Reaction products of diazotised sodium 2-amino-5-{[2-(sulfonatooxy)ethyl]sulfonyl}benzenesulfonate coupled with sodium 2-(4-aminobenzene-1-sulfonyl)ethyl sulfate, subsequently coupled with diazotised sodium 2-(4-aminobenzene-1-sulfonyl)ethyl sulfate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

Food black 2 (FB2) did not induce reversion of mutation when applied to Salmonella typhimurium strains 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:

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.

 

In vitro gene mutation study in mammalian cells:

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

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Remarks:

read across data

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Salmonella/microsome assay was performed to evaluate the mutagenic response for the test chemical Food black 2 (FB2)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material: Food black 2 (FB2)
- IUPAC name: tetrasodium (3E)-6-amino-4-oxo-3-(2-{7-sulfinato-4-[(E)-2-(4-sulfonatophenyl)diazen-1-yl] naphthalen-1-yl}hydrazin-1-ylidene)-3,4- dihydronaphthalene-2,7-disulfonate
- Molecular formula: C26H19N5O13S4.4Na
- Molecular weight: 809.651 g/mol
- Substance type: Organic
- Physical state: No data available
- Purity: 85%
- Impurities (identity and concentrations): 15%

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA1538 and TA98

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

The S9 liver homogenate was prepared from rats treated with Aroclor 1254 5 days prior to S9 preparation.

Test concentrations with justification for top dose:

Doses upto 5 mg/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

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: No data
- Expression time (cells in growth medium): No data
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available

SPINDLE INHIBITOR (cytogenetic assays): No data available

STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data available

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

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available

- Determination of endoreplication: No data available

- Other: No data available

OTHER: No data available

Rationale for test conditions:

No data

Evaluation criteria:

A dose-related increase (at least 2-fold) in revertant colonies was used to define a significant mutagenic response.

Statistics:

No data available

Species / strain:

S. typhimurium, other: TA1538 and TA98

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:

not specified

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Conclusions:

Food black 2 (FB2) did not induce reversion of mutation when applied to Salmonella typhimurium strains 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.

Executive summary:

Salmonella/microsome assay was performed to evaluate the mutagenic response for the read across substance tetrasodium (3E)-6 -amino-4 -oxo-3 -(2 -{7 -sulfinato-4 -[(E)-2 -(4 -sulfonatophenyl)diazen-1 -yl]naphthalen-1 -yl}hydrazin-1 -ylidene)-3,4 -dihydronaphthalene-2,7 -disulfonate (CAS no. 2118 -39 -0, E.C. no. 218 -326 -9). The test was performed using Salmonella typhimurium strainsTA1538 and TA98 in the presence and absence of S9 metabolic activation system upto a dose level of 5 mg/plate. The test chemical was dissolved in DMSO and a dose-related increase (at least 2 -fold) in revertant colonies was used to define a significant mutagenic response. Food black 2 (FB2)did not induce reversion of mutation when applied to Salmonella typhimurium strainsTA1538 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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Remarks:

Read across data

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Principles of method if other than guideline:

Equivalent or similar to OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-WBL

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 fractions (livers of Aroclor 1254-treated male Sprague-Dawley rats)

Test concentrations with justification for top dose:

0, 2500, 3850 or 5000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Medium
- Justification for choice of solvent/vehicle: The test chemical was dissolved in medium

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Medium

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): No data

DURATION
- Preincubation period: No data
- Exposure duration: Without S9: 8 hrs
With S9: 2 hrs
- Expression time (cells in growth medium): 20 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):

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa stain

NUMBER OF REPLICATIONS: No data

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No data

NUMBER OF CELLS EVALUATED: 100-200 cells from each of the three highest scorable doses were analyzed

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: Yes, endoreduplication was recorded
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: All aberrations were individually classified (e.g., chromatid breaks, chromosome breaks, triradials, etc.). These data were combined as the percent of cells with simple (deletions), complex (exchanges), and total (simple, complex and other) aberrations. Only the total percent cells with aberrations was considered in the statistical evaluation

Rationale for test conditions:

No data

Evaluation criteria:

The cell line was observed for chromatid breaks, chromosome breaks, triradials with simple (deletions), complex (exchanges), and total (simple, complex and other) aberrations.

Statistics:

A significant increase in the aberration assay was based on a binomial sampling assumption; the P values were adjusted according to Dunnett’s method to take into account multiple dose comparisons. The trend test used a linear regression analysis: the percentage of cells with aberrations vs. the log dose. Trials with two or more significant doses were considered positive (+), and trials with one significant dose and a significant trend were judged as having weak evidence of a positive response (+ W). The evaluation scheme used places emphasis on the number of doses that were positive rather than on the magnitude of the response or its progression with dose; all calls were based upon this scheme. Trials with a significant response at one dose and no significant trend, and trials with no significant responses but having a significant trend were considered equivocal (?).

If a positive or a weak-positive response was observed both with and without activation, generally only the trial without activation was repeated. When only one activation condition was positive or weak-positive, only that condition was repeated. If a weak-positive response was not repeatable, the chemical was generally concluded to be negative for that activation condition. When a positive response was not repeatable under conditions similar to those used in the original test, the chemical was concluded to be equivocal (?) for that activation condition. The terms positive and weak-positive were used to indicate the level of evidence for a significant response, and + W is considered positive in all evaluations of test performance. Equivocals were considered negative in all evaluations.

Species / strain:

Chinese hamster Ovary (CHO)

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: In the assays for chromosomal aberrations, the top dose (TD) was based on toxicity, solubility, or the upper testing limit (5 mg/ml).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

Mutagenic effects: Without S9

Dose (µg/mL)	Cells	Percent Cells with Aberrations
		Total	Simple	Complex
0	200	2.0	0.0	2.0
2500	200	2.0	1.0	1.0
3850	200	2.0	1.0	1.0
5000	200	2.0	1.0	1.0
Positive control
0.5	200	12.0	8.0	7.0
1.0	25	40.0	20.0	28.0

 

 Mutagenic effects: With S9

Dose (µg/mL)	Cells	Percent Cells with Aberrations
		Total	Simple	Complex
0	200	2.0	1.0	1.0
2500	200	3.0	1.0	2.0
3850	200	5.0	2.0	2.0
5000	200	4.0	1.0	2.0
Positive control
7.5	200	9.0	4.0	4.0
37.5	25	36.0	24.0	12.0

Conclusions:

Based on all the available data, it was concluded that the test chemical did not induce chromosome aberrations in Chinese hamster ovary cells (CHO) 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:

Chromosomal aberration tests were carried out to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in medium and used at dose levels of up to 5 mg/mL. In the assays for chromosomal aberrations, the top dose (TD) was based on toxicity, solubility, or the upper testing limit (5 mg/ml). In the chromosome aberration assay without activation, cells were exposed to the test chemical for 8 hr. The test chemical was washed off, and the cells were treated with 0.1 µg/ml Colcemid for 2-2.5 hr. With metabolic activation, the cells were exposed to the test chemical plus the metabolic activation mixture for 2 hr, washed, incubated for 8 hr, and then treated with Colcemid for 2-2.5 hr. A delayed harvest was used in the aberration assay. In these tests the cell growth period was extended to about 20 hr. Air-dried slides were coded and stained with Giemsa. One hundred to 200 cells from each of the three highest scorable doses were analyzed. All aberrations were individually classified (e.g., chromatid breaks, chromosome breaks, triradials, etc.). These data were combined as the percent of cells with simple (deletions), complex (exchanges), and total (simple, complex and other) aberrations. The test chemical did not induce chromosome aberrations in Chinese hamster ovary cells (CHO) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Remarks:

Read across data

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from safety assessment reports

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

The test chemical was assayed for gene mutations at the tk locus of mouse lymphoma cells both in the absence and presence of S9 metabolic activation

GLP compliance:

not specified

Type of assay:

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

Target gene:

tk locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Liver S9 fraction from Syrian golden hamsters was used as exogenous metabolic activation system.

Test concentrations with justification for top dose:

300 - 4800 μg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)] : Deionised water

Untreated negative controls:

yes

Remarks:

Negative controls were in accordance with the OECD guideline.

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Positive controls were in accordance with the OECD guideline.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : duplicate
- Number of independent experiments : 2 independent experiments

TREATMENT AND HARVEST SCHEDULE:

- Exposure duration/duration of treatment: cells were treated for 4 h (experiment I) or 24 h (experiment II)
- Harvest time after the end of treatment (sampling/recovery times):

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): expression period of 72 h

Evaluation criteria:

Toxicity was measured as percentage relative survival and total growth of the treated cultures relative to the survival of the solvent control cultures.

Statistics:

No data available.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Precipitation of the test chemical was not observed

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation: No precipitation was observed.
- Other confounding effects: The ratio of small colonies versus large colonies was not shifted as compared to the solvent controls.

RANGE-FINDING/SCREENING STUDIES: Test concentrations were based on the results of a pre-test measuring relative suspension growth. Since no toxic effects were observed in the pre-test, the test chemical was tested up to the prescribed maximum concentration of 10 mM (= 4800 μg/ml).

ADDITIONAL INFORMATION ON CYTOTOXICITY: Precipitation of the test chemical was not observed.

- Genotoxicity results: In both experiments in the absence and presence of S9 mix the appropriate level of toxicity (10-20% survival after the highest dose) was not reached.
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency : No reproducible or dose dependent increase in mutant colony number was observed in both experiments neither in the absence nor in the presence of S9 mix. The ratio of small colonies versus large colonies was not shifted as compared to the solvent controls.

Remarks on result:

other: not mutagenic

Conclusions:

No reproducible or dose dependent increase in mutant colony number was observed in both experiments neither in the absence nor in the presence of S9 mix. The ratio of small colonies versus large colonies was not shifted as compared to the solvent controls.Under the experimental conditions used, the test chemical was not genotoxic (mutagenic and/or clastogenic) in the mouse lymphoma assay at the tk locus.

Executive summary:

The test chemical was assayed for gene mutations at the tk locus of mouse lymphoma cells both in the absence and presence of S9 metabolic activation. The study was performed according to OECD 476 Guidelines. Test concentrations were based on the results of a pre-test measuring relative suspension growth. The study was performed in duplicates in 2 independent experiments. The test concentrations used were- Experiment I: 300 - 4800 μg/ml both without and with S9 mix; Experiment II: 150 - 4800 μg/ml without S9 mix. Liver S9 fraction from Syrian golden hamsters was used as exogenous metabolic activation system.Cells were treated for 4 h (experiment I) or 24 h (experiment II) followed by an expression period of 72 h to fix the DNA damage into a stable tk mutation. Toxicity was measured as percentage relative survival and total growth of the treated cultures relative to the survival of the solvent control cultures. Negative and positive controls were in accordance with the OECD Guideline. Precipitation of the test chemical was not observed. In both experiments in the absence and presence of S9 mix the appropriate level of toxicity (10-20% survival after the highest dose) was not reached.No reproducible or dose dependent increase in mutant colony number was observed in both experiments neither in the absence nor in the presence of S9 mix. The ratio of small colonies versus large colonies was not shifted as compared to the solvent controls. Under the experimental conditions used, the test chemical was not genotoxic (mutagenic and/or clastogenic) in the mouse lymphoma assay at the tk locus.

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 were reviewed to determine the mutagenic nature of the given test chemical. The studies are as mentioned below:

 

Ames assay:

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

 

Study 1:

Salmonella/microsome assay was performed to evaluate the mutagenic response for the read across substance tetrasodium (3E)-6 -amino-4 -oxo-3 -(2 -{7 -sulfinato-4 -[(E)-2 -(4 -sulfonatophenyl)diazen-1 -yl]naphthalen-1 -yl}hydrazin-1 -ylidene)-3,4 -dihydronaphthalene-2,7 -disulfonate (CAS no. 2118 -39 -0, E.C. no. 218 -326 -9). The test was performed using Salmonella typhimurium strainsTA1538 and TA98 in the presence and absence of S9 metabolic activation system upto a dose level of 5 mg/plate. The test chemical was dissolved in DMSO and a dose-related increase (at least 2 -fold) in revertant colonies was used to define a significant mutagenic response. Food black 2 (FB2)did not induce reversion of mutation when applied to Salmonella typhimurium strainsTA1538 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.

 Study 2 :

An in vitro bacterial gene mutation assay was performed to access the mutagenic potential of the read across substance C.I. Brilliant black (food black 1) (CAS no.: 2519-30-4). The test substance was tested by plate incorporation method using Salmonella thphimurium strains TA98, TA100, TA1535, TA1537 and TA1538. The test substance was used at dosage levels of the range 10-10000 µg /plate with and without metabolic activation. The test chemicals was exposed to the tester strains for a period of 48 hours. There was no increase in the number of revertant colonies observed at any of the dosage group. Hence, the test substance can be considered to be non-mutagenic.

 

 

In vitro mammalian chromosome aberration study:

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

 

Study 1:

Chromosomal aberration tests were carried out to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in medium and used at dose levels of up to 5 mg/mL. In the assays for chromosomal aberrations, the top dose (TD) was based on toxicity, solubility, or the upper testing limit (5 mg/ml). In the chromosome aberration assay without activation, cells were exposed to the test chemical for 8 hr. The test chemical was washed off, and the cells were treated with 0.1 µg/ml Colcemid for 2-2.5 hr. With metabolic activation, the cells were exposed to the test chemical plus the metabolic activation mixture for 2 hr, washed, incubated for 8 hr, and then treated with Colcemid for 2-2.5 hr. A delayed harvest was used in the aberration assay. In these tests the cell growth period was extended to about 20 hr. Air-dried slides were coded and stained with Giemsa. One hundred to 200 cells from each of the three highest scorable doses were analyzed. All aberrations were individually classified (e.g., chromatid breaks, chromosome breaks, triradials, etc.). These data were combined as the percent of cells with simple (deletions), complex (exchanges), and total (simple, complex and other) aberrations. The test chemical did not induce chromosome aberrations in Chinese hamster ovary cells (CHO) 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 2:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of test chemical. The study was performed using Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in serum free culture medium and used at dose level of 0, 1000, 1600, 3000 or 5000 µg/plate. Concurrent solvent and negative control chemicals were also included in the study. Approximately 24 hr prior to cell treatment, 1.2 x 10⁶cells were seeded per 75 cm² flask. For assays without metabolic activation, the cells were treated for about 10 hr. Colcemid was added 2-3 hr prior to cell harvest by mitotic shake-off. In the test protocol for assays with metabolic activation cells were harvested approximately 11 hr after removal of the S9 fraction. Colcemid was added 2 hr prior to harvest. Slides were stained in 6% Giemsa for 5-10 min. One hundred cells were scored for each dose in early studies and 200 cells per dose in later studies. All slides except high-dose positive controls were coded. Only metaphase cells in which the chromosome number was between 19 and 23 were scored. The test chemical did not induce aberrations at doses up to 5000µg/ml in either the presence or the absence of S9. Based on the observations made, the test chemical did not induce chromosome aberration in Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

 

In vitro gene mutation study in mammalian cells:

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

 

Study 1:

The test chemical was assayed for gene mutations at the tk locus of mouse lymphoma cells both in the absence and presence of S9 metabolic activation. The study was performed according to OECD 476 Guidelines. Test concentrations were based on the results of a pre-test measuring relative suspension growth. The study was performed in duplicates in 2 independent experiments. The test concentrations used were- Experiment I: 300 - 4800 μg/ml both without and with S9 mix; Experiment II: 150 - 4800 μg/ml without S9 mix. Liver S9 fraction from Syrian golden hamsters was used as exogenous metabolic activation system.Cells were treated for 4 h (experiment I) or 24 h (experiment II) followed by an expression period of 72 h to fix the DNA damage into a stable tk mutation. Toxicity was measured as percentage relative survival and total growth of the treated cultures relative to the survival of the solvent control cultures. Negative and positive controls were in accordance with the OECD Guideline. Precipitation of the test chemical was not observed. In both experiments in the absence and presence of S9 mix the appropriate level of toxicity (10-20% survival after the highest dose) was not reached.No reproducible or dose dependent increase in mutant colony number was observed in both experiments neither in the absence nor in the presence of S9 mix. The ratio of small colonies versus large colonies was not shifted as compared to the solvent controls. Under the experimental conditions used, the test chemical was not genotoxic (mutagenic and/or clastogenic) in the mouse lymphoma assay at the tk locus.

 

Study 2:

A L5178Y TK +/- mouse lymphoma assay was conducted to determine the mutagenic potential of the test chemical. L5178Y TK +/- mouse lymphoma cells were originally obtained from Dr. Donald Clive, Burroughs Wellcome Company, Research Triangle Park, NC. The cells were grown in Fischer's medium for leukemic cells of mice supplemented with 10% horse serum, antibiotics (50 IU penicillin/mi and 50 /ug streptomycin/ml), and 0.02% Pluronic F-68. All serum lots were pre-screened for their ability to support optimal growth. The cells were checked for the presence of mycoplasma by agar block isolation and Hoechst staining before and after cryopreservation. The toxicity of each chemical was first determined both with and without S9 prepared from Aroclor-1254-induced male Fischer 344 rats. S9 mix was prepared according to the procedure of Clive et al. (1979). Cells at a concentration of 6 × 105/ml were exposed for 4 h to a range of concentrations from 0.0 to 10000 microgram/ml or the limit of solubility. The cells were then washed, resuspended in growth medium, and incubated at 37 °C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity. For each assay there was a solvent control, a positive control for the test without metabolic activation and for the test with metabolic activation. The maximum solvent concentration was 1% for organic solvents and 10% for water. These levels had no effects on cell growth or spontaneous mutation frequency. Cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37 ° C; washed twice with growth medium; and maintained at 37 °C for 48 h in log phase growth to allow recovery and mutant expression. The cultures were adjusted to 0.3 × 106 cells/ml at 24-h intervals. They were then cloned in soft agar medium containing Fischer's medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% Pluronic F-68 and 0.35% Noble agar. Resistance to trifluorothymidine (TFT) was determined by adding 3 ug/ml TFT to one set of plates. The 100 × stock solution of TFT in saline was stored at -70°C and thawed immediately before use. Plates were incubated at 37°C in 5% CO 2 in air for 12 days, and then counted with an automatic colony counter. Mutant frequencies were expressed as mutants per 104 surviving cells. In general, a response was considered positive if there was a dose-related increase in the mutant frequency above the spontaneous control frequency, with a 2-fold increase at more than 1 dose and relative total growth greater than 10%.Based on all the available data, it was concluded that the results in all the assays with test chemical were negative. Hence, the test chemical can be considered to be non mutagenic to L5178Y TK +/- mouse lymphoma cells.

 

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 and Escherichia coli 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.

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