Silicic acid, ethyl ester

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:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

other: TA98, TA100, TA1535, TA1537, and TA1538

Remarks:

2

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Remarks:

3

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

2. No data
3. No data

Metabolic activation:

with and without

Metabolic activation system:

2. 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.
3. Metabolic activation system extracted from liver of Wistar rats, induced with Phenobarbital and beta-naphthoflavone.

Test concentrations with justification for top dose:

2. 100- 10000 µg/plate
3. 0, 50, 160, 500, 1600 and 5000 µg/plate

Vehicle / solvent:

2. - Vehicle(s)/solvent(s) used: Yes, no detailed data available
- Justification for choice of solvent/vehicle: No data available
3. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance is soluble in DMSO.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

not specified

Remarks:

2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-nitrofluorene

other: 2-aminoanthracene at 2.5 µg/plate

Remarks:

3

Details on test system and experimental conditions:

2. METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h
NUMBER OF REPLICATIONS: Triplicate
3. METHOD OF APPLICATION: A preincubation period test was followed by a plate incorporation test.
NUMBER OF REPLICATIONS: Triplicates

Rationale for test conditions:

2. No data
3. No data

Evaluation criteria:

2. The criteria used to evaluate a test were as follows: 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.
3. A doubling of the mean revertants per plate accompanied by a dose response constituted a positive response.

Statistics:

2. No data available
3. No data available

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538

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: TA 98, TA 100, TA 1535, and TA 1537

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 available
3. No data available

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:

The Ames salmonella typhimurium mutagenicity test was conducted for the given test chemical as per OECD Guideline 471 (Bacterial Reverse Mutation Assay) to evaluate its gene toxic effects when exposed to Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 in the presence and absence of S9 metabolic activation system Liver S9 homogenate, which was prepared from male Sprague-Dawley rats and Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight with dose concentration of 100-10000 µg/plate in plate incorporation assay. Based on the preliminary study conducted, the test compound was used at a five dose level from 100- 10000 µg/plate. 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. The given test chemical failed to induce mutation in the Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 both in the presence and absence of S9 activation system and hence is not likely to be a gene mutant.

In another study, the given test chemical was investigated for its ability to induce mutagenic activity when tested in an in vitro reverse mutagenicity test as per OECD Guideline 471 (Bacterial Reverse Mutation Assay). The test material was exposed to Salmonella typhimurium strains TA 98, TA 100, TA 1535, and TA 1537 in the presence and absence of metabolic activation S9 system which was extracted from liver of Wistar rats, induced, with Phenobarbital and beta-naphthoflavone at the concentrations of 0, 50, 160, 500, 1600 and 5000 µg/plate. DMSO was used as a solvent control. 2-aminoanthracene, 2.5 ug/plate, and 2-nitrofluorine, 2.5 micrograms/plate were used as positive controls. A preincubation period test was followed by a plate incorporation test. Three numbers of replicates were used. Evaluation was done by a doubling of the mean revertants per plate accompanied by a dose response constituted a positive response. Mutagenic effects were not observed in all strains, in the presence and absence of metabolic activation. From the above study conditions, the test chemical was considered to be non mutagenic in Salmonella typhimurium strains TA 98, TA 100, TA 1535, and TA 1537 by AMES test in the presence and absence of metabolic activation S9.

 

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:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

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:

Chinese hamster lung fibroblasts (V79)

Remarks:

5

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

6

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

5. No data
6. No data

Metabolic activation:

with and without

Metabolic activation system:

5. Metabolic activation system induced with phenonobarbital/B-Napthoflavone was used.
6. not specified

Test concentrations with justification for top dose:

5. 0, 50, 100, 200, 400, 600, 1000, 1800, 3000, 5000 µg/plate
6. 0, 125, 250, 500, 750, 1000, 1250, 1500, 2080 µg/plate

Vehicle / solvent:

5. - Vehicle(s)/solvent(s) used: MEM
- Justification for choice of solvent/vehicle: The test substance is soluble in MEM.
6. - Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The test substance is soluble in dimethylsulfoxide (DMSO).

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

MEM4 served as negative control without activation and MEMO plus S9 mix was used for the assay with metabolic activation

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:

dimethylsulfoxide (DMSO)

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Mitomycin without S9 activation system

Remarks:

6

Details on test system and experimental conditions:

5. METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 20 hours exposure
NUMBER OF REPLICATIONS: Duplicate
NUMBER OF CELLS EVALUATED: 2,000 cells (1,000 per slide)
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Nine concentrations of the test substance (50-5000 µg /ml) in the presence and absence of metabolic activation were used to determine cytotoxicity and to set doses for the chromosome aberration study.
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
6. NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 100 per duplicate flask
- Number of independent experiments: No data

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): CHO cells were seeded at approximately 5 x 105 cells/25 cm2 flask
- Test substance added - in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: No data
- Exposure duration/duration of treatment: 4 and 20 hours in the non activation system and 4 hours in the S9 activation system
- Harvest time after the end of treatment (sampling/recovery times): All the cells were
harvested at 20 hours after treatment initiation.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): No data
- Number of cells spread and analyzed per concentration (number of replicate cultures and total number of cells scored): 500 cells scored
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): No data
- Determination of polyploidy: No data
- Determination of endoreplication: No data

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method - mitotic index (MI): To ensure that a sufficient number of metaphase cells were present on the slides, the percentage of cells in mitosis per 500 cells scored (mitotic index) was determined for each treatment group. Metaphase cells with 20+/-2 centromeres were examined under oil immersion without prior knowledge of treatment groups.
- Any supplementary information relevant to cytotoxicity: No data
- OTHER: Initially, the non-activated and S9 activated 4 hour exposure groups were evaluated for chromosome aberrations and since a negative result was obtained in the non-activated 4 hour exposure group, the non-activated 20 hour continuous exposure group was then also evaluated for chromosome aberrations. A minimum of 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations.

Rationale for test conditions:

5. No data
6. Not specified

Evaluation criteria:

5. The test chemical is to be considered clastogenic in this assay if –
1. it induces chromosomal aberrations (excl. gaps) in statistically significant manner in one or more concentrations
2. The induced proportion of aberrant cells at such test substance concentrations exceeds the normal range of the test system (i.e.>>5%)
3. Positive results can be verified in an independent experiment.
6. The number and type of aberrations found the percentage of structurally and numerically damaged cells (% aberrant cells) in the total population examined and mean aberrations per cell were calculated for each group.

Statistics:

5. Chi-square test
6. Fisher's exact test was used to compare pair wise the percent aberrant cells of each treatment group with that of the solvent control. Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

5

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Only after 18 hours exposure without metabolic activation was cytotoxicity observed

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

6

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: With metabolic activation: 1250 ug/mL - 54%; Without metabolic activation: 1250 ug/mL - 52%

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. No data
6. RANGE-FINDING/SCREENING STUDIES: Selection of doses for microscopic analysis was based on toxicity (the lowest dose with at least 50% reduction in cell growth and the next two lower doses) in all harvests.

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 test was performed to evaluate the mutagenic nature of the test chemical as per OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test). The test material was exposed to V79 Chinese hamster lung cells in the presence and absence of metabolic activation S9 which was induced with phenonobarbital/B-Napthoflavone. The concentration of test material used were 0, 50, 100, 200, 400, 600, 1000, 1800, 3000, 5000 µg/plate. MEM is used as growth medium. Experiment was performed in duplicates. Per experimental point, at least 2,000 cells (1,000 per slide) were scored. MEM4 served as negative control without activation and MEMO plus S9 mix was used for the assay with metabolic activation. Positive controls were used as, Mitomycin C (MMC) without metabolic activation and Cyclophosphamide (CP) with metabolic activation. Nine concentrations of the test substance (50-5000 µg /ml) in the presence and absence of metabolic activation were used to determine cytotoxicity and to set doses for the chromosome aberration study. Without metabolic activation the cells treated with the test material revealed chromosomal aberrations frequencies excluding gaps of 0.0 to 2.0 % and with S9 mix was 1.0 to 3.5%. The frequency of polyploidy cells in both parts of the experiment was within the expected range (<10%) of historical controls. Under the condition of the test, the given test chemical did not induce statistically and biologically significant increases in the chromosomal aberration frequency of V79 Chinese hamster cells and is therefore judged to be not clastogenic in vitro.

 

In another study, the gene mutation study was conducted according to in vitro mammalian chromosome aberration test to determine the mutagenic nature of the given test chemical. The test material was exposed to Chinese hamster ovary cells in the presence and absence of metabolic activation S9. For the chromosome aberration assay, CHO cells were seeded at approximately 5 x 105 cells/25 cm2 flask and were incubated at 37+/- 1C in a humidified atmosphere of 5+/-1% CO2 in air for 16-24 hours. The concentration of test material used were 0, 125, 250, 500, 750, 1000, 1250, 1500, 2080 µg/plate. The solvent and negative control for all test article dose levels was dimethylsulfoxide (DMSO). Mitomycin was dissolved in water and used as the positive control in the non activated system. Cyclophosphamide was dissolved in water and used as the positive control in the S9 activation system. Selection of doses for microscopic analysis was based on toxicity (the lowest dose with at least 50% reduction in cell growth and the next two lower doses) in all harvests. Duration of treatment was 4 and 20 hours in the non activation system and 4 hours in the S9 activation system.All the cells were harvested at 20 hours after treatment initiation. To ensure that a sufficient number of metaphase cells were present on the slides, the percentage of cells in mitosis per 500 cells scored (mitotic index) was determined for each treatment group. Metaphase cells with 20+/-2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Initially, the non-activated and S9 activated 4 hour exposure groups were evaluated for chromosome aberrations and since a negative result was obtained in the non-activated 4 hour exposure group, the non-activated 20 hour continuous exposure group was then also evaluated for chromosome aberrations. A minimum of 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations. The number and type of aberrations found the percentage of structurally and numerically damaged cells (% aberrant cells) in the total population examined and mean aberrations per cell were calculated for each group.Fisher's exact test was used to compare pair wise the percent aberrant cells of each treatment group with that of the solvent control. Cytotoxicity was scored with metabolic activation at 1250µg/mL was 54% and without metabolic activation at 1250 µg/mL was 52%. Under the condition of the study, the given test chemical did not induce mutation structurally and numerically by in vitro mammalian chromosome aberration test in Chinese hamster ovary cells and hence is not likely to be mutagenic under the conditions of this study.

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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

Ames assay:

The Ames salmonella typhimurium mutagenicity test was conducted for the given test chemical to evaluate its gene toxic effects when exposed to Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 in the presence and absence of S9 metabolic activation system Liver S9 homogenate, which was prepared from male Sprague-Dawley rats and Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight with dose concentration of 100-10000 µg/plate in plate incorporation assay. Based on the preliminary study conducted, the test compound was used at a five dose level from 100- 10000 µg/plate. 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. The given test chemical failed to induce mutation in the Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 both in the presence and absence of S9 activation system and hence is not likely to be a gene mutant.

In another study, the given test chemical was investigated for its ability to induce mutagenic activity when tested in an in vitro reverse mutagenicity test as per OECD Guideline 471 (Bacterial Reverse Mutation Assay). The test material was exposed to Salmonella typhimurium strains TA 98, TA 100, TA 1535, and TA 1537 in the presence and absence of metabolic activation S9 system which was extracted from liver of Wistar rats, induced, with Phenobarbital and beta-naphthoflavone at the concentrations of 0, 50, 160, 500, 1600 and 5000 µg/plate. DMSO was used as a solvent control. 2-aminoanthracene, 2.5 ug/plate, and 2-nitrofluorine, 2.5 micrograms/plate were used as positive controls. A preincubation period test was followed by a plate incorporation test. Three numbers of replicates were used. Evaluation was done by a doubling of the mean revertants per plate accompanied by a dose response constituted a positive response. Mutagenic effects were not observed in all strains, in the presence and absence of metabolic activation. From the above study conditions, the test chemical was considered to be non mutagenic in Salmonella typhimurium strains TA 98, TA 100, TA 1535, and TA 1537 by AMES test in the presence and absence of metabolic activation S9.

In vitro mammalian chromosome aberration study:

In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical as per OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test). The test material was exposed to V79 Chinese hamster lung cells in the presence and absence of metabolic activation S9 which was induced with phenonobarbital/B-Napthoflavone. The concentration of test material used were 0, 50, 100, 200, 400, 600, 1000, 1800, 3000, 5000 µg/plate. MEM is used as growth medium. Experiment was performed in duplicates. Per experimental point, at least 2,000 cells (1,000 per slide) were scored. MEM4 served as negative control without activation and MEMO plus S9 mix was used for the assay with metabolic activation. Positive controls were used as, Mitomycin C (MMC) without metabolic activation and Cyclophosphamide (CP) with metabolic activation. Nine concentrations of the test substance (50-5000 µg /ml) in the presence and absence of metabolic activation were used to determine cytotoxicity and to set doses for the chromosome aberration study. Without metabolic activation the cells treated with the test material revealed chromosomal aberrations frequencies excluding gaps of 0.0 to 2.0 % and with S9 mix was 1.0 to 3.5%. The frequency of polyploidy cells in both parts of the experiment was within the expected range (<10%) of historical controls. Under the condition of the test, the given test chemical did not induce statistically and biologically significant increases in the chromosomal aberration frequency of V79 Chinese hamster cells and is therefore judged to be not clastogenic in vitro.

 

In another study, the gene mutation study was conducted according to in vitro mammalian chromosome aberration test to determine the mutagenic nature of the given test chemical. The test material was exposed to Chinese hamster ovary cells in the presence and absence of metabolic activation S9. For the chromosome aberration assay, CHO cells were seeded at approximately 5 x 105 cells/25 cm2 flask and were incubated at 37+/- 1C in a humidified atmosphere of 5+/-1% CO2 in air for 16-24 hours. The concentration of test material used were 0, 125, 250, 500, 750, 1000, 1250, 1500, 2080 µg/plate. The solvent and negative control for all test article dose levels was dimethylsulfoxide (DMSO). Mitomycin was dissolved in water and used as the positive control in the non activated system. Cyclophosphamide was dissolved in water and used as the positive control in the S9 activation system. Selection of doses for microscopic analysis was based on toxicity (the lowest dose with at least 50% reduction in cell growth and the next two lower doses) in all harvests. Duration of treatment was 4 and 20 hours in the non activation system and 4 hours in the S9 activation system.All the cells were harvested at 20 hours after treatment initiation. To ensure that a sufficient number of metaphase cells were present on the slides, the percentage of cells in mitosis per 500 cells scored (mitotic index) was determined for each treatment group. Metaphase cells with 20+/-2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Initially, the non-activated and S9 activated 4 hour exposure groups were evaluated for chromosome aberrations and since a negative result was obtained in the non-activated 4 hour exposure group, the non-activated 20 hour continuous exposure group was then also evaluated for chromosome aberrations. A minimum of 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations. The number and type of aberrations found the percentage of structurally and numerically damaged cells (% aberrant cells) in the total population examined and mean aberrations per cell were calculated for each group.Fisher's exact test was used to compare pair wise the percent aberrant cells of each treatment group with that of the solvent control. Cytotoxicity was scored with metabolic activation at 1250µg/mL was 54% and without metabolic activation at 1250 µg/mL was 52%. Under the condition of the study, the given test chemical did not induce mutation structurally and numerically by in vitro mammalian chromosome aberration test in Chinese hamster ovary cells and hence is not likely to be mutagenic under the conditions of this study.

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 and In vitro mammalian chromosome aberration study. Hence, the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

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