Tetrasodium [μ-[[3,3'-[(3,3'-dihydroxy[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicuprate(4-)

Genetic toxicity: in vitro

Administrative data

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:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Mutagenicity of Azo Dyes Following Metabolism by Different Reductive/Oxidative Systems

Author:

Thomas M. Reid, Kenneth C. Morton, Ching Y. Wang, and Charles M. King

Year:

1984

Bibliographic source:

Environmental Mutagenesis 6: 705-717 (1984)

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Gene mutation toxicity study was performed for Direct blue 218 to evaluate its mutagenic nature

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium [μ-[[3,3'-[(3,3'-dihydroxy[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicuprate(4-)

EC Number:

249-008-8

EC Name:

Tetrasodium [μ-[[3,3'-[(3,3'-dihydroxy[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicuprate(4-)

Cas Number:

28407-37-6

Molecular formula:

C32H16Cu2N6O16S4.4Na

IUPAC Name:

Tetrasodium [μ-[[3,3'-[(3,3'-dihydroxy[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicuprate(4-)

Test material form:

other: amorphous powder

Details on test material:

Name of the test chemical: Tetrasodium [μ-[[3,3'-[(3,3'-dihydroxy[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicuprate(4-)
Common Name: C.I Direct Blue 218
IUPAC name: tetrasodium (3E)-5-amino-3-{2-[4-(4-{2-[(2E)-8-amino-1-oxo-3,6-disulfonato-1,2-dihydronaphthalen-2-ylidene]hydrazin-1-yl}-3- hydroxyphenyl)-2-hydroxyphenyl]hydrazin-1-ylidene}-4-oxo-3,4-dihydronaphthalene-2,7-disulfonate dicopper
Molecular Formula: C32H20Cu2N6Na4O16S4
Molecular Weight: 1087.84 g/mol
SMILES Notation: c12c3c(c(S(=O)(=O)[O-])cc1cc(S(=O)(=O)[O])cc2N)N=Nc1ccc(cc1O[Cu]O3)c1cc2c(N=Nc3c(cc4c(c3O[Cu]O2)c(cc(c4)S(=O) (=O)[O-])N)S(=O)(=O)[O-])cc1.[Na+].[Na+].[Na+].[Na+]
InChI: 1S/C32H24N6O16S4.2Cu.4Na/c33-19-11-17(55(43,44)45)5-15-9-25(57(49,50)51)29(31(41)27(15)19)37-35-21-3-1-13(7-23(21)39) 14-2-4-22(24(40)8-14)36-38-30-26(58(52,53)54)10-16-6-18(56(46,47)48)12-20(34)28(16)32(30)42;;;;;;/h1-12,39-42H,33-34H2,(H,43,44,45)(H,46,47,48)(H,49,50,51)(H,52,53,54);;;;;;/q;2*+2;4*+1/p-8/b37-35-,38-36-;;;;;;
Substance Type: Organic
Physical State: Solid Deep purple to dark blue amorphous powder

Specific details on test material used for the study:

- Name of test material: Direct blue 218
- IUPAC name: Copper,[tetrahydrogen-3,3'-[(3,3'-dihydroxy-4,4'-biphenylylene)bis(azo)]bis[5-amino-4-hydroxy-2,7-naphthalenedisulfonato](4-)]di-,tetrasodium salt (7CI)
- Molecular formula: C32H16Cu2N6O16S4.4Na
- Molecular weight: 1087.82 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: >98%
- Impurities (identity and concentrations): <2%

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 was prepared from the livers of Aroclor-induced male Fisher rats and hamster S9 was prepared from uninduced female hamsters

Test concentrations with justification for top dose:

0, 0.25 or 0.50 µmole/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Benzidine congeners (Dimethoxybenzidine)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 72 hr
- Expression time (cells in growth medium): 72 hr
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

The plates were observed for revertants

Statistics:

No data

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1538

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:

No data

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

Table: Mutation data for the test chemical Direct blue 218 and the positive control chemical

Dose (µmole/plate)	Reduction system (activation system)
	Bacterial (rat)	None (rat)	FMN (hamster)	None(hamster)	Bacterial (hamster)
Direct blue 218
0	43	35	33	-	-
0.25	40 (63)	14	27	-	-
0.50	47 (47)	16	21	-	-
Dimethoxybenzidine
0	43	35	33	27	26
0.25	790	843	235	169	108
0.50	1073	1203	316	125	167
1.0	1491	1287	366	170	124

Applicant's summary and conclusion

Conclusions:

Direct blue 218 did not induce gene mutation in the Salmonella typhimurium strain TA1538 both in the presence and absence of rat and hamster liver S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene mutation toxicity study was performed for Direct blue 218 to evaluate its mutagenic nature. The study was performed as per the preincubation protocol and reduction of the test chemical using Salmonella typhimurium strain RA1538 both in the presence and absence of rat and hamster liver S9 metabolic activation system at doses of 0, 0.25 or 0.50 µmole/plate. DMSO was used at the vehicle. The plates were incubated for 48 hrs after 20 mins preincubation before the evaluation of the revertant colonies could be made. Concurrent solvent and negative control chemicals were also included in the study. Direct blue 218 did notinduce gene mutation in theSalmonella typhimurium strain TA1538 both in the presence and absence of rat and hamster liver S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.