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 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.

 

In vitro mammalian chromosome aberration study:

 

The test chemical did not induce chromosome aberration in Chinese hamster cultured cells (CHL/IU) 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:

 

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

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
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:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA1535, TA100, TA1537, TA1538, TA1978 and TA98
Remarks:
1
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other:
Species / strain / cell type:
S. typhimurium, other: TA97, TA98, TA100
Remarks:
2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
Strains TA1535, TA1537 and TA1538 have mutations hisG46, hisC3076 and hisD3052 in their respective histidine operons, resulting in a nutritional requirement for histidine. In addition they possess deletions in the uvr region of the genome removing their capacity for excision repair and contain rfa (deep rough) mutations resulting in the impaired synthesis of the polysaccharide side chains of their surface lipopolysaccharide. Strain TA1978 resembles TA1538 except that it possesses the normal uvr repair system and a mutation in gal E. Strains TA100 and TA98 are derived from TA1535 and TA1538 respectively by insertion of the R factor plasmid pKM101.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced S9 fraction was obtained from Female albino rats
Test concentrations with justification for top dose:
1. 100-200 µg/plate
2. 0, 5, 10, 50 or 200 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:
ethylmethanesulphonate
Remarks:
For TA1535 / 1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
For TA100 / 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
Remarks:
For TA1537 / 1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
TA1538 and TA98. / 1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Remarks:
Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA1978 and TA98 / 1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
2
Details on test system and experimental conditions:
1. METHOD OF APPLICATION: as impregnation on paper disk

DURATION
- Preincubation period: No data
- Exposure duration: 72 hrs
- Expression time (cells in growth medium): 72 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: ): 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

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

DURATION
- Preincubation period:No data
- Exposure duration:2 days (48 hrs)
- Expression time (cells in growth medium):2 days (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:Duplicate

NUMBER OF CELLS EVALUATED:No data

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

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

OTHER:No data
Rationale for test conditions:
No data
Evaluation criteria:
1. Growth inhibition was noted
2. The plates were observed for a dose dependent increase in the number of revertants/plate
Statistics:
No data
Species / strain:
S. typhimurium, other: TA1535, TA100, TA1537, TA1538, TA1978 and TA98
Remarks:
1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium, other: TA97, TA98, TA100
Remarks:
2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce mutation in Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence 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:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed by spot test using Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA1978 and TA98 with and without S9 metabolic activation system. 100-200 µg of the test material dissolved in 10-20µL DMSO/disc was applied to 6mm paper concentration discs and used for mutagenicity testing. From 1 to 4 discs were applied per plate. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did not induce mutation in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA1978 and TA98 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 performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA97, TA98, TA100 with and without PCB induced S9 metabolic activation system at dose levels of 0, 5, 10, 50 or 200µg/plate. The plates were incubated for 48 hrs and the number of dose dependent increase in the revertants was counted. The test chemical did not induce reversion of histidine gene mutation in Salmonella typhimurium strains TA97, TA98, TA100 both in the presence and absence of PCB induced rat liver S9 fraction 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 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.

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:
1. No data
Species / strain / cell type:
mammalian cell line, other: Chinese hamster cultured cells (CHL/IU)
Remarks:
1
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle MEM culture medium supplemented with 10% fetal bovine serum
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
mammalian cell line, other: Chinese hamster cultured cells (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 mix
Test concentrations with justification for top dose:
1. Continuous treatment: 0, 0.3, 0.7, 1.3 µg/mL
Short term treatment method: 0, 06, 1.1, 2.2 µg/mL

2. Continuous treatment: 0, 19.1, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/mL
Short term treatment method:

With S9: 0, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/mL

Without S9: 0, 19.1, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/mL
Vehicle / solvent:
1. - Vehicle(s)/solvent(s) used: 0.5% CMC Na
- Justification for choice of solvent/vehicle: The test chemical was soluble in CMC Na

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:
CMC Na
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:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
2
Details on test system and experimental conditions:
1. METHOD OF APPLICATION: in medium
Cells at the start of the experiment: 20000 cells

DURATION
- Preincubation period: No data
- Exposure duration: Direct method: 24 and 48 hrs
Short term treatment method with S9: 6 hrs
- Expression time (cells in growth medium):
Direct method: 24 and 48 hrs
Short term treatment method with S9: 18 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

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

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 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: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

2. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: Direct method: 24 and 48 hrs
Short term treatment method with S9: 6 hrs
- Expression time (cells in growth medium):
Direct method: 24 and 48 hrs
Short term treatment method with S9: 18 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

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

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: For structural abnormalities, 200 metaphase cells per group were analyzed.

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

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

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
The presence or absence of structural abnormality such as chromosome type or chromosome type gap, The presence or absence of cells (polyploid) was also observed.
Statistics:
1. A significant difference test (p <0.05) between the negative control group and the test substance treated group and between the negative control group and the positive control group was performed on the occurrence frequency of cells having chromosomal abnormality by Fisher's exact probability test method. According to the judgment criteria of Ishikan et al, the frequency of cells with chromosomal abnormality is negative, negative 5% or higher and less than 10% positive false positive 10% or higher Respectively.

2. No data
Species / strain:
mammalian cell line, other: Chinese hamster cultured cells (CHL/IU)
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: To determine the treatment concentration of the test substance used for the chromosomal aberration test, the influence of the test substance on cell proliferation was investigated. The growth inhibitory action of the test substance on CHL / IU cells was determined by measuring the proliferation degree of each group using a monolayer culture cell densitometer, and the negative control group was used as an index.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

2. No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce chromosome aberration in Chinese hamster cultured cells (CHL/IU) 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. The studies are mentioned below:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster cultured cells (CHL/IU) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in 0.5% CMC Na and used at dose level of 0, 0.3, 0.7, 1.3 µg/mL in continuous treatment method and 0, 06, 1.1, 2.2 µg/mL in short term treatment method. The doses for the main study were based on data from preliminary dose range finding study. Concurrent solvent and positive control plates were also included in the study. The cells were exposed to the test chemical for 24 or 48 hrs in the continuous treatment method and for 6 hrs in the short term treatment method. Two hours before the end of the culture, colcemid was added to the culture solution to a final concentration of about 0.1 μg / ml. Chromosome specimens were prepared according to a conventional method. Giemsa stained six slide specimens were prepared for each petri dish. The presence or absence of structural abnormality such as chromosome type or chromosome type gap, the presence or absence of cells (polyploid) was also observed.The test chemical did not induce chromosome aberration inChinese hamster cultured cells (CHL/IU) 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 study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster cultured cells (CHL) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 19.1, 38.3, 76.6, 153, 306, 613, 1230, 2450µg/mL without S9 and 0, 38.3, 76.6, 153, 306, 613, 1230, 2450µg/mL with S9 in the short term treatment method and 0, 19.1, 38.3, 76.6, 153, 306, 613, 1230, 2450µg/mL in continuous treatment method. The doses for the main study were based on data from preliminary dose range finding study. Concurrent solvent and positive control plates were also included in the study. The cells were exposed to the test chemical for 24 or 48 hrs in the continuous treatment method and for 6 hrs in the short term treatment method.The test chemical did not induce chromosome aberration inChinese hamster cultured cells (CHL) 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 aberration in Chinese hamster cultured cells (CHL/IU) 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
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 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:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
1. Thymidine kinase
2. HGPRT locus
Species / strain / cell type:
mouse lymphoma L5178Y 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 lung fibroblasts (V79)
Remarks:
2
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 fraction from phenobarbital/β-naphthoflavone induced rats was used as exogenous metabolic activation system
Test concentrations with justification for top dose:
1. Experiment I: 0, 1.6, 3.3, 6.5, 13.0 and 26.0 μg/ml without and with S9-mix
Experiment II: 0, 1.6, 3.3, 6.5, 13.0 and 26.0 μg/ml without S9-mix

2. No data
Vehicle / solvent:
1. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

2. No data
Untreated negative controls:
yes
Remarks:
As per OECD guideline
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: As per OECD guideline
Remarks:
1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
2
Details on test system and experimental conditions:
1. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: Experiment 1: 4 hrs
Experiment 2: 24 hrs
- Expression time (cells in growth medium):
Experiment 1: 72 hrs
Experiment 2: 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): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Toxicity was measured in the main experiments as percentage relative total growth of the treated cultures relative to the total growth of the solvent control cultures.

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

OTHER: No data

2. METHOD OF APPLICATION: in medium
Rationale for test conditions:
No data
Evaluation criteria:
The cell line was observed for gene mutation at the respective locus
Statistics:
No data
Species / strain:
mouse lymphoma L5178Y 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:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Remarks:
2
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
1. CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: Yes
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Test concentrations were based on the results of a pre-test on toxicity measuring relative suspension growth.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

2. No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce gene mutation in mammalian cell line 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. The studies are as 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 L5178Y tk+/- mouse lymphoma cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level 0, 1.6, 3.3, 6.5, 13.0 and 26.0 μg/ml in experiment 1 (with and without S9) and in experiment 2 (without S9). The doses for the main study were dependent on the dose range finding study conducted. At the end of the treatment at 13 and 26 μg/ml in all cultures of both main experiments precipitation was observed by the unaided eye. No substantial and reproducible increase of the mutant frequency was observed in both experiments. A minor increase exceeding the threshold of twice the mutant frequency of the corresponding solvent control was observed in the second culture of the second experiment. Since the historical range of negative and solvent controls was not exceeded, the effect occurred at precipitating concentrations and was not reproducible in the other culture it was considered an artificial effect due to the precipitation of the test item rather than indicating a possible mutagenic effect. Based on the observations made, thetest chemical did not induce gene mutation in L5178Y tk+/- mouse lymphoma 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 was performed to determine the mutagenic nature of the test chemical. The study was performed using V79 cells with and without co-cultivation with rat hepatocytes. A test is considered to be positive if a doubling of the background mutation frequency and/or a dose dependency is observed. Based on the observations made, thetest chemical did not induce gene mutation inV79 cells 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 gene mutation in mammalian cell line 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 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:  

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed by spot test using Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA1978 and TA98 with and without S9 metabolic activation system. 100-200 µg of the test material dissolved in 10-20µL DMSO/disc was applied to 6mm paper concentration discs and used for mutagenicity testing. From 1 to 4 discs were applied per plate. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did not induce mutation in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA1978 and TA98 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 performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA97, TA98, TA100 with and without PCB induced S9 metabolic activation system at dose levels of 0, 5, 10, 50 or 200µg/plate. The plates were incubated for 48 hrs and the number of dose dependent increase in the revertants was counted. The test chemical did not induce reversion of histidine gene mutation in Salmonella typhimurium strains TA97, TA98, TA100 both in the presence and absence of PCB induced rat liver S9 fraction and hence it is not likely to classify as a gene mutant in vitro.  

In vitro mammalian chromosome aberration study:  

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster cultured cells (CHL/IU) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in 0.5% CMC Na and used at dose level of 0, 0.3, 0.7, 1.3 µg/mL in continuous treatment method and 0, 06, 1.1, 2.2 µg/mL in short term treatment method. The doses for the main study were based on data from preliminary dose range finding study. Concurrent solvent and positive control plates were also included in the study. The cells were exposed to the test chemical for 24 or 48 hrs in the continuous treatment method and for 6 hrs in the short term treatment method. Two hours before the end of the culture, colcemid was added to the culture solution to a final concentration of about 0.1 μg / ml. Chromosome specimens were prepared according to a conventional method. Giemsa stained six slide specimens were prepared for each petri dish. The presence or absence of structural abnormality such as chromosome type or chromosome type gap, the presence or absence of cells (polyploid) was also observed.The test chemical did not induce chromosome aberration inChinese hamster cultured cells (CHL/IU) 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 in vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster cultured cells (CHL) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 19.1, 38.3, 76.6, 153, 306, 613, 1230, 2450µg/mL without S9 and 0, 38.3, 76.6, 153, 306, 613, 1230, 2450µg/mL with S9 in the short term treatment method and 0, 19.1, 38.3, 76.6, 153, 306, 613, 1230, 2450µg/mL in continuous treatment method. The doses for the main study were based on data from preliminary dose range finding study. Concurrent solvent and positive control plates were also included in the study. The cells were exposed to the test chemical for 24 or 48 hrs in the continuous treatment method and for 6 hrs in the short term treatment method.The test chemical did not induce chromosome aberration inChinese hamster cultured cells (CHL) 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 vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using L5178Y tk+/- mouse lymphoma cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level 0, 1.6, 3.3, 6.5, 13.0 and 26.0 μg/ml in experiment 1 (with and without S9) and in experiment 2 (without S9). The doses for the main study were dependent on the dose range finding study conducted. At the end of the treatment at 13 and 26 μg/ml in all cultures of both main experiments precipitation was observed by the unaided eye. No substantial and reproducible increase of the mutant frequency was observed in both experiments. A minor increase exceeding the threshold of twice the mutant frequency of the corresponding solvent control was observed in the second culture of the second experiment. Since the historical range of negative and solvent controls was not exceeded, the effect occurred at precipitating concentrations and was not reproducible in the other culture it was considered an artificial effect due to the precipitation of the test item rather than indicating a possible mutagenic effect. Based on the observations made, the test chemical did not induce gene mutation in L5178Y tk+/- mouse lymphoma 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 yet another study, In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using V79 cells with and without co-cultivation with rat hepatocytes. A test is considered to be positive if a doubling of the background mutation frequency and/or a dose dependency is observed. Based on the observations made, thetest chemical did not induce gene mutation inV79 cells and hence it is not likely to classify as a gene mutant in vitro.  

Based on the data available and applying the weight of evidence approach, the test chemical does not induce gene mutation in vitro. Hence the test chemical is not likely to be mutagenic 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 induce gene mutation in vitro. Hence the test chemical is not likely to be mutagenic as per the criteria mentioned in CLP regulation.