Quaternary ammonium compounds, C18 and C18 (unsaturated) alkyltrimethyl, chlorides

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of in vitro studies with the test as well as read across substance, the test substance is considered to have any no genotoxic potential.

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:

12 May 2017 - 26 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

d.d. 3 November 2015

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

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:

Rat liver S9-mix induced with Aroclor 1254
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight). Each S9 batch is characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

Preparation of S9-Mix
S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 ml: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 ml or 5.0 ml Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA., USA); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution (Merck); 1 ml 0.33 M KCl solution (Merck). The above solution was filter (0.22 μm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with 5% S9-mix) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study:
TA1535, TA1537 and TA98: without and with 5% S9-mix: 0.18, 0.55, 1.7, 5.4, 17 and 52 µg/plate
Additional:
TA1535 and TA98: with 5% S9-mix: 17, 52, 164 and 512 µg/plate

Experiment 2:
All strains: without S9-mix: 4.7, 8.5, 15, 27, 48 and 86 µg/plate
All strains: with 10% S9-mix: 8.5, 15, 27, 48, 86 and 154 µg/plate
Additional:
TA1535, TA1537, TA98 and TA100: without S9-mix: 0.83, 1.5, 2.6, 4.7, 8.5 and 15 µg/plate
WP2uvrA: with 10% S9-mix: 86, 154, 275 and 492 µg/plate

Vehicle / solvent:

- Vehicle used: Milli-Q Water
- Justification for choice of solvent/vehicle: Test compound was soluble in water and water has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9; 5 µg/plate in saline for TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

without S9; 2.5 µg/plate in DMSO for TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene

Remarks:

without S9; 10 µg/plate in DMSO for TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9; 650 µg/plate in DMSO for TA100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9; 10 µg/plate in DMSO for WP2uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

in DMSO; 2.5 μg/plate for TA1535 (5% + 10% S9-mix) and TA1537 (5% S9-mix); 5 μg/plate for TA1537 (10% S9-mix); 1 μg/plate for TA98 (5% + 10% S9-mix) and TA100 (5% S9-mix); 2 μg/plate for TA100 (10% S9-mix); 15 μg/plate for WP2uvrA (5% + 10% S9-mix).

Details on test system and experimental conditions:

Method of application: in agar (plate incorporation)

Duration
- Exposure duration: 48 ± 4 hour

Number of replications:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

Number of cells evaluated: 10E8 per plate

Determination of cytotoxicity
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

Other examinations:
- The presence of precipitation of the test compound on the plates was determined.

Additional experiments:
Since in the first experiment in the presence of S9-mix, no toxicity and no precipitate was observed at the highest dose levels tested in the tester strains TA1535 and TA98, an additional experiment was performed. In the second mutation experiment, due to the cytotoxicity, only two to three analyzable dose levels were left for the determination of the mutagenicity of the test substance in the tester strains TA1535, TA1537, TA98 and TA100 in the absence of S9-mix. Furthermore, in tester strain WP2uvrA no dose level with toxicity or precipitate on the plates was observed in the presence of S9-mix. Therefore an additional mutation experiment was performed.

Rationale for test conditions:

A dose-range finding test was performed and the highest concentration of the test item used in the subsequent mutation assay was 5000 μg/plate or the level at which the test item inhibited bacterial growth.

Evaluation criteria:

ACCEPTABILITY CRITERIA
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 S9-mix) 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.

INTERPRETATION
A test substance 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 substance is considered positive 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:

No formal hypothesis testing was done.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle 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:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Results

Dose-range Finding Test/First Mutation Experiment

Test substance (dried) 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: 0.18, 0.55, 1.7, 5.4, 17 and 52 μg/plate.

 

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

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, except for the tester strains TA1535 and TA98 in the presence of S9.

 

Mutagenicity

No increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

 

First Mutation Experiment (additional)

Since in the first experiment in the presence of S9-mix, no toxicity and no precipitate was observed at the highest dose levels tested in the tester strains TA1535 and TA98, an additional experiment was performed. Based on the results of the first mutation experiment, the following dose-range was selected for the additional mutation experiment with the tester strains, TA1535 and TA98 in the presence of S9-mix: 17, 52, 164 and 512 μg/plate. Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period.

 

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 both tester strains.

 

Mutagenicity

In the additional mutation experiment, no increase in the number of revertants was observed upon treatment with Test substance (dried) 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 substance was tested up to the dose levels of 86 and 154 µg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the absence and presence of S9 mix, respectively. Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period.

 

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. Except for tester strain WP2uvrA, where no cytotoxicity was observed in the presence of S9 mix.

 

Mutagenicity

In the second mutation assay, no increase in the number of revertants was observed upon treatment with Test substance (dried) under all conditions tested.

 

Second Mutation Experiment (additional)

In the second mutation experiment, due to the cytotoxicity, only two to three analyzable dose levels were left for the determination of the mutagenicity of the test substance in the tester strains TA1535, TA1537, TA98 and TA100 in the absence of S9-mix. Furthermore, in tester strain WP2uvrA no dose level with toxicity or precipitate on the plates was observed in the presence of S9-mix. Therefore an additional mutation experiment was performed. In this additional experiment, the test substance was tested at concentration ranges of 0.83 to 15 µg/plate in strains TA1535, TA1537, TA98 and TA100 in the absence of S9 mix and at 86 to 492 µg/plate in tester strain WP2uvrA in the presence of S9 mix.

 

Precipitate

No precipitate was observed at the start of the incubation period. Although in the tester strains TA1535 and TA1537, as well as in the second mutation experiment, no precipitation was observed, the test substance seemed to show slight precipitation at the highest tested concentration of 15 µg/plate in the tester strains TA98 and TA100 in the presence of a reduced bacterial background.

 

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 or presence of S9-mix. Except for tester strain TA1535, where no cytotoxicity was observed in the absence of S9 mix.

 

Mutagenicity

In the second mutation assay, no increase in the number of revertants was observed upon treatment with Test substance (dried) under all conditions tested.

 

Discussion

All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two experiments. The negative and strain-specific positive control values were within the laboratory historical control data. 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 TA98 in the 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 8 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.

 

Conclusion

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

Conclusions:

Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay, with or without metabolic activation.

Executive summary:

An in vitro study was conducted to determine the mutagenic potential of test substance, Oleyl TMAC (91.9% acive), according to OECD Guideline 471 and EU Method B.13/B.14, in compliance with GLP. In the dose-range finding test, the test substance was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Based on the results of the dose-range finding test, the test substance was tested in the first mutation assay at a concentration range of 0.18 to 52 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test substance did not precipitate on the plates at this dose level.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, except for the tester strains TA1535 and TA98 in the presence of S9. So, an additional experiment was performed with these two tester strains TA1535 and TA98, using concentration range of 17 to 512 μg/plate of the test substance. No precipitation on the plates was observed at this dose level, however, cytotoxicity was observed in both tester strains. In a follow-up experiment of the assay with additional parameters, the test substance was tested at concentration ranges of 4.7 to 86 μg/plate and 8.5 to 154 μg/plate in the absence and presence of 10% (v/v) S9-mix, respectively, in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test substance did not precipitate on the plates at this dose level. Cytotoxicity, was observed in all tester strains in the absence and presence of S9-mix. Except for tester strain WP2uvrA, where no cytotoxicity was observed in the presence of S9 mix. In the second mutation experiment, due to the cytotoxicity, only two to three analyzable dose levels were left for the determination of the mutagenicity of the test substance in the tester strains TA1535, TA1537, TA98 and TA100 in the absence of S9-mix. Furthermore, in tester strain WP2uvrA no dose level with toxicity or precipitate on the plates was observed in the presence of S9-mix. Therefore an additional mutation experiment was performed. In this additional experiment, the test substance was tested at concentration ranges of 0.83 to 15 μg/plate in strains TA1535, TA1537, TA98 and TA100 in the absence of S9 mix and at 86 to 492 μg/plate in tester strain WP2uvrA in the presence of S9 mix. The test substance seemed to show slight precipitation at the highest tested concentration of 15 μg/plate in the tester strains TA98 and TA100 in the presence of a reduced bacterial background. Cytotoxicity, was observed in all tester strains in the absence or presence of S9-mix. Except for tester strain TA1535, where no cytotoxicity was observed in the absence of S9 mix. 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 the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. In this study, acceptable responses were obtained for the negative and strain-specific positive control substances, indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay, with or without metabolic activation (Gijsbrechts, 2017).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From April 17, 1989 to September 15, 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.

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

Qualifier:

according to guideline

Guideline:

other: Toxicity test guideline, Japan 1984

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM medium supplemented with 10% foetal calf Serum (FCS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

without S9 mix: (7 hours: 1.0 µg/mL; 18 hours: 0.3, 1.0 and 3.0 µg/mL; 28 hours: 3.0 µg/mL)
with S9 mix: (7 hours: 10.0 µg/mL; 18 hours: 1.0, 3.0 and 10.0 µg/mL; 28 hours: 10.0 µg/mL)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h
- Fixation time (start of exposure up to fixation or harvest of cells): 7h (high dose), 18h (low, medium and high dose), and 28h (high dose)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (approx. 0.2 µg/mL/culture medium)

STAIN (for cytogenetic assays): Giemsa stains

NUMBER OF REPLICATIONS: Two

NUMBER OF CELLS EVALUATED: 100 cells of each cell culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

- A test article is classified as clastogenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points.
- A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant and reproducible positive response at any one of the test points is considered non-clastogenic in this system.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

- In the pre-experiments for toxicity the colony forming ability of the V79 cells was totally reduced after treatment with 6.0 µg/mL. Accordingly, one (for 7 and 28h time point) and three concentrations (for 18h time point) were selected to evaluate metaphases for cytogenetic damage. Mitotic index was reduced after treatment with the highest dose levels in the absence and presence of S9 mix, in the main test.
- Mutation results:
There was no relevant increase in cells with structural aberrations after treatment with the test substance at any fixation interval either without or with metabolic activation by S9 mix. Positive controls showed distinct increases in cells with structural chromosome aberrations. The sensitivity of the test system and efficacy of the S9 mix was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.
(COMPARISON WITH HISTORICAL CONTROL DATA: Yes)

Conclusions:

Under the study conditions, the read across substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line with and without metabolic activation.

Executive summary:

An in vitro study was conducted to investigate the potential of read across substance, C16 TMAC (24 -26% active in water) to induce chromosome aberrations in V79 Chinese hamster lung cells, according to OECD Guideline 473 and EU Method B.10, in compliance with GLP. The concentration range of the read across substance was determined in a pre-experiment using the plating efficiency assay as an indicator of toxicity response. Cells were exposed for 7, 18 or 28 h at concentrations levels of 0.3 to 10.0 µg a.i./mL read across substance with or without metabolic activation. Treatment with 3.0 µg/mL and 10.0 µg a.i./mL completely reduced the plating efficiency of the V79 cells. The mitotic index was reduced after treatment with the highest concentration at each fixation interval in the presence and absence of S9 mix. Positive controls showed a distinct increase in the number of cells with structural chromosome aberrations. There was no relevant increase in cells with structural aberrations after treatment with the read across substance at any fixation interval either without or with S9 mix. Under the study conditions; the read across substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line with and without metabolic activation (Heidemann, 1989). Based on the results of the read across study, the test substance is not clastrogenic in V79 chinese hamster lung cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From August 30, 2006 to March 12, 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4E

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) supplemented with 10% foetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I: without S9 mix: 0.2, 0.4, 0.8, 1.5 and 2.3 µg/mL, with S9 mix: 1.6, 3.1, 6.3, 12.5 and 18.8 µg/mL
Experiment II:without S9 mix: 0.4, 0.8, 1.6, 3.1 and 4.7 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water

Untreated negative controls:

yes

Remarks:

Untreated control

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

other: Untreated cells were cultivated without interruption throughout the assay and without addition of test item, in order to obtain the initial spontaneous mutation rate at the beginning of the experiments.

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

yes

Remarks:

Untreated control

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

other: Untreated cells were cultivated without interruption throughout the assay and without addition of test item, in order to obtain the initial spontaneous mutation rate at the beginning of the experiments.

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Seeded into plastic culture flasks
DURATION
- Preincubation period: 24h
- Exposure duration: 4h (in experiment I; with and without S9), 24h (in experiment II; without S9)
- Expression time (cells in growth medium): 7d
- Selection time (if incubation with a selection agent): Day 7

SELECTION AGENT (mutation assays): Thioguanine (6TG)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The test item was considered positive if (a) It reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment. (b) There is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from the enhancement factor for induced mutants. (c) Survival of the responding dose group is at least 30%. However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.

Statistics:

A linear regression was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT® statistics software.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effects
- Effects of osmolality: No effects
- Precipitation: No precipitation or phase separation of the test substance was observed up to the maximum concentration in both main experiments.

RANGE-FINDING/SCREENING STUDIES: Two range finding pre-tests were performed in the presence (4h treatment) and absence (4h and 24h treatment) of S9. In the first pre-test test substance concentrations between 25 and 3200 µg/mL (active substance) were used to evaluate toxicity. In this first pretest strong toxic effects were noted at all concentrations with and without metabolic activation. Therefore, a second pre-test was performed using concentrations of 0.2 to 25 µg/mL (active substance).
Following 4h treatment without S9 mix strong toxicity occurred at 1.58 µg/mL. The cell growth was completely stopped at the next higher concentration of 3.13 µg/mL and above. In the presence of S9 mix (4h treatment) strong toxicity was determined at the highest concentration of 25 µg/mL. After 24h of treatment a relevant toxic effect occurred at 3.13 µg/mL. At all higher concentrations the cell growth was also completely inhibited.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

The sensitivity of the test system and efficacy of the S9 mix was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.

Mutation results:

Main experiment:

- No relevant and reproducible increase of the mutation frequency occurred at any concentration with and without metabolic activation. All mutant frequencies remained well within the historical range of negative and solvent controls.

- A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in both cultures of experiment I with metabolic activation. However, a small increase of the mutation frequency at toxic concentrations is common in this assay system and does not indicate a possible mutagenic potential provided that the mutation frequency does not exceed the threshold of 3 times above the corresponding solvent control. Since the mutation frequency neither exceeded the historical range of negative and solvent controls nor the threshold as indicated above, the statistical results were considered as biologically irrelevant.

Conclusions:

Under the study conditions, the read across substance did not induce gene mutations in the HPRT locus in V79 Chinese hamster cells, either in the presence or absence of metabolic activation.

Executive summary:

An in vitro study was conducted to investigate the potential of read across substance, C16 TMAC (25% active in water) to induce gene mutations at the HPRT locus in V79 Chinese hamster cells, according to OECD Guideline 476, in compliance with GLP. The assay was performed in two independent experiments. The cells were exposed to the test substance for 4 h in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 h. The concentrations of the read across substance in experiment I ranged from 1.6 to 5 µg a.i./mL and 0.2 to 3 µg a.i./mL with and without S9 mix respectively and from 0.4 to 6.3 µg a.i./mL without S9 mix in experiment II. All positive controls showed a distinct increase in the number of mutant colonies. No substantial and reproducible concentration-dependent increases in the mutation frequency at the HPRT locus, was seen in read across substance-treatment cells either with or without metabolic activation. Under the study conditions, the read across substance did not induce gene mutations in the HPRT locus in V79 Chinese hamster cells, either in the presence or absence of metabolic activation (Wollny, 2007). Based on the results of the read across study, the test substance does not show mutagenic activity in mouse lymphoma assay

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Study 1: An in vitro study was conducted to determine the mutagenic potential of test substance, Oleyl TMAC (91.9% acive), according to OECD Guideline 471 and EU Method B.13/B.14, in compliance with GLP.In the dose-range finding test, the test substance was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA.Based on the results of the dose-range finding test, the test substance was tested in the first mutation assay at a concentration range of 0.18 to 52 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test substance did not precipitate on the plates at this dose level.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, except for the tester strains TA1535 and TA98 in the presence of S9. So, an additional experiment was performed with these two tester strains TA1535 and TA98, using concentration range of 17 to 512 μg/plate of the test substance. No precipitation on the plates was observed at this dose level, however, cytotoxicity was observed in both tester strains. In a follow-up experiment of the assay with additional parameters, the test substance was tested at concentration ranges of 4.7 to 86 μg/plate and 8.5 to 154 μg/plate in the absence and presence of 10% (v/v) S9-mix, respectively, in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test substance did not precipitate on the plates at this dose level. Cytotoxicity, was observed in all tester strains in the absence and presence of S9-mix. Except for tester strain WP2uvrA, where no cytotoxicity was observed in the presence of S9 mix.In the second mutation experiment, due to the cytotoxicity, only two to three analyzable dose levels were left for the determination of the mutagenicity of the test substance in the tester strains TA1535, TA1537, TA98 and TA100 in the absence of S9-mix. Furthermore, in tester strain WP2uvrA no dose level with toxicity or precipitate on the plates was observed in the presence of S9-mix. Therefore an additional mutation experiment was performed. In this additional experiment, the test substance was tested at concentration ranges of 0.83 to 15 μg/plate in strains TA1535, TA1537, TA98 and TA100 in the absence of S9 mix and at 86 to 492 μg/plate in tester strain WP2uvrA in the presence of S9 mix. The test substance seemed to show slight precipitation at the highest tested concentration of 15 μg/plate in the tester strains TA98 and TA100 in the presence of a reduced bacterial background. Cytotoxicity, was observed in all tester strains in the absence or presence of S9-mix. Except for tester strain TA1535, where no cytotoxicity was observed in the absence of S9 mix.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 the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.In this study, acceptable responses were obtained for the negative and strain-specific positive control substances, indicating that the test conditions were adequate and that the metabolic activation system functioned properly.Under the study conditions,it was concluded that the read acrosssubstance was not mutagenic in Salmonella typhimurium strain TA98, TA100, TA1535 or TA 1537 and E.Coli WP2uvrA, with or without metabolic activation(Gijsbrechts, 2017).

Study 2: An in vitro study was conducted to investigate the potential of read across substance, C16 TMAC (24 -26% active in water) to induce chromosome aberrations in V79 Chinese hamster lung cells, according to OECD Guideline 473 and EU Method B.10, in compliance with GLP. The concentration range of the read across substance was determined in a pre-experiment using the plating efficiency assay as an indicator of toxicity response. Cells were exposed for 7, 18 or 28 h at concentrations levels of 0.3 to 10.0 µg a.i./mL read across substance with or without metabolic activation. Treatment with 3.0 µg/mL and 10.0 µg a.i./mL completely reduced the plating efficiency of the V79 cells. The mitotic index was reduced after treatment with the highest concentration at each fixation interval in the presence and absence of S9 mix. Positive controls showed a distinct increase in the number of cells with structural chromosome aberrations. There was no relevant increase in cells with structural aberrations after treatment with the read across substance at any fixation interval either without or with S9 mix. Under the study conditions; the read across substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line with and without metabolic activation (Heidemann, 1989).Based on the results of the read across study, the test substance is not clastrogenic in V79 chinese hamster lung cells.

Study 3: An in vitro study was conducted to investigate the potential of read across substance, C16 TMAC (25% active in water) to induce gene mutations at the HPRT locus in V79 Chinese hamster cells, according to OECD Guideline 476, in compliance with GLP. The assay was performed in two independent experiments. The cells were exposed to the test substance for 4 h in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 h. The concentrations of the read across substance in experiment I ranged from 1.6 to 5 µg a.i./mL and 0.2 to 3 µg a.i./mL with and without S9 mix respectively and from 0.4 to 6.3 µg a.i./mL without S9 mix in experiment II. All positive controls showed a distinct increase in the number of mutant colonies. No substantial and reproducible concentration-dependent increases in the mutation frequency at the HPRT locus, was seen in read across substance-treatment cells either with or without metabolic activation. Under the study conditions, the read across substance did not induce gene mutations in the HPRT locus in V79 Chinese hamster cells, either in the presence or absence of metabolic activation (Wollny, 2007).Based on the results of the read across study, the test substance does not show mutagenic activity in mouse lymphoma assay.

Based on the results of the read across study, similar non-mutagenic and clastogenic potential can be expected for the test substance.

 

Justification for classification or non-classification

Based on the results from an in vitro Ames asay with the test substance as well as in vitro chromosomal aberration and mammalian mutagenicity assays with the read across substance, the test substance does not warrant a classification for genotoxicity according to the EU CLP criteria (Regulation 1272/2008/EC).​