Calcium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity In-vitro

AMES assay;

Various experimental studies were reviewed to determine the mutagenic nature of target substance calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate (5281-04-9) Common name; D and C red no. 7 or C.I. 15850:1. The studies are as mentioned below:

Gene mutation toxicity study was observed by National Institute of Technology and Evaluation (J check,2018) to determine the mutagenic nature of target substance calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate (5281-04-9) using Salmonella typhimurium strains. Genetic toxicity in vitro study was assessed for Calcium salt of 3-hydroxy-4 - [(4-methyl-2-sulfophenyl) azo] -2-naphthalenecarboxylic acid (5281-04-9). For this purpose AMES test was performed according to Guidelines for Screening Mutagenicity Testing of Chemicals (Japan).The test material was exposed to Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were 0, 312.5, 625, 1250, 2500, 5000 µg/plate. No mutagenic effects were observed in all strains, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA by AMES test. Hence the substance cannot be classified as gene mutant in vitro.

Chromosomal abbreviation;

Gene mutation toxicity study was observed by National Institute of Technology and Evaluation (J check,2018) to determine the mutagenic nature of target substance calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate (5281-04-9) using Mammalian cell line. This test was considered as confirmatory test for mutagenic potential. Hence the test was performed to conform its mutagenic nature. Genetic toxicity in vitro study was assessed for Calcium salt of 3-hydroxy-4 - [(4-methyl-2-sulfophenyl) azo] -2-naphthalenecarboxylic acid (5281-04-9). For this purpose chromosomal aberration test was performed according to Guidelines for Screening Mutagenicity Testing of Chemicals (Japan).The test material was exposed to Chinese hamster CHL/IU cells in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were mention as fallow

-S9 (continuous treatment): 0, 0.09, 0.19, 0.37 mg/ml

-S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml

+S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml

 No significant statistical mutagenic effects were observed in chromosome, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non mutagenic in Chinese hamster CHL/IU cells by chromosomal aberration test. Hence the substance cannot be classified as 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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Data is from publication.

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Principles of method if other than guideline:

To evaluate the mutagenic potential of test chemical in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA by AMES test.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-Naphthalenecarboxylic acid, 3-hydroxy-4-[(4-methyl-2-sulfophenyl)azo]-, calcium salt- Molecular formula (if other than submission substance): C18H14N2O6S.Ca- Molecular weight (if other than submission substance): 424.445 g/mole- Smiles notation (if other than submission substance): c12c(c(c(C(=O)[O-])cc1cccc2)O)\N=N\c1c(cc(C)cc1)S(=O)(=O)[O-].[Ca+2]- InChl (if other than submission substance): 1S/C18H14N2O6S.Ca/c1-10-6-7-14(15(8-10)27(24,25)26)19-20-16-12-5-3-2-4-11(12)9-13(17(16)21)18(22)23;/h2-9,21H,1H3, (H,22,23)(H,24,25,26);/q;+2/p-2/b20-19+;- Substance type: Organic - Physical state: Solid

Target gene:

Histidine for Salmonella typhimurium and tryptophan Escherichia coli

Species / strain / cell type:

S. typhimurium, other: TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA

Details on mammalian cell type (if applicable):

Not specified

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

0, 312.5, 625, 1250, 2500, 5000µg/plate

Vehicle / solvent:

Vehicle- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: -S9, AF-2 (TA100, WP2, TA98), sodium azide (TA1535) and 9- aminoacridine (TA1537) +S9, 2-aminoanthracene (all strains), trypane blue (TA100, TA98, azo reduction method)

Details on test system and experimental conditions:

Details on test system and conditionsAzo reduction methodMETHOD OF APPLICATION: Preincubation method (including azo dye method)DURATION- Preincubation period: 20 minutes - Exposure duration: 48 hoursNUMBER OF REPLICATIONS: Duplicate OTHER EXAMINATIONS: 3 plates per test were observed.

Rationale for test conditions:

Direct methodMETHOD OF APPLICATION: Preincubation method DURATION- Exposure duration: 48 hoursNUMBER OF REPLICATIONS: Duplicate OTHER EXAMINATIONS: 3 plates per test were observed.

Evaluation criteria:

Among the five test bacteria used, in the direct method or metabolic activation method of one or more test bacteria, the number of revertive mutant colonies on the flat plate containing the test substance is more than twice that of the negative control , And when the increase was found to be reproducible or dose-dependent, it was decided that the test substance had mutagenicity (positive) in this test system.

Statistics:

Yes, Mean ±standard deviation was observed.

Species / strain:

S. typhimurium, other: TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Additional information on resultsADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed up to a concentration of 5000 µg/plate with or without metabolic activation.

Remarks on result:

other: No mutagenic effect were observed.

Please refer attached background material

Conclusions:

Test chemical was evaluated for its mutagenic potential in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA by AMES test. The test result was considered to be negative in all strain in the presence and absence of metabolic activation S9.

Executive summary:

Genetic toxicity in vitro study was assessed for Calcium salt of 3-hydroxy-4 - [(4-methyl-2-sulfophenyl) azo] -2-naphthalenecarboxylic acid (5281-04-9). For this purpose AMES test was performed according to Guidelines for Screening Mutagenicity Testing of Chemicals (Japan).The test material was exposed to Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were 0, 312.5, 625, 1250, 2500, 5000 µg/plate. No mutagenic effects were observed in all strains, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA by AMES test. Hence the substance cannot be classified as gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Data is from jcheck

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Principles of method if other than guideline:

To evaluate the mutagenic potential of test chemical in Chinese hamster CHL/IU cells by chromosomal aberration test.

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-Naphthalenecarboxylic acid, 3-hydroxy-4-[(4-methyl-2-sulfophenyl)azo]-, calcium salt- Molecular formula (if other than submission substance): C18H14N2O6S.Ca- Molecular weight (if other than submission substance): 424.445 g/mole- Smiles notation (if other than submission substance): c12c(c(c(C(=O)[O-])cc1cccc2)O)\N=N\c1c(cc(C)cc1)S(=O)(=O)[O-].[Ca+2]- InChl (if other than submission substance): 1S/C18H14N2O6S.Ca/c1-10-6-7-14(15(8-10)27(24,25)26)19-20-16-12-5-3-2-4-11(12)9-13(17(16)21)18(22)23;/h2-9,21H,1H3, (H,22,23)(H,24,25,26);/q;+2/p-2/b20-19+;- Substance type: Organic

Target gene:

Not specified

Species / strain / cell type:

other: Chinese hamster CHL/IU cells

Details on mammalian cell type (if applicable):

CHL cells derived from Chinese hamster obtained from Research • Resource Bank (JCRB) (February 1988, obtained at passage 4) were used in the test within 10 years of thawing succession.

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

-S9 (continuous treatment): 0, 0.09, 0.19, 0.37 mg/ml-S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml+S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml

Vehicle / solvent:

Vehicle- Vehicle(s)/solvent(s) used: 0.5% Carboxymethyl cellulose sodium

Untreated negative controls:

yes

Remarks:

Not specified

Negative solvent / vehicle controls:

yes

Remarks:

0.5% Carboxymethyl cellulose sodium

True negative controls:

not specified

Positive controls:

yes

Remarks:

-S9, Mitomycin C +S9, Cyclophosph amide

Details on test system and experimental conditions:

Details on test system and conditionsCells usedCHL cells derived from Chinese hamster obtained from Research • Resource Bank (JCRB) (February 1988, obtained at passage 4) were used in the test within 10 years of thawing succession.2. Preparation of culture solutionEagle MEM culture medium supplemented with 10% fetal bovine serum (FCS: JRH BIOSCIENCES, lot number: 1C2073) was used for the culture.3. Culture conditions2 × 10 4 CHL cells were seeded in a dish (6 cm in diameter, Corning) containing 5 ml of the culture solution and cultured in a 37 ° C. CO 2 incubator (5% CO 2).In the direct method, test substances were added on day 3 of cell seeding and treated for 24 hours and 48 hours. In the metabolic activation method, the cells were treated for 6 hours in the presence and absence of S9mix on the third day of cell seeding, and after completion of the treatment, the cells were cultured for 18 hours with fresh culture medium.Method for preparing chromosome specimenTwo 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. Six slide specimens were prepared for each dish. The prepared specimens were stained with 3% Giemsa solution for about 10 minutes. OTHER EXAMINATIONS:- Determination of polyploidy: Yes

Rationale for test conditions:

Not specified

Evaluation criteria:

Among the five test bacteria used, in the direct method or metabolic activation method of one or more test bacteria, the number of revertive mutant colonies on the flat plate containing the test substance is more than twice that of the negative control, And when the increase was found to be reproducible or dose-dependent, it was decided that the test substance had mutagenicity (positive) in this test system.

Statistics:

Analysis results for untreated control, solvent, positive control group and test substance treated group are tabulated for the observed cell number, type and number of structural abnormality, and number of ploidy cells, and the values of each group are entered on the recording paper did. With regard to the frequency of occurrenceof chromosomal abnormal cells, a significant difference test between the solvent control group and the test substance treated group and between the solvent control group and the positive control group was carried out by Fisher's exact probability test method. According to the judgment criteria of Ishikan et al.2), the frequency ofcells with chromosomal abnormality is negative, less than 5% negative, less than 10% false positive, and more than 10% Positive.

Species / strain:

other: Chinese hamster CHL/IU cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Additional information on resultsLowest concentration producing cytogenetic effects in vitro:without metabolic activation (continuous treatment ): > 0.37 mg/mlwithout metabolic activation (short-term treatment): > 5.0 mg/mlwith metabolic activation (short-term treatment): > 5.0 mg/mlother;No polyploidy was also observed.

Remarks on result:

other: No mutagenic effect were observed.

Table: Chromosome analysis of Chinese hamster cells (CHL) treated with D and C Red NO.7** with and without S9 mix

Group	Concentration (mg/ml)	S9 mix	Time of exposure (hrs)	No. of cells analysed	No. of structural aberrations								others	No. of cells with abberations				Polyploid^4(%)
					gap	ctb	Cte	csb	cse	f	Mul^2	total		TAG	(%)	TA	(%)
Control			6-(18)	200	0	0	1	0	1	1	0	3	0	3	(1.5)	3	(1.5)	0.13
Solvent^1	0	-	6-(18)	200	0	0	0	0	0	0	0	0	0	0	(0.0)	0	(0.0)	0.50
D&C Red no.7	1.3	-	6-(18)	200	0	5	0	1	0	0	0	6	0	5	*(2.5)	5	(2.5)	0.13
D&C Red no.7	2.5	-	6-(18)	200	1	0	0	1	1	0	0	3	0	3	(1.5)	2	(1.0)	1.25
D&C Red no.7	5.0	-	6-(18)	200	1	0	0	0	0	0	0	1	0	1	(0.5)	0	(0.0)	4.75*
CPA	0.005	-	6-(18)	200	0	0	0	3	0	0	0	3	2	1	(0.5)	1	(0.5)	0.00
Solvent^1	0	+	6-(18)	200	0	1	0	0	0	0	0	1	0	1	(0.5)	1	(0.5)	0.13
D&C Red no.7	1.3	+	6-(18)	200	0	0	0	0	0	0	0	0	0	0	(0.0)	0	(0.0)	0.75
D&C Red no.7	2.5	+	6-(18)	200	0	0	0	3	0	0	0	3	0	1	(0.5)	1	(0.5)	0.25
D&C Red no.7	5.0	+	6-(18)	200	1	3	3	0	0	0	0	7	0	5	(2.5)	0	(2.5)	1.00*
CPA	0.005	+	6-(18)	200	20	58	187	8	0	6	10	289	1	131	*(65.5)	125	*(62.5)	0.63

 

Conclusions:

Test chemical was evaluated for its mutagenic potential in Chinese hamster CHL/IU cells by chromosomal aberration test. The test result was considered to be negative in Chinese hamster CHL/IU cells in the presence and absence of metabolic activation S9.

Executive summary:

Genetic toxicity in vitro study was assessed for Calcium salt of 3-hydroxy-4 - [(4-methyl-2-sulfophenyl) azo] -2-naphthalenecarboxylic acid (5281-04-9). For this purpose chromosomal aberration test was performed according to Guidelines for Screening Mutagenicity Testing of Chemicals (Japan).The test material was exposed to Chinese hamster CHL/IU cells in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation weremention as fallow

-S9 (continuous treatment): 0, 0.09, 0.19, 0.37 mg/ml

-S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml

+S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml

 

 No significant statistical mutagenic effects were observed in chromosome, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non mutagenic in Chinese hamster CHL/IU cells by chromosomal aberration test. Hence the substance cannot be classified as 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

Genetic toxicity In-vitro

AMES assay;

Various experimental studies were reviewed to determine the mutagenic nature of target substance calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate (5281-04-9) Common name; D and C red no. 7 or C.I. 15850:1. The studies are as mentioned below:

Gene mutation toxicity study was observed by National Institute of Technology and Evaluation (J check,2018) to determine the mutagenic nature of target substance calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate (5281-04-9) using Salmonella typhimurium strains. Genetic toxicity in vitro study was assessed for Calcium salt of 3-hydroxy-4 - [(4-methyl-2-sulfophenyl) azo] -2-naphthalenecarboxylic acid (5281-04-9). For this purpose AMES test was performed according to Guidelines for Screening Mutagenicity Testing of Chemicals (Japan).The test material was exposed to Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were 0, 312.5, 625, 1250, 2500, 5000 µg/plate. No mutagenic effects were observed in all strains, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA by AMES test. Hence the substance cannot be classified as gene mutant in vitro.

Supported by an experimental study conducted by Jeanne M et al.( Mutation Research, 1979) to determine the mutagenic nature of target substance. Test chemicalwas tested for mutagenic potential with the Salmonella/ mammalian-microsome test. The 2 ml of liquid top agar was cooled to 45°C and 0.1 ml of a broth culture of microorganism and test substance in volumes of ≤ 0.4 ml of DMSO was added prior to placing on minimal agar plates. After 48 h incubation at 37°C, the colonies which reverted to the prototroph were counted and compared to counts on the control plate (containing no test substance) to demonstrate mutagenicity or toxicity. Materials which caused a 2-fold increase of revertants, as compared to the number of spontaneous revertants on the control plates, were denoted as mutagens. Those which reduced the number of revertants were considered inhibitory.The test chemical failed to induce mutation inSalmonella typhimurium TA98, TA1537, TA100, TA1535 and hence is negative for gene mutation in vitro.

It is further supported by another experimental study conducted by MIYAGOSHI M et al(EISEI KAGAKU(J HYG CHEM),1983) ) to determine the mutagenic nature of target substance. Genetic toxicity in vitro study was assessed for test chemical .The test material was exposed to Salmonella typhimurium TA 98 and TA 100 in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were5, 50, 500 and 1000 μ/plate. No mutagenic effects were observed in all strains, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non mutagenic in Salmonella typhimurium TA 98 and TA 100 by AMES test. Hence the substance cannot be classified as gene mutant in vitro.

Chromosomal abbreviation;

Gene mutation toxicity study was observed by National Institute of Technology and Evaluation (J check,2018) to determine the mutagenic nature of target substance calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate (5281-04-9) using Mammalian cell line. This test was considered as confirmatory test for mutagenic potential. Hence the test was performed to conform its mutagenic nature. Genetic toxicity in vitro study was assessed for Calcium salt of 3-hydroxy-4 - [(4-methyl-2-sulfophenyl) azo] -2-naphthalenecarboxylic acid (5281-04-9). For this purpose chromosomal aberration test was performed according to Guidelines for Screening Mutagenicity Testing of Chemicals (Japan).The test material was exposed to Chinese hamster CHL/IU cells in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were mention as fallow

-S9 (continuous treatment): 0, 0.09, 0.19, 0.37 mg/ml

-S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml

+S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml

 No significant statistical mutagenic effects were observed in chromosome, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non mutagenic in Chinese hamster CHL/IU cells by chromosomal aberration test. Hence the substance cannot be classified as gene mutant in vitro.

 

Based on the experimental data , available for the target chemical, calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate (5281-04-9) Common name; D and C red no. 7 or C.I. 15850:1 does not induce gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Thus based on the above annotation and CLP criteria for the target chemical. Calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate (5281-04-9) Common name; D and C red no. 7 or C.I. 15850:1 does not induce gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.