Barium 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

Gene toxicity in-vitro:

The test result was considered to be negative in the presence and absence of metabolic activation.

Hence the substance cannot be classified as genetox in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data of structurally similar chemicals

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

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 and TA100 by AMES test.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

Target gene:

1.& 2. Histidine

Species / strain / cell type:

S. typhimurium, other: TA98 and TA100 bacteria

Remarks:

1

Details on mammalian cell type (if applicable):

not specified

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

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

Remarks:

2

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

The S-9 fraction (Organon Teknika, Durham, NC, USA) was from Sprague-Dawley rats induced with Aroclor-1254.

Test concentrations with justification for top dose:

1. 0,50-200 μg/plate2. 0,50,100and 500μg/plate

Vehicle / solvent:

1. - Vehicle(s)/solvent(s) used: Water2. - Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: DMSO, 1,6-dinitropyrene and benzo[a]pyrene

Remarks:

1.

Untreated negative controls:

yes

Remarks:

Historical value provided for comparison.

Negative solvent / vehicle 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 experimental conditions:

1. METHOD OF APPLICATION: Plate incorporation method2. 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:

S. typhimurium, other: TA98 and TA100. bacteria

Remarks:

1

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

Species / strain:

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

Remarks:

2.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

2. COMPARISON WITH HISTORICAL CONTROL DATA: Historical Negative control values were compared with the test substance value.

Remarks on result:

other: No mutagenic effect were observed.

Conclusions:

The test result was considered to be negative in the presence and absence of metabolic activation.

Executive summary:

Data available for the target chemical was reviewed to determine the mutagenic nature of the test chemical barium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate (CAS no 17852-98-1)The studies are as mentioned below:

Ames test:

Test chemical was assessed for its possible mutagenic potential. For this purpose AMES assay was performed on Salmonella typhimurium TA98 and TA100. The test material was exposed at the concentration of 50-200 µg/plate in the presence and absence of S9. No mutagenic effects were observed. Therefore, test chemical was considered to be non mutagenic in the Salmonella typhimurium TA98 and TA100 by AMES test. Hence the substance cannot be classified as genetox in vitro.

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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data of structurally similar chemicals

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

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

Target gene:

2. Thymidine kinase TK

Species / strain / cell type:

other: Chinese hamster CHL/IU cells

Remarks:

1.

Details on mammalian cell type (if applicable):

CHL cells derived from Chinese hamster obtained from Research • Resource Bank (JCRB) (February1988, obtained at passage 4) were used in the test within 10 years of thawing succession.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

other: mouse lymphoma L5178Y cells mammalian cell line

Remarks:

2.

Details on mammalian cell type (if applicable):

- Type and identity of media: The cells were grown in Fischer's medium for leukemic cells of mice supplemented 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 ofmycoplasma 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 / 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:

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/ml2. Test concentration without S9 mix:0.0, 3706.0, 4560.0, 4560.0, 5000.0 and 5000.0 μg/mLTest 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 / solvent:

1.- Vehicle(s)/solvent(s) used: 0.5% Carboxymethyl cellulose sodium2.- Vehicle(s)/solvent(s) used: The solvents used were water, dimethyl sulfoxide, and acetone (exact solvent details are not available)

Untreated negative controls:

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

Remarks:

10

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

2.

Details on test system and experimental conditions:

1.Cells 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: Yes2.METHOD OF APPLICATION: in mediumDURATION- 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 availableSELECTION AGENT (mutation assays): No data availableSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: No data availableNUMBER OF CELLS EVALUATED: No data availableDETERMINATION 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 availableOther: 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-dependent, 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 thefrequency 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 / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

mammalian cell line 2.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

1.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.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 availableRANGE-FINDING/SCREENING STUDIES: The toxicity of each chemical was first determined both withand without S9 prepared from Aroclor-1254-induced male Fischer 344 rats.COMPARISON WITH HISTORICAL CONTROL DATA: No data availableADDITIONAL INFORMATION ON CYTOTOXICITY: No data available

Remarks on result:

other: No mutagenic effect were observed.

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 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.

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 barium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate (CAS no 17852-98-1)The studies are as mentioned below:

Ames test:

Test chemical was assessed for its possible mutagenic potential. For this purpose AMES assay was performed on Salmonella typhimurium TA98 and TA100. The test material was exposed at the concentration of 50-200 µg/plate in the presence and absence of S9. No mutagenic effects were observed. Therefore, test chemical was considered to be non mutagenic in the Salmonella typhimurium TA98 and TA100 by AMES test. Hence the substance cannot be classified as genetox in vitro.

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 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.