N-phenyl-4-[[4-(phenylamino)phenyl][4-(phenylimino)cyclohexa-2,5-dien-1-ylidene]methyl]aniline monohydrochloride

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:

Further Study of The Genetic Toxicity Of Gentian Violet

Author:

William Au, Mary Ann Butler, Stephen E. Bloom and Thomas S. Matney

Year:

1979

Bibliographic source:

Mutation Research, 66 (1979) 103-112

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:

Ames assay was performed to determine the mutagenic nature of gentian violet

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[4-[4,4'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium chloride

EC Number:

208-953-6

EC Name:

[4-[4,4'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium chloride

Cas Number:

548-62-9

Molecular formula:

C25H30N3.Cl

IUPAC Name:

(4-(4,4'-Bis(dimethylamino)benzhydrylidene)cyclohexa-2,5-dien-1-ylidene)dimethylammonium, chloride

Test material form:

solid

Details on test material:

- Name of test material (as cited in study report):Gentian violet
- IUPAC name: N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride
- Molecular formula : C25H30N3.Cl
Molecular weight : 407.986 g/mol
- Smiles notation: C(\c1ccc(N(C)C)cc1)(c1ccc(N(C)C)cc1)=C1\C=C\C(=[N+](/C)C)C=C1.[ClH-]
- InChl:1S/C25H30N3.ClH/c1-26(2)22-13-7-19(8-14-22)25(20-9-15-23(16-10-20)27(3)4)21-11-17-24(18-12-21)28(5)6;/h7-18H,1-6H3;1H/q+1;/p-1
- Substance type: Organic
- Physical state: Solid

Specific details on test material used for the study:

- Name of test material: Gentian violet
- IUPAC name: 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride
- Molecular formula: C25H30ClN3
- Molecular weight: 407.986 g/mol
- Substance type: Organic
- Physical state:
- Purity: 97% dye
- Impurities (identity and concentrations): 3 %

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:

Liver preparations were obtained from 200-250 g male Long-Evans hooded rats pretreated with Aroclor 1254 in corn oil

Test concentrations with justification for top dose:

0, 0.1, 1.0, 10., 25.0 or 50.0 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:

other: 2-aminoanthracene

Details on test system and experimental conditions:

Details on test system and conditions
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: 2 days
- Expression time (cells in growth medium): 2 days
- 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: Duplicate

NUMBER OF CELLS EVALUATED: No data

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

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

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

His+ revertant colonies on the plates were counted

Statistics:

No data

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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 effect were observed

Any other information on results incl. tables

Table: Ames test data

Agent	Dose (µg/plate)	His+ revertant colonies/plate
		TA98				TA100
		-		+		-		+
		1	2	1	2	1	2	1	2
Crystal violet	0	11.0	20.0	88.5	30.5	158.5	160.5	127.5	161.5
	0.1	19.0	15.0	43.5	30.5	140.0	146.0	168.0	140.0
	1.0.	14.0	20.5	40.5	31.0	87.0	130.0	154.0	146.5
	5.0	8.5	0	54.0	35.0	0	0	186.5	200.5
	10.0	1.0	0	38.5	28.0	0	0	218.5	260.0
	50.0	0	0	39.5	32.0	0	0	165.0	155.0
Positive control	5.0	22.0	25.5	1898.1	3374.3	201.5	-	2148.6	2313.9
	10.0	-	-	-	-	-	-	-	-

 

Agent	Dose (µg/plate)	His+ revertant colonies/plate
		TA1535				TA1537
		-		+		-		+
		1	2	1	2	1	2	1	2
Crystal violet	0	31.5	32.5	19.0	21.5	4.5	10.0	7.5	6.0
	0.1	29.0	36.5	18.0	22.0	5.5	4.0	11.0	9.5
	1.0.	27.5	36.0	10.0	23.5	9.0	10.0	8.5	10.0
	5.0	2.0	0	16.0	25.5	0	0	12.0	16.0
	10.0	1.5	0	17.0	29.5	0	0	14.0	14.0
	50.0	0	0	10.5	16.0	0	0	14.0	14.0
Positive control	5.0	21.0	25.5	-	1489.2	-	-	-	-
	10.0	-	-	-	-	28.5	42.5	289.2	380.5

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

Ames mutagenicity assay was performed to study the mutagenic potential of gentian violet both in the presence and absence of metabolic activator S9 mix. The study was performed using Salmonella typhimurium TA98, TA100, TA1535, TA1537 at dose levels of 0, 0.1, 1.0, 10., 25.0 or 50.0 µg/plate. The test chemical was dissolved in DMSO and used for the study. Plates were incubated in dark at 37°C for 48 hrs before counting his⁺ revertant colonies and each dose point was determined from at least two plates. Gentian violet did not induce gene mutagenicity in the Salmonella typhimurium TA98, TA100, TA1537 and TA1535 strains in the presence and absence of S9 mix and hence it is not likely to classify as a gene mutant in vitro.