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

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Use of HPLC/UPLC-spectrophotometry for detection of formazan in in vitro Reconstructed human Tissue (RhT)-based test methods employing the MTT-reduction assay to expand their applicability to strongly coloured test chemicals

Author:

N. Alépée, J. Barroso , A. De Smedt, B. De Wever, J. Hibatallah, M. Klaric, K.R. Mewes,M. Millet, U. Pfannenbecker, M. Tailhardat, M. Templier, P. McNamee

Year:

2015

Bibliographic source:

Toxicology in Vitro, 29 (2015), 741–761

Materials and methods

Test guideline

Qualifier:

according to guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

221-831-7

EC Name:

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

Cas Number:

3248-91-7

Molecular formula:

C22H24ClN3

IUPAC Name:

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

Details on test material:

-[(4-amino-m-tolyl)(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride/ 3248-91-7
/ 221-831-7
Commen name-Basic Violet 2
mol.formula:C22H23N3 .HCl
smiles:C(\c1cc(c(N)cc1)C)(c1cc(c(N)cc1)C)=C1\C=C(C(=[NH+])C=C1)C.[ClH-]
InChI: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
Physical state: Solid Green crystalline powder
molecular weight:365.906 g/mol

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

In vitro test system

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

Results and discussion

In vitro

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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

Results 5

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

Results 6

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

Any other information on results incl. tables

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

159.0^b(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

99.6^b(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.

 

Applicant's summary and conclusion

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.