Hexadecyltrimethylammonium methyl sulphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of the in vitro genotoxicity studies with the test and read across substances, the test substance is not considered to be genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Study period:

From 05 December, 2002 to 13 January, 2003

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.

Reason / purpose for cross-reference:

read-across source

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

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other:

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and the cofactors necessary for their function. S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route. The S9 fraction was preserved in sterile tubes at -80°C, until use. The S9 mix was prepared at +4°C immediately before use and maintained at this temperature until added to the overlay agar.

Test concentrations with justification for top dose:

Preliminary test concentrations:10, 100, 500, 1000, 2500 and 5000 μg/plate for both with and without S9-mix

Main test concentrations: Experiments without S9 mix:
• 1.23, 3.7, 11.1, 33.3 and 100 μg/plate, for all tester strains in the first experiment,
• 0.41, 1.23, 3.7, 11.1 and 33.3 μg/plate, for all tester strains in the second experiment.

Experiments with S9 mix:
• 1.23, 3.7, 11.1, 33.3 and 100 μg/plate, for all tester strains in the first experiment,
• 3.7, 11.1, 33.3, 100 and 200 μg/plate, for all tester strains in the second experiment, except for the TA 1537 strain,
• 1.23, 3.7, 11.1, 33.3 and 100 μg/plate, for the TA 1537 strain in the second experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO), batch No. K30379650 214 (Merck Eurolab, Fontenay-Sous-Bois, France).

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

1 μg/plate: for TA 1535 and TA 100 in the absence of S-9 mix

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

50 μg/plate: for TA 1537 in the absence of S-9 mix

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

0.5 μg/plate: for TA 98 in the absence of S-9 mix

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.5 μg/plate: for TA 102 in the absence of S-9 mix

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-anthramine

Remarks:

2 μg/plate: for TA 1535, TA1537, TA 98 and TA 100 in the presence of S9 mix and 10 μg/plate: TA 102 in the presence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation method) as well as preincubation method

DURATION
- Incubation period: 48 to 72 h at approximately 37°C

NUMBER OF REPLICATIONS: Three/dose

DETERMINATION OF CYTOTOXICITY
- Method: Number of revertant colonies were counted, with an automatic counter (Artek counter, model 880, OSi, 75015 Paris, France or cardinal counter, Perceptive instruments, Suffolk CB9 7 BN, UK)

Treatment
The test item was tested in a preliminary test and two mutagenicity experiments.

The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the preincubation method. The direct plate incorporation method was performed as follows: test item solution (0.1 mL), S9 mix when required (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium. The preincubation method was performed as follows: test item solution (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C under shaking before adding the overlay agar and pouring onto the surface of a minimum agar plate.
After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk CB9 7 BN, UK).

Preliminary toxicity test
To assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100 and TA 102 strains, with and without S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Mutagenicity experiments
In two independent experiments, using three plates/dose-level, each strain was tested, with and without S9 mix, with:
• at least five dose-levels of the test item,
• the vehicle control,
• the appropriate positive control.
The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.

Evaluation criteria:

- Treatment of results
In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test item/mutants obtained in the presence of the vehicle), are presented in tabular form.

- Acceptance criteria
This study is considered valid if the following criteria are fully met:
• the number of revertants in the vehicle controls is consistent with the historical data of the testing facility (appendix 1),
• the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data of the testing facility.

- Evaluation criteria
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A moderate to strong toxicity was noted in all tester strains mainly at concentration-levels ≥ 33.3 μg/plate (experiments without S9). A moderate to strong toxicity was noted in all tester strains mainly at concentration-levels ≥ 100 μg/plate (with S9).

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

Remarks:

A moderate to strong toxicity was noted in all tester strains mainly at concentration-levels ≥ 33.3 μg/plate (experiments without S9). A moderate to strong toxicity was noted in all tester strains mainly at concentration-levels ≥ 100 μg/plate (with S9).

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

Remarks:

A moderate to strong toxicity was noted in all tester strains mainly at concentration-levels ≥ 33.3 μg/plate (experiments without S9). A moderate to strong toxicity was noted in all tester strains mainly at concentration-levels ≥ 100 μg/plate (with S9).

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

Remarks:

A moderate to strong toxicity was noted in all tester strains mainly at concentration-levels ≥ 33.3 μg/plate (experiments without S9). A moderate to strong toxicity was noted in all tester strains mainly at concentration-levels ≥ 100 μg/plate (with S9).

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A moderate to strong toxicity was noted in all tester strains mainly at concentration-levels ≥ 33.3 μg/plate (experiments without S9). A moderate to strong toxicity was noted in all tester strains mainly at concentration-levels ≥ 100 μg/plate (with S9).

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test: Yes
The test item was freely soluble in the vehicle (DMSO) at 50 mg/mL. Consequently, with a treatment volume of 100 μL/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 μg/plate. No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. Moderate to strong toxicity was induced in all tester strains at dose-levels ≥ 100 μg/plate, both with and without S9 mix.

Result

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix:

The selected treatment-levels were as follows:

• 1.23, 3.7, 11.1, 33.3 and 100 μg/plate, for all tester strains in the first experiment,

• 0.41, 1.23, 3.7, 11.1 and 33.3 μg/plate, for all tester strains in the second experiment.

A moderate to strong toxicity was noted in all tester strains mainly at dose-levels ≥ 33.3 μg/plate.

Experiments with S9 mix:

The selected treatment-levels were as follows:

• 1.23, 3.7, 11.1, 33.3 and 100 μg/plate, for all tester strains in the first experiment,

• 3.7, 11.1, 33.3, 100 and 200 μg/plate, for all tester strains in the second experiment, except for the TA 1537 strain,

• 1.23, 3.7, 11.1, 33.3 and 100 μg/plate, for the TA 1537 strain in the second experiment.

A moderate to strong toxicity was noted in all tester strains mainly at dose-levels ≥ 100 μg/plate. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Conclusions:

Under the study conditions, the test substance is not considered to be mutagenic in the presence and absence of exogenous metabolic activation.

Executive summary:

A study was conducted to determine the mutagenic potential of the read across substance, C12-14 ADMAES (95% active ingredient), according to OECD 471 Guideline and EU Method B.13/14, in compliance with GLP. The read across substance was examined for mutagenic activity in five strains of Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102, using the plate incorporation and pre-incubation methods. The studies were performed in the absence and presence of metabolic activation (S9-mix). The concentrations ranged from 1.23 to 100µg/plate in first experiment and 0.41 to 200 µg/plate in second experiment.No increase in reversion to prototrophy was obtained with any of the bacterial strain at any concentration either in the presence or absence of S9-mix. Further, inhibition of growth, observed as thinning of the background lawn of non-revertant cells, occurred in all strains following exposure to the read across substance at ≥100 µg/plate. Under the study conditions, the read across substance was not considered to be mutagenic in the presence and absence of exogenous metabolic activation (Haddouck, 2003).

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 April 17, 1989 to September 15, 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across source

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 test 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).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 09, 2017 to December 12, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

OECD Guideline 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016,

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

The test method was designed to be in alignment with the following Japanese Guidelines
1) Kanpoan No. 287 - - Environment Protection Agency
2) Eisei No. 127 - - Ministry of Health and Welfare
3) Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade & Industry

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burrough Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix [S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%].

Test concentrations with justification for top dose:

10 dose levels (0, 0.25, 0.5, 1, 2, 3, 6, 9, 12, 15, 18 µg/mL) in duplicate
4-h with and without S9 (2%): 0, 0.25, 0.5, 1, 2, 3, 6, 9, 12, 15, 18 µg/mL
24-h without S9: 0, 0.03, 0.06, 0.13, 0.25, 0.5, 1, 2, 3, 4, 5 µg/mL

The dose levels plated for viability and expression of mutant colonies were as follows:
4-h without S9: 0.5, 1, 2, 3, 6, 9 µg/mL
4-h with S9 (2%): 2, 3, 6, 9, 12, 15 µg/mL
24-h without S9: 0.06, 0.13, 0.25, 0.5, 1, 2 µg/mL

Vehicle / solvent:

Solvent: R0 media
Gibco batch: 1896455
Expiry: August 31, 2018

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent: R0 media, Gibco batch: 1896455, Expiry: August 31, 2018

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at approximately 37 deg C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Microsomal Enzyme Fraction
Lot No. PB/βNF S9 01/10/17 was used in this study, and was pre-prepared in-house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test. The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Test substance preparation
Following solubility checks performed in-house, the test substance was accurately weighed and formulated in R0 media prior to serial dilutions being prepared. The test substance was considered to be a UVCB; therefore the maximum recommended dose level was initially set at 5000µg/mL and no correction for purity was applied to formulations. There was no marked change in pH when the test substance was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al. 1991). No analysis was conducted to determine the homogeneity, concentration or stability of the test substance formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Statistics:

The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989). The statistical package used indicates the presence of statistically significant increases and linear trend events. The Delta building monitoring system was used during the course of the study.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Results

Preliminary Cytotoxicity Test

The dose range of the test substance used in the preliminary toxicity test was 0.16 to 20 µg/mL.

Table1: The results for the Relative Suspension Growth (%RSG) were as follows:

Dose (µg/mL)	% RSG (-S9) 4-H Exposure	% RSG (+S9) 4-H Exposure	% RSG (-S9) 24-H Exposure
0	100	100	100
0.16	95	101	98
0.31	94	110	67
0.63	99	122	70
1.25	89	101	27
2.5	72	75	6
5	45	51	1
10	7	27	0
15	0	5 p	0
20	0 p	2 p	0

p = precipitate of test substance at the end of exposure

In the all three exposure groups there was evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test substance when compared to the concurrent vehicle controls. A precipitate of the test substance was observed at 20 µg/mL in the 4-h –S9 exposure. A precipitate of the test substance was observed at 15 and 20 µg/mL in the 4-h +S9 exposure. In the subsequent mutagenicity experiments the maximum dose level was limited by a combination of test substance induced toxicity and precipitate.

Mutagenicity Test

A summary of the results from the test is presented in below table:

Table 2: Main Experiment

Treatment (µg/mL)		4-h-S-9				Treatment (µg/mL)		4-h+S-9
		%RSG	RTG	MF§				%RSG	RTG	MF§
0		100	1.00	97.82		0		100	1.00	141.33
0.25	Ø	90				0.25	Ø	104
0.5		87	0.90	119.83		0.5	Ø	104
1		73	0.80	109.62		1	Ø	93
2		68	0.65	125.14		2		94	0.97	133.05
3		55	0.49	117.06		3		79	0.92	100.97
6		31	0.22	112.32		6		48	0.48	144.28
9	X	8	0.07	118.40		9		41	0.48	111.37
12	Ø	1				12		24	0.28	94.32
15	Ø	0				15		12	0.10	124.90
18	Ø	0				18	Ø	1
MF threshold for a positive response= 223.82						MF threshold for a positive response= 267.33
EMS						CP
400		64	0.59	864.01		1.5		77	0.54	951.16

               

Treatment (µg/mL)		24-h-S-9
		%RSG	RTG	MF§
0		100	1.00	123.75
0.03	Ø	68
0.06		68	0.76	124.91
0.13		62	0.68	141.65
0.25		98	1.58	116.36
0.5		66	1.18	119.33
1		33	1.06	140.60
2		10	0.35	105.74
3	Ø	3
4	Ø	1
5	Ø	0
MF threshold for a positive response= 249.75
EMS
150		34	0.34	1786.40

 

Cell and 96-Well Plate Counts: Mutagenicity Test (-S9) 4-Hour Exposure

Treatment (µg/ml)		Cell counts $			Viability § after day 2 2 cells/well				Resistant mutants § after day 2 2000 cells/well
		0h	24h	48h
0	A B	11.52 12.03	5.94 5.59	7.97 8.24	75 89	77 88	79 87	87 91	18 14	15 22	18 21	16 18
0.25	A B	12.00 12.26	5.31 5.69	7.55 7.25	NP NP	NP NP			NP NP	NP NP
0.5	A B	12.87 10.84	5.03 6.17	7.47 7.03	81 86	86 86			19 23	21 24
1	A B	10.77 12.59	4.95 4.70	6.93 7.24	80 87	89 90			22 21	22 21
2	A B	11.26 11.44	4.50 3.61	8.59 7.59	83 85	74 90			20 23	19 23
3	A B	10.22 11.10	4.49 3.51	7.38 6.87	78 80	78 88			19 16	21 19
6	A B	7.60 8.48	3.46 2.86	6.20 7.23(2.86)	78 79	79 84			18 19	11 22
X      9	A B	4.42 3.91	2.10 2.14	4.96(2.10) 5.07(2.14)	81 83	84 78			21 20	15 21
12	A B	1.74 1.49	1.12(1.74) 1.32(1.49)	2.16(1.12) 1.96(1.32)	NP NP	NP NP			NP NP	NP NP
15	A B	1.58 1.08	0.30(1.58) 0.37(1.08)	0.36(0.30) 0.56(0.37)	NP NP	NP NP			NP NP	NP NP
18	A B	0.79 0.48	0.18(0.79) 0.18(0.48)	0.14(0.18) 0.32(0.18)	NP NP	NP NP			NP NP	NP NP
Positive Control EMS (µg/ml)
400	A B	11.23 11.77	4.09 4.51	7.17 7.17	80 86	74 86			73 86	75 75

 

Table 3: Summary Analysis: Mutagenicity Test (-S9) 4-H Exposure

Treatment (µg/mL)		SG	%RSG	%V	RTG	MF§
0		11.68	100	104.50	1.00	97.82
0.25	Ø	10.18	90
0.5		10.15	87	107.20	0.90	119.83
1		8.55	73	115.65	0.80	109.62
2		8.20	68	99.97	0.65	125.14
3		7.13	55	92.81	0.49	117.06
6		5.30	31	89.59	0.22	112.32
9	X	2.66	8	94.51	0.07	118.40
12	Ø	0.63	1
15	Ø	0.04	0
18	Ø	0.01	0
Positive Control EMS
Treatment (µg/mL)		SG	%RS	%V	RTG	MF§
400		7.71	64	94.51	0.59	864.01

GEF = 126, therefore MF threshold for positive response = 223.82

Large and Small Colonies Analysis: Mutagenicity Test (-S9) 4-Hour Exposure

Treatment (µg/ml)		Viability # after day 2				Small colonies # after day 2				Large colonies # after day 2
0	A B	75 89	77 88	79 87	87 91	7 4	8 8	10 11	12 12	11 10	7 14	8 10	4 6
0.5	A B	81 86	86 86			10 10	12 9			9 13	9 15
1	A B	80 87	89 90			13 12	10 8			9 9	12 13
2	A B	83 85	74 90			11 12	8 7			9 11	11 16
3	A B	78 80	78 88			10 9	12 10			9 7	9 9
6	A B	78 79	79 84			8 9	6 13			10 10	5 9
X      9	A B	81 83	84 78			11 15	6 5			10 5	9 16
400 EMS	A B	80 86	74 86			32 39	37 33			41 47	38 42

 

Mutation frequencies

Treatment (µg/ml)				Small colonies			Large Colonies			Proportion small colony mutants
		Viable		Mutants			Mutants
		Yv	Nv	Ym	Nm	MF§	Ym	Nm	MF§
0		95	768	696	768	47.1	698	768	45.7	0.51
0.5		45	384	343	384	52.7	338	384	59.5	0.47
1		38	384	341	384	51.3	341	384	51.3	0.50
2		52	384	346	384	52.1	337	384	65.3	0.45
3		60	384	343	384	60.8	350	384	49.9	0.55
6		64	384	348	384	54.9	350	384	51.7	0.51
9		58	384	347	384	53.6	344	384	58.2	0.48
400 EMS		58	384	243	384	242.1	216	384	304.4	0.46

 

Cell and 96-Well Plate Counts: Mutagenicity Test (+S9) 4-Hour Exposure

Treatment (µg/ml)		Cell counts $			Viability § after day 2 2 cells/well				Resistant mutants § after day 2 2000 cells/well
		0h	24h	48h
0	A B	9.07 8.87	8.45 7.73	6.94 7.36	76 78	72 86	75 81	77 81	23 18	22 20	21 18	22 19
0.25	A B	9.16 8.61	8.15 7.45	7.35 8.16	NP NP	NP NP			NP NP	NP NP
0.5	A B	9.15 8.64	7.16 7.49	8.57 8.06	NP NP	NP NP			NP NP	NP NP
1	A B	9.19 7.87	7.18 7.35	7.68 7.96	NP NP	NP NP			NP NP	NP NP
2	A B	8.05 7.94	7.25 7.16	8.08 8.90	80 77	78 81			20 17	21 21
3	A B	8.65 7.97	5.32 5.69	8.80 9.11	82 89	75 84			18 13	20 18
6	A B	8.63 7.42	4.57 4.76	6.70 6.62	78 84	76 75			19 21	21 22
9	A B	8.01 7.15	4.44 3.98	6.11 7.24	79 84	84 84			15 20	24 17
12	A B	6.31 5.34	3.52 3.51	5.97 5.98	84 81	80 87			11 16	18 21
15	A B	4.09 3.36	2.52 2.69	6.54(2.52) 6.69(2.69)	79 81	78 82			19 18	18 22
18	A B	1.96 1.65	1.09(1.96) 1.05(1.65)	1.98(1.09) 1.74(1.05)	NP NP	NP NP			NP NP	NP NP
Positive Control CP (µg/ml)
1.5	A B	8.23 8.84	5.93 5.56	7.31 9.06	66 68	58 74			62 67	73 57

Table 4: Summary Analysis: Mutagenicity Test (+S9) 4-H Exposure

Treatment (µg/mL)		SG	%RSG	%V	RTG	MF§
0		14.46	100	84.40	1.00	141.33
0.25	Ø	15.12	104
0.5	Ø	15.23	104
1	Ø	14.20	93
2		15.29	94	86.56	0.97	133.05
3		12.32	79	98.08	0.92	100.97
6		7.77	48	84.40	0.48	144.28
9		7.03	41	99.02	0.48	111.37
12		5.25	24	99.97	0.28	94.32
15		4.31	12	89.59	0.10	124.90
18	Ø	0.50	1
Positive Control CP (µg/mL)
Treatment (µg/mL)		SG	%RSG	%V	RTG	MF§
1.5		11.76	77	59.00	0.54	951.16

GEF = 126, therefore MF threshold for a positive response = 267.33

Large and Small Colonies Analysis: Mutagenicity Test (+S9) 4-Hour Exposure

Treatment (µg/ml)		Viability # after day 2				Small colonies # after day 2				Large colonies # after day 2
0	A B	76 78	72 86	75 81	77 81	13 12	13 11	12 11	9 10	10 6	9 9	9 7	13 9
2	A B	80 77	78 81			9 9	9 11			11 8	12 10
3	A B	82 89	75 84			7 4	8 11			11 9	12 7
6	A B	78 84	76 75			12 11	8 11			7 10	13 11
9	A B	79 84	84 84			8 11	11 8			7 9	13 9
12	A B	84 81	80 87			6 7	10 9			5 9	8 12
15	A B	79 81	78 82			10 12	9 11			9 6	9 11
1.5 CP	A B	66 68	58 74			45 41	57 39			17 26	16 18

 

Mutation frequencies

Treatment (µg/ml)				Small colonies			Large Colonies			Proportion small colony mutants
		Viable		Mutants			Mutants
		Yv	Nv	Ym	Nm	MF§	Ym	Nm	MF§
0		142	768	677	768	74.7	696	768	58.3	0.56
2		68	384	346	384	60.2	343	384	65.2	0.48
3		54	384	354	384	41.5	345	384	54.6	0.43
6		71	384	342	384	68.6	343	384	66.9	0.51
9		53	384	346	384	52.6	346	384	52.6	0.50
12		52	384	352	384	43.5	350	384	46.4	0.48
15		64	384	342	384	64.6	349	384	53.3	0.55
1.5 CP		118	384	202	384	544.4	307	384	189.7	0.70

 

Cell and 96-Well Plate Counts: Mutagenicity Test (-S9) 24-Hour Exposure

Treatment (µg/ml)		Cell counts $			Viability § after day 2 2 cells/well				Resistant mutants § after day 2 2000 cells/well
		0h	24h	48h
0	A B	13.16 12.14	5.74 5.58	7.24 5.85	69 77	66 76	70 82	80 72	18 16	19 17	20 14	11 13
0.03	A B	10.11 10.18	7.34 6.76	6.35 4.68	NP NP	NP NP			NP NP	NP NP
0.06	A B	10.34 10.65	7.58 6.01	5.94 4.85	69 73	68 73			13 16	16 14
0.13	A B	10.96 10.64	6.24 6.33	5.27 4.79	76 73	70 68			16 17	15 20
0.25	A B	9.10 8.73	7.78 9.26	9.17 7.95	75 78	81 80			17 13	18 21
0.5	A B	8.05 7.19	7.86 9.16	8.39 7.37	72 86	70 79			22 15	16 14
1	A B	6.38 5.81	5.98 6.86	8.14 8.07	81 76	75 72			18 19	18 21
2	A B	4.46 4.38	4.55 5.04	5.83 6.40	84 79	84 78			14 21	16 18
3	A B	3.04 3.37	3.53 3.66	4.72 4.78	NP NP	NP NP			NP NP	NP NP
4	A B	2.19 2.24	2.78(2.19) 3.08(2.24)	3.76(2.78) 3.00	NP NP	NP NP			NP NP	NP NP
5	A B	1.81 1.93	2.07(1.81) 2.28(1.93)	2.24(2.07) 2.11(2.28)	NP NP	NP NP			NP NP	NP NP
Positive Control EMS
150	A B	7.88 6.81	6.46 7.83	5.25 5.32	60 49	54 57			82 70	69 79

 

Table 5: Summary Analysis: Mutagenicity Test (-S9) 24-H Exposure

Treatment (µg/mL)		SG	%RSG	%V	RTG	MF§
0		78.10	100	73.67	1.00	123.75
0.03	Ø	65.74	68
0.06		64.12	68	66.78	0.76	124.91
0.13		56.90	62	68.80	0.68	141.65
0.25		108.36	98	85.11	1.58	116.36
0.5		85.16	66	80.34	1.18	119.33
1		52.86	33	78.43	1.06	140.60
2		21.60	10	93.66	0.35	105.74
3	Ø	9.12	3
4	Ø	3.66	1
5	Ø	1.47	0
Positive Control EMS
Treatment (µg/mL)		SG	%RSG	%V	RTG	MF§
150		46.23	34	42.54	0.34	1786.40

GEF = 126, therefore MF threshold for a positive response = 249.75

Large and Small Colonies Analysis: Mutagenicity Test (-S9) 24-Hour Exposure

Treatment (µg/ml)		Viability # after day 2				Small colonies # after day 2				Large colonies # after day 2
0	A B	69 77	66 76	70 82	80 72	7 4	10 10	10 3	7 3	11 12	9 7	10 11	4 10
0.06	A B	69 73	68 73			4 4	9 7			9 12	7 7
0.13	A B	76 73	70 68			8 9	8 3			8 8	7 17
0.25	A B	75 78	81 80			4 2	1 7			14 11	17 14
0.5	A B	72 86	70 79			11 3	6 7			11 12	10 7
1	A B	81 76	75 72			8 9	11 7			10 10	7 14
2	A B	84 79	84 78			4 11	6 9			10 10	10 9
150 EMS	A B	60 49	54 57			42 39	30 30			50 31	39 49

 

Mutation frequencies

Treatment (µg/ml)				Small colonies			Large Colonies			Proportion small colony mutants
		Viable		Mutants			Mutants
		Yv	Nv	Ym	Nm	MF§	Ym	Nm	MF§
0		176	768	714	768	49.5	694	768	68.8	0.42
0.06		101	384	360	384	48.3	349	384	71.6	0.41
0.13		97	384	356	384	55.0	344	384	79.9	0.41
0.25		70	384	370	384	21.8	328	384	92.6	0.20
0.5		77	384	357	384	45.4	344	384	68.5	0.40
1		80	384	349	384	60.9	343	384	72.0	0.46
2		59	384	354	384	43.4	345	384	57.2	0.43
150 EMS		164	384	243	384	537.8	215	384	681.7	0.45

 

Historical Vehicle and Positive Control Mutation Frequencies

Experiments –S9

Vehicle control	Positive control
MF	MF
125.29 167.03 151.47 126.39 122.55 134.46 119.60 127.28 83.55 137.68 144.00 166.01 153.99 176.14 188.96 165.68 137.56 131.08 156.04 182.88	977.54 1813.54 1405.39 956.03 1415.62 1527.79 1627.16 1344.78 458.34 1755.05 992.76 1579.00 1561.92 2123.10 1190.02 1601.92 784.61 1559.44 1285.29 1810.47
Mean:144.88 Minimum:83.55 Maximum:188.96 SD:25.46	Mean:1388.49 Minimum:458.34 Maximum:2123.1 SD:400.37

Experiments + S9

Vehicle control	Positive control
MF	MF
138.89 141.60 112.65 153.11 127.79 158.90 136.51 174.72 143.92 126.53 121.98 163.36 130.11 132.84 122.91 133.02 132.99 129.71 114.84 138.33	992.90 1153.69 918.06 1269.64 612.14 1029.27 1615.27 1081.10 798.45 1115.22 1231.42 879.70 928.69 809.90 1015.38 920.29 1304.16 871.86 571.16 1938.27
Mean:136.74 Minimum:112.65 Maximum:174.72 SD:15.87	Mean:1052.83 Minimum:571.16 Maximum:1938.27 SD:318.44

There was evidence of marked toxicity following exposure to the test substance in all three exposure groups, as indicated by the %RSG and RTG values, (Tables 3, 4, and 5). There was no evidence of reductions in viability (%V) in either of the three exposure groups, therefore indicating that residual toxicity had not occurred (Tables 3, 4 and 5). Optimum or very near to optimum to levels of toxicity were achieved in all three exposure groups. The dose levels of 12, 15 and 18 µg/mL in the 4-h exposure in the absence of metabolic activation, 18 µg/mL in the 4-h exposure in the presence of metabolic activation and 3, 4 and 5 µg/mL in the 24-h exposure in the absence of metabolic activation were not plated out for 5-TFT resistance and viability due to excessive toxicity. The dose level of 9 µg/mL in the 4-h exposure in the absence of metabolic activation was plated for 5-TFT resistance and % viability as the %RSG was only marginally too toxic. However this dose level was later excluded form analysis as the RTG value fell below the acceptable limit. Acceptable levels of toxicity were seen with both positive control substances (Tables 3, 4 and 5). The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional (Tables 3, 4, and 5). The test substance did not induce any toxicologically significant increases in the mutant frequency x 10^-6 per viable cell in either of the three exposure groups. The GEF value of the test substance dose levels were not exceeded in any of the three exposure groups, including a dose level (9 µg/mL) beyond the acceptable level of toxicity in the 4-h exposure in the absence of metabolic activation. A precipitate of the test substance was observed at 18 µg/mL in the 4-h exposure in the absence of metabolic activation and 15 and 18 µg/mL in the presence of metabolic activation.

Conclusion

The test substance did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6, consequently it is considered to be non-mutagenic in this assay.

Conclusions:

Under study conditions, the test substance was determined to be non-genotoxic with and without metabolic activation.

Executive summary:

A study was conducted to determine the genotoxic activity potential of the test substance, C16 TMA-MS (88.57% active), using the Thymidine Kinase Gene method, according to the OECD Guideline 490 and EU method B.17, in compliance with GLP. In main mutagenicity test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test substance at up to 10 dose levels (0, 0.25, 0.5, 1, 2, 3, 6, 9, 12, 15, 18 µg/mL) in duplicate, together with vehicle (R0), and positive controls using 4 h exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-h exposure group in the absence of metabolic activation. A precipitate of the test substance was observed at and above 15 µg/mL in the 4 -h -S9 exposure (absence of metabolic activation), 15 and 18 µg/mL in the 4 -h +S9 exposure (presence of metabolic activation). No precipitate was observed in the 24 -h –S9 exposure. There was evidence of marked toxicity following exposure to the test substance in all three exposure groups, as indicated by the %RSG (Relative suspension growth) and RTG (Relative total growth) values. There was no evidence of reductions in viability (%V) in either of the three exposure groups, therefore indicating that residual toxicity had not occurred. Optimum or very near to optimum to levels of toxicity were achieved in all three exposure groups. The dose levels of 12, 15 and 18 µg/mL in the 4 -h exposure in the absence of metabolic activation, 18 µg/mL in the 4 -h exposure in the presence of metabolic activation and 3, 4 and 5 µg/mL in the 24 -h exposure in the absence of metabolic activation were not plated out for 5 -TFT resistance and viability due to excessive toxicity. The dose level of 9 µg/mL in the 4 -h exposure in the absence of metabolic activation was plated for 5 -TFT resistance and % viability as the %RSG was only marginally too toxic. However this dose level was later excluded form analysis as the RTG value fell below the acceptable limit. Acceptable levels of toxicity were seen with both positive control substances. The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional. The test substance did not induce any toxicologically significant increases in the mutant frequency x 10^-6 per viable cell in either of the three exposure groups. The Global Evaluation Factor (GEF) value of the test substance dose levels were not exceeded in any of the three exposure groups, including a dose level (9 µg/mL) beyond the acceptable level of toxicity in the 4 -h exposure in the absence of metabolic activation. A precipitate of the test substance was observed at 18 µg/mL in the 4-h exposure in the absence of metabolic activation and 15 and 18 µg/mL in the presence of metabolic activation. The test substance did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6, consequently it is considered to be non-mutagenic in this assay. Under study conditions, the test substance was determined to be non-genotoxic with and without metabolic activation (Envigo, 2018).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Study 1: A study was conducted to determine the mutagenic potential of the read across substance, C12-14 ADMAES (95% active ingredient), according to OECD 471 Guideline and EU Method B.13/14, in compliance with GLP. The read across substance was examined for mutagenic activity in five strains of Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102, using the plate incorporation and pre-incubation methods. The studies were performed in the absence and presence of metabolic activation (S9-mix). The concentrations ranged from 1.23 to 100µg/plate in first experiment and 0.41 to 200 µg/plate in second experiment.No increase in reversion to prototrophy was obtained with any of the bacterial strain at any concentration either in the presence or absence of S9-mix. Further, inhibition of growth, observed as thinning of the background lawn of non-revertant cells, occurred in all strains following exposure to the read across substance at ≥100 µg/plate. Under the study conditions, the read across substance was not considered to be mutagenic in the presence and absence of exogenous metabolic activation (Haddouck, 2003).

Study 2: Anin vitrostudy 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).   

Study 3: A study was conducted to determine the genotoxic activity potential of the test substance, C16 TMA-MS (88.57% active),using the Thymidine Kinase Gene method,according to the OECD Guideline 490 and EU method B.17, in compliance with GLP. In main mutagenicity test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test substance at up to 10 dose levels (0, 0.25, 0.5, 1, 2, 3, 6, 9, 12, 15, 18 µg/mL) in duplicate, together with vehicle (R0), and positive controls using 4 h exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-h exposure group in the absence of metabolic activation.A precipitate of the test substance was observed at and above 15µg/mLin the 4 -h -S9 exposure (absence of metabolic activation), 15 and 18µg/mL in the 4 -h +S9 exposure (presence of metabolic activation). No precipitate was observed in the 24 -h –S9 exposure.There was evidence of marked toxicity following exposure to the test substance in all three exposure groups, as indicated by the %RSG (Relative suspension growth) and RTG (Relative total growth) values. There was no evidence of reductions in viability (%V) in either of the three exposure groups, therefore indicating that residual toxicity had not occurred. Optimum or very near to optimum to levels of toxicity were achieved in all three exposure groups. The dose levels of 12, 15 and 18 µg/mL in the 4 -h exposure in the absence of metabolic activation, 18 µg/mL in the 4 -h exposure in the presence of metabolic activation and 3, 4 and 5 µg/mL in the 24 -h exposure in the absence of metabolic activation were not plated out for 5 -TFT resistance and viability due to excessive toxicity. The dose level of 9 µg/mL in the 4 -h exposure in the absence of metabolic activation was plated for 5 -TFT resistance and % viability as the %RSG was only marginally too toxic. However this dose level was later excluded form analysis as the RTG value fell below the acceptable limit. Acceptable levels of toxicity were seen with both positive control substances. The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional. The test substance did not induce any toxicologically significant increases in the mutant frequency x 10^-6per viable cell in either of the three exposure groups. The Global Evaluation Factor (GEF) value of the test substance dose levels were not exceeded in any of the three exposure groups, including a dose level (9 µg/mL) beyond the acceptable level of toxicity in the 4 -h exposure in the absence of metabolic activation. A precipitate of the test substance was observed at 18 µg/mL in the 4-h exposure in the absence of metabolic activation and 15 and 18 µg/mL in the presence of metabolic activation. The test substance did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6, consequently it is considered to be non-mutagenic in this assay.Under study conditions, the test substance was determined to be non-genotoxic with and without metabolic activation(Envigo, 2018).

Based on the results of the in vitro genotoxicity studies with the test and read across substances, the test substance is not considered to be genotoxic.

 

Justification for classification or non-classification

Based on the available negative results from test substance and read across in vitro genotoxicity assays, the test substance does not warrant a classification for genotoxicity according to EU CLP criteria (Regulation EC 1272/2008).