Allergic contact dermatitis is to some extent a preventable disease. The evaluation of skin sensitization potential is an important component of the safety assessment of chemicals. Traditionally the identification and characterization of skin sensitizing chemicals has required the use of animal tests such as the murine Local Lymph Node Assay (LLNA) and/or guinea pig tests (such as Buehler Test and Guinea Pig Maximization Test). The LLNA was developed initially as an alternative to guinea pig test methods and offered a number of animal welfare (reduced numbers of animals required and reduced trauma) as well as other important benefits (reduced time and cost and greater objectivity). Importantly also, the LLNA uses an endpoint that is both causally and quantitatively associated with the acquisition of sensitization and can therefore be used for the measurement of relative sensitizing potency. These methods have been proven to be accurate in terms of the predictive identification of skin sensitizing chemicals. Accurate identification of hazard, an understanding of relative sensitizing potency, together with an appreciation of likely human exposure provides a sound basis for accurate risk assessment and risk management and protection of human health.
If the introduction of the LLNA significantly contributed to 2 of 3 Rs (refinement and reduction), the acceptance of the reduced LLNA further contributed to the reduction of animals as the rLLNA requires up to 40% fewer animals than the traditional LLNA. NICEATM and the ICCVAM Immunotoxicity Working Group developed a revision to OECD TG 429, adopted in July 2010, which incorporates the procedure for the rLLNA. This assay is particularly valuable in reducing animal use in large testing programs such as REACH. There is a pressing need in the EU for alternative non-animal methods to reduce and ultimately replace animal tests for this endpoint due to the testing needs of the Cosmetics Regulation (EC/1223/2009), REACH Regulation (EC/1907/2006), and others. The challenge is how to obtain the same quality of information using in silico or in vitro methods. Four goals can be identified for a full replacement of skin sensitization animal data:
In recent years important progress has been made in the development of alternative methods that could lead to the replacement of the existing animal models (Corsini and Roggen, 2009; Galbiati et al., 2010; Lankveld et al., 2010; Kimber et al., 2013). Cell-based assays for skin sensitization are being developed and refined. These methods use cultured cells to model the mechanism(s) of induction of skin sensitization. Such mechanisms include epidermal cell and dendritic cell interactions, alterations in dendritic cell phenotype and function following allergen exposure, and the human T cell priming assay developed within the EU project Sens-it-iv (Richter et al., 2013). With regard to potency assessment, Gibbs et al. (2013) explored the possibility of combining the epidermal equivalent potency assay with a test that assesses release of interleukin-18 to provide a combined approach for identification and classification of skin sensitizing chemicals, including chemicals of low water solubility or stability. (Quantitative) structure activity relationship ((Q)SAR) systems are also being used and refined, and some may become validated within the next five years. These models seek to identify allergens on the basis of physicochemical data and the reactivity parameters of functional groups.
As explained in the Regulatory Testing Overview section, three cell-based test methods are currently, or have recently, undergone formal validation review at EURL-ECVAM for their potential to predict skin sensitization potential, namely the Direct Peptide Reactivity Assay (DPRA), the human Cell Line Activation Test (hCLAT), and the KeratinosensTM. These methods cannot be used alone to replace animal testing, but may become part of an integrated testing strategy. Each of them represents biological mechanisms covered by the OECD Guidance Document on Developing and Assessing Adverse Outcome Pathways for skin sensitization (OECD GD 184, 2013).
ICCVAM is also developing a strategy for the evaluation of skin sensitization potential without the use of intact animals. They are working with US agency scientists and industry “to create an integrated testing strategy to combine information from multiple testing methods to identify potential skin sensitizers.”
Information generated by in vitro methods to assess skin sensitization can already be used in a weight-of-evidence approach to support regulatory decision making, e.g., to characterize equivocal responses in in vivo studies (e.g., conflicting results from multiple studies). For the purposes of some regulations (for example REACH in the EU), a positive result should be considered sufficient to classify a test material as a skin sensitizer.
While progress has been made with regard to hazard identification, there is no doubt that potency and risk assessment remain important challenges. In vitro methods may eventually contribute to potency categorization, e.g., GHS sub-category 1A (strong sensitizers) and 1B (other sensitizers) as defined in the fourth revised edition of GHS (UNECE, 2011).
Due to the complexity of allergic reactions, the chemical diversity of sensitizing chemicals, and the varying applicability domains of the different in vitro tests, the development of predictive integrated testing strategies remains the most realistic approach to reduce or replace animal testing for the assessment of skin sensitization potential (Jaworska et al., 2011, 2013). As toxicology in the 21st century progresses towards a future without the use of in vivo testing, the endpoint of skin sensitization is now to be found in the front line.
The following invited commentaries provide the AltTox community with new perspectives on skin sensitization testing:
Emanuela Corsini, PhD
Professor, University of Milan
Ian Kimber, PhD
Professor, University of Manchester
AltTox Editorial Board reviewer(s):
Sherry L. Ward, PhD, MBA
AltTox Contributing Editor