9,10-Anthracenedione, 1,4-diamino-, N,N'-mixed 2-ethylhexyl and hexyl and octyl derivs.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

In vitro mammalian chromosome aberration study:

The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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 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: As mentioned below

Principles of method if other than guideline:

WoE for the target CAS is summarized based on data from various test chemicals.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

2. Histidine
3. Histidine

Species / strain / cell type:

S. typhimurium, other: TA1535, TA100, TA1538 and TA98

Remarks:

2

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium, other: TA1535, TA100, TA1538 and TA98

Remarks:

3

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

2. No data
3. No data

Metabolic activation:

with and without

Metabolic activation system:

2. S9 mix was prepared from liver homogenate of rats treated with Aroclor 1254 plus adequate cofactors
3. S9 mix was prepared from liver homogenate of rats treated with Aroclor 1254 plus adequate cofactors

Test concentrations with justification for top dose:

2. 100, 500 or 1000 µg/plate
3. 100, 500 or 1000 µg/plate

Vehicle / solvent:

2. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
3. - 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

methylmethanesulfonate

other: Hycanthone (TA1538 and TA98)

Remarks:

2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

methylmethanesulfonate

other: Hycanthone (TA1538 and TA98)

Remarks:

3

Details on test system and experimental conditions:

2. METHOD OF APPLICATION: soft-agar overlay method

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 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: 4 experiments were performed

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
3. METHOD OF APPLICATION: soft-agar overlay method

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 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: 4 experiments were performed

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:

2. No data
3. No data

Evaluation criteria:

2. The plates were observed for number of revertants/plate
3. The plates were observed for number of revertants/plate

Statistics:

2. No data
3. No data

Species / strain:

S. typhimurium, other: TA1535, TA100, TA1538, and TA98

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

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA1535, TA100, TA1538, and TA98

Remarks:

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

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

2. No data
3. No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

Executive summary:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

Bacterial gene mutation test was performed to evaluate the mutagenic response for the given test chemical The test was performed using Salmonella typhimurium strainsTA1535, TA100, TA1538, and TA98 in the presence and absence of S9 metabolic activation system. The test compound was dissolved in DMSO and used at dose levels of 100, 500 or 1000µg/plate. Concurrent positive control chemicals were also included in the study. The given test chemical did not induce reversion of mutation when applied to Salmonella typhimurium strains TA1535, TA100, TA1538, 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.

 

In another study, the bacterial gene mutation test was performed to evaluate the mutagenic response for the given test chemical. The test was performed using Salmonella typhimurium strainsTA1535, TA100, TA1538, and TA98 in the presence and absence of S9 metabolic activation system. The test compound was dissolved in DMSO and used at dose levels of 100, 500 or 1000µg/plate. Concurrent positive control chemicals were also included in the study. The given test chemical did not induce reversion of mutation when applied to Salmonella typhimurium strainsTA1535, TA100, TA1538, 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.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on the 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: As mentioned below

Principles of method if other than guideline:

WoE for the target CAS is summarized based on data from various test chemicals.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

5. No data
6. No data

Species / strain / cell type:

mammalian cell line, other: Chinese hamster cultured cells (CHL/IU)

Remarks:

5

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:

6

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

5. No data
6. No data

Metabolic activation:

with and without

Metabolic activation system:

5. S9 mix
6. S9 mix

Test concentrations with justification for top dose:

5. Continuous treatment: 0, 0.3, 0.7, 1.3 µg/mL
Short term treatment method: 0, 06, 1.1, 2.2 µg/mL
6. 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:

5. - Vehicle(s)/solvent(s) used: 0.5% CMC Na
- Justification for choice of solvent/vehicle: The test chemical was soluble in CMC Na
6. - 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:

5

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:

6

Details on test system and experimental conditions:

5. 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
6. 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:

5. No data
6. No data

Evaluation criteria:

5. 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.
6. 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:

5. 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.
6. No data

Species / strain:

mammalian cell line, other: Chinese hamster cultured cells (CHL/IU)

Remarks:

5

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

mammalian cell line, other: Chinese hamster cultured cells (CHL/IU)

Remarks:

6

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

5. 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
6. No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

Executive summary:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using 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, and 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 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 another study, In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster 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, 2450 µg/mL without S9 and 0, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/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 in Chinese 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.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

Reference 3

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 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:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

8. Thymidine kinase
9. HGPRT locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

8

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:

9

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:

8. 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

9. No data

Vehicle / solvent:

8. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

9. 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:

8

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:

9

Details on test system and experimental conditions:

8. 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

9. 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:

8

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:

9

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:

8. 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

9. No data

Remarks on result:

other: No mutagenic potential

Conclusions:

Based on the observations and results from the available data, 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:

Study 8:

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.

Study 9:

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

Gene mutation in vitro:

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

Ames assay:

 

Bacterial gene mutation test was performed to evaluate the mutagenic response for the given test chemical The test was performed using Salmonella typhimurium strainsTA1535, TA100, TA1538, and TA98 in the presence and absence of S9 metabolic activation system. The test compound was dissolved in DMSO and used at dose levels of 100, 500 or 1000µg/plate. Concurrent positive control chemicals were also included in the study. The given test chemical did not induce reversion of mutation when applied to Salmonella typhimurium strains TA1535, TA100, TA1538, 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.

 

In another study, the bacterial gene mutation test was performed to evaluate the mutagenic response for the given test chemical. The test was performed using Salmonella typhimurium strainsTA1535, TA100, TA1538, and TA98 in the presence and absence of S9 metabolic activation system. The test compound was dissolved in DMSO and used at dose levels of 100, 500 or 1000µg/plate. Concurrent positive control chemicals were also included in the study. The given test chemical did not induce reversion of mutation when applied to Salmonella typhimurium strainsTA1535, TA100, TA1538, 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.

 

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, and 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 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 another study, In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster 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, 2450 µg/mL without S9 and 0, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/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 in Chinese 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 study:

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 L5178Ytk+/-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 L5178Ytk+/-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 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 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.

Justification for classification or non-classification

Based on the data available and applying weight of evidence approach, the given test chemical does not exhibit gene mutation in vitro by Ames assay, In vitro mammalian chromosome aberration study and in IN-vitro mammalian cell gene mutation study. Hence, the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.