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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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 usingSalmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538 at dose levels of 0, 0.1, 0.32, 1.0 or 3.2 µg/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 concentrations selected for initial testing. Crystal violet did not induce gene mutagenicity in theSalmonella 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.

Mammalian cell gene mutation assay was conducted to evaluate the genotoxic potential of Gentian violet. The study was performed using Chinese hamster ovary (CHO) cell strain CHO-K1-BH₄and CHO-AS52 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of0, 0.02, 0.04, 0.05, 0.06, 0.08, 0.10, 0.125, 0.25, 0.50, 1.00 µg/ml without S9 and 0, 0.10, 0.20, 0.25, 0.30, 0.40, 0.50, 1.00, 1.50 µg/ml with S9. 1X 10⁶cells/100 mm dish were exposed to the test chemical for 5 hrs in serum-free nutrient mixture F12. After treatment, cells were washed with Ca+ +/Mgt +-free phosphate-buffered saline (PBS) and allowed to recover overnight in fresh F12 medium with 5% fetal bovine serum (FBS). The cells were then plated in F12 medium with 5% FBS and incubated for 7 days with three passages to permit expression of induced mutants. Parallel cultures of 400 cells/60-mm dish were also established for cytotoxicity determination. At the end of the phenotypic expression period, 2 X 10⁶cells were plated at a concentration of 2 X 10⁵celIs/ 100-mm dish in hypoxanthine-free F12 containing 5% dialyzed FBS and 10 µM 6-thioguanine. Cloning efficiency cultures, grown in medium without 6-thioguanine, had 200 cells/60-mm plate. All cultures were incubated for 7 days before colonies were fixed, stained and counted. Results were expressed as 6-thioguanine resistant mutants/10⁶clonable cells. At the concentrations tested, the results were negative in CHO-K1-BH4 cells, while 66 mutants per 10⁶clonable cells were obtained in AS52 cells at a GV concentration producing a high level of toxicity. This positive response, however, was not consistently found in subsequent experiments. Toxicity was apparent in CHO cells, both with and without metabolic activation, although it was more evident in assays conducted without S9. GV-treated cells, especially AS52 cells, became tenaciously attached to the plates such that it required longer trypsinization times to dissociate them during subculture. When observed with an inverted microscope, the exposed cells appeared larger and more spindle-shaped than control cells. Gentian violet did not induce gene mutation in Chinese hamster ovary (CHO) cell strain CHO-K1-BH₄and CHO-AS52 in the presence and absence of S9 metabolic activation system and hence the test chemical is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

Link to relevant study records

Reference

Reference 1

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

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

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 %

Target gene:

Histidine

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

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

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

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Mammalian cell gene mutation assay was conducted to evaluate the genotoxic potential of Gentian violet

GLP compliance:

not specified

Type of assay:

other: Mammalian cell gene mutation assay

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 %

Target gene:

hprt locus in CHO-K1-BH4 cells and the gpt locus in AS52 cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1-BH4 and CHO-AS52

Details on mammalian cell type (if applicable):

- Type and identity of media: No data
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

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 Arocolor 1254

Test concentrations with justification for top dose:

Without S9: 0, 0.02, 0.04, 0.05, 0.06, 0.08, 0.10, 0.125, 0.25, 0.50, 1.00 µg/ml

With S9: 0, 0.10, 0.20, 0.25, 0.30, 0.40, 0.50, 1.00, 1.50 µg/ml

Vehicle / solvent:

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

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
Cells at the strat of experiment: 1 X 106 cells/100-mm dish

DURATION
- Preincubation period: No data
- Exposure duration: 5 hrs
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days

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: Yes, Parallel cultures
of 400 cells/60-mm dish were established for cytotoxicity determination

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:

6-thioguanine resistant mutants/106 clonable cells were noted

Statistics:

No data

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1-BH4 and CHO-AS52

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was apparent in CHO cells, both with and without metabolic activation, although it was more evident in assays conducted without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No data

TABLE. Mutagenicity and Cytotoxicity of Gentian Violet in CHO-Kl-BH, and CHO-AS52 Cells

Compound	Dose (µg/mL)	Cloning efficiency		Mutants/10^6cells
Gentian violet (-S9)	0	100	100	17	14
	0.02	131	144	20	15
	0.04	133	98	23	27
	0.05	96	-	8	-
	0.06	85	80	0	7
	0.08	53	13	3	6
	0.10	25	13	2	<1
	0.125	11	4	6	66
	0.25	2	-	-	-
	0.50	<0.9	-	-	-
	1.00	<0.5	-	-	-
Gentian violet (+S9)	0	100	100	1	27
	0.10	-	101	-	20
	0.20	-	84	-	12
	0.25	88	-	<1	-
	0.30	-	62	-	13
	0.40	-	48	-	21
	0.50	28	22	5	36
	1.00	23	-	7	-
	1.50	20	-	2	-
Benzo(a)pyrene (+S9)	5.00	35	34	419	219

Conclusions:

Gentian violet did not induce gene mutation in Chinese hamster ovary (CHO) cell strain CHO-K1-BH4 and CHO-AS52 in the presence and absence of S9 metabolic activation system and hence the test chemical is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

Executive summary:

Mammalian cell gene mutation assay was conducted to evaluate the genotoxic potential of Gentian violet. The study was performed using Chinese hamster ovary (CHO) cell strain CHO-K1-BH₄and CHO-AS52 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of0, 0.02, 0.04, 0.05, 0.06, 0.08, 0.10, 0.125, 0.25, 0.50, 1.00 µg/ml without S9 and 0, 0.10, 0.20, 0.25, 0.30, 0.40, 0.50, 1.00, 1.50 µg/ml with S9. 1X 10⁶cells/100 mm dish were exposed to the test chemical for 5 hrs in serum-free nutrient mixture F12. After treatment, cells were washed with Ca+ +/Mgt +-free phosphate-buffered saline (PBS) and allowed to recover overnight in fresh F12 medium with 5% fetal bovine serum (FBS). The cells were then plated in F12 medium with 5% FBS and incubated for 7 days with three passages to permit expression of induced mutants. Parallel cultures of 400 cells/60-mm dish were also established for cytotoxicity determination. At the end of the phenotypic expression period, 2 X 10⁶cells were plated at a concentration of 2 X 10⁵celIs/100-mm dish in hypoxanthine-free F12 containing 5% dialyzed FBS and 10 µM 6-thioguanine. Cloning efficiency cultures, grown in medium without 6-thioguanine, had 200 cells/60-mm plate. All cultures were incubated for 7 days before colonies were fixed, stained and counted. Results were expressed as 6-thioguanine resistant mutants/10⁶clonable cells.

 

At the concentrations tested, the results were negative in CHO-K1-BH4 cells, while 66 mutants per 10⁶clonable cells were obtained in AS52 cells at a GV concentration producing a high level of toxicity. This positive response, however, was not consistently found in subsequent experiments. Toxicity was apparent in CHO cells, both with and without metabolic activation, although it was more evident in assays conducted without S9. GV-treated cells, especially AS52 cells, became tenaciously attached to the plates such that it required longer trypsinization times to dissociate them during subculture. When observed with an inverted microscope, the exposed cells appeared larger and more spindle-shaped than control cells.

 

Gentian violet did not induce gene mutation inChinese hamster ovary (CHO) cell strain CHO-K1-BH₄and CHO-AS52 in the presence and absence of S9 metabolic activation system and hence the test chemical is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Nucleoid Sedimentation Analysis was performed in vivo to determine the DNA damaging effect of Gentian violet. The study was performed using B6C3F₁mice. The animals were pretreated for 1 hour with 0- 10µg/mL Gentian Violet administered by injection into the tail-vein. The spleen lymphocytes were isolated from the treated animals by lympho-plaque centrifugation. Animals treated with MMS were used as positive controls.The position of nucleoids generated was analyzed and results were expressed as the ratio of the distance traveled by nucleoids in the sample gradients to the reference gradient. No DNA damage was detected by nucleoid sedimentation analysis of lymphocytes isolated from B6C3F1 mice exposed to different concentrations of Gentian Violet and hence the test chemical does not exhibit gene mutation in vivo.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

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

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Nucleoid Sedimentation Analysis was performed in vivo to determine the DNA damaging effect of Gentian violet

GLP compliance:

not specified

Type of assay:

other: Nucleoid Sedimentation Analysis Assay

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 %

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

No data

Sex:

not specified

Details on test animals or test system and environmental conditions:

No data

Route of administration:

intravenous

Vehicle:

Vehicles
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
- Concentration of test material in vehicle: 0- 10 µg/mL
- Amount of vehicle (if gavage or dermal): No data
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data

Details on exposure:

Not applicable

Duration of treatment / exposure:

Duration of exposure: 1 hour

Frequency of treatment:

No data

Post exposure period:

No data

Remarks:

10 µg/mL

No. of animals per sex per dose:

No data

Control animals:

yes

Positive control(s):

Methylmethanesulphonate
- Route of administration: No data
- Doses / concentrations: No data

Tissues and cell types examined:

Spleen lymphocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: No data

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): No data

DETAILS OF SLIDE PREPARATION: No data

METHOD OF ANALYSIS: The position of nucleoids generated was analyzed

OTHER: Cells were washed with PBS and resuspended in PBS at a concentration of 2 to 10 X 106 cells/ml. A 50 µL sample was then added to 150 µL of lysing solution (2.5 M NaCl, 0.133 M EDTA, 2.67 mM Tris, and 0.67% Triton X-100, pH 8.0) layered on top of a 15-30% neutral (pH 8.0) sucrose gradient containing 1.95 M NaCl, 0.01 M Tris, 0.001 M EDTA, and 0.1 kg/ml Hoechst 33258 dye. Following a 30-minute lysis in the dark at room temperature, the gradients were centrifuged at 25,000 rpm for 75 minutes at 20°C in an SW41 rotor. Six gradients were run per rotor with one gradient serving as a reference. The position of the nucleoids in the gradients was visually determined with a UV light.

Evaluation criteria:

The position of nucleoids generated was analyzed and results were expressed as the ratio of the distance traveled by nucleoids in the sample gradients to the reference gradient

Statistics:

No data

Sex:

not specified

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Concentration more than 10 µg/mL were fatal to the mice during the 1 hour treatment period

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No data

Conclusions:

No DNA damage was detected by nucleoid sedimentation analysis of lymphocytes isolated from B6C3F1 mice exposed to different concentrations of Gentian Violet.

Executive summary:

Nucleoid Sedimentation Analysis was performed in vivo to determine the DNA damaging effect of Gentian violet. The study was performed using B6C3F₁mice. The animals were pretreated for 1 hour with 0- 10µg/mL Gentian Violet administered by injection into the tail-vein. The spleen lymphocytes were isolated from the treated animals by lympho-plaque centrifugation. Animals treated with MMS were used as positive controls.The position of nucleoids generated was analyzed and results were expressed as the ratio of the distance traveled by nucleoids in the sample gradients to the reference gradient. No DNA damage was detected by nucleoid sedimentation analysis of lymphocytes isolated from B6C3F1 mice exposed to different concentrations of Gentian Violet and hence the test chemical does not exhibit gene mutation in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Data available for the target chemical Gentian violet was reviewed to determine the mutagenic nature both in vitro and in vivo. The summary is as mentioned below:

Gene mutation in vitro:

Salmonella / mammalian microsome mutagenicity assay was performed by Bonin et al (Mutation Research, 1981) 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 of 0, 0.1, 0.32, 1.0 or 3.2 µg/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 concentrations 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.

In another study by Aidoo et al ( Teratogenesis. Carcinogenesis and Mutagenesis, 1990) Mammalian cell gene mutation assay was conducted to evaluate the genotoxic potential of Gentian violet. The study was performed using Chinese hamster ovary (CHO) cell strain CHO-K1-BH₄and CHO-AS52 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of0, 0.02, 0.04, 0.05, 0.06, 0.08, 0.10, 0.125, 0.25, 0.50, 1.00 µg/ml without S9 and 0, 0.10, 0.20, 0.25, 0.30, 0.40, 0.50, 1.00, 1.50 µg/ml with S9. 1X 10⁶cells/100 mm dish were exposed to the test chemical for 5 hrs in serum-free nutrient mixture F12. After treatment, cells were washed with Ca+ +/Mgt +-free phosphate-buffered saline (PBS) and allowed to recover overnight in fresh F12 medium with 5% fetal bovine serum (FBS). The cells were then plated in F12 medium with 5% FBS and incubated for 7 days with three passages to permit expression of induced mutants. Parallel cultures of 400 cells/60-mm dish were also established for cytotoxicity determination. At the end of the phenotypic expression period, 2 X 10⁶cells were plated at a concentration of 2 X 10⁵celIs/ 100-mm dish in hypoxanthine-free F12 containing 5% dialyzed FBS and 10 µM 6-thioguanine. Cloning efficiency cultures, grown in medium without 6-thioguanine, had 200 cells/60-mm plate. All cultures were incubated for 7 days before colonies were fixed, stained and counted. Results were expressed as 6-thioguanine resistant mutants/10⁶clonable cells.   At the concentrations tested, the results were negative in CHO-K1-BH4 cells, while 66 mutants per 10⁶clonable cells were obtained in AS52 cells at a GV concentration producing a high level of toxicity. This positive response, however, was not consistently found in subsequent experiments. Toxicity was apparent in CHO cells, both with and without metabolic activation, although it was more evident in assays conducted without S9. GV-treated cells, especially AS52 cells, became tenaciously attached to the plates such that it required longer trypsinization times to dissociate them during subculture. When observed with an inverted microscope, the exposed cells appeared larger and more spindle-shaped than control cells. Gentian violet did not induce gene mutation in Chinese hamster ovary (CHO) cell strain CHO-K1-BH₄and CHO-AS52 in the presence and absence of S9 metabolic activation system and hence the test chemical is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

In the same study by Aidoo et al (1990), Mutagenic study was conducted to evaluate the genotoxic potential of Gentian violet using bacterial system. The study was performed using Salmonella typhimurium strain TA97, TA98,TA100, and TA104 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of 0, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 or 10.0µg/plate. The study was performed as per the plate incorporation assay for a duration of 48 hrs. The plates were observed for a dose dependent increase in the number of revertants/plate.Gentian violet induced gene mutation in strains TA98 in the presence and absence of S9 metabolic activation system and in strain TA104 with S9. Weak mutagenic effects were observed for the strain TA100. No mutagenic activity could be detected in strain TA98 and in strain TA104 without S9.

Ames mutagenicity assay was performed by Au et al (Mutation Research, 1979) 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.

Gene mutation in vivo:

Nucleoid Sedimentation Analysis was performed by Aidoo et al ( Teratogenesis, Carcinogenesis, and Mutagenesis, 1990) in vivo to determine the DNA damaging effect of Gentian violet. The study was performed using B6C3F₁mice. The animals were pretreated for 1 hour with 0- 10µg/mL Gentian Violet administered by injection into the tail-vein. The spleen lymphocytes were isolated from the treated animals by lympho-plaque centrifugation. Animals treated with MMS were used as positive controls.The position of nucleoids generated was analyzed and results were expressed as the ratio of the distance traveled by nucleoids in the sample gradients to the reference gradient. No DNA damage was detected by nucleoid sedimentation analysis of lymphocytes isolated from B6C3F1 mice exposed to different concentrations of Gentian Violet and hence the test chemical does not exhibit gene mutation in vivo.

In another study by Mason et al ( Environmental and Molecular Mutagenesis, 1992), Drosophila SLRL test was performed in vivo to determine the gene mutation ability of Gentian violet. Test chemical was dissolved in water at dose levels of 0 or 500 ppm and fed to Canton S males by the oral feed route of exposure. Males to be injected were held on regular food for 1-3 days before treatment. They were injected with about 0.2-0.3µLof the test solution, held 24 h to recover and were then mated. In the SLRL assay, treated and control males were mated individually to three harems of virgin Basc females to produce broods of 3, 2, and 2 days. Thus, the great majority of germ cells tested were at post-meiotic stages at the time of treatment. Daughters of the treated and control males were allowed to mate with their brothers, then distributed to individual vials. To reduce the chance of recovering more than one lethal from a single male, no more than 40 females were mated from each brood of each male. Thus, no more than 120 chromosomes were tested from each P1 male. F2 cultures were scored as presumptive lethal-bearing if the number of wildtype males recovered was 0, 1 or fewer than 5% of the number of Basc males (or heterozygous Basc females). Presumptive lethals were confirmed by repeating the matings. Lethal-bearing cultures are defined as containing fewer than 5% of the expected number of wild type males when at least 20 Basc males or heterozygous Basc females are present. A chemically induced increase in the SLRL frequency of 0.2% over a background of 0.08% with a power of 75% was noted. Gentian Violet did not induce SLRL mutation in Drosophila melanogaster and hence it is not likely to classify as a gene mutant in vitro.

In the same study by Mason et al (1992), Drosophila SLRL test was performed in vivo to determine the gene mutation ability of Gentian violet. Test chemical was dissolved in water at dose levels of 0 or 500 ppm and fed to Canton S males by the oral feed route of exposure. Males to be fed were transferred into vials containing glass fiber filter material that was soaked with the feeding solution. At 24 and 48 h the flies were transferred to vials with freshly prepared feeding solution. At 72 h the males were removed from the feeding solution and mated. In the SLRL assay, treated and control males were mated individually to three harems of virgin Basc females to produce broods of 3, 2, and 2 days. Thus, the great majority of germ cells tested were at post-meiotic stages at the time of treatment. Daughters of the treated and control males were allowed to mate with their brothers, then distributed to individual vials. To reduce the chance of recovering more than one lethal from a single male, no more than 40 females were mated from each brood of each male. Thus, no more than 120 chromosomes were tested from each P1 male. F2 cultures were scored as presumptive lethal-bearing if the number of wildtype males recovered was 0, 1 or fewer than 5% of the number of Basc males (or heterozygous Basc females). Presumptive lethals were confirmed by repeating the matings. Lethal-bearing cultures are defined as containing fewer than 5% of the expected number of wild type males when at least 20 Basc males or heterozygous Basc females are present. A chemically induced increase in the SLRL frequency of 0.2% over a background of 0.08% with a power of 75% was noted. Gentian Violet did not induce SLRL mutation in Drosophila melanogaster and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available for the target chemical and its read across, Gentian violet did not induce gene mutation in vitro and in vivo. Hence the test chemical is not likely to classify as a gene mutant in vitro and in vivo as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the target chemical and its read across, Gentian violet did not induce gene mutation in vitro and in vivo. Hence the test chemical is not likely to classify as a gene mutant in vitro and in vivo as per the criteria mentioned in CLP regulation.