1,4-bis[(2-methylphenyl)amino]anthraquinone

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:

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:

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 to determine the mutagenic nature of crystal 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:

C25H30ClN3

IUPAC Name:

N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium 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.ClMolecular 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:

Details on test material
- Name of test material: Crystal 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, other: 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 was prepared from male Sprague Dawley rats pretreated with Aroclor 1254

Test concentrations with justification for top dose:

0, 0.1, 0.32, 1.0 or 3.2 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

Remarks:

Pooled data

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

other: β- propiolactone

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

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

Criteria for mutagenicity were (a) a dose-response and, (b) reproducibility of the result.

Statistics:

No data

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA98, TA100, 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

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

Table: Experimental controls

Agent	Dose (µg/plate)	S9	His^+revertant colonies/plate
			TA98	TA100	TA1535	TA1537	TA1538
Positive control: 2-acetylaminofluorene	1000	-	34±6	144±6	31±9	7±2	26±5
		+	10900±1344	2649±223	38±8	61±10	6232±259
	320	-	25±4	144±10	34±14	11±2	23±4
		+	8550±1047	4102±453	42±6	63±6	7960±791
	100	-	29±4	150±5	45±15	8±2	25±2
		+	1728±108	746±118	24±4	25±7	1231±95
	32	-	29±6	150±5	45±15	8±2	25±2
		+	1728±108	746±118	24±4	24±7	1231±95
Positive control:βpropiolactone	1000	-	28±3	150±5	45±16	8±2	25±2
		+	44±11	1979±457	1585±108	53±26	26±6
	320	-	28±2	764±224	642±126	12±1	24±3
		+	54±18	1297±260	646±405	17±2	29±8
	100	-	28±2	764±224	642±126	12±1	24±3
		+	49±12	640±146	355±102	14±6	33±15
	32	-	25±1	222±15	116±14	12±1	15±8
		+	43±18	212±42	119±29	16±3	30±9
Negative control (pooled data)	0	-	31±6	159±28	40±11	12±3	22±5
		+	51±10	137±16	22±5	16±4	37±8

 

Table: Ames test data

Agent	Dose (µg/plate)	His^+revertant colonies/plate
		TA98		TA100		TA1535		TA1537		TA1538
		-	+	-	+	-	+	-	+	-	+
Crystal violet	32	25	48	T	159	88	19	17	16	18	22
	1.0	35	51	82	145	96	18	16	13	17	20
	0.32	27	41	100	137	187	28	15	13	19	25
	0.1	33	43	108	140	61	20	10	13	18	21
	0	33	50	106	150	52	23	15	16	20	24

T: Toxicity

Applicant's summary and conclusion

Conclusions:

Crystal violet did not induce gene mutagenicity in the Salmonella typhimurium TA98, TA100, TA1537 and TA1538 strains in the presence and absence of S9 mix and in strain TA1535 in the presence of S9 mix. It however induced gene mutation in the strain TA1535 at low concentration in the absence of S9 mix and the response was not dose related. Hence the test chemical is considered to be negative for gene mutation as per the criteria mentioned in CLP regulation.

Executive summary:

Salmonella / mammalian microsome mutagenicity assay was performed to study the mutagenic potential of Crystal violet both in the presence and absence of metabolic activator S9 mix. The study was performed using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538 at dose levels of0, 0.1, 0.32, 1.0 or 3.2µg/plate. The test chemical was dissolved in DMSO and used for the study.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. Crystal violet did not induce gene mutagenicity in the Salmonella typhimurium TA98, TA100, TA1537 and TA1538 strains in the presence and absence of S9 mix and in strain TA1535 in the presence of S9 mix. It however induced gene mutation in the strain TA1535 at low concentration in the absence of S9 mix and the response was not dose related. Hence the test chemical is considered to be negative for gene mutation as per the criteria mentioned in CLP regulation.