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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

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

Target gene:

Histidine and Tryptophan

Species / strain / cell type:

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

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)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation for strain TA1535

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation for strain TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation for strain TA100

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

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

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

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

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.

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).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 27, 2014 to May 15, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: peripheral human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver

Test concentrations with justification for top dose:

First cytogenetic assay
Without and with S9-mix: 10, 100 and 1,000 µg/mL culture medium (3 h exposure time, 24 h fixation time).

Second cytogenetic assay
Without S9-mix : 10, 30, 100, 200, 300, 400 and 500 µg/mL culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time).
With S9-mix : 10, 100 and 1,000 µg/mL culture medium (3 h exposure time, 48 h fixation time).

Cytogenetic assay 2A (i.e., repeated experiment)
Without S9-mix :
10, 100, 300, 325, 350, 375, 400 and 450 μg/ml culture medium (24 h exposure time, 24 h fixation time).
10, 100, 250, 300, 325, 350, 375 and 400 μg/ml culture medium (48 h exposure time, 48 h fixation time).

Vehicle / solvent:

cell culture medium (i.e., RPMI 1640 medium)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

(with metabolic activation)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

(without metabolic activation)

Details on test system and experimental conditions:

Cytogenetic assay
Test substance was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate in two independent experiments. To be able to select appropriate dose levels for scoring of chromosome aberrations a repeat assay for the second cytogenetic assay had to be performed.

First cytogenetic assay
-Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:

-Without and with S9-mix: 10, 100 and 1000 μg/mL culture medium (3 h exposure time, 24 h fixation time).

-Lymphocytes were cultured for 48±2 h and thereafter exposed in duplicate to selected doses of test substance for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (i.e., 5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 mL culture medium and incubated for another 20 - 22 h (i.e., 24 h fixation time). Appropriate negative and positive controls were included in the first cytogenetic assay.

-Based on the mitotic index of the dose range finding test and the first cytogenetic assay and on the solubility of test substance in the culture medium appropriate dose levels were selected for the second cytogenetic assay. The independent repeat was performed with the following modifications of experimental conditions.

Second cytogenetic assay

-To obtain more information about the possible clastogenicity of test substance, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to test substance in the absence of S9-mix for 24 or 48 h. In the presence of S9-mix, cells were fixed after 48 h following a 3 h exposure to test substance. The following dose levels were selected for the second cytogenetic assay:
-Without S9-mix : 10, 30, 100, 200, 300, 400 and 500 μg/mL culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time).
-With S9-mix : 10, 100 and 1,000 μg/mL culture medium (3 h exposure time, 48 h fixation time).

-Lymphocytes were cultured for 48±2 h and thereafter exposed in duplicate to selected doses of test substance for 24 h and 48 h in the absence of S9-mix and for 3 h in the presence of S9-mix.

-After 3 h exposure, the cells exposed to test substance in the presence of S9-mix were separated from the exposure medium by centrifugation (i.e., 5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL of HBSS and incubated in 5 mL culture medium for another 44 - 46 h (i.e., 48 h fixation time).

-The cells that were treated for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately after 24 h and 48 h (i.e., 24 h and 48 h fixation time). Appropriate negative and positive controls were included in the second cytogenetic assay.

Since, in the absence of S9-mix no appropriate dose levels could be selected for scoring of chromosome aberrations. The experiment was repeated in cytogenetic assay 2A:
Without S9-mix :
10, 100, 300, 325, 350, 375, 400 and 450 μg/mL culture medium (24 h exposure time, 24 h fixation time).
10, 100, 250, 300, 325, 350, 375 and 400 μg/mL culture medium (48 h exposure time, 48 h fixation time)

Mitotic index/dose selection for scoring of the cytogenetic assay
The mitotic index of each culture was determined by counting the number of metaphases from at least 1,000 cells (i.e., with a maximum deviation of 5%). At least three analysable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analysed from those cultures with an inhibition of the mitotic index of about 50% or above whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations. Test substance was not cytotoxic and/or difficult to dissolve in aqueous solutions, the highest concentration analysed at the 3 h exposure time was determined by the solubility in the culture medium.

Evaluation criteria:

Acceptability of the assay
A chromosome aberration test is considered acceptable if it meets the following criteria:

a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.

b) The positive control substances should produce a statistically significant (i.e., Chi-square test, onesided, p < 0.05) increase in the number of cells with chromosome aberrations.

c) A homogeneous response between the replicate cultures is observed.

d) A possible precipitate present on the slides should not interfere with the scoring of chromosome aberrations.

Statistics:

Data evaluation and statistical procedures

A test substance was considered positive (i.e., clastogenic) in the chromosome aberration test if:

a) It induced a dose-related statistically significant (i.e., Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (i.e., not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (i.e., Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

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

The incidence of aberrant cells (i.e., cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics

Key result

Species / strain:

lymphocytes: peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: first cytogenetic assay and second cytogenetic assay (with metabolic activation): no (but tested up to precipitating concentrations); second cytogenetic assay(without metabolic activation): yes at ≥300 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose range finding test

-At a concentration of 1,000 μg/mL test substance precipitated in the culture medium and was used as the highest concentration.

First cytogenetic assay

-All dose levels were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix, test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

-Both in the absence and presence of S9-mix, test substance did not biologically relevant increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Second cytogenetic assay

-In the absence of S9-mix no appropriate dose levels could be selected for scoring of chromosome aberrations since at the concentration of 300 μg/mL not enough cytotoxicity was observed (i.e., 36% (24 h exposure time) and 43% (48 h exposure time)), whereas the next higher concentration of 400 μg/mL was too toxic for scoring (i.e., 69% (24 h exposure time) and 85% (48 h exposure time)). The experiment was repeated in cytogenetic assay 2A.

Based on the observations the following doses were selected for scoring of chromosome aberrations:
Without S9-mix :
10, 100 and 300 μg/mL culture medium (24 h exposure time, 24 h fixation time). :
10, 100, 250 and 300 μg/mL culture medium (48 h exposure time, 48 h fixation time).
With S9-mix :
10, 100 and 1,000 μg/mL culture medium (3 h exposure time, 48 h fixation time).

-Although at the 24 h continuous exposure time the inhibition of the mitotic index at the concentration of 300 μg/mL was only 41% the next higher concentration of 325 μg/mL had an inhibition of the mitotic index of 70%. Due to the narrow concentration range and since a quality control check of the mitotic index at the concentration of 300 μg/mL gave an inhibition of the mitotic index of 51% this concentration was used to score for chromosome aberrations.

-Both in the absence and presence of S9-mix, test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

-Both in the absence and presence of S9-mix, test substance did not biologically relevant increase the number of polyploid cells and cells with endoreduplicated chromosomes

- The positive control chemicals (i.e., Mitomycin C (MMC-C) and Cyclophosphamide (CP)) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

- Both in the absence and presence of S9-mix test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

- No biologically relevant effects of test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Conclusions:

Under the study conditions, the test substance did not induce structural chromosome aberrations in cultured peripheral human lymphocytes both in the presence and absence of a metabolic activation system.

Executive summary:

A study was conducted to determine the potential of the test substance to induce chromosome aberrations in cultured peripheral human lymphocytes according to OECD Guideline 473, in compliance with GLP. Two independent experiments were conducted. The test substance was dissolved in cell culture medium and the dose levels were selected on the basis of dose range finding test. In the first assay, the substance was tested up to a concentration of 1,000 μg/mL with and without metabolic activation (S9-mix). In the second assay, the concentrations used were up to 1,000 μg/mL and 300 μg/mL with and without metabolic activation, respectively. Positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay and the efficacy of the S9-mix. The test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in presence and absence of metabolic activation. Likewise, no biologically relevant effect on number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of metabolic activation. Under the study conditions, the test substance did not induce structural chromosome aberrations in cultured peripheral human lymphocytes both in the presence and absence of a metabolic activation system (Buskens, 2014).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 21, 2014 to March 18, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (i.e., IWGT)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine-kinase locus (i.e., TK locus)

Species / strain / cell type:

mouse lymphoma L5178Y cells

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:
3 h treatment (with/without metabolic activation): 33, 100, 333, 666 and 1,000 μg/mL
24 h treatment (without metabolic activation): 33, 100, 333, 666 and 1,000 μg/mL

First mutagenicity test:
Without S9-mix: 1, 3, 10, 33, 66, 100, 125, 150, 160, 170, 180 and 190 μg/mL exposure medium
With 4% (v/v) S9-mix: 10, 33, 100, 150, 170, 200, 225, 235, 250 and 265 μg/mL exposure medium

Second mutagenicity test:

Without S9-mix: 3, 10, 33, 50, 65, 85, 100, 115, 130, 150, 170 and 185 μg/mL exposure medium
With 8% (v/v) S9-mix: 10, 33, 100, 150, 200, 225, 235, 250, 265 and 280 μg/mL exposure medium

Vehicle / solvent:

- Vehicle for test substance: RPMI 1640 (exposure medium, Hepes buffered medium (Dutch modification).
- Solvent(s) for reference substances:
Methyl methanesulfonate (i.e., MMS): Dimethyl sulphoxide (i.e., DMSO)
Cyclophosphamide (i.e., CP): Hanks’ balanced salt solution (i.e., HBSS) without calcium and magnesium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Dose range finding test:

In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 10E6 cells (i.e., 10E6 cells/mL for 3 h treatment) or 5 x 10E6 cells (i.e., 1.25 x 10E5 cells/mL for 24 h treatment) with a number of test substance concentrations increasing with approximately half log steps. The cell cultures for the 3 h treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0±1.0°C and 145 spm. The cell cultures for the 24 h treatment were placed in sterile 75 cmE2 culture flasks at 37.0±1.0°C. Test substance was tested in the absence and presence of 4% (v/v) S9-fraction. The highest tested concentration was 1,000 μg/mL exposure medium. Although test substance was soluble in the exposure medium up to 120 mg/mL, at test substance concentrations of 666 μg/mL and above the test substance formed a suspension in the exposure medium.

Cell cultures were exposed to test substance in exposure medium for 3 h in the presence of S9-mix and for 3 and 24 h in the absence of S9-mix. After exposure, the cells were separated from treatment solutions by 2 centrifugation steps (i.e., 216 g, 8 minutes) each followed by removal of the supernatant. The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the final centrifugation step the cells were resuspended in R10 medium. The cells in the final suspension were counted with the coulter particle counter. For determination of the cytotoxicity, the surviving cells of the 3 h treatment were subcultured twice. After 24 h of subculturing, the cells were counted (Day 1) and subcultured again for another 24 h, after which the cells were counted (Day 2). The surviving cells of the 24 h treatment were subcultured once. After 24 h of subculturing, the cells were counted. If less than 1.25x10E5 cells/mL were counted no subculture was performed. The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 h or only 24 h cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests.

Mutagenicity test:
Eight doses of test substance were tested in the mutation assay, both in the absence and presence of S9-mix. The highest doses that were tested gave a cell survival of approximately 10-20% and the survival in the lowest doses was approximately the same as the cell survival in the solvent control. Also some intermediate doses were tested.

Treatment of the cells:
Per culture 8x10E6 cells (i.e, 106 cells/mL for 3 h treatment) or 5 x 10E6 cells (i.e., 1.25x10E5 cells/mL for 24 h treatment) were used. The cell cultures for the 3 h treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0±1.0°C and 145 spm. The cell cultures for the 24 h treatment were placed in sterile 75 cmE2 culture flasks at 37.0±1.0°C. Solvent and positive controls were included and the solvent control was tested in duplicate.

In the first experiment, cell cultures were exposed for 3 h to test substance in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to test substance in exposure medium for 24 h in the absence of S9-mix and for 3 h in the presence of S9-mix. After exposure, the cells were separated from treatment solutions by 2 centrifugation steps (i.e., 216 g, 8 minutes) each followed by removal of the supernatant. The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the final centrifugation step the cells were resuspended in R10 medium. The cells in the final suspension were counted with the coulter particle counter.

Expression period:
For expression of the mutant phenotype, the remaining cells were cultured for 2 d after the treatment period. During this culture period at least 4x10E6 cells (where possible) were subcultured every day in order to maintain log phase growth. 2 d after the end of the treatment with the test substance the cells were plated for determination of the cloning efficiency (i.e., CEday2) and the mutation frequency (MF).

Evaluation criteria:

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

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (i.e., mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (i.e., questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (i.r., not mutagenic) in the mutation assay if:

a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Acceptability of the assay:
 
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (i.e., CE Day2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106survivors and ≤ 170 per 106survivors.
c) The growth rate (i.e., GR) over the 2 d expression period for the negative controls should be between 8 and 32 (3 h treatment) and between 32 and 180 (24 h treatment).
d) The mutation frequency of MMS should not be below 500 per 106 survivors, and for CP not below 700 per 106 survivors.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9-mix: 170 μg/mL and above; With 4% (v/v) S9-mix: 250 μg/mL and above; With 8% (v/v) S9-mix: 200 μg/mL and above

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility:
Test substance precipitated in the exposure medium at concentrations of 666 μg/mL and above.

Dose range finding test:
Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to test substance concentrations of 100 μg/mL compared to the suspension growth of the solvent control. No cell survival was observed at test substance concentrations of 333 μg/mL and above.

In the absence of S9-mix, the relative suspension growth was 64% at the test substance concentration of 100 μg/mL compared to the relative suspension growth of the solvent control. Hardly any or no cell survival was observed at test substance concentrations of 333 μg/mL and above.

Mutation experiments:

First mutagenicity test:

Evaluation of toxicity:

In the absence of S9-mix, the dose levels of 1 to 125 μg/mL showed no cytotoxicity. The dose levels of 170, 180 and 190 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

In the presence of S9-mix, the dose levels of 10 to 235 μg/mL showed no cytotoxicity.

The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 1, 3, 10, 33, 100, 125, 150 and 160 μg/mL exposure medium.
With S9-mix: 10, 100, 170, 200, 225, 235, 250 and 265 μg/mL exposure medium.

In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 65% compared to the total growth of the solvent controls.

In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 90% compared to the total growth of the solvent controls.

Evaluation of the mutagenicity:
No significant increase in the mutation frequency at the TK locus was observed after treatment with test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test substance treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second mutagenicity test:

Evaluation of toxicity:
In the absence of S9-mix, the dose levels of 3 to 130 μg/mL showed no cytotoxicity.

In the presence of S9-mix, the dose levels of 10 to 150 μg/mL showed no cytotoxicity and the dose levels of 200 to 265 showed similar cytotoxicity.

The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 3, 10, 33, 50, 100, 150, 170 and 185 μg/mL exposure medium.
With S9-mix: 33, 100, 150, 225, 235, 250, 265 and 280 μg/mL exposure medium.

In the absence of S9-mix, the relative total growth of the highest test substance was reduced by 90% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 94% compared to the total growth of the solvent controls.

Evaluation of mutagenicity:
No significant increase in the mutation frequency at the TK locus was observed after treatment with test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test substance treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Results of control groups:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range. The growth rate over the 2 d expression period for cultures treated with the vehicle control was between 14 and 22 (3 h treatment) and 98 and 105 (24 h treatment).

Mutation frequencies in cultures treated with positive control chemicals were increased by 11-fold for MMS in the absence of S9-mix, and by 34- and 19-fold for CP in the presence of S9-mix, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay.

Conclusions:

Under the study conditions, the test substance did not induce a significant increase in the mutation frequency in the absence and presence of the metabolic activation and was therefore considered to be non-mutagenic in the mouse lymphoma assay.

Executive summary:

A study was performed to investigate the potential of the test substance to induce gene mutations in L5178Y mouse lymphoma cells according to OECD Guideline 476, in compliance with GLP. The study was performed in two independent experiments in the presence and absence of S9-mix derived from rat liver homogenate. In the first experiment, the substance was evaluated at concentrations up to 160 and 265 μg/mL in the absence and presence of 4% v/v S9. The incubation time was 3 h. In the second experiment, the substance was tested at concentrations up to 185 and 280 μg/mL in the absence and presence of 8% v/v S9. The incubation time was 24 h in the absence of S9 and 3 h in the presence of S9. The negative control values were within the laboratory historical control data ranges and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased 11-fold for methyl methanesulfonate in the absence of S9-mix, and 34- and 19-fold for cyclophosphamide in the presence of S9-mix, indicating that the test conditions were appropriate and that the metabolic activation functioned properly. Under the study conditions, the test substance did not induce a significant increase in the mutation frequency in the absence and presence of the metabolic activation and was therefore considered to be non-mutagenic in the mouse lymphoma assay (Verspeek, 2014).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

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).

Mouse lymphoma assay

A study was performed to investigate the potential of the test substance to induce gene mutations in L5178Y mouse lymphoma cells according to OECD Guideline 476, in compliance with GLP. The study was performed in two independent experiments in the presence and absence of S9-mix derived from rat liver homogenate. In the first experiment, the substance was evaluated at concentrations up to 160 and 265 μg/mL in the absence and presence of 4% v/v S9. The incubation time was 3 h. In the second experiment, the substance was tested at concentrations up to 185 and 280 μg/mL in the absence and presence of 8% v/v S9. The incubation time was 24 h in the absence of S9 and 3 h in the presence of S9. The negative control values were within the laboratory historical control data ranges and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased 11-fold for methyl methanesulfonate in the absence of S9-mix, and 34- and 19-fold for cyclophosphamide in the presence of S9-mix, indicating that the test conditions were appropriate and that the metabolic activation functioned properly. Under the study conditions, the test substance did not induce a significant increase in the mutation frequency in the absence and presence of the metabolic activation and was therefore considered to be non-mutagenic in the mouse lymphoma assay (Verspeek, 2014).

Chromosome aberration test

A study was conducted to determine the potential of the test substance to induce chromosome aberrations in cultured peripheral human lymphocytes according to OECD Guideline 473, in compliance with GLP. Two independent experiments were conducted. The test substance was dissolved in cell culture medium and the dose levels were selected on the basis of dose range finding test. In the first assay, the substance was tested up to a concentration of 1,000 μg/mL with and without metabolic activation (S9-mix). In the second assay, the concentrations used were up to 1,000 μg/mL and 300 μg/mL with and without metabolic activation, respectively. Positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay and the efficacy of the S9-mix. The test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in presence and absence of metabolic activation. Likewise, no biologically relevant effect on number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of metabolic activation. Under the study conditions, the test substance did not induce structural chromosome aberrations in cultured peripheral human lymphocytes both in the presence and absence of a metabolic activation system (Buskens, 2014).

Justification for classification or non-classification

Based on the available negative in vitro data on 9-Octadecenoic acid (Z)-, sulfonated, potassium salts, no classification is required for genotoxicity according to CLP (EC 1272/2008) criteria.