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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data is from SCCS report

Data source

Reference
Reference Type:
secondary source
Title:
Opinion on Basic Violet 2 Colipa N° B115
Author:
Scientific Committee on Consumer Safety
Year:
2011
Bibliographic source:
European Commission, SCCS/1340/10, 2011

Materials and methods

Test guideline
Qualifier:
according to
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 Basic violet 2
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Basic Violet 2
- IUPAC name: 4,4'-[(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methylene]bis(2-methylaniline) hydrochloride
- Molecular formula: C22H23N3ClH
- Molecular weight: 365.906 g/mol
- Substance type: Organic
- Physical state: No data
Specific details on test material used for the study:
- Name of test material: Basic Violet 2
- IUPAC name: 4,4'-[(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methylene]bis(2-methylaniline) hydrochloride
- Molecular formula: C22H23N3ClH
- Molecular weight: 365.906 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: HPLC: 94.2%; NMR: 96.8%
- Impurities (identity and concentrations): No data

Method

Target gene:
Histidine
Species / strain
Species / strain:
other: TA98, TA100, TA102, TA1535, TA1537
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 fraction from Aroclor 1254-induced rats was used as exogenous metabolic activation system.
Test concentrations with justification for top dose:
Experiment 1: 1, 10, 100, 300, 1000 and 5000 μg/plate with and without S9-mix

Experiment 2: 3, 10, 30, 100, 300, 1000 and 3000 μg/plate with and without S9-mix
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Controls
Negative controls:
yes
Remarks:
in accordance with the OECD guideline
Solvent controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Remarks:
in accordance with the OECD guideline
Positive control substance:
not specified
Details on test system and conditions:
Details on test system and conditions
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 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: triplicates in two independent experiments

NUMBER OF CELLS EVALUATED: No data

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

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

OTHER: No data

Rationale for test conditions:
No data
Evaluation criteria:
The plates were observed for a dose dependent doubling of an increase in the number of revertants
Statistics:
No data

Results and discussion

Test results
Species / strain:
other: TA98, TA100, TA102, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: No mutagenic potential
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
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: No data

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: Cytotoxicity observed as growth inhibition and clearing of the background lawn was found in the first experiment in the absence of S9-mix for TA98 and TA102 (1000 and 5000 μg/plate), TA100, TA1535 and TA1537 (100, 1000 and 5000 μg/plate); in the presence of S9-mix for TA98 (5000 μg/plate), TA100 and TA1535 (100, 1000 and 5000 μg/plate) and TA1537 and TA102 (1000 and 5000 μg/plate). This toxicity was observed also in the second experiment.

Applicant's summary and conclusion

Conclusions:
Basic violet 2 did not induce gene mutation in Salmonella typhimurium strains TA98, TA100, TA102, TA1535, TA1537 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Ames assay was performed to determine the mutagenic nature of Basic violet 2. The study was performed using Salmonella typhimurium strains TA98, TA100, TA102, TA1535, TA1537 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of 1, 10, 100, 300, 1000 and 5000 μg/plate in experiment 1 and 3, 10, 30, 100, 300, 1000 and 3000 μg/plate in experiment 2. Direct plate incorporation method with 48 h incubation without and with S9-mix was performed. Toxicity observed as growth inhibition, clearing of the background lawn and strong reduction in the number of spontaneous revertants, was found in the first experiment in the absence of S9-mix for TA98 and TA102 (1000 and 5000 μg/plate), TA100, TA1535 and TA1537 (100, 1000 and 5000 μg/plate); in the presence of S9-mix for TA98 (5000 μg/plate), TA100 and TA1535 (100, 1000 and 5000 μg/plate) and TA1537 and TA102 (1000 and 5000 μg/plate). This toxicity was observed also in the second experiment. An increase in the number of revertants was observed on TA102 strain only in the presence of S9-mix at non-toxic concentrations. However, the effect never reached a factor of 2 compared to the control. This effect was therefore not considered biologically relevant. Under the experimental conditions used Basic Violet 2 was not mutagenic in this gene mutation tests in bacteria.