Human evidence for the skin sensitization of chemicals derives from clinical experience, diagnostic patch testing, and other tests designed to confirm the absence of sensitization potential.
The Globally Harmonized System for Classification and Labeling of Chemicals (GHS) defines a skin sensitizer as “a substance that will lead to an allergic response following skin contact” (UNECE, 2015, p. 39). GHS classifies a substance as a skin sensitizer if: (a) “there is evidence in humans that the substance can lead to sensitization by skin contact in a substantial number of persons,” or (b) “there are positive results from an appropriate animal test” (p. 41). Skin sensitizers are classified as GHS hazard Category 1 “where sub-categorization is not required by a competent authority or where data are not sufficient for sub-categorization” (p. 41). When data are sufficient and required, skin sensitizers can be classified as strong sensitizers (1A) or other sensitizers (1B).
Contact dermatitis, including both allergic and irritant contact dermatitis, is the second most commonly reported occupational illness, accounting for 10% to 15% of all occupational diseases. Allergic contact dermatitis (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, which 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, an aggressive immune response will be elicited resulting in a 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 (2006), provides an excellent overview of the types and mechanisms of allergies. The key biological events of skin sensitization have also been described in the Organisation for Cooperation and Development (OECD) report The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins (OECD, 2014).
A diagram of the mechanism of skin sensitization (Gildea, 2005)
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) Test Guideline (TG) 406, 1992; EPA OPPTS 870.2600 Skin Sensitization, 1996).
Currently, the mouse LLNA is the preferred and accepted method for assessing skin sensitization of most substances (ICCVAM Evaluations; ICCVAM Protocols; ECVAM ESAC Statement, 2000; OECD TG 429, 2010). 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 evaluates 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 the 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).
The reduced mouse Local Lymph Node Assay (rLLNA), which provides an additional reduction in numbers of animals used, is a validated version also described in OECD TG 429. The rLLNA, 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 an alternative to the traditional LLNA, which requires the use of 3H-thymidine or 125I-iodiodeoxyuridine to assess lymph node proliferation, the LLNA:DA (OECD TG442A) and the LLNA:BrdU-ELISA (OECD TG442B) were also adopted by the OECD in 2010. The nonradioactive LLNA methods measure cell proliferation by assessing the level of ATP using 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, and having a good appreciation of these limitations is necessary for making the best use of the information derived from any method.
The GHS recommends using the OECD Test Guidelines for skin sensitization, but notes that “other methods may be used provided that they are well-validated and scientific justification is given” (UNECE, 2015, p. 42).
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). EPA, FDA, and OSHA accepted the LLNA in 1999 as an alternative to the GPMT (ICCVAM, 2007). The EPA’s revised test guideline, OPPTS 870.2600 Skin Sensitization, incorporates the LLNA as an alternative method for assessing skin sensitization under the appropriate circumstances, and it has been harmonized with the OECD TG 429. More recently, FDA has claimed the LLNA test methods are not appropriate for some of the types of products they regulate.
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 is the initial requirement for sensitization testing within REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances).
ARE-Nrf2 luciferase test method1: KeratinoSens™
In vitro skin sensitization
EURL ECVAM (2014)
OECD TG 442D (2015)
Direct Peptide Reactivity Assay (DPRA)2
In chemico skin sensitization
EURL ECVAM (2013)
OECD TG 442C (2015)
Human Cell Line Activation Test (h-CLAT)3
In vitro skin sensitization
EURL ECVAM (2015)
Draft OECD TG (2015)
1 In vitro method endorsed as valid for supporting the discrimination between skin sensitizers and non-sensitizers in accordance with the UN GHS. The guidelines explain it is likely that combinations of non-animal methods using integrated testing approaches will be needed to substitute for the animal tests. The only ARE-Nrf2 luciferase test method covered currently by this TG is the KeratinoSens™ test method.
2 In chemico method endorsed as valid for supporting the discrimination between skin sensitizers and non-sensitizers in accordance with the UN GHS. The guidelines explain it is likely that combinations of non-animal methods using integrated testing approaches will be needed to substitute for the animal tests.
3 In vitro method endorsed as valid for supporting the discrimination between skin sensitizers and non-sensitizers in accordance with the UN GHS. The guidelines explain it is likely that combinations of non-animal methods using integrated testing approaches will be needed to substitute for the animal tests.
Scientists generally agree that non-animal methods, individually, which focus on only a part of the biological events involved in a skin irritation response, are not sufficient to replace the animal test methods. Adler et al. (2011), Hartung et al. (2011), and others, however, have proposed that integrated testing strategies (ITS) composed of some combination of (Q)SAR, molecular, and/or cell-based assays could come to be used to form predictive test batteries to identify skin sensitizers and replace animal testing.
Since that time, three non-animal test methods, the Direct Peptide Reactivity Assay (DPRA), the KeratinoSens™, and the human Cell Line Activation Test (hCLAT), have been endorsed as valid by EURL ECVAM for their potential to predict skin sensitization.
In November 2013, EURL ECVAM recommended the DPRA for use as part of a testing strategy as follows:
The DPRA addresses a key mechanism (haptenation) in the development of skin sensitization/allergic contact dermatitis. Overall the provided data support transferability and reproducibility of the test to qualified laboratories. The predictive capacity of the test is not defined yet, but the preliminary data profiles the test as a useful tool for early decision making during product development (screening) and a component in a weight-of-evidence approach or ITS for safety/hazard assessment (p. 21).
In February 2014, EURL ECVAM recommended the KeratinoSens™ assay for use as part of a testing strategy as follows:
…the KeratinoSens™ appears to be a reliable test method that provides information on the ability of a chemical to activate the Nrf2 electrophilic and oxidative-stress response signalling pathway which has been shown to be a relevant pathway in the induction of skin sensitisation ….Therefore, Nrf2–dependent luciferase induction measurements in the KeratinoSens™ assay when combined with information from other non-animal methods in the context of a Weight-of-Evidence (WoE) approach or Integrated Testing Strategy (ITS) may provide useful information about the sensitisation potential of chemicals….it is plausible that KeratinoSens™ data may also contribute to characterisation of skin sensitisation potency within integrated approaches… (p. 11).
In March 2015, EURL ECVAM published its recommendation on the human Cell Line Activation Test (h-CLAT) for skin sensitisation testing; summarized as follows:
Within [the OECD] AOP the activation of dendritic cells (DC), typically assessed by expression of cell surface markers, chemokines and cytokines, is considered to be a key event….The human Cell Line Activation Test (h-CLAT) measures the upregulation of the CD86 and CD54 markers of DC activation in THP-1 cells, a human monocytic leukemia cell line. …EURL ECVAM concluded that the h-CLAT test method should prove valuable within Integrated Approaches to Testing and Assessment (IATA) for hazard assessment. The h-CLAT may also be able to contribute to the assessment of sensitising potency, however it is recognised that further efforts are required to explore how h-CLAT data may contribute to potency assessment (p. ii).
The three endorsed non-animal methods represent key biological mechanisms covered by the OECD Guidance Document (GD) on Developing and Assessing Adverse Outcome Pathways (AOPs) (OECD GD 184, 2013). EURL ECVAM experts, however, continue to recommend that “Due to the complexity of the mechanisms underlying skin sensitisation, it is likely that information from different methods (in silico, in chemico, in vitro) is needed to reduce or replace the need for animal testing, both for hazard identification and potency characterisation purposes” (p. 9).
More on recent developments in non-animal methods for skin sensitization testing can be found at:
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
The information provided here is intended only as an overview, and is neither guidance or a comprehensive review of the laws and regulations on skin sensitization testing. Individual countries/regions and their regulatory authorities usually provide specific guidance on hazard/toxicity testing requirements.