9-Octadecenoic acid (Z)-, sulfonated, potassium salts

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:

From February 06, 2014 to February 17, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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:

9-Octadecenoic acid (Z)-, sulfonated, potassium salts

EC Number:

271-843-1

EC Name:

9-Octadecenoic acid (Z)-, sulfonated, potassium salts

Cas Number:

68609-93-8

Molecular formula:

A generic formula cannot be provided for this UVCB substance. The alkyl chain length of the sulfonated fatty acids range from C12-C22, however the major alkyl chain is C18.

IUPAC Name:

9-Octadecenoic acid (Z)-, sulfonated, potassium salts

Test material form:

other: powder/flakes with lumps

Details on test material:

- Name of test material (as cited in study report): 9-Octadecenoic acid (Z)-, sulfonated, potassium salts
- Physical state: Beige-yellow powder/flakes with lumps (i.e., determined at WIL Research Europe B.V.)
- Analytical purity: 100%
- Water content: 0.5%
- pH: 6 at concentration of 1g/L
- Lot/batch No.: 7495382
- Expiration date of the lot/batch: December 31, 2015
- Storage condition of test material: At room temperature in the dark
- Stability in vehicle: Water: Yes
- Solubility in vehicle: Water: Completely miscible

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 from rat liver microsomal enzymes (i.e., S9 homogenate)

Test concentrations with justification for top dose:

Dose range finding test (i.e., in the absence and presence of S9-mix):
3, 10, 33, 100, 333, 1000, 3330 and 5,000 μg/plate

Mutation assay (i.e., in the absence and presence of S9-mix):
100, 333, 1000, 3330 and 5,000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Milli-Q water (Millipore Corp., Bedford, MA., USA)
- Solvent(s) for reference substances: Saline and dimethyl sulphoxide (i.e., DMSO)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation for strain TA1535

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

without metabolic activation for strain TA1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation for strain TA98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation for strain TA100

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation for strain WP2uvrA

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (i.e., 5% rat liver S 9 mix) for all strains

Controls 7

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (i.e., 10% rat liver S 9 mix) for all strains

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 WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1,000, 3,330 and 5,000 μg/plate were tested in triplicate. The highest concentration of test substance used in the subsequent mutation assay was 5,000 μg/plate. Results of this dose range finding test were reported as part of the mutation assay.

Mutation assay (i.e., plate incorporation method):
Five different doses (increasing with approximately half-log steps) (i.e., 100, 333, 1,000, 3,330 and 5,000 μg/plate) of the test substance were tested in triplicate in each strain.

Experiment-1:
Test substance was tested both in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98.

Experiment-2:
An independent repeat of the assay with additional parameters was conducted in the absence and presence of 10% (v/v) S9-mix in all tester strains.

- The negative control (i.e., vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

Assay procedure:
Top agar in top agar tubes was melted by heating to 45°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (i.e., 109 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test substance in Milli-Q water and either 0.5 mL S9-mix (i.e., in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (i.e., 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.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:

a) The total number of revertants in tester strain TA100 is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three times the concurrent vehicle control.

b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:

a) The total number of revertants in tester strain TA100 is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three times the concurrent vehicle 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 independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

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 negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.

b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean.

c) 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.

Statistics:

No formal hypothesis testing was done

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose range finding test and/or first mutation test (i.e., experiment 1):
Precipitate: No precipitation on test substance was observed.
Toxicity: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Mutagenicity: No increase in the number of revertants was observed upon treatment with test substance under all conditions tested.

Based on the results of the first mutation assay, test substance was tested up to the dose level of 5000 μg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the absence and presence of S9-mix (i.e., 10% (v/v)).

Second mutation test (i.e. experiment 2):
Precipitate: No precipitation on test substance was observed.
Toxicity: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Mutagenicity: No increase in the number of revertants was observed upon treatment with test substance under all conditions tested. In strain TA1535, fluctuations in the number of revertant colonies above the laboratory historical control data range were observed in the absence of S9-mix at dose levels of 100 and 333 μg/plate. However, since the increases were less than the concurrent vehicle control, these increases were not considered to be relevant.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance in bacteria, according to OECD Guideline 471, plate incorporation method, in compliance with GLP. The study was performed in two independent experiments in the presence and absence of metabolic activation (S9-mix derived from a rat liver homogenate). Mutagenicity of the test substance was evaluated at five concentrations in the range of 100 to 5,000 µg/plate with S9 (5% v/v in Experiment 1 and 10% v/v in Experiment 2). The negative control values were within the laboratory historical control data ranges, except for TA1535 in the absence of S9-mix (second experiment). However, since this value was just outside the limit of the range, the validity of the test was considered not affected. Further, the strain-specific positive control values were at least three times the concurrent vehicle control group mean, indicating that the test conditions were adequate. Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay (Verspeek-Rip, 2014).