Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the available Ames test with the test substance as well as in vitro genotoxicity data with the read across substance, Oleyl TMAC is not considered to have any genotoxic potential.

Link to relevant study records

Referenceopen allclose all

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
d.d. 3 November 2015
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Oleyltrimethylammonium chloride (dried)
- Batch No.: 1439581 (superdry)
- Expiration date of the batch: 09 September 2018
- Appearance: yellow paste
- Storage conditions: At room temperature container flushed with nitrogen, desiccated
- Handlings with test material: in glove box (nitrogen environment)
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
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 item 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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed except in the second experiment where the test item 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.

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 17 μg/plate and above and 52 μg/plate and above in the absence and presence of S9-mix, respectively. In tester strain WP2uvrA, toxicity was observed at dose levels of 52 μg/plate and above and 164 μg/plate and above in the absence and presence of S9-mix, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
- 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, except the response for TA98 in the presence of S9-mix in the second experiment. Since the value (230 ± 69) was more than 8 times greater than the concurrent solvent control values (27 ± 6), this deviation in the mean plate count of the positive control had no effect on the results of the study.

HISTORICAL CONTROL DATA
- Positive historical control data:
TA1535 TA1537 TA98
S9-mix - + - + - +
Range 125 - 1381 78 - 1058 55 – 1324 55 – 1051 410 – 1995 250 - 1977
Mean 839 220 736 382 1369 929
SD 153 112 331 150 310 345
n 2065 1967 1740 1933 1920 2014

TA100 WP2uvrA
S9-mix - + - +
Range 537 – 1848 408 - 2651 93 – 1951 93 - 1359
Mean 908 1330 1128 422
SD 178 324 484 151
n 2007 2020 1679 1728

- Negative (solvent/vehicle) historical control data:
TA1535 TA1537 TA98
S9-mix - + - + - +
Range 3 - 36 3 - 32 3 – 20 3 – 23 8 - 41 9 - 55
Mean 11 11 6 7 16 23
SD 4 4 3 3 5 7
n 2057 2039 1950 1931 2023 2083

TA100 WP2uvrA
S9-mix - + - +
Range 66 - 161 63 - 160 10 – 59 9 - 69
Mean 105 105 25 31
SD 19 20 7 8
n 2027 2033 1739 1745


ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 27 µg/plate and above; with 5% S9: 164 µg/plate and above; with 10% S9: 154 µg/plate and above
TA1537: without S9: 8.5 µg/plate and above; with 5%S9: 52 µg/plate and above; with 10% S9: 154 µg/plate and above
TA98: without S9: 15 µg/plate and above; with 5% S9: 164 µg/plate and above; with 10% S9: 86 µg/plate and above
TA100: without S9: 15 µg/plate and above; with 5% S9: 52 µg/plate and above; with 10% S9: 86 µg/plate and above
WP2uvrA: without S9: 48 µg/plate and above; with 5% S9: 164 µg/plate and above; with 10% S9: 275 µg/plate and above
Conclusions:
Under the study conditions, the test substance was not mutagenic in Salmonella typhimurium strain TA98, TA100, TA1535 or TA 1537 and E.Coli WP2uvrA, with or without metabolic activation.
Executive summary:

An in vitro study was conducted to determine the mutagenic potential of test substance, Oleyl TMAC, according to OECD Guideline 471 and EU Method B.13/B.14, in compliance with GLP. 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 test substance was not mutagenic in Salmonella typhimurium strain TA98, TA100, TA1535 or TA 1537 and E.Coli WP2uvrA, with or without metabolic activation (Gijsbrechts, 2017).

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. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
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:
Based on the results of the read across study, the test substance, Oleyl TMAC, is not considered to induce clastogenicity in the V79 Chinese hamster cell line.
Executive summary:

An in vitro study was conducted to investigate the potential of the read across substance, C16 TMAC (active: 26%), 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 test 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 hours at concentrations levels of 0.0 to 10.0 µg/L test substance with or without metabolic activation. Treatment with 3.0 µg/mL and 10.0 µg/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 test substance at any fixation interval either without or with S9 mix. Under the conditions of the study, the test 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, Oleyl TMAC, is not considered to induce clastogenicity in the V79 Chinese hamster cell line, with or without metabolic activation.

.

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. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4E
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
Based on the results of the read across study, the test substance, Oleyl TMAC, is also not considered to induce mutagenicity in the HPRT locus in V79 Chinese hamster cells, with or without metabolic activation.
Executive summary:

An in vitro study was conducted to investigate the potential of the read across substance, C16 TMAC (active: 25%), to induce gene mutations at the HPRT locus in V79 Chinese hamster cells, according to OECD Guideline 476, in compliance with GLP. 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 test substance-treatment cells either with or without metabolic activation. Under the study conditions, the test substance was not found to 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, Oleyl TMAC, is also not considered to induce mutagenicity in the HPRT locus in V79 Chinese hamster cells, with or without metabolic activation.

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, according to OECD Guideline 471 and EU Method B.13/B.14, in compliance with GLP. 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 thetest substance 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 the read across substance, C16 TMAC (active: 26%), 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 test 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 hours at concentrations levels of 0.0 to 10.0 µg/L test substance with or without metabolic activation. Treatment with 3.0 µg/mL and 10.0 µg/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 test substance at any fixation interval either without or with S9 mix. Under the conditions of the study, the test 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, Oleyl TMAC, is not considered to induce clastogenicity in the V79 Chinese hamster cell line, with or without metabolic activation.

Study 3: An in vitro study was conducted to investigate the potential of the read across substance, C16 TMAC (active: 25%), to induce gene mutations at the HPRT locus in V79 Chinese hamster cells, according to OECD Guideline 476, in compliance with GLP. 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 test substance-treatment cells either with or without metabolic activation. Under the study conditions, the test substance was not found to 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, Oleyl TMAC, is also not considered to induce mutagenicity in the HPRT locus in V79 Chinese hamster cells, with or without metabolic activation.

Justification for classification or non-classification

Based on the available Ames test with the test substance as well asin vitrogenotoxicity data with the read across substance, Oleyl TMAC does not warrant classification for genotoxicity according to EU CLP criteria (Regulation 1272/2008/EC).