tetrasodium 3-amino-4-{[4-({4-fluoro-6-[phenyl(2-{[2-(sulfonatooxy)ethyl]sulfonyl}ethyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-5-hydroxynaphthalene-2,7-disulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No evidence for genotoxicity was observed in the bacterial reverse mutation assay and/or in the mammalian chromosomal aberration assay in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 19, 2004 to October 29, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Substance Control Law (JSCL) Test Guideline 111.1 Gene Mutation Test with bacteria

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:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver (plate incorporation test) and S9-mix from hamster liver (preincubation test)

Test concentrations with justification for top dose:

Plate incorporation test:
with metabolic activation(i.e.,10% rat liver):
50, 160, 500, 1,600 and 5,000 µg/plate
without metabolic activation:
50, 160, 500, 1,600 and 5,000 µg/plate
Preincubation test:
with metabolic activation (i.e., 30% hamster liver):
50, 160, 500, 1,600 and 5,000 µg/plate
without metabolic activation:
50, 160, 500, 1,600 and 5,000 µg/plate

Vehicle / solvent:

Deionized water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation for strain TA 100 and TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation for strain TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation for strain TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation for strain WP2uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (10% rat liver) for all strains

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (30% syrian golden hamster liver) for strain TA 100, TA 1535, TA 1537 and WP2uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

congo red

Remarks:

with metabolic activation (30% syrian golden hamster liver) for strain TA 98

Details on test system and experimental conditions:

ASSAY PROCEDURE

An independent mutation test was performed using the plate incorporation method as results were negative, a second test was conducted. This included a preincubation step which involved incubating the test substance, S9-mix and bacteria for a short period before pouring this mixture onto plates of minimal agar.

Each test was performed in both the presence and absence of S9-mix using all bacterial tester strains and a range of concentrations of the test substance. Positive and negative controls as well as solvent controls were included in each test. Triplicate plates were used. The highest concentration in the first mutation experiment was 50 mg/mL of the test substance in the chosen solvent, which provided a final concentration of 5,000 µg/plate. Further dilutions of 1,600, 500, 160 and 50 µg/plate were also used. Dose levels used in the second experiment were based on findings, including toxicity, in the first experiment. Toxicity was assessed after microscopic thinning of the bacterial lawn and/or reduction of the number of spontaneously occurring mutants compared to the corresponding solvent control value.

In both tests top agar was prepared which, for the Salmonella strains, contained 100 mL agar (i.e., 0.6% (w/v) agar, 0.5% (w/v) NaCI) with 10 mL of a 0.5 mM histidine-biotin solution. For E. coli histidine was replaced by tryptophan (i.e., 2.5 mL, 2.0 mM). The following ingredients were added (in the following order) to 2 mL of molten top agar at approximately 48°C:

0.5 mL S9-mix (if required) or buffer
0.1 mL of an overnight nutrient broth culture of the bacterial tester strain
0.1 mL test substance solution (i.e., dissolved in deionized water)

In the second mutagenicity test if appropriate these top-agar ingredients were preincubated by shaking for approximately 20 to 30 minutes at approximately 30°C.

After mixing, and preincubation if appropriate, the liquid was poured into a petri dish containing a 25 mL layer of minimal agar (i.e., 1.5% (w/v) agar, Vogel-Bonner E medium with 2% (w/v) glucose). After incubation for approximately 48 h at approximately 37°C in the dark, colonies (i.e., his+ or trp+ revertants) were counted by hand or by a suitable automatic colony counter. The counter was calibrated for each test by reading a test pattern plate to verify the manufacturer's requirements for sensitivity.

Evaluation criteria:

Criteria for a valid assay

The assay is considered valid if the following criteria are met:

- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency. Slight deviations from the laboratory's normal control range can be accepted if the positive controls show no correlated deviation and if requirements for a negative response are clearly fulfilled.

- the positive controls induce increases in the mutation frequency which are significant and within the laboratory's normal range. Slight deviations from the laboratory's normal control range can be accepted if the negative/solvent controls show no correlated deviation and if requirements for a positive response are clearly fulfilled.

Criteria for a positive response

A test substance is classified as mutagenic if it has either of the following effects:

a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn

b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test substance at complete bacterial background lawn

If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(in plate incorporation test and preincubation test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(in plate incorporation test and preincubation test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

SOLUBILITY AND TOXICITY

Test substance was dissolved in deionized water and a stock solution of 50 mg/mL was prepared for the highest concentration, which provided a final concentration of 5,000 µg/plate. Further dilutions of 1,600, 500, 160 and 50 µg/plate were used in all experiments.

Test substance did not precipitate on the plates up to the highest investigated dose of 5,000 µg/plate.

Test substance proved to be not toxic to the bacterial strains in all experiments.

MUTAGENICITY

In both independent mutation tests test substance was tested for mutagenicity with the stated concentrations. The number of colonies per plate with each strain as well as mean values of 3 plates were given.

Plate incorporation test:

The test substance did not cause a significant increase in the number of revertant colonies at any dose level with any of the tester strains either in the absence or presence of rat liver S9-mix in either mutation test. No dose-dependent effect was obtained.

Preincubation test:

In the presence and absence of hamster liver S9-mix (i.e., 30 % (v/v)) using the preincubation method according to Prival the test substance did not cause a significant increase in the number of revertant colonies under the experimental conditions described.

All positive controls produced significant increases in the number of revertant colonies. Thus the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated.

STERILITY CHECKS AND CONTROL PLATES

 

Sterility of S9-mix and the test substance were indicated by the absence of contamination on the test substance and S9-mix sterility check plates. Control plates (i.e., background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control range

 

In the plate incorporation test the number of revertant colonies of the solvent control with the strain TA 1537 in the absence of S9-mix was marginally above the historical control data range, which did not influence the validity of the study. The number of revertant colonies of the solvent control in the absence of S9-mix (i.e., plate incorporation test) and of the solvent and negative control in the presence of S9-mix (i.e., plate and preincubation test) with the strain WP2uvrA was below the historical control data range. This indicated rather higher sensitivity of this strain than usual, because all positive controls fulfill the required criteria completely and were within the expected range. The validity of the study is therefore not influenced.

Conclusions:

Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay with and without metabolic activation.

Executive summary:

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to OECD Guideline 471, EPA OPPTS 870.5100, EU Method B.13/14 and Japanese Guideline, in compliance with GLP.

Two independent mutagenicity studies were conducted, one as the standard plate test with the plate incorporation method and the other as a modified preincubation test (i.e., Prival test). The studies were performed in the absence and presence of a metabolizing system derived from a rat or hamster liver homogenate. The substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 50 - 5,000 µg/plate in both assays.

Positive controls showed an expected increase in the number of revertant colonies, thus indicating the sensitivity of the assay and the efficacy of the S9-mix. In the plate incorporation test, the test substance exposure did not result in relevant increases in the number of revertants in any of the bacterial strains in the absence and presence of the metabolic activation (i.e., rat liver S9-mix (10% (v/v)). Also, in the preincubation test no relevant increase in the number of revertants was observed in any of the bacterial strains in the absence and presence of the metabolic activation (i.e., hamster liver S9 -mix (30% (v/v)).

Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Oct. 11, 2004 to Jan. 27, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Substance Control Law (JSCL) Test Guideline 111.2 - Chromosome Aberration Test with Mammalian Cells in Culture

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver

Test concentrations with justification for top dose:

First experiment with 3 h treatment time/ 20 h sampling time
Without S9-mix: 312.5 #, 625.0 #, 1,250.0 $Ω¥, 2,500.0 $Ω¥ and 5,000.0 $Ω¥ µg/mL, Positive control (ethylmethane sulfonate): 1500.0 µg/mL
With S9-mix: 312.5 #, 625.0 #, 1,250.0 $, 2,500.0 $ and 5,000.0 $Ω¥ µg/mL, Positive control (cyclophosphamide): 1500.0 µg/mL

Second Experiment 20 h treatment time/ 20 h sampling time
Without S9-mix: 312.5 $, 625.0 $, 1,250.0 $Ω¥, 2,500.0 £Ω¥, 3,750.0 £Ω¥ and 5,000 £Ω¥ µg/mL, Positive control (ethylmethane sulfonate): 400.0 µg/mL
Second Experiment 3 h treatment time/28 h sampling time
With S9-mix: 312.5 $, 625.0 $, 1,250.0 $, 2,500.0 £, 3,750.0 £ and 5,000 £Ω¥ µg/mL, Positive control (cyclophosphamide): 7.5 µg/mL

Second Experiment 28 h treatment/28 h sampling time
Without S9-mix: 39.1#, 78.2#, 156.3#, 312.5 $, 625.0 $, 1,250.0 $Ω¥, 2,500.0 £ µg/mL, Positive control (ethylmethane sulfonate): 400.0 µg/mL

# = not used because higher concentrations were evaluated
$ = concentrations at which metaphase analysis was conducted
Ω = macroscopic precipitation after end of treatment
¥ = microscopic precipitation after end of treatment
£ = not evaluable because of high toxicity

Vehicle / solvent:

- Vehicle/solvent used: MEM (minimal essential medium) with Earle's salts and L-glutamine

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

without metabolic activation: ethyl methane sulfonate, with metabolic activation: cyclophosphamide

Details on test system and experimental conditions:

Details on test system and conditions:
- Formulation of test substance: dissolved in MEM (minimum essential medium) at appropriate concentrations immediately before use
- Formulation of positive controls: Ethylmethanesulphonate dissolved in cell culture medium on the day of treatment: final concentration: 1.5 mg/mL (3/20 h treatment/sampling), final concentration: 0.4 mg/mL (20/20 h treatment/sampling), final concentration: 0.4 mg/mL (28/28 h treatment/sampling). Cyclophosphamide was dissolved in cell culture medium on the day of treatment, final concentration in cell culture medium: 7.5 µg/mL
- Experimental conditions in vitro: approx. 37°C and approx. 4% CO2 in plastic flasks

METHOD OF APPLICATION: in medium

ASSAY PROCEDURE:
Evaluation of cell number was performed in the first experiment with the 3/20 h treatment/sampling time with and without S9-mix.
In the second experiment cell evaluation was performed with the 20/20h and 28/28h treatment/sampling time without S9-mix and with 3/28h treatment/sampling time with S9-mix. Using a 500 fold microscopic magnification the cells were counted in 10 fields of the slides. The cell number of the treatment groups was given as % cells in relation to the control. Treatments were performed both in the presence and absence of the S9-mix metabolic activation system using a duplicate cell culture at each test point.

Rationale for dose selection: The evaluated concentrations for mutagenicity were based on the results of cell counting.

Mutagenicity test: Two independent experiments were conducted using duplicate cultures of cells seeded onto slides (i.e. 2 per dose level) and at least three dose levels, as no positive results were obtained in the first test. The experiments included exposure of the cell culture to test concentrations in the presence and absence of metabolic activation system. Colcemide was added to each culture 2 h before sampling in order to arrest cell division. Chromosome preparations were made, fixed, stained and examined. Before treatment, the pH values and osmolality of the treatment medium were determined. If necessary the pH was adjusted to pH 7.3 with NaOH or HCl. Exponentially growing cultures which were more than 50% confluent were trypsinated by an approx. 0.25% (v/v) trypsin solution ready for use (mfr. Gibco). A single cell suspension (culture) was prepared. The trypsin concentration was approx. 0.25% (v/v) in Ca-Mg-free salt solution.
Two slides were placed in Quadriperm® dishes which were then seeded with cells to yield 3-4 x 10000 cells/slide. Thus for each dose level and treatment time, duplicate cultures slides were used. The Quadriperm® dishes contained 6 mL MEM (minimum essential medium) with approx. 10% (v/v) FCS (fetal calf serum) and approx. 0.1% (w/v) neomycinsulfate.
After 48 h, the medium was replaced with one containing approx. 10% (v/v) FCS and the test substance, or positive control, or solvent and in the presence of metabolic activation additionally 2% (v/v) S9-mix.
For the 3 h treatment time, the medium was replaced by normal medium following two rinses.
In the second experiment the cells were exposed to the treatment medium without S9-mix for 20 h and 28 h.
18 hand 26 h after the start of the treatment, Colcemide was added (approx. 0.05 µg/mL/culture medium) to the cultures to arrest mitosis and 2 h later (20 h and 28 h after the start of treatment) metaphase spreads were prepared as follows: The cultures were made hypotonic by adding about 5 mL of approx. 0.075 M potassium chloride solution at around 37°C. The cells were then incubated for 20 minutes at approx. 37°C. The next step was the addition of 2 mL fixative. Then the liquid was replaced by 6 mL fixative (methanol: glacial acetic acid, 3: 1). After 10 minutes the procedure was repeated. After at least another 10 minutes, the slides were taken out and air-dried for 24 h. The chromosomes were stained as follows:
- staining for 10 minutes in approx. 2% (w/v) orcein solution
- rinsing 3 times in distilled water
- rinsing twice in acetone
- brief rinsing in acetone/xylene
- 2 minutes in acetone/xylene
- 5 minutes in xylene
- 10 minutes in xylene
- embedding in Entellan® or Corbit®

Evaluation criteria:

Analysis of metaphases
The slides were coded and 25-100 metaphases per experimental group and cell culture were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed and classified. Only metaphases with 22 +/- chromosomes were included in the analysis. The metaphases were examined for the following aberrations: chromatid gap, chromosome gap, chromatid break, chromosome break, acentric chromatids (chromosomes), chromatid deletion, chromosome deletion, chromatid exchanges, chromosome exchanges including intrachanges, dicentrics and ring formation, pulverization, and multiple aberrations. Furthermore the incidence of polyploid metaphases was determined in 500 metaphase cells of each cell culture. The rate of the polyploid cells is expressed as a percentage. Additionally the mitotic index was determined by counting the number of cells undergoing mitosis in a total of 1,000 cells. After the metaphases had been evaluated, the code was broken. For each experiment the results from the dose groups were compared with those of the control group and the positive control at each sampling time.
The assay was considered valid if the solvent control data were within the laboratory's normal control range for the spontaneous mutant frequency or the positive controls induced increases in the mutation frequency which were statistically significant and within or slightly above the laboratory's normal range.
Criteria for clastogenicity:
A test substance was classified as non-clastogenic if: the no. of induced structural chromosome aberrations in all evaluated dose groups was in the range of historical control data and/or no significant increase in the no. of structural chromosome aberrations was observed. A test substance was classified as clastogenic if: the number of induced aberrations was above the range of historical control data and either a dose related or a significant increase in the no. of aberrations was seen.

Statistics:

The Biometry of the results was performed with a one-sided Fisher's exact test. Statistical significance was confirmed by means of the Fisher's exact test.

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

Positive controls validity:

valid

Additional information on results:

SOLUBILITY AND TOXICITY TESTING
Evaluation of the solubility in MEM Earle's cell culture medium showed that 5,000 μg/mL was the highest practicable concentration and produced macroscopic and microscopic precipitation of the test substance at 5,000 μg/mL with (S9 mix) metabolic activation and 1,250 μg/mL and above without metabolic activation after treatment time. Evaluation of cytotoxicity by cell counting showed that test substance was toxic to the V79 cells in the presence and in the absence of metabolic activation (S9-mix) after 20 and 28 h treatment times. In absence of metabolic activation, slight toxicity was observed at 5,000 μg/mL (cell survival = 71.9% of the control). In the presence and absence of metabolic activation, moderate toxicity was observed reaching 48.6% (-S9 mix; after 28 h treatment period), 50% (+S9 mix; 3 h treatment period) and 57.6% (-S9 mix; after 20 h treatment period). The highest evaluated concentration group (1,250 μg/mL) also produced a distinct lowering of the mitotic index in the second experiment reaching survival of 44.7% (20/20 h treatment/sampling), 48.8% (3/28 h treatment /sampling) and 41.6% (28/28 h treatment/sampling). The addition of test substance solutions did not have any relevant effect on pH and osmolality of the treatment media.
MUTAGENICITY TEST
After treatment with the test substance there was no relevant increase in the number of polyploid cells as compared with the solvent controls. No relevant or dose-dependent increase in the number of metaphases with aberrations outside the range of the solvent control was found with any of the concentrations used, either with or without metabolic activation by S9-mix. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control items.

Conclusions:

Under the test conditions, the test substance did not induce structural chromosome aberrations in V79 Chinese hamster cells at cytotoxic concentrations in the presence and absence of a metabolic activation system.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce chromosome aberrations in V79 cells of the Chinese hamster lung according to OECD Guideline 473, EPA OPPTS 870.5375, EU Method B.10 and Japanese Guideline 111.2, in compliance with GLP.

The test substance was dissolved in cell culture medium and tested at the concentrations ranging from 39.1-5,000 µg/mL with and without metabolic activation, based on the results of preliminary testing for solubility and toxicity.

The test substance did not induce a significant and dose-dependent increase in the number of aberrant metaphases and chromosomal aberrations. However, moderate cytotoxicity was demonstrated by cell counting (survival rate between 54 and 70%) at concentrations 5,000 and 1,250 µg/mL with and without metabolic activation. Positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay and the efficacy of the S9-mix.

Under the test conditions, the test substance did not induce structural chromosome aberrations in V79 Chinese hamster cells at cytotoxic concentrations in the presence and absence of a metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Additional information from genetic toxicity in vitro:

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to OECD Guideline 471, EPA OPPTS 870.5100, EU Method B.13/14 and Japanese Guideline, in compliance with GLP. Two independent mutagenicity studies were conducted, one as the standard plate test with the plate incorporation method and the other as a modified preincubation test (i.e., Prival test). The studies were performed in the absence and presence of a metabolizing system derived from a rat or hamster liver homogenate. The substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 50 - 5,000 µg/plate in both assays. Positive controls showed an expected increase in the number of revertant colonies, thus indicating the sensitivity of the assay and the efficacy of the S9-mix. In the plate incorporation test, the test substance exposure did not result in relevant increases in the number of revertants in any of the bacterial strains in the absence and presence of the metabolic activation (i.e., rat liver S9-mix (10% (v/v)). Also, in the preincubation test no relevant increase in the number of revertants was observed in any of the bacterial strains in the absence and presence of the metabolic activation (i.e., hamster liver S9 -mix (30% (v/v)). Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay.

A study was conducted to investigate the potential of the test substance to induce chromosome aberrations in V79 cells of the Chinese hamster lung according to OECD Guideline 473, EPA OPPTS 870.5375, EU Method B.10 and Japanese Guideline 111.2, in compliance with GLP. The test substance was dissolved in cell culture medium and tested at the concentrations ranging from 39.1-5,000 µg/mL with and without metabolic activation, based on the results of preliminary testing for solubility and toxicity. The test substance did not induce a significant and dose-dependent increase in the number of aberrant metaphases and chromosomal aberrations. However, moderate cytotoxicity was demonstrated by cell counting (survival rate between 54 and 70%) at concentrations 5,000 and 1,250 µg/mL with and without metabolic activation. Positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay and the efficacy of the S9-mix. Under the test conditions, the test substance did not induce structural chromosome aberrations in V79 Chinese hamster cells at cytotoxic concentrations in the presence and absence of a metabolic activation system.

Justification for classification or non-classification

As no evidence of genotoxicity was observed in bacterial reverse mutation assay and in vitro mammalian chromosomal aberration assay, the test substance does not require classification for this endpoint according to the EU CLP criteria (EC 1272/2008).