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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

Ames assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98 and TA100 with and without metabolic activation. The test chemical was dissolved in DMSO and used at dose level of 0, 1, 10 or 100 µg/plate. Concurrent solvent and positive control plates was also included in the study. The test chemical in DMSO from doses 1-100 µg/plate was not mutagenic in Salmonella typhimurium strains TA98 and TA100 with and without metabolic activation and hence it is not likely to classify as a genem utant in vitro.

In vitro mammalian chromosome aberration study:

The test chemical did not induce chromosome aberrations in Chinese hamster ovary cell line (CHO) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation assay:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM and S9-induced metabolic activation for 3 hours.The results showed that there was no evidence of cytotoxicity after treatment. Independently of tested concentration, the results showed no evidence of gene toxicity. Therefore, it is considered that the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.

Link to relevant study records

Referenceopen allclose all

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:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data is from Japanese publication
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
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:
S9 mix
Test concentrations with justification for top dose:
0, 1, 10 or 100 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): No data

DURATION
- Preincubation period: No data
- Exposure duration: No data
- Expression time (cells in growth medium): No data
- 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

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No data

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
The plates were observed for an increase in number of histidine revertants
Statistics:
No data
Species / strain:
S. typhimurium TA 98
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 TA 100
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
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential
Conclusions:
In an Ames test , the test chemical in DMSO from doses 1-100 µg/Plate was not mutagenic in Salmonella typhimurium strains TA98 and TA100 with and without metabolic activation and hence it is not likely to classify as a genem utant in vitro.
Executive summary:

Ames assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98 and TA100 with and without metabolic activation. The test chemical was dissolved in DMSO and used at dose level of 0, 1, 10 or 100 µg/plate. Concurrent solvent and positive control plates was also included in the study. The test chemical in DMSO from doses 1-100 µg/plate was not mutagenic in Salmonella typhimurium strains TA98 and TA100 with and without metabolic activation and hence it is not likely to classify as a genem utant in vitro.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
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:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
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 study
Target gene:
No data
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
1 / 2
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy’s 5A medium
- 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
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague Dawley rat liver S9 and cofactor mix
Test concentrations with justification for top dose:
1. Without S9: 0, 50, 100, 200, 300, 400 or 500 µg/mL
With S9: 0, 150, 160, 170, 180, 190 or 200 µg/mL

2. 0, 50, 108, 233 or 500 µg/mL
Vehicle / solvent:
1/2. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
1/2
Details on test system and experimental conditions:
1. METHOD OF APPLICATION:in medium

DURATION
- Preincubation period:No data available
- Exposure duration: Without S9: 10 hrs
With S9: 2 hrs
- Expression time (cells in growth medium): Without S9: 2 hrs
With S9: 10 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: Two hundred first-division metaphase cells were scored at each dose level

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

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

OTHER: No data available

2. METHOD OF APPLICATION:in medium

DURATION
- Preincubation period:No data available
- Exposure duration: Without S9: 14.7 hrs
With S9: 2 hrs
- Expression time (cells in growth medium): Without S9: 2 hrs
With S9: 10.5 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: Two hundred first-division metaphase cells were scored at each dose level

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

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

OTHER: No data available
Rationale for test conditions:
No data
Evaluation criteria:
1. The cell line was observed for classes of aberrations including simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

2. The cell line was observed for classes of aberrations including simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).
Statistics:
To arrive at a statistical call for a trial, analyses were conducted on both the dose response curve and individual dose points. For a single trial, a statistically significant (P≤ 0.05) difference for one dose point and a significant trend (P≤ 0.015) were considered weak evidence for a positive response; significant differences for two or more doses indicated the trial was positive. A positive trend test in the absence of a statistically significant increase at any one dose resulted in an equivocal call
Species / strain:
Chinese hamster Ovary (CHO)
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
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce chromosome aberrations in Chinese hamster ovary cell line (CHO) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a 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 mentioned below:

In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster ovary cell line (CHO) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of0, 50, 100, 200, 300, 400 or 500µg/mL without S9 and 0, 150, 160, 170, 180, 190 or 200µg/mL with S9. Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. Each test consisted of concurrent solvent and positive controls and of at least three doses of the test chemical; the high dose was limited by toxicity. A single flask per dose was used. In the Abs test without S9, cells were incubated in McCoy’s 5A medium with the test chemical for 10 hours; Colcemid was added, and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with the test chemical and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 10 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9. Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ±2 chromosomes). All slides were scored blind, and those from a single test were read by the same person. Two hundred first-division metaphase cells were scored at each dose level. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations). Based on the observations made, the test chemical didnot induce chromosome aberrations in Chinese hamster ovary cell line (CHO) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster ovary cell line (CHO) in the presence and absence of S9 metabolic activation system. The test chemical was , or dissolved in DMSO and used at dose level of0, 50, 108, 233 or 500 µg/mL without and with S9. Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. Each test consisted of concurrent solvent and positive controls and of at least three doses of the test chemical; the high dose was limited by toxicity. A single flask per dose was used. In the Abs test without S9, cells were incubated in McCoy’s 5A medium with the test chemical for 14.7 hours; Colcemid was added, and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with isoeugenol and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 10.5 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9. Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ±2 chromosomes). All slides were scored blind, and those from a single test were read by the same person. Two hundred first-division metaphase cells were scored at each dose level. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations). Based on the observations made, the test chemical didnot induce chromosome aberrations in Chinese hamster ovary cell line (CHO) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the observations made, the test chemical did not induce chromosome aberrations in Chinese hamster ovary cell line (CHO) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical in the presence of S9 metabolic activation system
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable
Additional strain / cell type characteristics:
other: Hypodiploid, modal No. 20
Cytokinesis block (if used):
No data
Metabolic activation:
with
Metabolic activation system:
S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)
Test concentrations with justification for top dose:
0, 0.5, 1.0, 2.5 or 5.0mM
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle: The test chemical was easily dissolved in ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium with pre-incubation

DURATION
Pre-incubation
One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.

Exposure duration
3 hours

Expression time
7 days

Selection time
14 days

Fixation time
7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)

SPINDLE INHIBITOR (cytogenetic assays): Not applicable

STAIN (for cytogenetic assays): Crystal violet

NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.
Rationale for test conditions:
No data
Evaluation criteria:
Chinese Hamster Ovary Cells (CHO) were observed for gene mutation caused by the test compound
Statistics:
No data
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
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
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Completed without S9 metabolic activation. A range of test concentrations (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0 or 5.0mM) was applied 24 hours after seeding to single cultures in fresh medium in 96-well plates. The cell population (control and treated cells) were assessed 24 and 48 hours after treatment using the colorimetric assay MTT and the BCA assay to assess cell viability and total protein concentration, respectively. From the basis of these results, the test concentrations of the chemical was chosen to be included in the gene toxicity test. Since cytotoxicity was evident at the tested concentration in this preliminary dose-finding test further testing concentrations were adapted to have a maximum test concentration of 5.0mM.Since the test chemical was dissolved in ethanol, higher concentrations of the test chemical than the concentration mentioned above would result in a toxic effect of ethanol. The test chemical could only be dissolved in 99.5% ethanol.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable:No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0mM did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical.
Executive summary:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM and S9-induced metabolic activation for 3 hours.The results showed that there was no evidence of cytotoxicity after treatment. Independently of tested concentration, the results showed no evidence of gene toxicity. Therefore, it is considered that the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.

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 target chemical ans its various other read across chemicals was reviewed to determinet he mutagenic nature of the test chemical. The studies are as mentioned below:

Ames assay:

Ames assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98 and TA100 with and without metabolic activation. The test chemical was dissolved in DMSO and used at dose level of 0, 1, 10 or 100 µg/plate. Concurrent solvent and positive control plates was also included in the study. The test chemical in DMSO from doses 1-100 µg/plate was not mutagenic in Salmonella typhimurium strains TA98 and TA100 with and without metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

In another study, bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activation. Based on the observations made, the test chemical from doses 0.1 , 1 , 10 , 100 , 1000 micrograms/litre was not mutagenic in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activation.

Agar Overlay Assay was also performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activation. The test chemical was dissolved in DMSO and used at dose level of 0.001, 0.01, 0.1, 1 or 5 μL/plate. In an Ames test , the test chemical from doses 0.001, 0.01, 0.1, 1 or 5 μL/plate was not mutagenic in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and with and without metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration study:

In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster ovary cell line (CHO) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of0, 50, 100, 200, 300, 400 or 500µg/mL without S9 and 0, 150, 160, 170, 180, 190 or 200µg/mL with S9. Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. Each test consisted of concurrent solvent and positive controls and of at least three doses of the test chemical; the high dose was limited by toxicity. A single flask per dose was used. In the Abs test without S9, cells were incubated in McCoy’s 5A medium with the test chemical for 10 hours; Colcemid was added, and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with the test chemical and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 10 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9. Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ±2 chromosomes). All slides were scored blind, and those from a single test were read by the same person. Two hundred first-division metaphase cells were scored at each dose level. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations). Based on the observations made, the test chemical didnot induce chromosome aberrations in Chinese hamster ovary cell line (CHO) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster ovary cell line (CHO) in the presence and absence of S9 metabolic activation system. The test chemical was , or dissolved in DMSO and used at dose level of0, 50, 108, 233 or 500 µg/mL without and with S9. Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. Each test consisted of concurrent solvent and positive controls and of at least three doses of the test chemical; the high dose was limited by toxicity. A single flask per dose was used. In the Abs test without S9, cells were incubated in McCoy’s 5A medium with the test chemical for 14.7 hours; Colcemid was added, and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with isoeugenol and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 10.5 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9. Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ±2 chromosomes). All slides were scored blind, and those from a single test were read by the same person. Two hundred first-division metaphase cells were scored at each dose level. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations). Based on the observations made, the test chemical didnot induce chromosome aberrations in Chinese hamster ovary cell line (CHO) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the observations made, the test chemical did not induce chromosome aberrations in Chinese hamster ovary cell line (CHO) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation assay:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM and with and without S9-induced metabolic activation for 3 hours.The results showed that there was no evidence of cytotoxicity after treatment. Independently of tested concentration, the results showed no evidence of gene toxicity. Therefore, it is considered that the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence and absence of metabolic activation.

Based on the data available 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 in vitro as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available 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 in vitro as per the criteria mentioned in CLP regulation.