The Globally Harmonized System for Classification and Labeling of Chemicals (GHS) defines a skin sensitizer as "a substance that will induce an allergic response following skin contact" (UNECE, 2004, p. 151). A substance is classified as a skin sensitizer when human data show it can induce a sensitization response following skin contact "in a substantial number of persons" or when "there are positive results from an appropriate animal test" (UNECE, 2004, p. 152). Human evidence for the skin sensitization of chemicals derives from clinical experience, diagnostic patch testing, and tests designed to confirm the absence of sensitization potential.
Allergic contact dermatitis (ACD) is an important occupational and environmental disease caused by topical exposure to chemical allergens. Contact dermatitis, including bother allergic and irritant contact dermatitis, is the second most commonly reported occupational illness, accounting for 10% to 15% of all occupational diseases. ACD - in common with other forms of allergic disease - develops in two phases. In the first phase, skin exposure of susceptible individuals to a chemical allergen causes immunological priming that results in the acquisition of sensitization. A sensitized subject has the capacity then to mount a more accelerated secondary response to the same chemical. Thus, if exposure occurs again, at the same or a different skin site, then an aggressive immune response will be elicited resulting in local inflammatory reaction. ACD is a T lymphocyte mediated allergic reaction (Cavani et al., 2007; Martin, 2012). Chemicals are normally too small to elicit an immune response. The successful acquisition of skin sensitization requires, therefore that the chemical is able to associate with protein. Sensitizing chemicals are as a consequence naturally protein-reactive, or can be converted locally into protein-reactive species. The induction of skin sensitization requires activation of both the innate and adaptive immune responses, and a variety of cells and molecules have pivotal roles to play in the initiation and orchestration of cutaneous immune responses to chemical allergens. Common examples of chemicals that are associated with skin sensitization and ACD include metals in jewelry and chemicals in cosmetics or in latex gloves. More than 3700 substances have been identified as contact allergens. The article Allergies Caused by Consumer Products and Foods provides an excellent overview of the types and mechanisms of allergies.
A diagram of the mechanism of skin sensitization (Gildea, 2005)
The Animal Test(s)
Prior to review of the mouse Local Lymph Node Assay (LLNA) by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the European Centre for the Validation of Alternative Methods (ECVAM) in 1999, the traditional tests accepted by regulatory authorities for the assessment of a substance's skin sensitization potential were two guinea pig tests, the Guinea Pig Maximization Test (GPMT) and the Buehler Test (OECD Guideline 406, originating in 1992).
Currently, the mouse LLNA is the preferred and accepted method for assessing skin sensitization of most substances (ICCVAM, 1999; ECVAM, 1999; ICCVAM, 2001 [the protocol]; OECD, 2002; EPA, 2003). Compared with the GPMT, the LLNA reduces animal use by 17% as well as pain and suffering associated with a positive response to an allergen as it evaluated the induction phase and doesn't require the use of irritating adjuvants; it is also faster (1 week vs 4 weeks). Furthermore, the LLNA provides several benefits over other tests for sensitization because it provides a quantitative endpoint, dose-responsive data, and allows for prediction of potency. The latter is particularly valuable because it is now appreciated that contact allergens differ by up to 5 orders of magnitude with respect to their relative skin sensitizing potential. An understanding of potency is, therefore, essential for the development of accurate risk assessments (Kimber et al., 2011). Recently, the reduced Local Lymph Node Assay (rLLNA), which provides an additional reduction in numbers of animals used, has been validated (OECD Test Guideline 429). The reduced LLNA, however, can currently only be used for negative classification and not if dose-response data are required. If a positive/equivocal response is obtained additional testing may be needed. As alternative to the traditional LLNA, which requires the use of 3H-thymidine or 125I-iodiodeoxyuridine to assess lymph node proliferation, the LLNA:DA and the LLNA:BrdU-ELISA have been recently adopted by the OECD (TG442A, LLNA:DA and TG442B, LLNA:BrdU-ELISA). The nonradioactive LLNA methods measure cell proliferation by assessing the level of ATP by a bioluminescence assay, or by the incorporation of BrdU followed by ELISA. Both assays have the same limitations of the traditional LLNA (i.e. false negative findings with certain metals, false positive findings with certain skin irritants, such as some surfactants, or solubility issues of the test substance, as for certain medical devices), but allow for broad use, with reduced hazard for the environment and laboratory workers.
The LLNA is overall appropriate for testing most types of substances. Nevertheless, and in common with all predictive test methods, concerns have been raised with this assay including: levels of false positive responses, variability due to vehicle, and predictivity (Anderson et al., 2011). No toxicology test is perfect, including the LLNA (Basketter et al., 2012). A key perspective is that no predictive test is without limitations; having a good appreciation of these limitations is necessary for making the best use of the information derived from any methods.
Regulatory Requirements & Test Guidelines
The OECD Test Guideline 429 (TG 429), Skin Sensitisation: Local Lymph Node Assay, was adopted in 2002 (OECD, 2002); the OECD TG 406 describes the traditional animal test methods (OECD, 1992). 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" (UNECE, 2004, p. 153).
US agencies that may require sensitization testing include the Environmental Protection Agency (EPA), Consumer Product Safety Commission (CPSC), Food and Drug Administration (FDA), and Occupational Safety and Health Administration (OSHA). The EPA, FDA, and OSHA accepted the LLNA in 1999 as an alternative to the GPMT (ICCVAM, 2007a). The EPA's revised test guideline, OPPTS 870.2600 Skin Sensitization, "incorporates the LLNA for use as an alternative method for assessing skin sensitization under the appropriate circumstances," and has been harmonized with the OECD TG 429 (EPA, 2003).
In Europe, the UK was the first country to introduce the LLNA for regulatory purposes and it has now replaced guinea pig tests in dossiers submitted to it under the Notification of New Substances Regulations. Currently the LLNA is the preferred assay for the predictive identification of skin-sensitizing chemicals, and it is the initial requirement for sensitization testing within REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances).
The GHS guidelines and most countries do not differentiate strong from weak sensitizers. However, only substances considered to be strong sensitizers are regulated under the Federal Hazardous Substances Act (FHSA) in the US. The OECD Scientific Issue Paper on Strong vs. Weak Sensitizers describes current approaches of different countries and regions to potency determination, as well as harmonization issues needing further consideration for classification purposes (OECD, 2006).
Non-animal Alternative Methods
There are currently no validated in vitro or in silico methods to replace animal testing for the identification of skin sensitizing chemicals. However, promising methods are in various stages of development and use. It is expected that a predictive method to totally replace animal testing may require a test battery comprising molecular, cell-based, and/or computational methods. It has been anticipated, however, that it will take at least another 3-5 years for the full replacement of the animal models currently used to assess sensitization (Adler et al., 2011).
(Quantitative) structure activity relationship [(Q)SAR]: Structure activity systems are 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.
Peptide reactivity (or depletion) assay: This approach is based on the understanding that for a chemical to induce skin sensitization it must form stable associations with proteins. Such assays therefore measure the inherent or acquired ability of chemicals for binding model peptides.
Cell-based assays: 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, and alterations in dendritic cell phenotype and function following allergen exposure. More recently, a human T cell priming assay has been developed within the EU project Sens-it-iv (Richter et al., 2013). This multiparametric flow cytometry based assay identifies chemical specific T cells based on their frequency and antigen-specific production of the cytokines IFN-γ and TNF-α at the single cell level. With regard to potency assessment, very recently Gibbs et al. (2013) have explored the possibility of combining the epidermal equivalent (EE) potency assay with a test that assesses release of interleukin-18 (IL-18) to provide a combined approach for identification and classification of skin sensitizing chemicals, including chemicals of low water solubility or stability. Results to date are encouraging.
Integrated testing Strategy: It is anticipated that (Q)SAR and molecular and/or cell-based assays may in the future be used in concert to form predictive test batteries to identify skin sensitizers and replace animal testing.
More about recent developments in non-animal test methods can be found in Emerging Research, Methods & Policies.
Validation and Acceptance of Non-animal Alternative Methods
Validated non-animal methods are not yet available for assessing skin sensitization. The only validated alternative methods are the mouse LLNA and the mouse rLLNA.
At present, three non-animal test methods, namely the Direct Peptide Reactivity Assay (DPRA), the KeratinosensTM and the human Cell Line Activation Test (hCLAT) are under formal validation at EURL-ECVAM for their potential to predict skin sensitization potential. Results are expected late 2013/early 2014. It has been anticipated, however, that it will take at least another 3-5 years for the full replacement of the in vivo animal models currently used to assess sensitization (Adler et al., 2011).