1,3-Benzenedisulfonic acid, 4-[[5-(aminocarbonyl)-1-ethyl-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridinyl]azo]-6-[[4-[[3-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-sulfophenyl]amino]-6-chloro-1,3,5-triazin-2-yl]amino]-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

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:

Salmonella Mutagenicity Tests: II. Results From The Testing Of 270 Chemicals

Author:

Mortelmans,K, Haworth,S, Lawlor,T, Speck,W, Tainer,B And Zeiger,E

Year:

1986

Bibliographic source:

Environ. Mutagen. 8(Suppl. 7):1 -119, 1986

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 25 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:

Reference substance 001

Cas Number:

2150-54-1

Molecular formula:

C34H26N4O16S4.4Na

Details on test material:

- Name of test material: Direct Blue 25
- IUPAC name: Tetrasodium 3,3'-[(3,3'-dimethyl[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[4,5-dihydroxynaphthalene-2,7-disulphonate]
- Molecular formula: C34H26N4O16S4.4Na
- Molecular weight: 962.7838 g/mol
- Substance type: Organic

Specific details on test material used for the study:

- Name of test material: Direct Blue 25
- IUPAC name: Tetrasodium 3,3'-[(3,3'-dimethyl[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[4,5-dihydroxynaphthalene-2,7-disulphonate]
- Molecular formula: C34H26N4O16S4.4Na
- Molecular weight: 962.7838 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: No data
- Impurities (identity and concentrations): No data

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

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:

Male Sprague-Dawley rats and male Syrian hamsters were routinely used for the S9 preparation of the liver fractions

Test concentrations with justification for top dose:

0, 100.0, 333.0, 1000.0, 3333.0, 10000.0 µg/plate

Vehicle / solvent:

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

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine (TA98; -S9), 2-aminoanthracene (all strains, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hr
- Expression time (cells in growth medium): 48 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: At least five dose levels of the chemicals were tested, with three plates per dose level.

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:

1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold;
2) nomutagenic response: when no increase in the number of revertants was elicited by the chemical;
3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible;
or when the response was of insufficient magnitude to support a determination of mutagenicity

Statistics:

Mean and Standard error of mean

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA100, TA1535, TA1537, TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

For strain 1537, at 10000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The chemical was initially tested with strain TA100 in the presence and the absence of the metabolic activation systems, over a wide dose range with an upper limit of 10 mg/plate, or less when solubility problems were encountered. Toxicity was evidenced by one or more of the following phenomena: appearance of his+ pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn. Nontoxic chemicals were tested in the initial experiment up to the 10 mg/plate dose level, or to a level determined by their solubility. Toxic chemicals were tested up to a high dose which exhibited some degree of toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

Remarks on result:

other: No mutagenc potential

Any other information on results incl. tables

Table: Mutation data for the test chemical Direct Blue 25

Dose (µg/plate)	TA100
	NA		10% HLI		10% RLI
	Mean	SEM	Mean	SEM	Mean	SEM
0	101	2.4	160	9.6	156	10.2
100	105	4.4	162	1.7	166	6.3
333	128	2.6	163	10.7	187	14.2
1000	101	18.1	173	13.9	136	6.8
3333	72	5.9	142	2.9	125	10.1
10000	82	3.8	99	6.1	87	3.0
Positive control	782	27.7	1136	43.5	1146	58.1

 

Dose (µg/plate)	TA1535
	NA		10% HLI		10% RLI
	Mean	SEM	Mean	SEM	Mean	SEM
0	20	0.9	13	1.9	15	1.2
100	24	2.7	16	2.1	17	1.0
333	17	0.3	12	0.9	15	2.6
1000	15	2.0	13	0.3	17	2.1
3333	16	2.9	10	1.7	19	2.3
10000	17	2.7	8	1.2	10	2.0
Positive control	1037	70.4	447	84.8	319	24.9

 

Dose (µg/plate)	TA1537
	NA		10% HLI		10% RLI
	Mean	SEM	Mean	SEM	Mean	SEM
0	8	1.2	13	2.3	13	3.3
100	10	3.2	15	1.2	17	4.3
333	11	1.8	11	0.0	16	2.0
1000	9	0.9	11	0.3	7	1.2
3333	6	0.6	7	1.8	7	1.5
10000	T		T		T
Positive control	165	11.8	293	8.5	307	9.0

 

Dose (µg/plate)	TA98
	NA		10% HLI		10% RLI
	Mean	SEM	Mean	SEM	Mean	SEM
0	18	1.0	37	5.0	31	3.3
100	20	5.0	45	3.7	28	3.0
333	23	3.1	41	5.5	33	1.5
1000	27	2.7	37	1.0	19	3.7
3333	15	2.6	42	3.5	21	1.5
10000	15	0.3	20	4.5	12	1.2
Positive control	465	36.1	1035	27.4	1026	47.0

 

T: Complete clearing of background lawn

Applicant's summary and conclusion

Conclusions:

Direct Blue 25 did not induce mutation in the Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.c v

Executive summary:

Gene mutation toxicity study was performed for direct blue 25 to evaluate its mutagenic nature. The study was performed as per the preincubation protocol using Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system at doses of 0, 100.0, 333.0, 1000.0, 3333.0 or 10000.0 µg/plate. Water was used as the vehicle. The plates were incubated for 48 hrs after 20 mins preincubation before the evaluation of the revertant colonies could be made. Direct blue 25 did not induce mutation in the Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.