Dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

The test chemical did not induce mutation in Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In vitro chromosome aberration study:

The test chemical did not induce chromosome aberration in the mammalian cell line both in the presence and absence of S9 activation system and hence it is not likely to be gene mutant 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:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on the data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE derived based on the experimental data from various test chemicals

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA97, TA98, TA100, TA1535, TA1537

Remarks:

1

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium, other: TA98 and TA100

Remarks:

2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

The S-9 (9,000 x g supernatant) fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers

Test concentrations with justification for top dose:

1. 0, 1, 3, 10, 100, 166, 333, 1000, 1666 or 3333 µg/plate
2. 0, 0.05, 0.1, 0.5, 1, 5 or 10 µg/plate

Vehicle / solvent:

1. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

2. - Vehicle(s)/solvent(s) used: Distilled water or DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO or distilled water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

TA1535 and TA100

Positive control substance:

sodium azide

Remarks:

Without S9/1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

TA 1537

Positive control substance:

9-aminoacridine

Remarks:

Without S9/1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

yes

Positive controls:

not specified

Remarks:

TA98 and TA1538

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

Without S9/1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9/1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water or DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

4-nitroquinoline-N-oxide

benzo(a)pyrene

other: N-Methyl-N’-nitro-N-nitrosoguanidine and N-Nitrosodimethylamine

Remarks:

2

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: Plates were machine counted unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the agar.

2. METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

1. The plates were observed for a dose dependent increase in the number of Histidine- independent (his+) colonies.

Evaluations were made at both the individual trial and chemical levels.

Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase in his+ revertants, and the shape of the dose response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “+ W”, if only a single dose was elevated over the control, or if a weak increase was not dose-related. The distinctions between a questionable response and a nonmutagenic or weakly mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach two-fold over background for a trial to be judged mutagenic.

A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible, dose-related response over the solvent control, under a single metabolic activation condition, in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials.Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response.

2. The plates were observed or a dose dependent increase in the number of revertants/plate

Statistics:

No data

Species / strain:

S. typhimurium, other: TA97, TA98, TA100, TA1535, TA1537

Remarks:

1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98 and TA100

Remarks:

2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce mutation in Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO and used at dose levels of 0, 1, 3, 10, 100, 166, 333, 1000, 1666 or 3333 µg/plate by the preincubation method. Concurrent solvent and negative control plates were also included in the study. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In another study, gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strainsTA98 and TA100 both in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of 0, 0.05, 0.1, 0.5, 1, 5 or 10 µg/plate by the preincubation method. COncurrent solvent and positive control plates were also included in the study. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Based on the observations made, the test chemical did not induce mutation in Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is based on data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE derived based on the experimental data from various test chemicals

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

mammalian cell line, other: CHL/IU

Remarks:

1

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle MEM culture broth supplemented with 10 vol% of inactivated calf serum was used for the culture
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Remarks:

2

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 metabolic activation system

Test concentrations with justification for top dose:

1. Main test: (without S9) : 0, 50, 75, 100, 125 or 150 µg/mL
Additional test (without S9): 0, 100, 110, 120, 130, 140 or 150 µg/mL
With and without S9: 0, 100, 125, 150, 175 or 200 µg/mL

2. Without metabolic activation: 0.3, 1.0 or 3.0 µg/mL for 4 hrs
Without metabolic activation: 1.0 or 3.07 µg/mL for 7 or 18 hrs
With metabolic activation: 1.0, 3.0 or 10.0 µg/mL for 18 hrs
With metabolic activation: 10.0 µg/mL for 7 and 28 hrs

Vehicle / solvent:

1. - Vehicle(s)/solvent(s) used: Physiological saline
- Justification for choice of solvent/vehicle: The test chemical was soluble in physiological saline

2. No data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Physiological saline

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Remarks:

1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

2

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: in medium
No. of cells at the start of the experiment: 20000 cells

DURATION
- Preincubation period: No data
- Exposure duration: Continuous treatment: 24 hrs
Short term treatment: 6 hrs
- Expression time (cells in growth medium):
Continuous treatment: 24 hrs
Short term treatment: 18 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 200 groups of metaphase cells per group for structural abnormalities and ploidy cells

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, cell proliferation was measured

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: No data
- Other:

OTHER: No data

2. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: 4 hrs
- Expression time (cells in growth medium):
Cells were washed in glucose-containing saline and cultured in normal medium for 7, 18 and 28 hours.
- Selection time (if incubation with a selection agent): Two hours (7 hour interval) or 2.5 hours (18 and 28 hours intervals)
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 100 metaphase cells were examined

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, cell proliferation was measured

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: No data
- Other:

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

1. Chromosomes were analyzed based on the classification method by the Japan Society for Environmental Mutagenesis and Mammalian Testing, and structural abnormalities such as breakage and exchange of chromosome type or chromosome type, presence and absence of gap, and ploidy The presence or absence of cells (polyploid) was observed. The gap was not included in structural abnormality. The frequency of cells with chromosomal abnormality is negative, less than 5% negative, less than 10% false positive, more than 10% Positive.

2. Chromosomes were analyzed for structural chromosomal aberrations (breaks, fragments, deletions, exchanges and chromosomal disintegrations). Chromosomal gaps were recorded separately.

Statistics:

No data

Species / strain:

mammalian cell line, other: CHL/IU

Remarks:

1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

1. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To determine the treatment concentration of the test substance used for the chromosomal aberration test, the influence of the test substance on cell proliferation was investigated. In the presence and absence of S9 mix in the short-time treatment method, 7 concentrations are similarly in the concentration range of 50 to 500 μg / mL, and 6 concentrations in the concentration range of 12.5 to 200 μg / mL in the 24-hour treatment in the continuous treatment method Respectively. The growth inhibitory effect of the test substance on CHL / IU cells was determined by counting the cells of each group using a hemocytometer and using the relative growth rate relative to the negative (solvent) control group as an index. The concentration showing 50% inhibition of cell proliferation was calculated from the linear equation connecting the two doses with 50% of the cell proliferation rate. As a result, the 50% cell proliferation inhibitory concentration was 242 μg / mL in the presence of S9 mix in the short-time treatment method, 146 μg / mL in the absence of S9 mix, 107 μg / mL in 24-hour treatment with the continuous treatment method

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Dose range finding study was performed to determine the mutagenic nature of the test chemical

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce chromosome aberration in the mammalian cell line both in the presence and absence of S9 activation system and hence it is not likely to be gene mutant in vitro.

Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using CHL/IU cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in physiological saline and used at dose level of 0, 100, 125, 150, 175 or 200 µg/mL (short term treatment method) and 0, 50, 75, 100, 125 or 150 µg/mL (main test) and 0, 100, 110, 120, 130, 140 or 150 µg/mL (additional test) without S9 in the continuous treatment method. The doses for the main study were based on the preliminary dose range finding study. The test was performed by continuous treatment method for 24 hrs and short term treatment method for 6 hrs. The cells were allowed to express for 18 hrs in short term treatment method. Two hours before the end of the culture, colcemid was added to the culture solution to a final concentration of about 0.1 μg / ml. Chromosome specimens were prepared according to a conventional method. Two slide specimens were prepared for each dish. The prepared specimen was stained with 3% Giemsa solution for 20 mins. Chromosomes were analyzed based on the classification method by the Japan Society for Environmental Mutagenesis and Mammalian Testing, and structural abnormalities such as breakage and exchange of chromosome type or chromosome type, presence and absence of gap, and ploidy. The presence or absence of cells (polyploid) was observed. The gap was not included in structural abnormality. The frequency of cells with chromosomal abnormality is negative, less than 5% negative, less than 10% false positive, more than 10% Positive. Based on the observations made, the test chemical did not induce chromosome aberration in the CHL/IU cells both in the presence and absence of S9 activation system and hence it is not likely to be gene mutant in vitro.

In another study, in vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster V79 cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in suitable solvent and used. The doses for the main study were based on the preliminary dose range finding study. Evaluated dose levels were 1.0 μg/ml without S9-mix and 10.0 μg/ml with S9-mix for 7 hours; 0.3, 1.0 and 3.0 μg/ml without S9-mix and 1.0, 3.0 and 10.0 μg/ml with S9-mix for 18 hours; and 3.0 μg/ml without S9-mix and 10.0 μg/ml with S9-mix for 28 hours.Logarithmically growing cells were incubated with the test substance in serum-free culture medium for 4 hrs. Cells were subsequently washed in glucose-containing saline and cultured in normal medium for 7, 18 and 28 hours. Ethylmethanesulfonate and cyclophosphamide were used as positive controls in 18-hour cultures without and with S9-mix, respectively. Two hours (7 hour interval) or 2.5 hours (18 and 28 hours intervals) before the end of the incubation period, colcemid was added to the cultures. The cells were put onto glass slides, treated with hypotonic potassium chloride solution, fixed in methanol and acetic acid and stained with Giemsa solution. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations (breaks, fragments, deletions, exchanges and chromosomal disintegrations). Chromosomal gaps were recorded separately. There were no biologically relevant and statistically significant increases in cells with structural aberrations after treatment with the test substance at any fixation interval either with or without metabolic activation. Based on the observations made, the test chemical did not induce chromosome aberration in the Chinese hamster V79 cells both in the presence and absence of S9 activation system and hence it is not likely to be gene mutant in vitro.

Based on the data available, the test chemical did not induce chromosome aberration in the mammalian cell line both in the presence and absence of S9 activation system and hence it is not likely to be gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Ames test:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO and used at dose levels of 0, 1, 3, 10, 100, 166, 333, 1000, 1666 or 3333 µg/plate by the preincubation method. Concurrent solvent and negative control plates were also included in the study. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In another study, gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strainsTA98 and TA100 both in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of 0, 0.05, 0.1, 0.5, 1, 5 or 10 µg/plate by the preincubation method. COncurrent solvent and positive control plates were also included in the study. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Based on the observations made, the test chemical did not induce mutation in Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In vitro chromosome aberration study:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using CHL/IU cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in physiological saline and used at dose level of 0, 100, 125, 150, 175 or 200 µg/mL (short term treatment method) and 0, 50, 75, 100, 125 or 150 µg/mL (main test) and 0, 100, 110, 120, 130, 140 or 150 µg/mL (additional test) without S9 in the continuous treatment method. The doses for the main study were based on the preliminary dose range finding study. The test was performed by continuous treatment method for 24 hrs and short term treatment method for 6 hrs. The cells were allowed to express for 18 hrs in short term treatment method. Two hours before the end of the culture, colcemid was added to the culture solution to a final concentration of about 0.1 μg / ml. Chromosome specimens were prepared according to a conventional method. Two slide specimens were prepared for each dish. The prepared specimen was stained with 3% Giemsa solution for 20 mins. Chromosomes were analyzed based on the classification method by the Japan Society for Environmental Mutagenesis and Mammalian Testing, and structural abnormalities such as breakage and exchange of chromosome type or chromosome type, presence and absence of gap, and ploidy. The presence or absence of cells (polyploid) was observed. The gap was not included in structural abnormality. The frequency of cells with chromosomal abnormality is negative, less than 5% negative, less than 10% false positive, more than 10% Positive. Based on the observations made, the test chemical did not induce chromosome aberration in the CHL/IU cells both in the presence and absence of S9 activation system and hence it is not likely to be gene mutant in vitro.

In another study, in vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster V79 cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in suitable solvent and used. The doses for the main study were based on the preliminary dose range finding study. Evaluated dose levels were 1.0 μg/ml without S9-mix and 10.0 μg/ml with S9-mix for 7 hours; 0.3, 1.0 and 3.0 μg/ml without S9-mix and 1.0, 3.0 and 10.0 μg/ml with S9-mix for 18 hours; and 3.0 μg/ml without S9-mix and 10.0 μg/ml with S9-mix for 28 hours.Logarithmically growing cells were incubated with the test substance in serum-free culture medium for 4 hrs. Cells were subsequently washed in glucose-containing saline and cultured in normal medium for 7, 18 and 28 hours. Ethylmethanesulfonate and cyclophosphamide were used as positive controls in 18-hour cultures without and with S9-mix, respectively. Two hours (7 hour interval) or 2.5 hours (18 and 28 hours intervals) before the end of the incubation period, colcemid was added to the cultures. The cells were put onto glass slides, treated with hypotonic potassium chloride solution, fixed in methanol and acetic acid and stained with Giemsa solution. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations (breaks, fragments, deletions, exchanges and chromosomal disintegrations). Chromosomal gaps were recorded separately. There were no biologically relevant and statistically significant increases in cells with structural aberrations after treatment with the test substance at any fixation interval either with or without metabolic activation. Based on the observations made, the test chemical did not induce chromosome aberration in the Chinese hamster V79 cells both in the presence and absence of S9 activation system and hence it is not likely to be gene mutant in vitro.

Based on the data available, the test chemical did not induce chromosome aberration in the mammalian cell line both in the presence and absence of S9 activation system and hence it is not likely to be gene mutant in vitro.

Based on the observations made and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentoned in CLP regulation.

Justification for classification or non-classification

Based on the observations made and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentoned in CLP regulation.