Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation toxicity study was performed by Lynnette R. Fergusonet al (Mutation Research, 1988) to determine the mutagenic nature of target substance Methyl blue ;IUPAC name: Disodium [[4-[bis[4-[(sulphonatophenyl)amino] phenyl]methylene] cyclohexa-2,5-dien-1-ylidene]amino]ben(28983-56-4)using Salmonella typhimurium strains. In Genetic toxicity in vitro study, Methyl Blue was assessed for its possible mutagenic potential. For this purpose AMES assay was performed in Salmonella typhimurium TA1537, TA1598 and TA100 by plate-incorporation assay. The test substance was exposed at the concentration of than 100µl of 50% ethanol, or 50% ethanol alone as negative control. Plates were allowed to harden, and then incubated for 3 days at 37°C before scoring colonies for reversion to Histidine independence. A dose range of each compound was tested, and each experimental point performed in triplicate on at leasttwo separate occasions. No mutagenic effects were observed at this dose .Therefore Methyl Blue was considered to be non mutagenic for AMES test. Hence the substance cannot be classified as gene mutant in vitro.

Link to relevant study records

Reference

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:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

To evaluate the mutagenic potential of Methyl Blue in Salmonella typhimurium TA1537, TA1598 and TA100 by AMES assay.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material :Methyl Blue
- Molecular formula : C37H26N3Na2O9S3
- Molecular weight : 800.8182 g/mol
- Smiles notation : c1cc(ccc1C(=C2C=CC(=[NH+]c3ccc (cc3)S(=O) (=O)[O-])C=C2)c4ccc(cc4)Nc5ccc(cc5)S (=O)(=O)[O-])Nc6ccc(cc6)S(=O)(=O)O.[Na+].[Na+]
- InChl : 1S/C37H29N3O9S3.2Na/c41-50(42,43)34-19-13-31(14-20-34)38-28-7-1-25(2-8-28)37(26-3-9-29(10-4-26)39-32-15-21-35(22-16-32)51(44,45 46)27-5-11-30(12-6-27)40-33-17-23-36(24-18-33)52(47,48)49;;/h1-24,38-39H,(H,41,42,43)(H,44,45,46)(H,47,48,49);;/q;2*+1/p-1
- Substance type: Organic
- Physical state: Solid

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA1537, TA1598 and TA100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

not specified

Test concentrations with justification for top dose:

100 µl

Vehicle / solvent:

50% of ethanol

Untreated negative controls:

yes

Remarks:

ethanol

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

not specified

Positive controls:

no

Details on test system and experimental conditions:

Details on test system and conditions
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 3days

NUMBER OF REPLICATIONS: triplicate

OTHER: Reversion characteristics of each strain were tested in each experiment using the disc method of Zieger et al. (1982). The background number of revertants was 8 ± 3 for TA1537, 34 ±7 for TA98, and 171 ± 10 for TAI00.

Rationale for test conditions:

Not specified

Evaluation criteria:

Mutagenicity is expressed as revertants colonies/µg compound added to the plate, and is derived from the slope of the corresponding regression equation.

Statistics:

Mutagenicity data were analyzed according to the method of Moore and Felton (1983). A regression line was fitted for the number of mutants versus
drug concentration for the linear part of the curve. Mutagenicity was scored as negative if the regression coefficient was non-significant. To determine whether verapamil had a significant effect on mutagenicity, the slopes ( +__ SD) of the corresponding regression lines with and without verapamil were determined, and the significance of the difference was calculated using Student's t test.

Key result

Species / strain:

S. typhimurium, other: TA1537, TA1598 and TA100

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

All mutagenicity data, calculated as revertant colonies per /µg of added drug in the presence or absence of verapamil.

Remarks on result:

other: NO mutagenic effct were observerd

Sr no	Compound	Mutagenicity^bin bacterial strain^c
		TA1537	TA98	TA100
1	Methyl Blue	0	0	0

 

b Mutagenicity is expressed as revertant colonies/tzg compound added to the plate, and is derived from the slope of the corresponding

regression equation,.

c Bacterial strains are described in Maron and Ames (1983).

d Statistical significance of the differences: **p < 0.001; *p < 0.05.

e A value of 0 means that the correlation coefficient of the regression equation is not statistically significant.

Conclusions:

Methyl Blue (28983-56-4) was evaluated for its mutagenic potential in Salmonella typhimurium TA1537, TA1598 and TA100. The test result was considered to be negative for the test.

Executive summary:

In Genetic toxicity in vitro study, Methyl Blue was assessed for its possible mutagenic potential. For this purpose AMES assay was performed in Salmonella typhimurium TA1537, TA1598 and TA100 by plate-incorporation assay. The test substance was exposed at the concentration of than 100µl of 50% ethanol, or 50% ethanol alone as negative control. Plates were allowed to harden, and then incubated for 3 days at 37°C before scoring colonies for reversion to Histidine independence. A dose range of each compound was tested, and each experimental point performed in triplicate on at leasttwo separate occasions. No mutagenic effect were observed at this dose .Therefore Methyl Blue was considered to be non mutagenic for AMES 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

Various experimental studies were reviewed to determine the mutagenic nature of target substance Methyl blue ; IUPAC name: Disodium [[4-[bis[4-[(sulphonatophenyl)amino] phenyl]methylene] cyclohexa-2,5-dien-1-ylidene]amino]ben(28983-56-4). The studies are as mentioned below:

Gene mutation toxicity study was performed by Lynnette R. Fergusonet al (Mutation Research, 1988) to determine the mutagenic nature of target substance Methyl blue ;IUPAC name: Disodium [[4-[bis[4-[(sulphonatophenyl)amino] phenyl]methylene] cyclohexa-2,5-dien-1-ylidene]amino]ben(28983-56-4)using Salmonella typhimurium strains. In Genetic toxicity in vitro study, Methyl Blue was assessed for its possible mutagenic potential. For this purpose AMES assay was performed in Salmonella typhimurium TA1537, TA1598 and TA100 by plate-incorporation assay. The test substance was exposed at the concentration of than 100µl of 50% ethanol, or 50% ethanol alone as negative control. Plates were allowed to harden, and then incubated for 3 days at 37°C before scoring colonies for reversion to Histidine independence. A dose range of each compound was tested, and each experimental point performed in triplicate on at leasttwo separate occasions. No mutagenic effects were observed at this dose .Therefore Methyl Blue was considered to be non mutagenic for AMES test. Hence the substance cannot be classified as gene mutant in vitro.

Supported by an experimental study conducted by William Au (Environmental Mutagenesis, 1979) to determine the mutagenic nature of target substance Methyl blue IUPAC name: Disodium [[4-[bis[4-[(sulphonatophenyl)amino] phenyl]methylene] cyclohexa-2,5-dien-1-ylidene]amino]ben(28983-56-4) . In Genetic toxicity in vitro was performed study,Methyl blue wasassessed for its possible clastogenic potential. For this purpose Invitro chromosome breakage assay was performed in Chinese hamster ovary cell line (CHO). The test material was exposed at the concentration of 1-20 µM in the presence and absence of metabolic activation. The 50 well-spread metaphases were scored for chromosome aberration for each experimental point. The average number of breaks per metaphase was calculated and was used for comparing the clastogenic activities of different compounds. The average number of chromosome breaks ranged from 0 to 0.16 per cell for the water-treated control. No chromosome breaks were observed for target substance compare to control. No statically significant value was also observed for test substance. There to for Methyl blue was considered to be non clastogenic in Chinese hamster ovary cell line (CHO) by In vitro chromosome breakage assay in the presence and absence of metabolic activation. Hence the substance cannot be classified as gene mutant in vitro.

 

It is further supported by another experimental study conducted by Zev Leifer 2et al(Mutation Research,1981) ) to determine the mutagenic nature of target substance Methyl blue IUPAC name: Disodium [[4-[bis[4-[(sulphonatophenyl)amino] phenyl]methylene] cyclohexa-2,5-dien-1-ylidene]amino]ben(28983-56-4). In Genetic toxicity in vitro was performed study, Methyl blue was assessed for its possible mutagenic potential. For this purpose REPAIR-DEFICIENCY ASSAYS was performed in bacteria E.coli and Bacillus SubtilisH17A. The test was performed by Diffusion tests and Suspension method. The zone of growth inhibition in the agar diffusion procedure and viability in suspension method was observed for test substance. Due to lack of zone of growth inhibition in the agar diffusion procedure and no viability in suspension method were taken as a No Test result for test substance. Therefore the test result can be considered as ambiguous.

 

Based on the experimental data , available for the target chemical, Methyl blue ; IUPAC name: Disodium [[4-[bis[4-[(sulphonatophenyl)amino] phenyl]methylene] cyclohexa-2,5-dien-1-ylidene]amino]ben(28983-56-4)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

Based on above annotation for the target chemical, Methyl blue ; IUPAC name: Disodium [[4-[bis[4-[(sulphonatophenyl)amino] phenyl]methylene] cyclohexa-2,5-dien-1-ylidene]amino]ben(28983-56-4)does not induce gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.