Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

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

In vitro mammalian chromosome aberration study:

The test chemical did not induce chromosome aberration in CHL / IU cells 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, 12.5, 25, 50 or 100 µM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. Independently of treatment concentration, the results showed no evidence of gene toxicity when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentration of 0, 12.5, 25, 50 or 100 µM does not cause genetic mutation(s) 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:
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 TA 1535, TA 1537, TA 98 and TA 100
Remarks:
1
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Remarks:
1
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
LT-2 strains / 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:
S9 metabolic activation system isolated from 7 week old male Sprague-Dawley male rats administered concomitant administration of phenobarbital (PB) and 5,6-benzoflavone (BF).
Test concentrations with justification for top dose:
1. 0, 313, 625, 1250, 2500 or 5000 µg/plate
2. 0 or 3 µmole/plate
Vehicle / solvent:
1. - Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test chemical was soluble in acetone


2. - Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The test chemical was soluble in ethanol
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide and 2-aminoanthracene
Remarks:
1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidin (without metabolic activation) and 2-aminoanthracene (with activation)
Remarks:
2
Details on test system and experimental conditions:
1. METHOD OF APPLICATION: in agar (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: Triplicate

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:Yes
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

2. METHOD OF APPLICATION: Spot test (in agar)

DURATION
- Preincubation period: No data available
- Exposure duration: No data available
- Expression time (cells in growth medium): No data available
- 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): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: No data available

NUMBER OF CELLS EVALUATED: No data available

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. When the increase in the number of revertants/plate increased more than 2 times and the increase was found to be reproducible or dose dependent, it was decided that the test substance has mutagenicity (positive) in this test system.
2. 1. Increase in the number of spontaneous revertants
2. The presence of the rfa-mutation was checked by crystal violet inhibition;
3. The presence of the plasmid pKM 101 in strains TA 98 and TA 100 was checked by resistance to ampicillin
Statistics:
No data
Species / strain:
S. typhimurium, other: TA100 and TA98
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:
not specified
Species / strain:
S. typhimurium, other: LT-2 strains TA 98, TA 100, TA 1535, and TA 1537
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
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: When the test was carried out with a common ratio of about 3 in the range of 50 to 5000 μg / plate for the test chemical, it was confirmed that in all test bacteria, no antibacterial activity was observed in any of the addition tests.

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 reversion of mutations in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likley 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 mentioned below:

Bacterial gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537, and Escherichia coli WP 2 uvrA in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in acetone and used at dose level of 0, 313, 625, 1250, 2500 or 5000µg/plate. No increase in the number of mutant colonies that were more than twice the solvent control value in the S9 mix non-addition test and addition test of the five test bacteria used was observed. Based on the observations made, thetest chemical did not induce gene mutation inSalmonella typhimurium TA 100, TA 1535, TA 98, TA 1537, and Escherichia coli WP 2 uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Gene mutation toxicity study was also performed to determine the mutagenic nature of the test chemical. The material was dissolved in ethanol and applied at a concentration of 0 or 3 µmole/plate in the spot test performed. The plates were observed for an increase in the number of spontaneous revertants. The test chemical did not induce an increase in the number of spontaneous revertants and hence is not mutagenic in the bacteriumSalmonella typhimuriumLT-2 strains TA98, TA100, TA1537 and TA1535 with and without S9 metabolic activation system and hence is not likely to classify as gene mutant in vitro.

Based on the observations made, the test chemical did not induce reversion of mutations in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likley to classify as a gene mutant 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 for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
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 cells
Remarks:
1
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle MEM culture broth supplemented with 10% fetal bovine serum was used for the culture.
- Properly maintained: Yes
- 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:
mammalian cell line, other: CHL/IU
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. Continuous treatment:
0, 0.0020, 0.0040, 0.0080 mg/mL

Short term treatment:
With S9: 0, 0.0075, 0.015 or 0.030 mg/mL
Without S9: 0, 0.00050, 0.0010 or 0.0020 mg/mL

2. 0, 250, 500 or 1000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test chemical was soluble in acetone
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 1-methyl-3-nitro-1-nitrosoguanidine, 3,4-benzo[a]pyrene
Details on test system and experimental conditions:
1. METHOD OF APPLICATION: in medium
Cells at the start of experiment: 20000 cells

DURATION
- Preincubation period: No data
- Exposure duration:
Continuous treatment: 24 or 48 hrs
Short term treatment: 6 hrs
- Expression time (cells in growth medium): 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): 3% Giemsa solution

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: For structural abnormalities, 200 metaphase cells per group and 800 division metastatic cells for multiplicative cells were analyzed.

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

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

OTHER: No data

2. METHOD OF APPLICATION: in medium
Cells at the start of experiment: 4000 cells

DURATION
- Preincubation period: No data
- Exposure duration:
Continuous treatment: 24 hrs
Short term treatment: 6 hrs
- Expression time (cells in growth medium): 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): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 200 metaphase cells were evaluated

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

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

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
1. The cell line was observed for the presence or absence of structural abnormality such as chromosome type or chromosome type gap, The presence or absence of cells (polyploid) was observed.
2. The clastogenicity was judged to be positive when the frequency of the structural chromosomal aberration detected was 10% or more.
Statistics:
1. Yes, With reference to the method of Hayashi, with reference to the method of the forest, the Fisher's direct probability method between the solvent background data and the test substance treated group (refer to the groupware significance level in consideration of multiplicity 5%), a significant difference test was carried out. In addition, when significant differences were found by Fisher's exact stochastic method, Cochran-Armitage's trend test (p <0.05) was carried out for dose dependency. In principle, a case where a significant difference was observed in both of the above two tests was regarded as positive. When there was no significant difference in the trend test, it was judged as false positives. The case where the number of observed cells was less than 100 for structural abnormality and less than 400 for polyploid cells was determined as undetectable due to cytotoxicity.

2. No data
Species / strain:
mammalian cell line, other: CHL / IU cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
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: The doses for the main study were based on cell proliferation inhibition test

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: A preliminary cell proliferation inhibition test was conducted using doses of 15.6-1000 ug/mL and two dishes/dose. Based on the results of the cell proliferation inhibition test, the highest dose level of the test substance was established at 1000 µg/mL (upper limit of the preparable dose of the test solution) with a total of three doses including 500 and 250 µg/mL at a common ratio of two both in the short-term and continuous treatment methods.

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 CHL / IU cells 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 test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The test was performed using CHL / IU cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in acetone as solvent and used at dose level of 0, 0.0020, 0.0040, 0.0080 mg/mL in continuous treatment and 0, 0.0075, 0.015 or 0.030 mg/mL with S9 and 0, 0.00050, 0.0010 or 0.0020 mg/mL without S9 in short term treatment method. The study was performed by short term treatment method for 6 hrs and continuous treatment method for 24 and 48 hrs. The cell line was allowed to express further for 18 hrs in shirt 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. Six slide specimens were prepared for each dish. The prepared specimens were stained with 3% Giemsa solution. The cell line was observed for the presence or absence of structural abnormality such as chromosome type or chromosome type gap, The presence or absence of cells (polyploid) was observed. Based on the observations made, the test chemicaldid not induce chromosome aberration inCHL / IU cells 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 chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The test was performed using CHL / IU cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in acetone as solvent and used at dose level of 0, 250, 500 or 1000µg/mL.The study was performed by short term treatment method for 6 hrs and continuous treatment method for 24 hrs. The cell line was allowed to express further for 18 hrs in short term treatment method. According to the results of the study, no increase in the number of chromosomally aberrant cells was observed in the short-term and continuous treatment method with and without S9 mix. Hence thetest chemical did not induce chromosome aberration inCHL / IU cells 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 data available, the test chemical did not induce chromosome aberration in CHL / IU cells 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
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
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.
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
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, 12.5, 25, 50 or 100 µM
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle: The test chemical was soluble in ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N-nitrosourea (ENU)
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)

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 CYTOTOXICITYAfter 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.

OTHER: No data available
Rationale for test conditions:
No data available
Evaluation criteria:
The number of mutant colonies were counted and compared to the negative and positive controls. The mutation frequency was calculated.
Statistics:
The Standard deviation (± SD) was calculated.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
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:
No data available
Remarks on result:
other: No mutagenic potential
Conclusions:
When CHO cells were exposed to the test chemical in the concentration of 0, 12.5, 25, 50 or 100 µM the results did not show any evidence of gene toxicity.
Executive summary:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 12.5, 25, 50 or 100 µM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. Independently of treatment concentration, the results showed no evidence of gene toxicity when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentration of 0, 12.5, 25, 50 or 100 µM does not cause genetic mutation(s) 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

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

Ames assay:

Bacterial gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537, and Escherichia coli WP 2 uvrA in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in acetone and used at dose level of 0, 313, 625, 1250, 2500 or 5000µg/plate. No increase in the number of mutant colonies that were more than twice the solvent control value in the S9 mix non-addition test and addition test of the five test bacteria used was observed. Based on the observations made, thetest chemical did not induce gene mutation inSalmonella typhimurium TA 100, TA 1535, TA 98, TA 1537, and Escherichia coli WP 2 uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Gene mutation toxicity study was also performed to determine the mutagenic nature of the test chemical. The material was dissolved in ethanol and applied at a concentration of 0 or 3 µmole/plate in the spot test performed. The plates were observed for an increase in the number of spontaneous revertants. The test chemical did not induce an increase in the number of spontaneous revertants and hence is not mutagenic in the bacteriumSalmonella typhimuriumLT-2 strains TA98, TA100, TA1537 and TA1535 with and without S9 metabolic activation system and hence is not likely to classify as gene mutant in vitro.

In vitro mammalian chromosome aberration study:

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

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The test was performed using CHL / IU cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in acetone as solvent and used at dose level of 0, 0.0020, 0.0040, 0.0080 mg/mL in continuous treatment and 0, 0.0075, 0.015 or 0.030 mg/mL with S9 and 0, 0.00050, 0.0010 or 0.0020 mg/mL without S9 in short term treatment method. The study was performed by short term treatment method for 6 hrs and continuous treatment method for 24 and 48 hrs. The cell line was allowed to express further for 18 hrs in shirt 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. Six slide specimens were prepared for each dish. The prepared specimens were stained with 3% Giemsa solution. The cell line was observed for the presence or absence of structural abnormality such as chromosome type or chromosome type gap, The presence or absence of cells (polyploid) was observed. Based on the observations made, the test chemicaldid not induce chromosome aberration inCHL / IU cells 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 chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The test was performed using CHL / IU cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in acetone as solvent and used at dose level of 0, 250, 500 or 1000µg/mL.The study was performed by short term treatment method for 6 hrs and continuous treatment method for 24 hrs. The cell line was allowed to express further for 18 hrs in short term treatment method. According to the results of the study, no increase in the number of chromosomally aberrant cells was observed in the short-term and continuous treatment method with and without S9 mix. Hence thetest chemical did not induce chromosome aberration inCHL / IU cells 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 data available, the test chemical did not induce chromosome aberration in CHL / IU cells 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, 12.5, 25, 50 or 100 µM and with and without S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. Independently of treatment concentration, the results showed no evidence of gene toxicity when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentration of 0, 12.5, 25, 50 or 100 µM does not cause genetic mutation(s) 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 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 as per the criteria mentioned in CLP regulation.