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Genetic toxicity in vitro

Description of key information

Ames assay:

The test chemical s expected to not induce gene mutation in the Salmonella typhimurium strains used in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

In vitro mammalian chromosome aberration study:

The test chemical did not induce gene mutation in the mammalian cell line 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, 1.25, 2.5, 5 or 10 µM 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, 1.25, 2.5, 5 or 10 µM 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:
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 of read across substances
Justification for type of information:
Data for the target chemical is summarized based on the structurally similar read across chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
WoE derived based on the experimental data from structurally and functionally similar read across chemicals
GLP compliance:
not specified
Type of assay:
bacterial gene mutation assay
Target gene:
Histidine
Species / strain / cell type:
other: TA98, TA100, TA1535, TA1537, and TA1538
Remarks:
1/ 2 / 3
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:
Liver S9 homogenate was prepared from male Sprague-Dawley rats and Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight
Test concentrations with justification for top dose:
1. 0.3- 100 µg/plate
2/3. Maximum nontoxic dose tested was 10 µg/plate
Vehicle / solvent:
1. - Vehicle(s)/solvent(s) used: No data
- Justification for choice of solvent/vehicle: No data

2/3. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
No specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
not specified
Remarks:
1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Remarks:
2/ 3
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: Five doses of test chemical were tested in triplicate on each tester strain without and with metabolic activation.

NUMBER OF CELLS EVALUATED: No data

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

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

OTHER: No data

2. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 mins
- 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):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: No data

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

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

OTHER: The revertant colonies were counted by using a hand-held tally.

3. METHOD OF APPLICATION: in agar (plate incorporation)

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):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: No data

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

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

OTHER: No data
Rationale for test conditions:
1. No data
2. No data
3. No data
Evaluation criteria:
1. For a test article to be considered positive, it had to induce at least a doubling (TA98, TA100, and TA1535) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test chemical. If the study showed a dose response with a less than 3-fold increase on TA1537 or TA1538, the response had to be confirmed in a repeat experiment.

2/3. A compound was considered mutagenic when the number of revertants above background was at least twice the value of the historical control mean or twice the value of the current control mean, whichever was greater
Statistics:
1. No data
2. No data
3. No data
Species / strain:
other: TA98, TA100, TA1535, TA1537, and TA1538
Remarks:
1/ 2 /3
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: The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

2/3. No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical does not induce gene mutation in Salmonella typhimurium strains 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 for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical are as mentioned below:

 

Salmonella/Mammalian-Microsome Mutagenicity Assay was performed to determine the mutagenic nature of the test chemical. The study was performed at dose levels of 0.3-100 µg/plate using Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 with and without of metabolic activation system. Concurrent solvent and positive controls were used in the study. The test chemical did not induce mutation in the Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 with and without of metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

 

In another study, gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed by the preincubation and standard plate incorporation assay using Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 with and without S9 metabolic activation system. The liquid preincubation assays were timed for 30 min at 37°C in a Dri-block. The test chemical was dissolved in DMSO and used upto a maximum nontoxic dose of 10 µg/plate. Concurrent solvent and positive controls were also included in the study. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 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 summarized, the test chemical is expected to not induce gene mutation in the Salmonella typhimurium strains used in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
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:
Experimetal data from various test chemicals
Justification for type of information:
Data for the target chemical is summarized based data from various test chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1
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:
in vitro mammalian chromosome aberration test
Target gene:
1. No data
2. No data
Species / strain / cell type:
other: Chinese hamster lung (CHL/IU) cells
Remarks:
1
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The substance been used is a read across chemical and hence forth it will be referred to as test chemical
CAS no: 1552-42-7
Title: Studies on the Clastogenic Effects of Biologic Stains and Dyes
Author: William Au and T. C. Hsu
Bibliographic source: Environmental Mutagenesis 1: 27-35
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. Dosage : -S9 mix (continuous treatment); 0, 1.1, 2.1, 4.2 mg/mL
-S9 mix (short-term treatment); 0, 1.1, 2.1, 4.2 mg/mL
+S9 mix (short-term treatment); 0, 1.1, 2.1, 4.2 mg/mL

2. 20 μM
Vehicle / solvent:
1. 0.5 % Sodium carboxymethylcellulose
2. - Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: No data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Sodium carboxymethylcellulose
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 / 1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Sodium carboxymethylcellulose
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9 / 1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
1. METHOD OF APPLICATION: in medium

2. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: 5 hrs
- Expression time (cells in growth medium): 5 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): Giemsa
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 50 well-spread metaphases were scored for chromosome aberration

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

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data
Rationale for test conditions:
No data
Evaluation criteria:
1. The cell line was observed for chromosome aberrations
2. Chromosome damage was observed as chromatid breaks, fragments, and gaps. The average number of breaks per metaphase was calculated and was used for comparing the clastogenic activities of different compounds
Statistics:
No data
Species / strain:
other: Chinese hamster lung (CHL/IU) cells
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 Ovary (CHO)
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:
not specified
Additional information on results:
1. No data
2. No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce gene mutation in the mammalian cell line 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 as mentioned below:

In vitro mammalian chromosome aberration study was performed to determine the mutagenc nature of the test chemical. The study was performed using Chinese hamster lung (CHL/IU) cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in Sodium carboxymethylcellulose and used at dose level of 0, 1.1, 2.1, 4.2 mg/mL. Cells with structural chromosomal aberrations were not significantly increased at any dose. Polyploidy was significantly induced at 4.2 mg/mL with the 24 hr continuous treatment. However, the test chemical was judged with the result as negative because of the low frequency (1.93 %).

Gene mutation toxicity study was performed to evaluate the mutagenic nature of the test chemical. The chemical was applied at dosage level of 20 μM for 5 hrs to Chinese hamster ovary cells maintained in McCoy 5a medium. Colcemid (0.04 µg / L final concentration) was added to each culture during the last hour of incubation and all cultures were harvested for conventional cytogenetic preparations, stained with Giemsa, and coded. 50 well-spread metaphases were scored for chromosome aberration. The average number of breaks per metaphase was calculated and was used for comparing the clastogenic activities. The test chemical did not induce chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells incubated for 5 hours at a dose of 20 μM and hence is not likely be mutagenic in vitro.

Based on the data available, the test chemical did not induce gene mutation in the mammalian cell line 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
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, 1.25, 2.5, 5 or 10 µM
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Phosphate-buffered saline (PBS)
Justification for choice of solvent/ vehicle: The test chemical was easily dissolved in PBS.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
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: A preliminary dose-finding study was conducted prior to the main study. A range of different methyl violet base concentrations were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bicinchoninic acid (BCA) assay to assess cell viability and protein concentration, respectively. From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test.

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, 1.25, 2.5, 5 or 10 µM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.
Executive summary:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 1.25, 2.5, 5 or 10 µM 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, 1.25, 2.5, 5 or 10 µM 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

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

Ames assay:

Salmonella/Mammalian-Microsome Mutagenicity Assay was performed to determine the mutagenic nature of the test chemical. The study was performed at dose levels of 0.3-100 µg/plate using Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 with and without of metabolic activation system. Concurrent solvent and positive controls were used in the study. The test chemical did not induce mutation in the Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 with and without of metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

 

In another study, gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed by the preincubation and standard plate incorporation assay using Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 with and without S9 metabolic activation system. The liquid preincubation assays were timed for 30 min at 37°C in a Dri-block. The test chemical was dissolved in DMSO and used upto a maximum nontoxic dose of 10 µg/plate. Concurrent solvent and positive controls were also included in the study. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

The test chemical s expected to not induce gene mutation in the Salmonella typhimurium strains used in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

In vitro mammalian chromosome aberration study:

In vitro mammalian chromosome aberration study was performed to determine the mutagenc nature of the test chemical. The study was performed using Chinese hamster lung (CHL/IU) cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in Sodium carboxymethylcellulose and used at dose level of 0, 1.1, 2.1, 4.2 mg/mL. Cells with structural chromosomal aberrations were not significantly increased at any dose. Polyploidy was significantly induced at 4.2 mg/mL with the 24 hr continuous treatment. However, the test chemical was judged with the result as negative because of the low frequency (1.93 %).

Gene mutation toxicity study was performed to evaluate the mutagenic nature of the test chemical. The chemical was applied at dosage level of 20 μM for 5 hrs to Chinese hamster ovary cells maintained in McCoy 5a medium. Colcemid (0.04 µg / L final concentration) was added to each culture during the last hour of incubation and all cultures were harvested for conventional cytogenetic preparations, stained with Giemsa, and coded. 50 well-spread metaphases were scored for chromosome aberration. The average number of breaks per metaphase was calculated and was used for comparing the clastogenic activities. The test chemical did not induce chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells incubated for 5 hours at a dose of 20 μM and hence is not likely be mutagenic in vitro.

Based on the data available, the test chemical did not induce gene mutation in the mammalian cell line 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, 1.25, 2.5, 5 or 10 µM 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, 1.25, 2.5, 5 or 10 µM 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 for the target chemical 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, the test chemical did not induce gene mutation in the mammalian cell line and hence it is not likely to classify as a gene mutant in vitro.