Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, aluminium salt

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

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 journal

Data source

Reference

Reference Type:

publication

Title:

Mutagenicity of Arylmethane dyes in Salmonella

Author:

Antonio M. Bonin, Janice R. Farquharson And Robert S.U. Baker

Year:

1981

Bibliographic source:

Mutation Research, 89 (1981) 26-34

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Patent Blue V was tested in the Salmonella/ mammalian microsome mutagenicity assay to check for its mutagenic potential.

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt

EC Number:

204-934-1

EC Name:

Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt

Cas Number:

129-17-9

Molecular formula:

C27H32N2O6S2.Na

IUPAC Name:

hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt

Details on test material:

- Name of test material: Patent Blue V
- Molecular formula: C27H31N2O6S2.Na
C27H32N2O6S2.Na
- Molecular weight (if other than submission substance): 566.672 g/mol
- Substance type: Organic
- Physical state: Solid
- Purity: No data available
- Impurities (identity and concentrations): No data available

Specific details on test material used for the study:

- Name of test material: Patent Blue V
- Molecular formula: C27H31N2O6S2.Na
C27H32N2O6S2.Na
- Molecular weight: 566.672 g/mol
- Substance type: Organic
- Physical state: Solid
- Purity: No data available
- Impurities (identity and concentrations): No data available

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538

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:

S9 fraction was prepared from male Sprague Dawley rats pretreated with Aroclor 1254

Test concentrations with justification for top dose:

0, 32, 100, 320, 1000 µg/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:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2- acetylaminoflourene, β- propiolactone

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data available
- Exposure duration: 72 hrs
- Expression time (cells in growth medium): 72 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: Duplicate

NUMBER OF CELLS EVALUATED: No data available

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

OTHER EXAMINATIONS:
- Determination of polyloidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available

Rationale for test conditions:

No data

Evaluation criteria:

Criteria for mutagenicity were (a) a dose-response and, (b) reproducibility of the result. Dose-responses were not always evident at concentratrations selected for initial testing.

Statistics:

No data

Results and discussion

Test results

Species / strain:

other: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538

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:

RANGE-FINDING/SCREENING STUDIES: Dose ranges for mutagenesis were determined first by preliminary pour-plate toxicity tests at 10, 1, 0.1, 0.01 mg/plate.

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

Table: Ames test data for Patent Blue V

Concentration	His+ revertant colonies/plate
	TA98		TA100		TA1535		TA1537		TA1538
S9	-	+	-	+	-	+	-	+	-	+
10000	28	52	175	140	38	16	6	10	25	42
3200	29	48	150	125	33	17	8	10	19	42
1000	24	48	135	111	39	13	8	13	19	40
320	24	42	152	105	31	17	7	12	23	39
0	24	68	180	167	30	15	16	13	23	42

Applicant's summary and conclusion

Conclusions:

Paten Blue V did induce mutagenicity in the Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538 strains in the presence and absence of S9 mix and hence it is not likely to classify as a gene mutant.

Executive summary:

Salmonella / mammalian microsome mutagenicity assay was performed to study the mutagenic potential of Patent Blue V both in the presence and absence of metabolic activator S9 mix. To each 2 ml of top agar at 42°C was added 100 µL bacterial broth culture, 100 µL test compound dissolved in DMSO various concentrations ranging from 0, 32, 100, 320, 1000 µg/plate, and 500 µL S9 mix as required. Plates were incubated at 37°C for 72 hrs before counting his⁺revertant colonies and each dose point was determined from at least two plates, unless indicated otherwise. Criteria for mutagenicity were (a) a dose-response and, (b) reproducibility of the result. Dose-responses were not always evident at concentratrations selected for initial testing. Paten Blue V did induce mutagenicity in the Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538 strains in the presence and absence of S9 mix and hence it is not likely to classify as a gene mutant.