Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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

Constituent 1
Chemical structure
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 resultsopen allclose all
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

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.