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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene toxicity in-vitro:

The test result was considered to be negative in the presence and absence of metabolic activation for the test substance 2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[[2-methoxy-5-methyl-4-[[2-(sulfooxy)ethyl]sulfonyl] phenyl]azo]-8-[[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, tetrasodium salt.Hence the substance cannot be classified as genetox in vitro

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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 of structurally similar chemicals
Justification for type of information:
Data for the target chemical is summarized based on the structurally similar chemicals
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: as below
Principles of method if other than guideline:
WoE report is based on two in vitro gene toxicity studies
1.To evaluate the mutagenic potential of test chemical in Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538.
2. To evaluate the mutagenic potential of test chemical in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98 by Salmonella microsome assay.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
1.& 2. Histidine
Species / strain:
other: TA98, TA100, TA1535, TA1537, and TA1538.
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
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.
Species / strain:
other: strains TA1535, TA100, TA1537, TA1538 and TA98 bacteria
Remarks:
2
Details on mammalian cell lines (if applicable):
Not applicable.
Additional strain characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
Liver-microsome preparation from rats injected with Aroclor 1254, 0.25 ml "S-9 mix
Test concentrations with justification for top dose:
1.of 10-10000 µg /plate
2. 0,50,100and 500μg/plate
Vehicle:
1. - Vehicle(s)/solvent(s) used: Not specified
2. - Vehicle(s)/solvent(s) used: DMSO
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
not specified
Negative controls:
yes
Remarks:
Historical value provided for comparison.
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Ethylmethanesulfonate Methylmethanesulfonate 9-Aminoacridine Anthragallol 2-Anthramine
Remarks:
2
Details on test system and conditions:
1. METHOD OF APPLICATION: Plate incorporation method
2. METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period:
- Exposure duration: 3 days
Rationale for test conditions:
Not specified.
Evaluation criteria:
1. A positive result in the Ames test, which is defined as a reproducible, dose-related, at least twofold increase in the number of revertants over background was not observed with any of the azo dyes or their metabolites either before or after incubation with S-9.
2. Number of HIS + Revertants/plate were observed for test substance and compared with positive and negative control.
Statistics:
1. Standard deviation was observed.
2.Rounded mean _+ standard deviation was observed.
Species / strain:
other: TA98, TA100, TA1535, TA1537, and TA1538.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: No mutagenic effect were observed
Species / strain:
other: strains TA1535, TA100, TA1537, TA1538 and TA98 bacteria
Remarks:
2.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: No mutagenic effect were observed.
Additional information on results:
2. COMPARISON WITH HISTORICAL CONTROL DATA: Historical Negative control values were compared with the test substance value.
Conclusions:
The test result was considered to be negative in the presence and absence of metabolic activation for test substance.
Executive summary:

Data available for the target chemical was reviewed to determine the mutagenic nature of the test chemical 2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[[2-methoxy-5-methyl-4-[[2-(sulfooxy)ethyl]sulfonyl] phenyl]azo]-8-[[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, tetrasodium salt.The studies are as mentioned below:

The mutagenic potency of test chemicalwas tested by the plate incorporation method usingSalmonella typhimuriumstrainTA98, TA100, TA1535, TA1537, and TA1538. When the test bacterial strain is exposed with the test chemical for 48hrs, no mutagenic response was seen in any of the strains of Salmonella typhimurium(with and without metabolic activation system).Therefore the test substance was considered to be not classified for gene mutation.

Test chemical was assessed for its possible mutagenic potential. For this purpose bacterial reverse mutation assay was performed on Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98. The test material was exposed at the concentration of 0, 50,100and 500µg/plate in the presence and absence of S9 mix. Number of HIS + Revertants/plate was observed for test substance and compared with positive and negative control. No mutagenic effect were observed in any of the strain, both in the presence and absence of S9.Therefore, test chemical was considered to be non mutagenic in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98 by bacterial reverse mutation assay. Hence the substance cannot be classified as gene mutant in vitro.

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 of structurally similar chemicals
Justification for type of information:
Data for the target chemical is summarized based on the structurally similar chemicals
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: as below
Principles of method if other than guideline:
WoE report is based on two in vitro gene toxicity studies
1. To evaluate the mutagenic potential of test chemical in Chinese hamster CHL/IU cells by chromosomal aberration test.
2. Mutagenicity and cytotoxicity activity was studied in the mouse lymphoma cell line for the test chemical according to the L5178Y TK +/- mouse lymphoma assay
GLP compliance:
not specified
Type of assay:
other: 1. In vitro mammalian chromosome aberration test; 2. mammalian cell gene mutation assay
Test material information:
Composition 1
Target gene:
2. Thymidine kinase TK
Species / strain:
other: Chinese hamster CHL/IU cells
Remarks:
1.
Details on mammalian cell lines (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 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
Species / strain:
other: mouse lymphoma L5178Y cells mammalian cell line
Remarks:
2.
Details on mammalian cell lines (if applicable):
- Type and identity of media: The cells were grown in Fischer's medium for leukemic cells of mice su
pplemented with 10% horse serum, antibiotics and 0.02% Pluronic F-68.
- Properly maintained: No data available
- Periodically checked for Mycoplasma contamination: yes, The cells were checked for the presence of
mycoplasma by agar block isolation and Hoechst staining before and after cryopreservation.
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available
Additional strain characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Test concentrations with justification for top dose:
1. -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
2. Test concentration without S9 mix:
0.0, 3706.0, 4560.0, 4560.0, 5000.0 and 5000.0 μg/mL
Test concentration with S9 mix:
0, 685.0, 685.0, 1072.0, 1072.0, 1456.0, 1845.0, 2231.0 and 2231.0 μg/mL
Vehicle:
1.- Vehicle(s)/solvent(s) used: 0.5% Carboxymethyl cellulose sodium
2.- Vehicle(s)/solvent(s) used: The solvents used were water, dimethyl sulfoxide, and acetone (exact solvent details are not available)
Negative controls:
not specified
Solvent controls:
yes
Remarks:
0.5% Carboxymethyl cellulose sodium
True negative controls:
not specified
Positive controls:
yes
Remarks:
-S9, Mitomycin C +S9, Cyclophosph amide
Remarks:
10
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
2.
Details on test system and conditions:
1.Cells used
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.
2. Preparation of culture solution
Eagle MEM culture medium supplemented with 10% fetal bovine serum (FCS: JRH BIOSCIENCES, lot number: 1C2073) was used for the culture.
3. Culture conditions
2 × 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 specimen
Two hours before the end of the culture, Colcemid was added to the culture solution to a final concentration of about 0.1 μg / ml. Chromosome specimens were prepared according to a conventional method.
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

2.METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: No data available
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 48 hrs
- 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 available
NUMBER OF CELLS EVALUATED: No data available
DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency, Relative total growths were observed.
OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available
Other: Relative suspension growth was also measured.
Evaluation criteria:
1. 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-dep
endent, it was decided that the test substance had mutagenicity (positive) in this test system.
2.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%.
Statistics:
1. 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 occurrence of 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 of cells with chromosomal abnormality is negative, less than 5% negative, less than 10% false positive, and more than 10% Positive.
2. No data avaialble
Species / strain:
other: Chinese hamster CHL/IU cells
Remarks:
1.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: No mutagenic effect were observed.
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
mammalian cell line 2.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested Migrated from field 'Test system
Additional information on results:
1.Additional information on results
Lowest concentration producing cytogenetic effects in vitro:
without metabolic activation (continuous treatment ): > 0.37 mg/ml
without metabolic activation (short-term treatment): > 5.0 mg/ml
with metabolic activation (short-term treatment): > 5.0 mg/ml
other;
No polyploidy was also observed.
2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available
RANGE-FINDING/SCREENING STUDIES: The toxicity of each chemical was first determined both with
and without S9 prepared from Aroclor-1254-induced male Fischer 344 rats.
COMPARISON WITH HISTORICAL CONTROL DATA: No data available
ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available
Conclusions:
The test chemical did not induce mutation and hence was negative (with and without S9 mix)
Executive summary:

Genetic toxicity in vitro study was assessed for test chemical. For this purpose chromosomal aberration test was performed according to Guidelines for Screening Mutagenicity Testing of Chemicals. 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 -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.

L5178Y TK +/- mouse lymphoma assay was performed using test chemical at different concentrations both with and without S9 metabolic activation system. 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 µg/ml TFT to one set of plates. The 100 X 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%. The test chemical did not induce mutation and hence was negative (with and without S9 mix) in L5178Y TK +/- mouse lymphoma assay.

Based on the data available for the test chemical, 2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[[2-methoxy-5-methyl-4-[[2-(sulfooxy)ethyl]sulfonyl] phenyl]azo]-8-[[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, tetrasodium salt does not exhibit gene toxicity in in the presence and absence of metabolic activation in mammalian cell. Hence the substance cannot be classified as genetox in vitro.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene toxicity in-vitro:

Gene mutation in vitro:

Data available for the target chemical was reviewed to determine the mutagenic nature of the test chemical 2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[[2-methoxy-5-methyl-4-[[2-(sulfooxy)ethyl]sulfonyl] phenyl]azo]-8-[[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, tetrasodium salt (503155 -49 -5).The studies are as mentioned below:

Ames test:

The mutagenic potency of test chemicalwas tested by the plate incorporation method usingSalmonella typhimuriumstrainTA98, TA100, TA1535, TA1537, and TA1538. When the test bacterial strain is exposed with the test chemical for 48hrs, no mutagenic response was seen in any of the strains of Salmonella typhimurium(with and without metabolic activation system).Therefore the test substance was considered to be not classified for gene mutation.

Test chemical was assessed for its possible mutagenic potential. For this purpose bacterial reverse mutation assay was performed on Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98. The test material was exposed at the concentration of 0, 50,100and 500µg/plate in the presence and absence of S9 mix. Number of HIS + Revertants/plate was observed for test substance and compared with positive and negative control. No mutagenic effect were observed in any of the strain, both in the presence and absence of S9.Therefore, test chemical was considered to be non mutagenic in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98 by bacterial reverse mutation assay. Hence the substance cannot be classified as gene mutant in vitro.

Chromosome aberration:

Genetic toxicity in vitro study was assessed for test chemical. For this purpose chromosomal aberration test was performed according to Guidelines for Screening Mutagenicity Testing of Chemicals. 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 -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.

L5178Y TK +/- mouse lymphoma assay was performed using test chemical at different concentrations both with and without S9 metabolic activation system. 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 µg/ml TFT to one set of plates. The 100 X 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%. The test chemical did not induce mutation and hence was negative (with and without S9 mix) in L5178Y TK +/- mouse lymphoma assay.

Based on the data available from the test chemical and read across, barium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate does not exhibit gene toxicity in in the presence and absence of metabolic activation.. Hence the substance cannot be classified as genetox in vitro.

Justification for classification or non-classification

Based on the data available from the test chemical 2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[[2-methoxy-5-methyl-4-[[2-(sulfooxy)ethyl]sulfonyl] phenyl]azo]-8-[[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, tetrasodium salt (503155 -49 -5)does not exhibit gene toxicity in in the presence and absence of metabolic activation.. Hence the substance cannot be classified as genetox in vitro.