Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, gene mutation was predicted for Basic violet 2. The study assumed the use of Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 with S9 metabolic activation system. Basic violet 2 was predicted to not induce gene mutation in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence of S9 metabolic activation system and hence, according to the prediction made, it is not likely to classify as a gene mutant in vitro.

Based on the predicted result it can be concluded that the substance is considered to be not toxic as per the criteria mentioned in CLP regulation.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification
Justification for type of information:
Data is from OECD QSAR Toolbox version 3.3 and the supporting QMRF report has been attached
Reference:
Composition 1
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Prediction is done using OECD QSAR Toolbox version 3.3, 2017
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
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: No data
- Impurities (identity and concentrations): No data
Target gene:
Histidine
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with
Metabolic activation system:
S9 metabolic activation system
Test concentrations with justification for top dose:
No data
Vehicle:
No data
Negative controls:
not specified
Solvent controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and conditions:
No data
Rationale for test conditions:
No data
Evaluation criteria:
Prediction is done considering a dose dependent increase in the number of revertants/plate
Statistics:
No data
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Additional information on results:
No data

The prediction was based on dataset comprised from the following descriptors: "Gene mutation"
Estimation method: Takes highest mode value from the 5 nearest neighbours
Domain  logical expression:Result: In Domain

((((((((((("a" or "b" or "c" or "d" or "e" )  and ("f" and ( not "g") )  )  and ("h" and ( not "i") )  )  and ("j" and ( not "k") )  )  and "l" )  and "m" )  and "n" )  and "o" )  and "p" )  and "q" )  and ("r" and "s" )  )

Domain logical expression index: "a"

Referential boundary: The target chemical should be classified as Anilines (Acute toxicity) AND Dianilines AND Not categorized AND Triarylmethane Pigments/Dyes with Non-solubilizing Groups by US-EPA New Chemical Categories

Domain logical expression index: "b"

Referential boundary: The target chemical should be classified as Anilines (Hindered) AND Inorganic Compound by Aquatic toxicity classification by ECOSAR

Domain logical expression index: "c"

Referential boundary: The target chemical should be classified as Alkene OR Alkyl arenes OR Allyl OR Aniline OR Aryl OR Dianilines OR Ketimine OR No functional group found OR Precursors quinoid compounds by Organic Functional groups ONLY

Domain logical expression index: "d"

Referential boundary: The target chemical should be classified as Alkene OR Alkyl arenes OR Allyl OR Dianilines OR Ketimine OR No functional group found OR Overlapping groups OR Precursors quinoid compounds by Organic Functional groups (nested) ONLY

Domain logical expression index: "e"

Referential boundary: The target chemical should be classified as Aliphatic Carbon [CH] OR Aliphatic Carbon [-CH2-] OR Aliphatic Carbon [-CH3] OR Aliphatic Nitrogen, one aromatic attach [-N] OR Aromatic Carbon [C] OR No functional group found OR Olefinic carbon [=CH- or =C<] by Organic functional groups (US EPA) ONLY

Domain logical expression index: "f"

Referential boundary: The target chemical should be classified as No alert found AND SN1 AND SN1 >> Nitrenium Ion formation AND SN1 >> Nitrenium Ion formation >> Primary aromatic amine by DNA binding by OECD

Domain logical expression index: "g"

Referential boundary: The target chemical should be classified as Michael addition OR Michael addition >> P450 Mediated Activation to Quinones and Quinone-type Chemicals OR Michael addition >> P450 Mediated Activation to Quinones and Quinone-type Chemicals >> Alkyl phenols OR Michael addition >> P450 Mediated Activation to Quinones and Quinone-type Chemicals >> Arenes OR Michael addition >> P450 Mediated Activation to Quinones and Quinone-type Chemicals >> Hydroquinones OR Michael addition >> P450 Mediated Activation to Quinones and Quinone-type Chemicals >> Polycyclic (PAHs) and heterocyclic (HACs) aromatic hydrocarbons-Michael addition OR SN1 >> Carbenium Ion Formation OR SN1 >> Carbenium Ion Formation >> Allyl benzenes OR SN1 >> Carbenium Ion Formation >> Hydrazine OR SN1 >> Carbenium Ion Formation >> Polycyclic (PAHs) and heterocyclic (HACs) aromatic hydrocarbons-SN1 OR SN1 >> Nitrenium Ion formation >> Aromatic nitroso OR SN1 >> Nitrenium Ion formation >> Primary (unsaturated) heterocyclic amine OR SN1 >> Nitrenium Ion formation >> Secondary aromatic amine OR SN1 >> Nitrenium Ion formation >> Tertiary aromatic amine OR SN2 OR SN2 >> Direct Acting Epoxides and related OR SN2 >> Direct Acting Epoxides and related >> Epoxides OR SN2 >> Direct Acting Epoxides and related >> Sulfuranes by DNA binding by OECD

Domain logical expression index: "h"

Referential boundary: The target chemical should be classified as No alert found AND Non-specific AND Non-specific >> Incorporation into DNA/RNA, due to structural analogy with  nucleoside bases    AND Non-specific >> Incorporation into DNA/RNA, due to structural analogy with  nucleoside bases    >> Specific Imine and Thione Derivatives AND Radical AND Radical >> Radical mechanism via ROS formation (indirect) AND Radical >> Radical mechanism via ROS formation (indirect) >> Specific Imine and Thione Derivatives AND SN1 AND SN1 >> Nucleophilic substitution on diazonium ions AND SN1 >> Nucleophilic substitution on diazonium ions >> Specific Imine and Thione Derivatives by DNA binding by OASIS v.1.3

Domain logical expression index: "i"

Referential boundary: The target chemical should be classified as AN2 OR AN2 >> Carbamoylation after isocyanate formation OR AN2 >> Carbamoylation after isocyanate formation >> N-Hydroxylamines OR Non-covalent interaction OR Non-covalent interaction >> DNA intercalation OR Non-covalent interaction >> DNA intercalation >> Aminoacridine DNA Intercalators OR Non-covalent interaction >> DNA intercalation >> Fused-Ring Primary Aromatic Amines OR Radical >> Generation of reactive oxygen species OR Radical >> Generation of reactive oxygen species >> Thiols OR Radical >> Radical mechanism via ROS formation (indirect) >> C-Nitroso Compounds OR Radical >> Radical mechanism via ROS formation (indirect) >> Fused-Ring Primary Aromatic Amines OR Radical >> Radical mechanism via ROS formation (indirect) >> N-Hydroxylamines OR Radical >> Radical mechanism via ROS formation (indirect) >> p-Aminobiphenyl Analogs OR Radical >> Radical mechanism via ROS formation (indirect) >> Single-Ring Substituted Primary Aromatic Amines OR SN1 >> Alkylation after metabolically formed carbenium ion species OR SN1 >> Alkylation after metabolically formed carbenium ion species >> Polycyclic Aromatic Hydrocarbon Derivatives OR SN1 >> Nucleophilic attack after metabolic nitrenium ion formation OR SN1 >> Nucleophilic attack after metabolic nitrenium ion formation >> Fused-Ring Primary Aromatic Amines OR SN1 >> Nucleophilic attack after metabolic nitrenium ion formation >> N-Hydroxylamines OR SN1 >> Nucleophilic attack after metabolic nitrenium ion formation >> p-Aminobiphenyl Analogs OR SN1 >> Nucleophilic attack after metabolic nitrenium ion formation >> Single-Ring Substituted Primary Aromatic Amines OR SN1 >> Nucleophilic substitution after glutathione-induced nitrenium ion formation OR SN1 >> Nucleophilic substitution after glutathione-induced nitrenium ion formation >> C-Nitroso Compounds OR SN2 OR SN2 >> Alkylation, direct acting epoxides and related after P450-mediated metabolic activation OR SN2 >> Alkylation, direct acting epoxides and related after P450-mediated metabolic activation >> Polycyclic Aromatic Hydrocarbon Derivatives by DNA binding by OASIS v.1.3

Domain logical expression index: "j"

Referential boundary: The target chemical should be classified as Non binder, non cyclic structure AND Strong binder, NH2 group by Estrogen Receptor Binding

Domain logical expression index: "k"

Referential boundary: The target chemical should be classified as Moderate binder, NH2 group OR Non binder, impaired OH or NH2 group OR Non binder, MW>500 OR Non binder, without OH or NH2 group OR Strong binder, OH group OR Weak binder, NH2 group OR Weak binder, OH group by Estrogen Receptor Binding

Domain logical expression index: "l"

Referential boundary: The target chemical should be classified as Bioavailable by Lipinski Rule Oasis ONLY

Domain logical expression index: "m"

Referential boundary: The target chemical should be classified as Alkene AND Alkyl arenes AND Allyl AND Aniline AND Aryl AND Dianilines AND Ketimine AND No functional group found AND Precursors quinoid compounds by Organic Functional groups ONLY

Domain logical expression index: "n"

Referential boundary: The target chemical should be classified as Alkene AND Alkyl arenes AND Allyl AND Dianilines AND Ketimine AND No functional group found AND Overlapping groups AND Precursors quinoid compounds by Organic Functional groups (nested) ONLY

Domain logical expression index: "o"

Referential boundary: The target chemical should be classified as Aliphatic Carbon [CH] AND Aliphatic Carbon [-CH2-] AND Aliphatic Carbon [-CH3] AND Aliphatic Nitrogen, one aromatic attach [-N] AND Aromatic Carbon [C] AND No functional group found AND Olefinic carbon [=CH- or =C<] by Organic functional groups (US EPA) ONLY

Domain logical expression index: "p"

Referential boundary: The target chemical should be classified as Amine AND Aromatic compound AND No functional group found AND Primary amine AND Primary aromatic amine by Organic functional groups, Norbert Haider (checkmol) ONLY

Domain logical expression index: "q"

Similarity boundary:Target: CC1=CC(=C(c2ccc(N)c(C)c2)c2ccc(N)c(C)c2)C=CC1=N_Cl
Threshold=20%,
Dice(Atom centered fragments)
Atom type; Count H attached; Hybridization

Domain logical expression index: "r"

Parametric boundary:The target chemical should have a value of log Kow which is >= 1.77

Domain logical expression index: "s"

Parametric boundary:The target chemical should have a value of log Kow which is <= 3.29

Conclusions:
Basic violet 2 was predicted to not induce gene mutation in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence of S9 metabolic activation system and hence, according to the prediction made, it is not likely to classify as a gene mutant in vitro.
Executive summary:

Based on the prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, gene mutation was predicted for Basic violet 2. The study assumed the use of Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 with S9 metabolic activation system. Basic violet 2 was predicted to not induce gene mutation in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence of S9 metabolic activation system and hence, according to the prediction made, it is not likely to classify as a gene mutant in vitro.

Based on the predicted result it can be concluded that the substance is considered to be not toxic 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

Based on the prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, gene mutation was predicted for Basic violet 2. The study assumed the use of male Sprague Dawley rats. Basic Violet 2 was predicted to not induce gene mutation in male Sprague Dawley rats and hence, according to the prediction made, it is not likely to classify as a gene mutant in vivo.

Based on the predicted result it can be concluded that the substance is considered to not toxic as per the criteria mentioned in CLP regulation

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification
Justification for type of information:
Data is from OECD QSAR Toolbox version 3.3 and the supporting QMRF report has been attached
Reference:
Composition 1
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Prediction is done using OECD QSAR Toolbox version 3.3, 2017
GLP compliance:
not specified
Type of assay:
micronucleus assay
Test material information:
Composition 1
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%
- Impurities (identity and concentrations): No data
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
No data
Sex:
male
Details on test animals and environmental conditions:
No data
Route of administration:
oral: gavage
Vehicle:
No data
Details on exposure:
No data
Duration of treatment / exposure:
No data
Frequency of treatment:
No data
Post exposure period:
No data
Remarks:
No data
No. of animals per sex per dose:
No data
Control animals:
not specified
Positive control(s):
No data
Tissues and cell types examined:
No data
Details of tissue and slide preparation:
No data
Evaluation criteria:
No data
Statistics:
No data
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Additional information on results:
No data

The prediction was based on dataset comprised from the following descriptors: "chromosome aberration"
Estimation method: Takes mode value from the 7 nearest neighbours
Domain  logical expression:Result: In Domain

((((((((("a" or "b" or "c" or "d" or "e" )  and ("f" and ( not "g") )  )  and ("h" and ( not "i") )  )  and ("j" and ( not "k") )  )  and "l" )  and "m" )  and "n" )  and ("o" and ( not "p") )  )  and ("q" and "r" )  )

Domain logical expression index: "a"

Referential boundary: The target chemical should be classified as Anilines (Acute toxicity) AND Dianilines AND Not categorized AND Triarylmethane Pigments/Dyes with Non-solubilizing Groups by US-EPA New Chemical Categories

Domain logical expression index: "b"

Referential boundary: The target chemical should be classified as Amine OR Aromatic compound OR No functional group found OR Primary amine OR Primary aromatic amine by Organic functional groups, Norbert Haider (checkmol) ONLY

Domain logical expression index: "c"

Referential boundary: The target chemical should be classified as Aliphatic Carbon [CH] OR Aliphatic Carbon [-CH2-] OR Aliphatic Carbon [-CH3] OR Aliphatic Nitrogen, one aromatic attach [-N] OR Aromatic Carbon [C] OR No functional group found OR Olefinic carbon [=CH- or =C<] by Organic functional groups (US EPA) ONLY

Domain logical expression index: "d"

Referential boundary: The target chemical should be classified as Alkene OR Alkyl arenes OR Allyl OR Dianilines OR Ketimine OR No functional group found OR Overlapping groups OR Precursors quinoid compounds by Organic Functional groups (nested) ONLY

Domain logical expression index: "e"

Referential boundary: The target chemical should be classified as Alkene OR Alkyl arenes OR Allyl OR Aniline OR Aryl OR Dianilines OR Ketimine OR No functional group found OR Precursors quinoid compounds by Organic Functional groups ONLY

Domain logical expression index: "f"

Referential boundary: The target chemical should be classified as No alert found AND Non-specific AND Non-specific >> Incorporation into DNA/RNA, due to structural analogy with  nucleoside bases    AND Non-specific >> Incorporation into DNA/RNA, due to structural analogy with  nucleoside bases    >> Specific Imine and Thione Derivatives AND Radical AND Radical >> Radical mechanism via ROS formation (indirect) AND Radical >> Radical mechanism via ROS formation (indirect) >> Specific Imine and Thione Derivatives AND SN1 AND SN1 >> Nucleophilic substitution on diazonium ions AND SN1 >> Nucleophilic substitution on diazonium ions >> Specific Imine and Thione Derivatives by DNA binding by OASIS v.1.3

Domain logical expression index: "g"

Referential boundary: The target chemical should be classified as AN2 OR AN2 >> Schiff base formation OR AN2 >> Schiff base formation >> Dicarbonyl compounds OR SN1 >> Alkylation after metabolically formed carbenium ion species OR SN1 >> Alkylation after metabolically formed carbenium ion species >> Polycyclic Aromatic Hydrocarbon Derivatives OR SN2 OR SN2 >> Alkylation, direct acting epoxides and related OR SN2 >> Alkylation, direct acting epoxides and related >> Epoxides and Aziridines OR SN2 >> Alkylation, direct acting epoxides and related after P450-mediated metabolic activation OR SN2 >> Alkylation, direct acting epoxides and related after P450-mediated metabolic activation >> Polycyclic Aromatic Hydrocarbon Derivatives OR SN2 >> Direct acting epoxides formed after metabolic activation OR SN2 >> Direct acting epoxides formed after metabolic activation >> Quinoline Derivatives OR SN2 >> SN2 at an activated carbon atom OR SN2 >> SN2 at an activated carbon atom >> Quinoline Derivatives by DNA binding by OASIS v.1.3

Domain logical expression index: "h"

Referential boundary: The target chemical should be classified as No alert found AND SN1 AND SN1 >> Nitrenium Ion formation AND SN1 >> Nitrenium Ion formation >> Primary aromatic amine by DNA binding by OECD

Domain logical expression index: "i"

Referential boundary: The target chemical should be classified as Michael addition OR Michael addition >> P450 Mediated Activation to Quinones and Quinone-type Chemicals OR Michael addition >> P450 Mediated Activation to Quinones and Quinone-type Chemicals >> Alkyl phenols OR Michael addition >> P450 Mediated Activation to Quinones and Quinone-type Chemicals >> Arenes OR Michael addition >> P450 Mediated Activation to Quinones and Quinone-type Chemicals >> Hydroquinones by DNA binding by OECD

Domain logical expression index: "j"

Referential boundary: The target chemical should be classified as Non binder, non cyclic structure AND Strong binder, NH2 group by Estrogen Receptor Binding

Domain logical expression index: "k"

Referential boundary: The target chemical should be classified as Non binder, without OH or NH2 group OR Weak binder, NH2 group OR Weak binder, OH group by Estrogen Receptor Binding

Domain logical expression index: "l"

Referential boundary: The target chemical should be classified as Class 2 (less inert compounds) AND Class 5 (Not possible to classify according to these rules) by Acute aquatic toxicity classification by Verhaar (Modified) ONLY

Domain logical expression index: "m"

Referential boundary: The target chemical should be classified as Bioavailable by Lipinski Rule Oasis ONLY

Domain logical expression index: "n"

Referential boundary: The target chemical should be classified as Alkene AND Alkyl arenes AND Allyl AND Aniline AND Aryl AND Dianilines AND Ketimine AND No functional group found AND Precursors quinoid compounds by Organic Functional groups ONLY

Domain logical expression index: "o"

Referential boundary: The target chemical should be classified as Not categorized by OECD HPV Chemical Categories

Domain logical expression index: "p"

Referential boundary: The target chemical should be classified as Zinc metal and salts by OECD HPV Chemical Categories

Domain logical expression index: "q"

Parametric boundary:The target chemical should have a value of log Kow which is >= -0.834

Domain logical expression index: "r"

Parametric boundary:The target chemical should have a value of log Kow which is <= 2.97

Conclusions:
Basic Violet 2 was predicted to not induce gene mutation in male Sprague Dawley rats and hence, according to the prediction made, it is not likely to classify as a gene mutant in vivo.
Executive summary:

Based on the prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, gene mutation was predicted for Basic violet 2. The study assumed the use of male Sprague Dawley rats. Basic Violet 2 was predicted to not induce gene mutation in male Sprague Dawley rats and hence, according to the prediction made, it is not likely to classify as a gene mutant in vivo.

Based on the predicted result it can be concluded that the substance is considered to not toxic as per the criteria mentioned in CLP regulation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Prediction model based estimation and existing data for the target chemical and its read across was reviewed to determine the mutagenic nature of Basic violet 2. The studies are as mentioned below:

Gene mutation in vitro:

Based on the prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, gene mutation was predicted for Basic violet 2. The study assumed the use of Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 with and without S9 metabolic activation system. Basic violet 2 was predicted to not induce gene mutation in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence, according to the prediction made, it is not likely to classify as a gene mutant in vitro.

Ames assay was performed to determine the mutagenic nature of Basic violet 2 (SCCS, 2011). 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.

In another study mentioned in SCCS (2011), In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of Basic violet 2. The study was performed using mouse lymphoma assay at the tk locus in the presence and absence of S9 metabolic activation system as per the OECD guideline 476. The test chemical was dissolved in DMSO and used at dose levels 0.0625, 0.125, 0.25, 0.5, 0.75, 1 and 1.5 μg/ml without S9-mix and 0.156, 0.313, 0.625, 1.25, 2.5 and 2.75 with S9-mix in experiment 1 and 0.025, 0.05, 0.1, 0.2, 0.3 and 0.4 μg/ml without S9-mix and 1.5, 2, 2.5, 2.6 and 2.7 μg/ml with S9-mix in experiment 2.The doses were selected on the basis of dose range finding study.In the main test cells were treated for 3 h or 24 h (experiment 2 with S9-mix only) followed by an expression period of 7-9 days to fix the DNA damage into a stable tk mutation. No biologically relevant and dose dependent increases in the mutant frequency following treatment with Basic Violet 2 were found at any dose level, in the absence or presence of S9-mix at any treatment time. Under the experimental conditions used, Basic Violet 2 was not mutagenic in the mouse lymphoma assay at the tk locus.

Disk-diffusion procedure was performed by Rosenkranz and (Chemical mutagens, 1980) to determine the mutagenic nature of Magenta ABN (C.I. 42520). Filter disc impregnated with known amount of test agent dissolved in water or DMSO was deposited on the surface of the agar. After incubation at 37˚C in the dark for 7 -12 hr, the diameters of the zones of growth inhibition are determined. On occasion, the edges of the zones of growth inhibition are not well delineated; for this reason, it is convenient to allow the plates to remain at room temperature (~23°C) for an additional 24 hr and to measure zones of growth inhibition once more. Magenta ABN did not induce gene mutation in E. coli pol A+and pol A1-strain and hence is negative for gene mutation in vitro.

The data for the target chemical was supported by using structurally and functionally similar read across chemical. In one such study by Mortelmans et al (Environmental Mutagenesis, 1986), Gene mutation toxicity study was performed for n-hexane (RA CAS no 110 -54 -3) to evaluate its mutagenic nature. The study was performed as per the preincubation protocol using Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system at doses of 0, 1, 3, 10, 33, 100, 333 µg/plate. 95% ethanol was used as the vehicle. The plates were incubated for 48 hrs after 20 mins preincubation before the evaluation of the revertant colonies could be made. n- Hexane did notinduce mutation in theSalmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

In another study for structurally and functionally similar read across chemical, Gene mutation toxicity study by Araki et al (Mutation Research, 1994) was performed to determine the mutagenic nature of read across chemical 2-butene (RA CAS no 107 -01 -7). The study was peformed as per the gas exposure method using S. typhimurium TA98, TA100, TA1535 and TA1537 and E. coli WP2 uvrA in the presence and absence of S9 metabolic activation system. Self-combustible 2-butene was diluted using HEPA-filtered air. The test substance in a gas cylinder was collected in a 20-L gas sampling bag. Another 20-L gas sampling bag was connected to a flow meter and a pump using vinyl tubing in order to collect a fixed amount of dilution gas. A fixed volume of the test substance gas was pumped out from the gas sampling bag which contained the test substance gas, and pumped into another gas sampiing bag (gas dilution bag), which had been filled with a fixed amount of dilution gas, using the flow meter and the pump. The gas dilution bag which then contained both the test substance gas and the dilution gas was squeezed by hand in order to mix both gases thoroughly. The concentration used in the test was calculated from the volume of test substance vapor and dilution gas in the gas exposure bag at the exposure temperature. The bacterial plates were separately placed, upside down without their lids, in a plate holder according to their concentration level and the method used (metabolic activation or the direct method). The plate holder was placed in a 10-1 gas sampling bag through an opening on one side of the bag made by scissors. Then the bag was closed by folding the opening 2 or 3 times and sealed with adhesive tape. The air in the bag was removed using a pump in order to check that the bag was air-tight. A flow meter and a pump were connected to the gas dilution bag, and about 1 liter of the test substance gas at an adjusted concentration level was pumped into the gas exposure bag and then the gas in the gas exposure bag was sucked out by pump quickly and the remaining air in the gas exposure bag washed out. After washing out the air, the gas exposure bag was filled with the test substance gas at an adjusted concentration level at a fixed amount per plate. The bacterial plates in the gas exposure bag filled with the test substance gas were kept at 25 or 37°C for a fixed period. After the termination of exposure, the test substance gas in the gas exposure bag was removed. Then sterile air was pumped into the gas exposure bag through a HEPA filter in order to completely replace the gas with air, the seal was peeled off and the plate holder was taken out of the bag. The plates were left to stand in a safety cabinet for 30 min in order to evaporate the test substance, and then the lids of the plates were replaced.The plates with lids were turned upside down, transferred to a container or vinyl bag separately according to their concentration levels and the method used (metabolic activation or direct method) and were then incubated further for a total exposure period and incubation period of 48 h. After incubation revertant colonies were counted. 2-butene did not induce gene mutation in S. typhimurium TA98, TA100, TA1535 and TA1537 and E. coli WP2 uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Gene mutation in vivo:

Based on the prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, gene mutation was predicted for Basic violet 2. The study assumed the use of male Sprague Dawley rats. Basic Violet 2 was predicted to not induce gene mutation in male Sprague Dawley rats and hence, according to the prediction made, it is not likely to classify as a gene mutant in vivo.

In vivo mammalian erythrocytes micronucleus test was performed to determine the mutagenic nature of Basic violet 2 (SCCS, 2011). The study was performed using 5 males and 5 females Swiss CD-1 Mice at dose levels of 0, 3, 6 or 12 mg/Kg bw for 24 and 48 hrs. Three toxicity studies with decreasing doses were performed in order to identify the doses to be used in the main study. Animals were inspected for signs of reaction to treatment daily throughout the study. Bone marrow cells were collected 24 h or 48 h (control and highest dose only) after dosing and stained with May-Gruenwald and Giemsa. Toxicity and thus exposure of the target cells was determined by measuring the ratio between polychromatic and total erythrocytes (PCE/PCE+NCE) over the negative control value. In the micronucleus test no animals died. Animals from the 6 mg/Kg bw treatment group showed piloerection; those from the high dose group hunched posture, swollen abdomen, closed eyes, piloerection and pink spots in the cage litter indicative for excretion of coloured urine. A reduction in the PCE/(PCE+NCE) ratio was observed for both sexes and both time points. Biological relevant increases in the number of bone marrow cells with micronuclei compared to the concurrent vehicle controls were not found at any dose tested, either 24 or 48 h after treatment or for males and females.Basic Violet 2 did not induce an increase in bone marrow cells with micronuclei and, consequently, Basic Violet 2 is not genotoxic (clastogenic and/or aneugenic) in bone marrow cells of mice.

Based on the data available for the target chemical and its read across, Basic violet 2 does not exhibit gene mutation in vitro and in vivo. Hence the test chemical is not likely to classify as a gene mutant 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, Basic violet 2 (CAS no 3248 -91 -7) does not exhibit gene mutation in vitro and in vivo. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.