Registration Dossier

Administrative data

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

Referenceopen allclose all

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:
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:
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:
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.

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:
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

 

Polyploid4(%)

gap

ctb

Cte

csb

cse

f

Mul2

total

 

TAG

(%)

TA

(%)

 

Control

 

 

6-(18)

200

0

0

1

0

1

1

0

3

0

3

(1.5)

3

(1.5)

0.13

Solvent1

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Solvent1

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.