2-[[4-(dimethylamino)phenyl]azo]-1,3-dimethyl-1H-imidazolium chloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Sep 2014 to 13 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Principles of method if other than guideline:

The objective was to assess the skin sensitizing potential of Vibracolor ruby red. A combination of several in vitro methods addressing key events of the adverse outcome pathway (AOP) for skin sensitization1 as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy:
*protein reactivity (DPRA),
*activation of keratinocytes (LuSens), and
*activation of dendritic cells (h-CLAT).

There are no official national or international guidelines for In Vitro Sensitization; however, the studies were performed according to the methods described in the following publications:
*Bauch C, Kolle SN, Fabian E, Pachel C, Ramirez T, Wiench B, Wruck CJ, van Ravenzwaay B, Landsiedel R. Intralaboratory validation of four in vitro assays for the prediction of the skin sensitizing potential of chemicals. Toxicology in Vitro 25, 1162 –1168, 2011.

*Maxwell G, Aeby P, Ashikaga T, Bessou-Touya S, Diembeck W, Gerberick F, Kern P, Marrec-Fairley M, Ovigne JM, Sakaguchi H, Schroeder K, Tailhardat M, Teissier S, Winkler P. Skin sensitisation: the Colipa strategy for developing and evaluating nonanimal test methods for risk assessment. ALTEX 28(1): 50-5, 2011.

*Bauch C, Kolle SN, Ramirez T, Eltze T, Fabian E, Mehling A, Teubner W, van Ravenzwaay B, Landsiedel R, (2012), Putting the parts together: Combining in vitro methods to test for skin sensitizing potentials, Regul Toxicol Pharmacol, 63(3):489-504.

*Urbisch D, Mehling A, Guth K, Ramirez T, Honarvar N, Kolle SN, Landsiedel R, Jaworska J, Kern P, Gerberick F, Natsch A, Emter R, Ashikaga T, Miyazawa M, Sakaguchi H. Assessing skin sensitization hazard in mice and men using non-animal test methods. Regulatory Toxicology and Pharmacology 71(2), 135-352. 2015.

For the DPRA test the following publications apply additionally:
The study is conducted based on the following draft test guideline:
*oECD: Draft Proposal for a New Guideline on In Vitro Skin Sensitization: Direct Peptide Reactivity Assay (DPRA), accessed on 13 Nov 2013 at http://www.oecd.org

In addition the study is performed according to the methods described in the following
publications:
*Gerberick GF, Vassallo JD, Bailey RE, Chaney JG, Morrall SW, Lepoittevin JP. Development of a Peptide Reactivity Assay for Screening Contact Allergens. Toxicological Sciences 81,332-343, 2004.

*Gerberick GF, Vassallo JD, Foertsch LM, Price BB, Chaney JG, Lepoittenvin JP. Quantificationn of Chemical Peptide Reactivity for Screening Contact Allergens: A Classification Tree Model Approach. Toxicological Sciences 97(2), 417-427, 2007.

Based on the results of an ECVAM (European Center for Validation of Alternative Methods) led validation study on the DPRA, it was concluded by the EURL (EU Reference Laboratory for Alternatives to Animal Testing) ECVAM Scientific Advisory Committee (ESAC) that the DPRA is suitable to be used in a weight-of-evidence approach or integrated testing strategy for distinguishing between skin sensitizers and non-skin sensitizers (ECVAM: EURL ECVAM Recommendation on the Direct Peptide Reactivity Assay (DPRA) of 12 Dec 2013).

For the LuSens the following publications apply additionally:

The study is conducted based on the following draft test guideline:
*OECD: Draft Proposal for a New Guideline on In Vitro Skin Sensitization: In vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method, accessed on 03 Sep 2014 at http://www.oecd.org

In addition the study is performed according to the methods described in the following publications:
*Ramirez T, Mehling A, Kolle SN, Wruck CJ, Teubner W, Eltze T, Aumann A, Urbisch D, Ravenzwaay BV, Landsiedel R.., LuSens: A keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification. Toxicol In Vitro. 2014 Dec;28(8).

For the h-CLAT test the following publications apply additionally:
The study is conducted based on the following draft test guideline:
*OECD: Draft Proposal for a New Guideline on In Vitro Skin Sensitization: human Cell Line Activation Test (h-CLAT), accessed on 01 Sep 2014 at http://www.oecd.org

In addition the study is performed according to the methods described in the following publications:
*Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa , Ito Y, Nishiyama N, Itagaki H. (2010) A comparative evaluation of in vitro skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern Lab Anim. 38(4):275-84.

*sakaguchi H, Ryan C, Ovigne JM, Schroeder KR, Ashikaga T. (2010) Predicting skin sensitization potential and inter-laboratory reproducibility of a human Cell Line Activation Test (h-CLAT) in the European Cosmetics Association (COLIPA) ring trials. Toxicol In Vitro. 24(6):1810-20.

GLP compliance:

yes

Type of study:

other: Study investigating: protein reactivity (DPRA), activation of keratinocytes (LuSens), activation of dendritic cells (h-CLAT).

Test material

Test material information

Constituent 1

Reference substance name:

2-[[4-(dimethylamino)phenyl]azo]-1,3-dimethyl-1H-imidazolium chloride

EC Number:

278-601-4

EC Name:

2-[[4-(dimethylamino)phenyl]azo]-1,3-dimethyl-1H-imidazolium chloride

Cas Number:

77061-58-6

Molecular formula:

C13H18N5.Cl

IUPAC Name:

2-[[4-(dimethylamino)phenyl]azo]-1,3-dimethyl-1H-imidazolium chloride

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Name of test substance: Vibracolor ruby red
Test-substance No.: 10/0383-3
Batch identification: 0009051093
CAS No.: 77061-58-6
Purity: 98% total color (for information see Certificate of Analysis no. 4186 in the appendix)
Homogeneity: The test substance is homogeneous by visual inspection.
Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.

ADDITIONAL TEST-SUBSTANCE INFORMATION
Molecular weight: 279.77 g/mol.
Physical state / color: solid / violet

In vitro test system

Details on the study design:

DPRA:
Synthetic peptides:
Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)

The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA and RS Synthesis, Louisville KY, USA) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive centre.

LuSens:
Cell line: LuSens
transgenic keratinocyte cell line derived from HaCaT cells, prepared in collaboration with Christoph J. Wruck, RWTH Aachen

h-CLAT:
Cell line: THP-1 cells
The human monocytic leukemia cell line was obtained from “American Type Culture Collection, Manassas, USA” (ATCC, TIB-202).

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Direct Peptide Reactivity Assay (DPRA); cysteine peptide depletion

Parameter:

other: EC 6.38% (the concentration resulting in a mean peptide depletion of 6.38%)

Value:

4.24

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

The EC 6.38% was calculated to be 4.24 mM

Results 2

Key result

Run / experiment:

other: Direct Peptide Reactivity Assay (DPRA); lysine peptide depletion

Parameter:

other: EC 6.38% (the concentration resulting in a mean peptide depletion of 6.38%)

Value:

7.93

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

The EC 6.38% was calculated to be 7.93 mM

Results 3

Key result

Run / experiment:

other: Keratinocyte Activation Assay - LuSens

Parameter:

other: EC1.50

Value:

0.24

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

In at least two consecutive concentrations of at least two independent experiments ARE-dependent luciferase activity induction above 1.50-fold of statistic significance at test substance concentrations that did not reduce-cell viability below 70% was observed. EC1.50 was calculated to be 0.24 μg/mL

Results 4

Key result

Run / experiment:

other: Dendritic Cell Line Activation Assay Human Cell Line Activation Test (h-CLAT)

Parameter:

other: EC150 for CD86

Value:

6.04

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

The EC150 for CD86 was calculated to be 8.3 μg/mL or 6.04 μg/mL.

Results 5

Key result

Run / experiment:

other: Dendritic Cell Line Activation Assay Human Cell Line Activation Test (h-CLAT)

Parameter:

other: The EC200 for CD54

Value:

5.68

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

The EC200 for CD54 was calculated to be 5.68 μg/mL and 5.79 μg/mL, respectively.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Vibracolor ruby red is peptide reactive, activates keratinocytes and activates dendritic cells. Applying the evaluation criteria Vibracolor ruby red is predicted to be a skin sensitizer.