Ammonium 2-mercaptopropionate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 01, 1995 to April 19, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

see 'Principles of method if other than guideline'

Qualifier:

according to guideline

Guideline:

other: EEC Directive 92/69, L 383 A

Deviations:

yes

Remarks:

see 'Principles of method if other than guideline'

Principles of method if other than guideline:

a) The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanibm (BRL, CH-4414 Fiillinsdorf; body weight approx. 220 - 320 g).
b) "Chemikaliengesetz ("Chemicals Act") of the Federal Republic of Germany, Anlage 1 ("Annex l"), dated July 25, 1994 (BGBL I S. 1703).

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Test concentrations with justification for top dose:

The following concentrations were evaluated, without S9 mix (exposure period 22 h): 100.0; 300.0; 1000.0 µg/mL and with S9 mix (exposure period 4 h): 300.0; 1000.0; 3000.0 µg/mL.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Blood Collection and Delivery:

For this study (for both experiments) blood was collected only from a single donor (male; age: 34 years) to reduce inter individual variability.
Blood samples were drawn by venous puncture and collected in heparinized tubes by Dr. med. V. Theodor (Odenwaldring 4, 64380 Rofldorf). The tubes were sent to CCR to initiate cell cultures within 24 hours after blood collection. Before use the blood was stored under sterile conditions at 4 °C.

Mammalian microsomal fraction S9 mix:

S9 (Preparation by CCR):

The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar HanIbm in deviation to the protocol (BRL, CH-4414 Flillinsdorf; body weight approx. 220 -320 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D-76275 Karlsruhe) in olive oil 5 days previously.
After cervical dislocation, the livers of the animals were removed, washed in 150 mM KCl and homogenized. The homogenate, diluted 1+3 with KCl was centrifuged twice at 9,000 g for 10 minutes (4 °C).
A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 3 ml and stored at -70 °C. Small numbers of the ampoules were kept at -20 °C for only several weeks before use. The protein content was determined using the analysis kit of Bio-Rad. Laboratories, D-80939 Munchen.
The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml. The protein concentration of the S9 used was 26.2 mg/ml.

S9 mix:

An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.74 mg/ml in the cultures. Cofactors were added to the S9 mix to reach the following concentrations:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-orthophosphate buffer, pH 7.4.
During the experiment, the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Evaluation criteria:

A test substance is classified as mutagenic if it induces reproducibly either a concentration-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 substance producing reproducibly neither a concentration­related increase in the number of structural chromosomal aberrations nor a significant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the chi-square test. However, both biological and statistical significance should be considered together.

Key result

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Without S9 mix the reduction of the mitotic· index was less pronounced (solvent control = 100 %, 1000.0 µg/ml = 74.1 %). However, preparation of higher concentrations could not be evaluated due to low mitotic index combined with poor metaphase quality.
- In the absence as well as in the presence of metabolic activation the substance did not increase the frequency of structural chromosomal aberrations as compared to the frequency of the corresponding solvent controls. Biometric evaluation of the results was done by means of the chi-square test. The aberration frequencies after treatment with the test substance proved not to be statistically significant different from the solvent control frequencies.
- EMS (440.0 µg/ml) and CPA (45.0 µg/ml) were used as positive controls and induced statistically significant increases in cells with structural chromosomal aberrations.
- The proliferation index of the lymphocytes in solvent control cultures was checked by analyzing the proportion of mitotic cells in the 1st, 2nd and 3rd metaphase (Ml, Ml+, M2 and M3) and showed that the lymphocytes divided adequately within the fixation interval.
- The test substance induced no structural chromosomal aberrations in human lymphocytes in vitro.

Conclusions:

Under the study conditions, the test substance did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, the test substance is considered to be non-mutagenic in this chromosome aberration test.

Executive summary:

A study was conducted to determine the genotoxic potential of the test substance to induce formation of micronuclei in human lymphocytes cultured in vitro, according to OECD Guideline 473 and EEC Directive 92/69, L 383 A, in compliance with GLP. The experiment was performed with a preparation time of 22 h after treatment with the test substance which was dissolved in culture medium. In each experimental group two parallel cultures were analyzed. Per culture 100 metaphases were scored for structural chromosomal aberrations. The following concentrations were evaluated, without S9 mix (exposure period 22 h): 100.0; 300.0; 1000.0 µg/mL and with S9 mix (exposure period 4 h): 300.0; 1000.0; 3000.0 µg/mL. In the absence as well as in the presence of S9 mix, the test substance was tested up to cytotoxic concentrations. Appropriate reference mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosomal aberrations. Under the study conditions, the test substance did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, the test substance is considered to be non-mutagenic in this chromosome aberration test (Czich, 1995).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 08, 1994 to June 22, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Concentrations up to 200 µL

Vehicle / solvent:

DSMO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene, 2-aminofluorene

Details on test system and experimental conditions:

All Salmonella typhimurium strains used in this test were supplied by Dr. B.N. Ames, University of California, Berkeley (USA). The tester strains, whose genotype is regularly confirmed were stored at -196 °C (liquid nitrogen) as frozen permanents (1 mL in 1.2 mL-cryotubes) prepared from fresh overnight Difeo nutrient broth cultures (ca. 2+E9 bacteria per mL) to which DMSO is added (8.25 % v/v) as cryoprotective agent. For each mutagenicity assay, a 20 mL broth culture is inoculated with 80 µL of a thawed permanent for each tester strain and incubated for 10 h at 37 °C in a gyratory incubator to reach a density of ca. 2.5+E9 bacteria per mL. The fresh overnight cultures are stored at 4 °C until they are needed for the assay (but not longer than 6 h).

S9 - homogenate:
The 9000 g supernatant (S9) of a KCl liver homogenate from rats induced with Aroclor 1254 is purchased from Molecular Toxicology (USA) and stored frozen at –80 °C. For the tests with metabolic activation 0.5 mL of S9-mix containing 25 – 50 µL S9 is added to each agar plate to reach a final concentration of approximately 1 mg S9-protein per plate. The components of the standard S9-mix are:
8 mM MgC12
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
100 mM Na-phosphate buffer pH 7.4
2 mg protein/ml S9

Evaluation criteria:

A test substance is considered mutagenic if there is a reproducibly increasing dose-response curve of induced revertant colonies for at least three test concentrations. The minimal criteria for a positive response are a two- to threefold increase in the number of revertants over the spontaneous number for the strains TA 1535, TA 1537, TA 1538 and TA 98, and a 50 % increase for TA100. Moreover, to avoid a misinterpretation of the mutagenicity data, the positive response should not be obtained only at concentrations near to toxic dose levels.

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

Untreated negative 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

Untreated negative 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

Untreated negative 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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- The mean numbers of spontaneous revertant colonies on untreated and/or solvent treated plates (= negative control values or background revertant counts) are within the normal range previously established in our laboratory and agree with the values from the literature. Similarly, the results with the positive control substances confirm the sensitivity of the tester strains as well as the activity of the metabolizing system.
- The test substance was not mutagenic, in the absence of S9-mix up to 0.03 - 0.1 µL per plate for TA 98, TA 1537 and TA 1538, and up to 1 - 6 µL per plate for TA 1535 and TA 100. In the presence of S9-mix the test substance was not mutagenic up to the concentration of 10 µL per plate, for TA 98, and up to 3 µL per plate for all other tester strains. Toxic effects were generally observed at higher concentrations with all tester strains.
- Very slight increases in the number of revertant colonies (1.3-1.4 x background revertant count) observed with TA 100 in the absence of S9-mix at concentrations ranging from 0.3 to 3 µL per plate do not fulfil the criteria for a positive response and are to be considered as biologically not relevant.
- Toxic effects of the test substance were observed with all tester strains. These effects, detected as clearing of the background lawn and/or strong reduction in the number of spontaneous revertants, were most evident in the absence of S9-mix, appearing at dose levels of 0.1 to 200 µL per plate without S9-mix and only from 10/30 to 200 µL per plate with S9-mix.

Conclusions:

Under the study conditions, the test substance was not mutagenic, in the absence of S9-mix up to 0.03 - 0.1 µL per plate for TA 98, TA 1537 and TA 1538, and up to 1 - 6 µL per plate for TA 1535 and TA 100. In the presence of S9-mix the test substance was not mutagenic up to the concentration of 10 µL per plate, for TA 98, and up to 3 µL per plate for all other tester strains. Toxic effects were generally observed at higher concentrations with all tester strains.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471, in compliance with GLP. The test substance was examined using five strains ofSalmonella typhimurium(TA 1535, TA 1537, TA 1538, TA 98 and TA 100). The strains were exposed on minimal agar plates to a range of concentrations (up to 200 µL/plate) both in the presence and absence of an exogenous metabolic activation system S9 and in three replicates. Under the study conditions, the test substance was not mutagenic, in the absence of S9-mix up to 0.03 - 0.1 µL per plate for TA 98, TA 1537 and TA 1538, and up to 1 - 6 µL per plate for TA 1535 and TA 100. In the presence of S9-mix, the test substance was not mutagenic up to the concentration of 10 µL per plate, for TA 98, and up to 3 µL per plate for all other tester strains. Toxic effects were generally observed at higher concentrations with all tester strains (Faller, 1994).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 01, 2007 to May 04, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Preliminary test: 4.81 to 1232 µg/ml,
Experiment 1: 154 to 1232 µg/ml,
Experiment 2: 77 to 1232 µg/ml in the absence of S9-mix and 154 to 1232 µg/ml in the presence of S9-mix.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

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, 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 -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 RIO media) at 37 °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) and without serum (RO) are used during the course of the study.

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 RIO medium.

Preparation of test and control substance:
The test substance was accurately weighed and diluted with R0 medium. The maximum concentration level was 1232 µg/ml, which was equivalent to 10 mM. The content of the active ingredient was 70.0 % and was accounted for in the 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.

Metabolic activation:
PB/βNF S9 was prepared in-house from the livers of male Sprague-Dawley rats weighing approx. 250 g.

Rationale for test conditions:

Results from the preliminary toxicity test were used to set the test substance concentration levels for the mutagenicity experiments. Maximum concentration levels were selected using the following criteria:
i) Maximum recommended concentration level, 5000 µg/ml or 10 mM.
ii) The presence of excessive precipitate where no test substance-induced toxicity was observed.
iii) Test substance-induced toxicity, where the maximum concentration level used should produce 10 to 20 % survival (the maximum level of toxicity required).

Evaluation criteria:

For a test substance to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Any test substance concentration level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126-E6 will be considered positive. However, if a test substance produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a concentration-related response, then it may be considered to have no toxicological significance.

Statistics:

The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test:

In the 4-hour exposure groups, both in the absence and presence of metabolic activation (S9), there was no marked reduction in the %RSG of cells treated with the test substance when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of S9 there was a clear concentration-related reduction of the %RSG values of cells treated with test substance indicating optimum levels of toxicity at the 1232 µg/ml concentration level.

Experiment 1:

- There was no evidence of toxicity following exposure to the test material in the 4-hour exposure group in the presence of metabolic activation and only a slight reduction in the 4-hour exposure group in the absence of metabolic activation as indicated by the %RSG and RTG values.
- The test substance did not induce any statistically significant or concentration related (linear-trend) increases in the mutant frequency in either the absence or presence of metabolic activation.

Experiment 2:

- The test substance did not induce any statistically significant or concentration-related increases in the mutant frequency in the 4-hour exposure group in the presence of metabolic activation.
- In the 24-hour exposure group in the absence of metabolic activation, the test substance induced a small but statistically significant increase in the mutant frequency at the upper surviving concentration level. However, the increase did not exceed the GEF value and was at a concentration level approaching the toxic limit. Therefore, it was considered that the response was due to a cytotoxic rather genotoxic mechanism and was, therefore, of no toxicological significance. This interpretation is supported by the fact that at a higher concentration of the test substance after treatment for 4-hours (with or without metabolic activation) an increase in induced mutations was not observed in the absence of marked cytotoxicity.

In Experiment 1 and 2:
- Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200-E6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.
- Precipitate of test substance was not observed at any of the concentration levels.

Conclusions:

Under the study conditions, the test substance was considered to be non-mutagenic to L5178Y cells.

Executive summary:

A study was conducted to determine the potential mutagenicity of the test substance on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line according to OECD 476 Guideline and EU Method B.17, in compliance with GLP. Two independent experiments were performed. The exposure groups were 4-hours in the presence of metabolic activation and 24-hours in the absence of metabolic activation. The concentration range of the active ingredient was selected following the results of a preliminary toxicity test and was 154 to 1232 µg/mL for the first experiment. For the second experiment the concentration range of the active ingredient was 154 to 1232 µg/mL with activation and 77 to 1232 µg/mL without activation. The maximum concentration level used, 1232 µg/mL, was the 10 mM limit concentration. Precipitate of test substance was not observed at any of the concentration levels. The vehicle (solvent) controls had acceptable mutant frequency values that that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any concentration level, either with or without metabolic activation, in either the first or the second experiment at concentration levels up to and including the 10 mM limit concentration. Under the study conditions, the test substance was considered to be non-mutagenic to L5178Y cells (Flanders, 2007).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in vitro

Study 1:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471, in compliance with GLP. The test substance was examined using five strains ofSalmonella typhimurium(TA 1535, TA 1537, TA 1538, TA 98 and TA 100). The strains were exposed on minimal agar plates to a range of concentrations (up to 200 µL/plate) both in the presence and absence of an exogenous metabolic activation system S9 and in three replicates. Under the study conditions, the test substance was not mutagenic, in the absence of S9-mix up to 0.03 - 0.1 µL per plate for TA 98, TA 1537 and TA 1538, and up to 1 - 6 µL per plate for TA 1535 and TA 100. In the presence of S9-mix, the test substance was not mutagenic up to the concentration of 10 µL per plate, for TA 98, and up to 3 µL per plate for all other tester strains. Toxic effects were generally observed at higher concentrations with all tester strains (Faller, 1994).

 

Study 2:

A study was conducted to determine the genotoxic potential of the test substance to induce formation of micronuclei in human lymphocytes culturedin vitro, according to OECD Guideline 473 and EEC Directive 92/69, L 383 A, in compliance with GLP. The experiment was performed with a preparation time of 22 h after treatment with the test substance which was dissolved in culture medium. In each experimental group two parallel cultures were analyzed. Per culture 100 metaphases were scored for structural chromosomal aberrations. The following concentrations were evaluated, without S9 mix (exposure period 22 h): 100.0; 300.0; 1000.0 µg/mL and with S9 mix (exposure period 4 h): 300.0; 1000.0; 3000.0 µg/mL. In the absence as well as in the presence of S9 mix, the test substance was tested up to cytotoxic concentrations. Appropriate reference mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosomal aberrations. Under the study conditions, the test substance did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, the test substance is considered to be non-mutagenic in this chromosome aberration test (Czich, 1995).

Study 3:

A study was conducted to determine the potential mutagenicity of the test substance on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line according to OECD 476 Guideline and EU Method B.17, in compliance with GLP. Two independent experiments were performed. The exposure groups were 4-hours in the presence of metabolic activation and 24-hours in the absence of metabolic activation. The concentration range of the active ingredient was selected following the results of a preliminary toxicity test and was 154 to 1232 µg/mL for the first experiment. For the second experiment the concentration range of the active ingredient was 154 to 1232 µg/mL with activation and 77 to 1232 µg/mL without activation. The maximum concentration level used, 1232 µg/mL, was the 10 mM limit concentration. Precipitate of test substance was not observed at any of the concentration levels. The vehicle (solvent) controls had acceptable mutant frequency values that that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any concentration level, either with or without metabolic activation, in either the first or the second experiment at concentration levels up to and including the 10 mM limit concentration. Under the study conditions, the test substance was considered to be non-mutagenic to L5178Y cells (Flanders, 2007).

Justification for classification or non-classification

Based on the results of in vitro testing, the test substance does not require classification for mutagenicity according to EU CLP (EC 1272/2008) criteria.