Registration Dossier

Administrative data

Description of key information

Skin irritation :

The in vitro skin irritation test was performed for test material 4-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine mono hydrochloride) by Alépée N, Adriaens E, Grandidier MH, Meloni M, Nardelli L, Vinall CJ, Toner F, Roper CS, Van Rompay AR, Leblanc V, Cotovio J.on Reconstructed human epidermis according to the guideline 439 ''In vitro skin irritation:Reconstructed human epidermis test method.

The evaluation of direct MTT reduction involved the incubation of test material with MTT solution. The solution absorbed 560nm or became coloured following incubation with chemical. The percentage viability was calculated on the basis of OD analysis by L'Oreal R&I , UPLC analysis VITO, HPLC UPLC spectrophotometery SD (3lab) 4-[(4-amino-m-tolyl)(4 -imino-3 -methylcyclohexa-2,5 -dien-1 -ylidene)methyl]-o-toluidine mono hydrochloride)

On the Basis of MTT reduction test using Formazan as a colour indicator .The percentage viability was calculated on the basis of OD analysis by L'Oreal R&I , UPLC analysis VITO, HPLC UPLC spectrophotometry SD (3lab) 4-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine mono hydrochloride) was considered to be Not irritating.

Eye irritation :

An eye irritation study in rabbits was conducted to assess the irritation potential of 4-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride.The study was performed according to Draize method. Undiluted test chemical was instilled in the eyes of 1 rabbit and observed for signs of irritation till 7 days. The reactions observed were scored according to Draize method.Mean scores are calculated for each animal from gradings at 24, 48, and 72 h after instillation of the test chemical and these “severity scores” are then used to determine the classification of the test chemical.

The study was terminated after day 1 since color staining of the cornea was observed.

Based on these observations,4-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloridewas considered to highly irritating and causing irreversible damage to rabbit eyes, was classified under the category “Category 1”.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
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 journals
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Principles of method if other than guideline:
EpiSkin™ in vitro test method was performed in 3 different labs to assess the skin irritation potential of 4-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material: C.I. Basic Violet 2 / (4-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride)
- IUPAC name: 4-[(4-amino-3-methylphenyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-2-methylaniline hydrochloride
- Molecular formula: C22H24ClN3
- Molecular weight: 365.906 g/mole
- Smiles :C(\c1cc(c(N)cc1)C)(c1cc(c(N)cc1)C)=C1\C=C(C(=[NH+])C=C1)C.[ClH-]
- Inchl: 1S/C22H23N3.ClH/c1-13-10-16(4-7-19(13)23)22(17-5-8-20(24)14(2)11-17)18-6-9-21(25)15(3)12-18;/h4-12,23H,24-25H2,1-3H3;1H
- Substance type: Organic
- Physical state: Solid Green crystalline powder
Test system:
other: Reconstructed human epidermis test method
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Not specified
Source strain:
other: human skin
Details on animal used as source of test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin™ in vitro test method
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: -20 deg C
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g., spectrophotometer); - 570 nm
- MTT concentration: as per OECD Guidelines
Justification for test system used:
Reconstructed Human tissue(Rht) most recently adapted on july 26th 2013
Vehicle:
water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin™ in vitro test method
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: -20 deg C
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: as per OECD Guidelines
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
Not specified
Duration of treatment / exposure:
according to the guidelines
Duration of post-treatment incubation (if applicable):
No data.
Number of replicates:
No data
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
114.2
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
when applied as solid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
119.1
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
when applied as solid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
2.5
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
when applied as solid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
108.3
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
when applied as a 1% (w/v) aqueous solution
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
107.2
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
when applied as a 1% (w/v) aqueous solution
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
109.8
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
when applied as a 1% (w/v) aqueous solution
Other effects / acceptance of results:
Acceptance criteria based on positive and negative controls in accordance with test guidelines

Table 3:EpiSkin™% tissue viability quantification from formazan tissue extracts by OD (L’Oréal R&I only) and HPLC/UPLC-spectrophotometry (3 laboratories) for the test chemicals

Name of the test chemical

In vivo Classification

Pre checks

EpiSkin™ (skin irritation) Tissue viability (%)

In vitro skin irritation classification

OD-HPLC/UPLC spectrophotometry viability

concordant Y/N

MTT

reducer

(Y/N)

 

Colour

interference

(Y/N)

OD analysis

L’Oréal R&I

HPLCanalysis L’Oréal R&I

HPLCanalysis

L’Oréal R&I

UPLC

analysis

VITO

HPLC/UPLC spectrophotometry

SD (3 Labs)*

Difference (OD-HPLC)

L’Oréal R&I

OD

HPLC/

UPLC spectrophotometry

Benzenamine, 4-((4-amino-3-methyl-phenyl)(4-imino-3-methyl-2,5-cyclohexadiene-1-ylidene)methyl-2-methyl HCl

NC

N

Y

159.0b(228.6)

114.2 (114.2)

119.1 (119.1)

2.5

44.8

119.1 (119.1)

Not compatible

NC

NA

Benzenamine, 4-((4-amino-3-methyl-phenyl)(4-imino-3-methyl-2,5-cyclohexadiene-1-ylidene)methyl-2-methyl HCl solution 1% (w/v) aqueous

NC

N

Y

99.6b(233.4)

108.3 (108.3)

107.2 (107.2)

109.8 (109.8)

1.3

8.7

Not compatible

NC

NA

Where

Uncorrected viability values in parentheses.

Not compatible: Using the OD measurement (% NSC and/or % NSMTT≥50%).

Y: Yes.

N: No.

NA: Not applicable.

NK: Not known.

NT: Not tested.

ND: Could not be determined.

Cat 1: Skin Corrosive.

NC: Not Corrosive.

NC: Not Classified.

%NSMTT≥50%

a- SD between three replicate tissues ≥18%

b - NSC≥50%.

c - NSMTTP50%.

** OD-HPLC/UPLC-spectrophotometry > 20%.

*** NT based on a decision by the participating laboratory not to proceed to further testing of this test chemical.

**** Difference between 2 laboratories.

 

Interpretation of results:
other: Not irritating
Conclusions:
The mean % tissue viability of C.I. Basic Violet 2 when evaluated as a solid in 3 different laboratories were 114.2, 119.1, 2.5 respectively and 108.3, 107.2, 109.8 respectively when evaluated as a 1% (w/v) aqueous solution. The results obtained from the test indicated a very strong possibility of Basic Violet 2 being not irritating to skin.
Executive summary:

EpiSkin™ in vitro test method was performed in 3 different labs to assess the skin irritation potential of4-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride. Since, 4-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride[C.I. Basic Violet 2] exhibited extreme color interference when tested undiluted, hence it was also evaluated as a 1% (w/v) aqueous dilution. This was done to determine how strong the colour interference °remained with dilution and the relevance of the two endpoint detection systems (OD and HPLC/UPLC-spectrophotometry).

EpiSkin™ in vitro test method was performed using the SOPs in accordance with the relevant OECD test guidelines i.e. Updated OECD TG439.Both positive and negative controls were run in parallel with the test substances. Acceptance criteria based on positive and negative controls were fulfilled as mentioned in the SOPs. The evaluation of direct MTT reduction involved the incubation of the test chemical with MTT solution. If the solution turned blue, killed tissue adapted controls incubated with MTT to determine non-specific MTT reduction (% NSMTT).Since the test chemical was both colored and MTT reducer, living tissue adapted controls to define non-specific colour (% NSC) and killed tissue adapted controls to determine non-specific MTT reduction (% NSMTT) were deemed necessary. Also a third set of adapted controls using killed tissues incubated with medium instead of MTT were used to define chemical binding to the killed tissue (% NSCkilled). In this case, the final corrected % tissue viability for the test chemical was obtained by subtracting % NSC and % NSMTT and adding % NSCkilled to the % tissue viability obtained with the chemical treated living tissues incubated with MTT.

The study was performed by L’Oreal R&I.The resulting formazan tissue extracts were analysed by photometry (OD) in accordance with the SOP for each test method after testing. After shipment of the formazan tissue extracts stored at -20°C, measurement of the formazan by HPLC/UPLC-spectrophotometry in the three different laboratories (L’Oréal R&I, Pierre Fabre Laboratories and VITO) was performed within one week of each other.Furthermore, photometry (OD) was performed in parallel with the HPLC/UPLC-spectrophotometric analysis of the -20°C stored samples by the laboratory that conducted the in vitro biological test method. Correlation analyses for both formazan detection methods were performed.

The mean % tissue viability of C.I. Basic Violet 2 when evaluated as a solid in 3 different laboratories were 114.2, 119.1, 2.5 respectively and 108.3, 107.2, 109.8 respectively  when evaluated as a 1% (w/v) aqueous solution. The results obtained from the test indicated a very strong possibility of Basic Violet 2 being not irritating to skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
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 journals
Qualifier:
according to
Guideline:
other: Draize method
Principles of method if other than guideline:
To assess the ocular irritation potential of 4-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride in rabbits according to Draize method
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material: C.I. Basic Violet 2 / (4-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride)
- IUPAC name: 4-[(4-amino-3-methylphenyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-2-methylaniline hydrochloride
- Molecular formula: C22H24ClN3
- Molecular weight: 365.906 g/mole
- Smiles :C(\c1cc(c(N)cc1)C)(c1cc(c(N)cc1)C)=C1\C=C(C(=[NH+])C=C1)C.[ClH-]
- Inchl: 1S/C22H23N3.ClH/c1-13-10-16(4-7-19(13)23)22(17-5-8-20(24)14(2)11-17)18-6-9-21(25)15(3)12-18;/h4-12,23H,24-25H2,1-3H3;1H
- Substance type: Organic
- Physical state: Solid Green crystalline powder
- Obtained from: Sigma Aldrich
- Purity: Analytical Grade
Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
no data available
Vehicle:
unchanged (no vehicle)
Controls:
not specified
Amount / concentration applied:
no data available
Duration of treatment / exposure:
single exposure
Observation period (in vivo):
From 1 hour till 21 days after instillation of test chemical
Duration of post- treatment incubation (in vitro):
no data available
Number of animals or in vitro replicates:
1
Details on study design:
SCORING SYSTEM: Draize method
Mean scores are calculated for each animal from gradings at 24, 48, and 72 h after instillation of the test chemical and these “severity scores” are then used to determine the classification of the test chemical
Other effects / acceptance of results:
no data available
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
24 h
Reversibility:
not specified
Remarks on result:
positive indication of irritation
Irritant / corrosive response data:
The study was terminated after day 1 since color staining of the cornea was observed.

Table: Draize eye test reference database

Name of the test chemical

CAS number

Substance class

Physical state

Purity

Commercial source

UN GHS category

Number of animals

Comments

4-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride

3248-91-7

Allyl|

Anilines|

Benzyl|

Conjugated hydrocarbon|

Dianilines|

Ketimine

Solid

Analytical standard

Sigma –Aldrich

CAT 1

1/1

The study was terminated after day 1 since color staining of the cornea was observed.

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The study was terminated after day 1 since color staining of the cornea was observed.
Based on these observations, 4-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride was considered to highly irritating and causing irreversible damage to rabbit eyes, was classified under the category “Category 1”.
Executive summary:

An eye irritation study in rabbits was conducted to assess the irritation potential of 4-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride.The study was performed according to Draize method. Undiluted test chemical was instilled in the eyes of 1 rabbit and observed for signs of irritation till 7 days. The reactions observed were scored according to Draize method.Mean scores are calculated for each animal from gradings at 24, 48, and 72 h after instillation of the test chemical and these “severity scores” are then used to determine the classification of the test chemical.

The study was terminated after day 1 since color staining of the cornea was observed.

Based on these observations,4-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloridewas considered to highly irritating and causing irreversible damage to rabbit eyes, was classified under the category “Category 1”.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Various studies have been investigated for assessing the dermal irritation potential of 4-[(4-amino-3-methylphenyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-2-methylaniline hydrochloride to a greater or lesser extent. The studies are based on in vivo experiments in rabbits along with in vitro data for target chemical.

EpiSkin™ in vitro test method was performed (Toxicology in Vitro, 29 (2015), 741–761) in 3 different labs to assess the skin irritation potential of 4-[(4-amino-m-tolyl)(4-imino-3 -methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride. Since, 4-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride[C.I. Basic Violet 2] exhibited extreme color interference when tested undiluted, hence it was also evaluated as a 1% (w/v) aqueous dilution. This was done to determine how strong the colour interference °remained with dilution and the relevance of the two endpoint detection systems (OD and HPLC/UPLC-spectrophotometry).

EpiSkin™ in vitro test method was performed using the SOPs in accordance with the relevant OECD test guidelines i.e. Updated OECD TG439.Both positive and negative controls were run in parallel with the test substances. Acceptance criteria based on positive and negative controls were fulfilled as mentioned in the SOPs. The evaluation of direct MTT reduction involved the incubation of the test chemical with MTT solution. If the solution turned blue, killed tissue adapted controls incubated with MTT to determine non-specific MTT reduction (% NSMTT).Since the test chemical was both colored and MTT reducer, living tissue adapted controls to define non-specific colour (% NSC) and killed tissue adapted controls to determine non-specific MTT reduction (% NSMTT) were deemed necessary. Also a third set of adapted controls using killed tissues incubated with medium instead of MTT were used to define chemical binding to the killed tissue (% NSCkilled). In this case, the final corrected % tissue viability for the test chemical was obtained by subtracting % NSC and % NSMTT and adding % NSCkilled to the % tissue viability obtained with the chemical treated living tissues incubated with MTT.

The study was performed by L’Oreal R&I. The resulting formazan tissue extracts were analysed by photometry (OD) in accordance with the SOP for each test method after testing. After shipment of the formazan tissue extracts stored at -20°C, measurement of the formazan by HPLC/UPLC-spectrophotometry in the three different laboratories (L’Oréal R&I, Pierre Fabre Laboratories and VITO) was performed within one week of each other. Furthermore, photometry (OD) was performed in parallel with the HPLC/UPLC-spectrophotometric analysis of the -20°C stored samples by the laboratory that conducted the in vitro biological test method. Correlation analyses for both formazan detection methods were performed.

The mean % tissue viability of C.I. Basic Violet 2 when evaluated as a solid in 3 different laboratories were 114.2, 119.1, 2.5 respectively and 108.3, 107.2, 109.8 respectively  when evaluated as a 1% (w/v) aqueous solution. The results obtained from the test indicated a very strong possibility of Basic Violet 2 being not irritating to skin.

This result is ably supported by the in vitro study performed(Toxicology in Vitro, 29 (2015), 741–761) in 3 different labs to assess the skin irritation potential of 4-[(4-amino-m-tolyl)(4-imino-3 -methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride.SkinEthic™ RHE in vitro test method was performed using the SOPs in accordance with the relevant OECD test guidelines i.e. TG 431: ‘‘In vitro skin corrosion: Reconstructed Human Epidermis (RHE) test method. The chemicals were tested for 2 exposure durations i.e 3 min and 1 h exposure. Both positive and negative controls were run in parallel with the test substances. Acceptance criteria based on positive and negative controls were fulfilled as mentioned in the SOPs. The evaluation of direct MTT reduction involved the incubation of the test chemical with MTT solution. If the solution turned blue, killed tissue adapted controls incubated with MTT to determine non-specific MTT reduction (% NSMTT).Since the test chemical was both colored and MTT reducer, living tissue adapted controls to define non-specific colour (% NSC) and killed tissue adapted controls to determine non-specific MTT reduction (% NSMTT) were deemed necessary. Also a third set of adapted controls using killed tissues incubated with medium instead of MTT were used to define chemical binding to the killed tissue (% NSCkilled). In this case, the final corrected % tissue viability for the test chemical was obtained by subtracting % NSC and % NSMTT and adding % NSCkilled to the % tissue viability obtained with the chemical treated living tissues incubated with MTT.

The study was performed by L’Oreal R&I.The resulting formazan tissue extracts were analysed by photometry (OD) in accordance with the SOP for each test method after testing. After shipment of the formazan tissue extracts stored at -20°C, measurement of the formazan by HPLC/UPLC-spectrophotometry in the three different laboratories (L’Oréal R&I, Pierre Fabre Laboratories and VITO) was performed within one week of each other. Furthermore, photometry (OD) was performed in parallel with the HPLC/UPLC-spectrophotometric analysis of the -20°C stored samples by the laboratory that conducted the in vitro biological test method. Correlation analyses for both formazan detection methods were performed.

The results obtained from the test indicate a very strong possibility of Basic Violet 2 being not irritating and non corrosive to skin.

These results are supported by the experimental study summarized inScientific Committee on Cosmetology (seventh series), 1988 for Basic Violet 2.0.5ml 10% aqueous solution was applied on the rabbit skin under occlusion. The above test did not induce any skin reaction. Hence, Basic violet 2 was considered non- skin irritant.

The above results are further supported by the experimental study summarized in Scientific Committee on Cosmetic Products and Non-Food Products (SCCNFP), 2004 for Basic Violet 2. 3 females New Zealand albino white rabbit were used as test animals. A gauze square with 0.5 g test material mixed to a paste with sterile water was placed on the shaved skin of three female rabbits and covered with a semi-occlusive dressing for 4 hours.

After the 4-hour application time, the area was wiped with cotton wool soaked with water. The animals were checked daily for mortality and systemic symptoms. Skin reactions were evaluated 1, 24, 48, and 72 hours after removing the patches according to the Draize scoring system.

No irritation was recorded in any test animals. Erythema could not be assessed due to intense staining of the skin. No oedema was noted in any of the test animals. Measurement of skin-fold thickness after 72 hours revealed no differences between treated skin and untreated (naive) skin. Erythema assessments after a longer period (when discolouration of the skin had disappeared) have not been performed.

Basic Violet 2 was considered to be skin non –irritant as the mean irritation score 24 to 72 hours after application were below the thresholds defined in Commission Directive 2001/59/EC.

The results of the in vivo studies and in vitro studies are in agreement with each other, thereby indicating a strong possibility of Basic Violet 2 being not irritating to skin.

Hence, by applying the weight of evidence approach, 4-(4-aminophenyl)(4-iminocyclohexa-2,5-dienylidene)methyl)-2-methylaniline hydrochloride can be considered to be not irritating to skin.Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified.”

Eye Irritation:

Various studies have been investigated for assessing the ocular irritation potential of 4-[(4-amino-3-methylphenyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-2-methylaniline hydrochloride to a greater or lesser extent. The studies are based on in vivo experiments in rabbits along with in vitro data for target chemical.

An eye irritation study in rabbits was conducted (Archives of toxicology, 91(2), 521-547,2017) to assess the irritation potential of4-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride.The study was performed according to Draize method. Undiluted test chemical was instilled in the eyes of 1 rabbit and observed for signs of irritation till 7 days. The reactions observed were scored according to Draize method.Mean scores are calculated for each animal from gradings at 24, 48, and 72 h after instillation of the test chemical and these “severity scores” are then used to determine the classification of the test chemical.

The study was terminated after day 1 since color staining of the cornea was observed.

 

Based on these observations,4-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloridewas considered to highly irritating and causing irreversible damage to rabbit eyes, was classified under the category “Category 1”.

EpiOcular™ EIT test method was performed (Toxicology in Vitro, 29 (2015), 741–761) in 3 different labs to assess the eye irritation potential of4-[(4-amino-m-tolyl)(4-imino-3 -methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride. Since, 4-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride[C.I. Basic Violet 2] exhibited extreme color interference when tested undiluted, hence it was also evaluated as a 1% (w/v) aqueous dilution. This was done to determine how strong the colour interference °remained with dilution and the relevance of the two endpoint detection systems (OD and HPLC/UPLC-spectrophotometry).

EpiOcular™ EIT test method was performed in accordance with the relevant OECD test guidelines i.e. updated TG 492, i.e 30 ± 2 min exposure time followed by a 120 ± 15 min post-exposure incubation period for liquid test chemicals and 90 ± 5 min exposure time followed by an 18 h ± 15 min post-exposure incubation period for solid test chemicals. Acceptance criteria based on positive and negative controls were fulfilled as mentioned in the SOPs. The evaluation of direct MTT reduction involved the incubation of the test chemical with MTT solution. If the solution turned blue, killed tissue adapted controls incubated with MTT to determine non-specific MTT reduction (% NSMTT).Since the test chemical was both colored and MTT reducer, living tissue adapted controls to define non-specific colour (% NSC) and killed tissue adapted controls to determine non-specific MTT reduction (% NSMTT) were deemed necessary. Also a third set of adapted controls using killed tissues incubated with medium instead of MTT were used to define chemical binding to the killed tissue (% NSCkilled). In this case, the final corrected % tissue viability for the test chemical was obtained by subtracting % NSC and % NSMTT and adding % NSCkilled to the % tissue viability obtained with the chemical treated living tissues incubated with MTT.

The study was performed by L’Oreal R&I. The resulting formazan tissue extracts were analysed by photometry (OD) in accordance with the SOP for each test method after testing. After shipment of the formazan tissue extracts stored at -20°C, measurement of the formazan by HPLC/UPLC-spectrophotometry in the three different laboratories (L’Oréal R&I, Pierre Fabre Laboratories and VITO) was performed within one week of each other. Furthermore, photometry (OD) was performed in parallel with the HPLC/UPLC-spectrophotometric analysis of the -20°C stored samples by the laboratory that conducted the in vitro biological test method. Correlation analyses for both formazan detection methods were performed. Themean % tissue viability of C.I. Basic Violet 2 when evaluated as a solid in 3 different laboratories were  63.1, 58.6, 54.9 respectively and 124.3, 114.4, 120.4 when evaluated as a 1% (w/v) aqueous solution. The results obtained from the test indicated a very strong possibility of Basic Violet 2 causing serious eye damage when exposed in undiluted form.

The above studies are supported by the experimental results summarized in Opinion of the SCCNFP on B115, Basic Violet 2, Scientific Committee on Cosmetic Products and Non-Food Products (SCCNFP), 2002 for assessing the eye irritation potential of Basic Violet 2 in rabbits. The study was performed as per OECD 405 Guidelines.

100mg of the undiluted test chemical was placed into the conjunctival sac of the right eye of 1 female New Zealand White rabbit. The eyelids were then held together for a few seconds. The untreated left eye served as control. Ocular reactions were evaluated approximately 1 and 24 hours after instillation of the test article.

Intense staining of the eye and adnexa occurred. Chemosis score 2 of the conjunctiva was reported 1 hour after dosing, increasing to score 4 at 24h. Discolouration of the nictitating membrane, possibly indicating corrosive actions of the test substance, was also noted at the 24h observation time. Marked to severe corneal opacity was apparent after 24 hours. The study was terminated after 24h. No indication of a systemic effect could be detected

Basic Violet 2 was considered to be severe eye irritant with a potential to cause irreversible damage to eyes.

The results of the in vivo studies and in vitro studies are in agreement with each other, thereby indicating a very strong possibility of Basic Violet 2 causing irreversible damage to the eyes.Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Category 1.”

Justification for classification or non-classification

Available data for 4-[(4-amino-3-methylphenyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-2-methylaniline hydrochloride suggests that it is likely to cause severe irritation to eyes and is not likely not cause any irritation to skin.

4-[(4-amino-3-methylphenyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-2-methylaniline hydrochloride can be classified under the category “Category 1” for eye irritation and "Not Classified" for skin irritation as per CLP regulation.