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Skin irritation / corrosion

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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:
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

Reference
Name:
Unnamed
Type:
Constituent
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

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
114.2
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
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 valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
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 valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
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 valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
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 valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
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 valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
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

159.0b(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.

 

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.