA98 in the absence of S9-mix at the highest tested concentration, as evidenced by a decrease in the number of revertants. Mutagenicity: no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.
- Second experiment: pre-incubation assay
A pre-incubation experiment was performed in the absence and presence of S9-mix. Based on the results of the first mutation assay, the test substance was tested up to the dose level of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Precipitation of the test substance on the plates was observed at the start of the incubation period at the top concentration of 5000 µg/plate and at 1600 and 5000 µg/plate at the end of the incubation period. Cytotoxicity, as evidenced by a decrease in the number of revertants was only observed in tester strain TA100 in the absence of S9-mix at concentrations of 1600 and 5000 µg/plate. There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all other tester strains in the absence and presence of S9-mix. No increase in the number of revertants was observed under all conditions tested.

Remarks on result:

other: no increase in the number of revertants

Any other information on results incl. tables

Acceptability of the assay:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:

a) The vehicle control and positive control plates from each tester strain (with or without S9mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.

b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance did not induce significant dose-related increases in the number of revertants in Salmonella typhimurium and Escherichia coli with and without metabolic activation and was therefore considered to be not mutagenic.

Executive summary:

A study was conducted to determine the genotoxicity of the test substance according to OECD Guideline 471 and EU Method B. 13/14 (Ames test). Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2 uvr A were exposed at concentrations ranging from 1.7 to 5,000 µg/plate (based on a dose range finding test). Several tests were performed: 1) a direct plate assay and 2) a pre-incubation assay. In the direct plate assay, precipitation of the test substance on the plates was observed at the start of the incubation period at the highest concentration of 5000 µg/plate. No precipitation was observed at the end of the incubation period. 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 observed. Cytotoxicity, as evidenced by a decrease in the number of revertants was observed in tester strains TA100, TA1537 and TA98 in the absence of S9-mix at the highest tested concentration. No increase in the number of revertants was observed upon treatment with the test substance under all conditions tested. In the pre-incubation assay, precipitation of the test substance on the plates was observed at the start of the incubation period at the top concentration of 5000 µg/plate and at 1600 and 5000 µg/plate at the end of the incubation period. Cytotoxicity was only observed in strain TA100 in the absence of S9-mix at concentrations of 1600 and 5000 µg/plate. There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all other tester strains in the absence and presence of S9-mix. No increase in the number of revertants was observed under all conditions tested. The negative and 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. In conclusion, the test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Under the study conditions, the test substance was not considered to be mutagenic with and without metabolic activation (Verspeek-Rip, 2016).