Skin Sensitisation

What is the definition of skin sensitisation?

Skin sensitisation is defined as allergic response to a substance after skin contact. Substances are classified as skin sensitisers if there is evidence in humans that the substance can lead to sensitisation by skin contact or if there are positive results from an appropriate animal test. Predictive testing for classification of sensitising substances are so far based on animal tests. Whereas many tests are able to distinguish between sensitisers and non-sensitisers, only the mouse local lymph node assay (LLNA) is capable to predict the relative potency of skin sensitising chemicals (1). So far there are no validated in vitro methods available to replace animal testing for identification of skin sensitising substances.

History*:

  • 1992 OECD 406 GPMT and Buehler Test validation (2)
  • 1999 LLNA accepted by ICCVAM and ECVAM (3,4)
  • 2002 OECD Test Guideline 429 adopted LLNA (5)
  • 2012 DPRA, MUSST, hCLAT, and Keratinosens evaluated as part of an integrated testing strategy for in vitro skin sensitisation testing by EURL ECVAM / ESAC WG (6)
  • 2013 EU Cosmetics Deadline
  • 2015 OECD Test Guideline 442C (DPRA) and 442D (ARE-Nrf2 Luciferase Test Method)

*Abbreviations:
DPRA: Direct Peptide Reactivity Assay
ECVAM: European Centre for the Validation of Alternative Methods
ESAC WG: ECVAM Scientific Advisory Committee Working Group
EURL ECVAM: European Union Reference Laboratories of ECVAM
GPMT: Guinea Pig Maximisation Test
hCLAT: human Cell Line Activation Test
ICCVAM: Interagency Coordinating Committee on the Validation of Alternative Methods
MUSST: MYELOID U937 Skin Sensitisation Test
LLNA: Local Lymph Node Assay

Allergic contact dermatitis is an occupational and environmental disease which can be easily prevented. This disease develops in two major steps: The first phase comprises immunological priming of the exposed skin tissue with the acquisition of sensitisation. After a second exposure to the same chemical an aggressive immune response might be elicited resulting in a local inflammatory reaction. The mechanisms by which a chemical interacts with the immune system comprise a direct chemical-protein reaction or an indirect reaction after conversion of the chemical into protein-reactive molecules. More than 3700 substances have been identified as contact allergens. Currently, the mouse LLNA is the preferred and accepted method for assessing skin sensitisation of most substances. The test system is dose-responsive and allows for prediction of potency, although there are still some limitations regarding predictivity and variability (7).

Regulatory Requirements & Test Guidelines for Skin Sensitisation

OECD TG 406 describes traditional animal test methods (2)
OECD TG 429 describes the Local Lymph Node Assay (5)

For animal data, the GHS recommends using the OECD Guidelines, but notes that "other methods may be used provided that they are well-validated and scientific justification is given" (8). Currently the LLNA is the preferred assay for the predictive identification of skin sensitising chemicals and it is the initial requirement for sensitisation testing within REACH (Registration, Evaluation, Authorisation and Restriction of Chemical substances). The GHS guidelines and most countries do not differentiate strong from weak sensitisers. However, only substances considered to be strong sensitisers are regulated under the Federal Hazardous Substances Act (FHSA) in the US. The OECD Scientific Issue Paper on strong vs. weak sensitisers describes current approaches to harmonise potency determination in different countries and regions (9).

What non-animal alternative methods are available?

Currently, two validated in vitro methods exist to partially replace animal testing for the identification of skin sensitising chemicals.

The Direct Peptide Reactivity Assay (DPRA) and the ARE-Nrf2 luciferase test method.

Several cell-based, molecular or computational methods will be needed in Integrated Approaches to Testing and Assessment (IATA) to be able to fully Substitute for the animal tests currently in use.

 QSAR (Quantitative Structure Activity Relationship) techniques are used to identify allergens on the basis of physicochemical data focusing on functional groups, the DPRA (Direct Peptide Reactivity Assay) investigates the ability of a chemical to directly bind to a model peptide. The basis for this approach is the understanding that a chemical forms stable associations with skin proteins to induce an inflammatory/allergenic response.

The ARE-Nrf2 luciferase test method addresses the induction of genes that are regulated by antioxidant response elements (ARE) by skin sensitisers.
Cell-based assays which are under development make use of target cells to mimic the mechanisms which play a key role in induction of skin sensitisation including keratinocytes, T-cells and dendritic cells. Keratinocytes are the first cells that get in contact with a chemical and are able to directly secrete specific cytokines after stimulation by contact allergens. Dendritic cells are investigated for their alterations in cell phenotype and function following allergen exposure. T-cells are used in a newly developed human T cell priming assay measuring chemical induced antigen-specific production of cytokines by FACS analysis (10). A great challenge for an in vitro assay is the attempt to rank chemicals according to their potency (extreme, strong, moderate and weak sensitising potency) and not only to obtain a yes/no answer. Efforts to develop those methods were made within the FP6 European financed project Sens-it-iv 2005-2011 to reach this goal.

Gibbs et al. (11)  explored the possibility to use the reconstructed epidermis epiCS for identification of skin sensitisers and to rank sensitisers according to their potency.  It combines viability measurement (MTT-Assay) with the detection of Interleukin-18 secretion from epiCS.This Skin Sensitisation and Potency Test method (epiCS - SSPT) can be used for water soluble and insoluble substances. It is easy to perform (Fig. 1) and results strongly correlate with LLNA and human DSA data (Fig. 2)(13).

Currently epiCS-SSPT method is part of a multicentre international validation study that proves scientific validity and will lead to official acceptance.

epiCS-SSPT method  proves to be used for mixtures and possibly particulate substances, which cannot be tested by traditional cell culture based test systems (as KeratinoSens or hCLAT).

Protocol for epiCS SSPT test method

To get familiar with the test system, CellSystems has developed the SSPT Training kit. It contains all essential parts to carried out the test method: sensitiser, non-sensitiser, IL-18 ELISA kit, solvent, media, filter discs etc.

Protocol for epiCS SSPT Training Kit

You will also get great support form the CellSystems' team.


Fig. 1 epiCS SSPT method

Fig. 2 Potency: Correlation epiCS-SSPT method EC50 data with LLNA. Data were obtained in two independent experimental approaches (red and grey).

Are non-animal alternative methods already validated?

At present two non-animal test methods are validated an OECD Test Guidelines have been adopted in February 2015:

The Direct Peptide Reactivity Assay (DPRA) (OECD TG 442C) and the ARE-Nrf2 luciferase test method (OECD TG 442D). The latter only covers the KeratinoSensTM  test method.

The human Cell Line Activation Test (hCLAT), is under formal validation at EURL-ECVAM (6). Results are expected soon. It has been anticipated, however, that it will take at least another 3-5 years for full replacement of in vivo animal models currently used to assess sensitisation (12).

Source: AltTox (Non-animal Methods for Toxicity Testing) www.alttox.org.

References

  1. Basketter, D.A., Gerberick, F. & Kimber, I. (2007). The local lymph node assay and the assessment of relative potency: status of validation. Contact Derm. 57, 70-75.
  2. OECD TG 406 (1992) Skin sensitisation.
  3. ICCVAM (1999). Peer Review Report. The murine local lymph node assay: A test method for assessing the allergic contact dermatitis potential of chemicals/compounds. NIH Publication No. 99-4494.
  4. ECVAM (1999). ESAC Statement, March 21, 1999: Local Lymph Node Assay for skin sensitisation (LLNA).
  5. OECD (2002). Skin sensitisation: Local Lymph Node Assay. TG 429.
  6. ESAC WG report on the ECVAM study on the DPRA assay for skin sensitisation testing,14 May 2012, 1-36.
  7. Anderson, S.E., Siegel, P.D., Meade, B.J. (2011). The LLNA: A Brief Review of Recent Advances and Limitations. J. Allergy. 2011, 424203.
  8. United Nations Economic Commission for Europe (UNECE), (2004). Globally Harmonized System of Classification and Labeling of Chemicals (GHS). Respiratory or Skin Sensitisation, Chapter 3.4. pp. 151-158.
  9. OECD (2006). Scientific issue paper on strong vs. weak sensitisers.
  10. Richter, A., Schmucker, S.S., Esser, P.R., et al. (2013). Human T cell priming assay (hTCPA) for the identification of contact allergens based onnaive T cells and DC--IFN-γ and TNF-α readout. Toxicol. In Vitro. 27, 1180-1185.
  11. Gibbs,S., Corsini, E., Spiekstra, S.W., Galbiati, V., Fuchs, H.W., DeGeorge, G.,Troese, M., Hayden, P., Deng, W., Roggen, E. (2013). An epidermal equivalent assay for identification and ranking potency of contact sensitisers. Toxicol. Appl. Pharmacol. 272, 529-541.
  12. Adler, S., Basketter, D., Creton S., et al. (2011). Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010. Arch. Toxicol. 85, 367-485.
  13. Teunis M.A.T., Spiekstra S.W., Smits M. et al. (2014). International ring trial of the epidermal equivalent sensitizer potency assay: reproducibility and predictive capacity, ALTEX 2014 31, 3/14, 251-268
  14. OECD (2015). In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method. TG 442 D.
  15. OECD (2015). In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA). TG 442C.