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Genetic toxicity in vitro

Description of key information

Ames test:

Test substance failed to induce mutation in Salmonella typhimurium strains and E. coli strains in the gradient plate assay performed both in the presence and absence of metabolic activation system and hence is not likely to classiify 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

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Gene mutation toxicity study was performed to determine the mutagenic nature of test substance.

GLP compliance:
no
Type of assay:
bacterial gene mutation assay
Specific details on test material used for the study:

- Impurities (identity and concentrations): No data available
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: LT2 strains G46 ,TA1535 ,TA100 ,C3076 ,D3052 ,TA1538 and TA98
Remarks:
In special cases, 4 additional strains are used for supplemental testing. These strains are TA92 and TA94
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli, other: WP2 and WP2 uvrA
Remarks:
In special cases, 4 additional strains are used for supple mental testing. These strains are CM881 and CM891
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: WP2 (E. coli tryptophan) and WP2 uvrA (E. coli tryptophan with uvrA deletion). CM881 (WP2 with the addition of R factor pKM1O1), and CM891 (WP2 with uVrA deletion and with the addition of A-factor pKM1O1).
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Enzymatic activation of test compound was achieved by use of a mixture of rat liver enzymes
Test concentrations with justification for top dose:
0.1-1000 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide. When appropriate, water or dimethoxyethane is used instead of dimethyl sulfoxide.
- Justification for choice of solvent/vehicle: No data
Untreated negative controls:
yes
Remarks:
Plates containing no compound are also prepared to serve as negative controls.
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
no
Positive control substance:
other: streptozotocin (Without metabolic activation) and 2-acetylaminofluorene (With metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: agar medium (plate incorporation)

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: Yes, auxotrophs were observed for growth

Rationale for test conditions:
No data
Evaluation criteria:
Dose dependent increase in the number of revertants/plate
Statistics:
No data
Species / strain:
S. typhimurium, other: LT2 strains G46 ,TA1535 ,TA100 ,C3076 ,D3052 ,TA1538 and TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli, other: WP2 and WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
no data
Remarks on result:
other: No mutagenic potential was observed
Conclusions:
Test substance failed to induce mutation in Salmonella typhimurium strains and E. coli strains in the gradient plate assay performed both in the presence and absence of metabolic activation system and hence is not likely to classiify as a gene mutant in vitro.
Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of test substance. The study was performed using Salmonella typhimurium LT2 strains G46, TA1535, TA100, C3076, D3052, TA1538 and TA98 and E. coli strains WP2 and WP2 uvrA. In special cases, 4 additional strains are used for supplemental testing. These strains were TA92, TA94, CM881, and CM891. The test chemical was dissolved in DMSO. When appropriate, water or dimethoxyethane is used instead of dimethyl sulfoxide. Agar plates containing a concentration gradient of test com pound was prepared. Ten ml of minimal agar medium (not containing test compound) was poured into a square Petri dish (9 x 9 cm) which is tilted at a slight angle. The agar was then allowed to solidify into a wedge-shaped layer by standing at room temperature. Meanwhile, a 1000 -µg/ml mixture of test compound in agar was prepared by adding 10 ml of minimal agar to 0.1 ml of a 100-mg/mI solution of test compound in dimethyl sulfoxide. When appropriate, water or dimethoxyethane was used instead of dimethyl sulfoxide. The cooled agar plates are then placed on a level surface, and an overlay of the 10 ml of agar containing the test compound was poured onto the plate to form a reversed wedge of agar on top of the first wedge. A concentration gradient of compound was produced by allowing the compound in the upper wedge to diffuse into the lower layer for 2 hr at room temperature. The concentration range in this plate is approximately 100 to 1000µg/ml. Three additional plates with concentration ranges of 10 to 100µg/ml, 1 to 10µg/ml and 0.1 to 1µg/ml are prepared. A streaking device consisting of 10 sterile 50µL pipets is next dipped into suspensions of the 10 test strains and allowed to fill by capillary action. The pipettes were then touched to the upper edge of the gradient and drawn across the plate. A wet trail of inoculum was observed. The plates were then incubated for 48 hr at 37°C. The study was performed in the presence and absence of rat liver enzyme metabolic activation system. Concurrent negative and positive controls were included in the study. Test substance failed to induce mutation in Salmonella typhimurium strains and E. coli strains in the gradient plate assay performed both in the presence and absence of metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

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 test substance. The study was performed using Salmonella typhimurium LT2 strains G46, TA1535, TA100, C3076, D3052, TA1538 and TA98 and E. coli strains WP2 and WP2 uvrA. In special cases, 4 additional strains are used for supplemental testing. These strains were TA92, TA94, CM881, and CM891. The test chemical was dissolved in DMSO. When appropriate, water or dimethoxyethane is used instead of dimethyl sulfoxide. Agar plates containing a concentration gradient of test com pound was prepared. Ten ml of minimal agar medium (not containing test compound) was poured into a square Petri dish (9 x 9 cm) which is tilted at a slight angle. The agar was then allowed to solidify into a wedge-shaped layer by standing at room temperature. Meanwhile, a 1000 -µg/ml mixture of test compound in agar was prepared by adding 10 ml of minimal agar to 0.1 ml of a 100-mg/mI solution of test compound in dimethyl sulfoxide. When appropriate, water or dimethoxyethane was used instead of dimethyl sulfoxide. The cooled agar plates are then placed on a level surface, and an overlay of the 10 ml of agar containing the test compound was poured onto the plate to form a reversed wedge of agar on top of the first wedge. A concentration gradient of compound was produced by allowing the compound in the upper wedge to diffuse into the lower layer for 2 hr at room temperature. The concentration range in this plate is approximately 100 to 1000µg/ml. Three additional plates with concentration ranges of 10 to 100µg/ml, 1 to 10µg/ml and 0.1 to 1µg/ml are prepared. A streaking device consisting of 10 sterile 50µL pipets is next dipped into suspensions of the 10 test strains and allowed to fill by capillary action. The pipettes were then touched to the upper edge of the gradient and drawn across the plate. A wet trail of inoculum was observed. The plates were then incubated for 48 hr at 37°C. The study was performed in the presence and absence of rat liver enzyme metabolic activation system. Concurrent negative and positive controls were included in the study. Test substance failed to induce mutation in Salmonella typhimurium strains and E. coli strains in the gradient plate assay performed both in the presence and absence of metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

 

Supported by other study. 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.

 

Supported by 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.

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 data available , the test chemical not likiy to 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 above annatation, the test chemical not likly to 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.