In 2014, are we closer than ever to a replacement for animal-based skin sensitization tests?

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In 2014, are we closer than ever to a replacement for animal-based skin sensitization tests?

Marilyn Matevia, Humane Society of the United States

Published: April 17, 2014

While there are a number of validated and widely-used non-animal methods for assessing a chemical’s potential to cause skin irritation or skin corrosion, the technical challenges of modeling skin sensitization in vitro have proven harder to surmount.  But 2013 and early 2014 have seen several important advances, including the first formally EURL ECVAM-validated non-animal sensitization assay.

Background

Although skin corrosion and irritation are acute responses and are observable relatively quickly after exposure to a substance, skin sensitization (allergic contact dermatitis, or ACD) is a more complex, two-step stage biological process by which a chemical exposure first potentiates the immune system and subsequent exposure elicits an allergic response.

The sequence of events comprising both stages is well-understood (see, e.g., Kimber et al., 2001; Lepoittevin, 2001; Maxwell et al., 2012; van der Veen et al., 2011; Vocanson et al., 2009).  For a substance to cause an immune response, it must be able to penetrate the skin and react with proteins in the epidermis.  Chemical-protein conjugates on the surface membrane of epidermal dendritic cells (Langerhans cells) are processed and carried to nearby lymph nodes where they are presented to immature T cells.  In response, the T cells in the lymph nodes begin differentiating into allergen-specific effector cells, as well as “memory” T cells.  At the same time, chemical-protein conjugates that form on epidermal keratinocytes cause these cells to secrete cytokines that further stimulate the immune response.  This sequence of events comprises the first phase of skin sensitization, called “induction” or “initiation.” On subsequent exposures to the allergen, the now-primed immune system quickly mounts an allergic response: memory T cells migrate to the point of contact, where they signal keratinocytes to again produce cytokines and chemokines that formulate a protective reaction – the rash or inflammation known as dermatitis – at the surface of the skin. This second phase of skin sensitization is called “elicitation.”

Since the elicitation of the full allergic sequence is entirely dependent on provoking the response, predicting sensitization can be accomplished – and simplified – by predicting just the initiation phase.  This is the logic behind the now commonly used in vivo Local Lymph Node Assay (LLNA). However, because even the initiation phase involves several cell types, reproducing the entire sequence in vitro has historically presented significant technical challenges to researchers.  An evolving approach is to use combinations of single assays in an Integrated Testing Strategy (ITS), with each assay targeting a key event in the sensitization process.  When informed by an adverse outcome pathway, an ITS can lead to increased confidence in prediction of sensitization (OECD, 2012).

New Developments

Over the previous year and the beginning of 2014, we have seen several new methods and strategies that could expand the sensitization testing toolbox.  Here are some highlights:

  • Activities at EURL ECVAM:
    • Early in 2013, EURL ECVAM issued its 5-year Strategy for Replacement of Animal Testing for Skin Sensitization Hazard Identification and Classification, recommending that three methods then in formal validation – Direct Peptide Reactivity Assay (DRPA), KeratinoSens, and the human Cell Line Activation Test (hCLAT) – be incorporated into an ITS for hazard identification. The DPRA is an in chemico assay that mixes potentially sensitizing chemicals with synthetic peptides and measures the extent of peptide reactivity; high reactivity correlates with high skin sensitization potential. The KeratinoSens assay uses a luminescent reporter gene to track activation of the Keap1-Nrf2-ARE pathway that regulates keratinocytes following exposure to sensitizers. The hCLAT measures changes in gene expression on surrogates for dendritic cells (such as human leukemia cell lines), following exposure to sensitizers. Each of the three recommended methods targets different key events identified in the OECD’s “Adverse Outcome Pathway (AOP) for Skin Sensitisation Initiated by Covalent Binding to Proteins” (OECD, 2012).
    • The EURL-ECVAM strategy document noted that, while full regulatory replacement of animal testing for skin sensitization will ultimately require development of additional non-animal tests capable of generating both hazard and potency information, adopting non-animal approaches for identifying skin sensitizers would satisfy several EU regulatory requirements and “would have the biggest impact on the saving of animals for regulatory testing in the short and medium term” (Executive Summary).
    • EURL ECVAM then issued a final recommendation on the DPRA (December 2013), endorsing it for inclusion in an ITS for hazard identification, and supporting the development of a DPRA OECD test guideline. “When employed within an integrated approach, the DPRA may be useful to satisfy information requirements for Cosmetics (Regulation EC/1223/2009), Chemicals (Regulation EC/1907/2006), Biocides (EC/528/2012), and Plant Protection Products (Regulation EC/1107/2009).”
    • In February 2014, EURL ECVAM issued a recommendation on KeratinoSens, also endorsing its inclusion in an ITS, and supporting development of an OECD test guideline, while also recommending additional evaluation of this method.
  • Researchers in Portugal developed a method that measures changes in gene expression and intracellular signaling activity of chemically-exposed dendritic cells to determine whether a chemical is a sensitizer or non-sensitizer. Bruno Neves et al. (2013) first established the translation and signal transduction profiles of cultured mouse fetal skin-derived dendritic cells exposed to a set of reference “training” chemicals. They then validated their model on a set of test chemicals, and compared its performance to existing LLNA data and human evidence.Twelve of 13 sensitizers were correctly classified (sensitivity=92%) and 5 of 5 non-sensitizers were identified (specificity=100%), leading the authors to propose that this model could be one component of an integrated test battery.
  • BASF and Promega Corporation collaborated on a new human skin cell line that forms the basis for an in vitro protein reactivity assay, LuSens, which they have submitted to EURL ECVAM for validation. The human skin cell line incorporates a luminescent reporter gene developed by Promega. When the skin cell exhibits stress reactions (irritation) in the presence of a potential sensitizer, those reactions produce a light signal that can be detected by a luminometer. LuSens’ predictivity is comparable to KeratinoSens, and when used as part of a comprehensive test battery, predicts human skin sensitizers as well as the LLNA.
  • Proteome Sciences announced that its SensiDerm Multiplex assay was prioritized for further development by Cosmetics Europe, after the assay performed well on a set of blinded samples that had been exposed to ten unknown sensitizers. The SensiDerm assay measures the expression of eight different proteins produced by cultured human skin cells following exposure to chemicals. The concentration of these proteins forms a response profile that has been correlated with the sensitizing potential of known chemicals.
  • Anne Dickinson and colleagues at Newcastle University developed an in vitro assay, Skimune, that involves isolating dendritic cells from the blood of healthy human donors, exposing the dendritic cells to potential allergens to activate an immune response, and then co-culturing the activated dendritic cells with skin cells from the same donor to assess how the skin cells respond to the immune signals. As Dickinson explained, they are “looking for an adverse immune response, rather than a toxic response,” and the test is meant to complement existing skin tests, “to give more confidence and more information.”
  • Researchers at the Kao Corporation published promising results on EpiSensA, an in vitro skin sensitization assay that measures gene expression in 3-dimensional reconstructed human epidermis (RhE) exposed to potential sensitizers. Using the expression profiles to classify sensitizers, non-sensitizers, and pre-/pro-haptens, three genes agreed completely or very closely with results from an LLNA: ATF3 (100%), DNAJB4 (93.8%), and GCLM (87.5%).
  • In a competition co-sponsored by the UK’s Technology Strategy Board and Innovation Norway, a collaboration between ScandiDerma and Epistem won funding to develop a new living (human) skin equivalent (LSE) that includes immune cells and is capable of reproducing inflammation. Results are expected in 2014.
  • Susan Gibbs et al. (2013) found that release of the cytokine interleukin-18 (IL-18) in human epidermal equivalents (EE) exposed to chemical compounds could be used not just to identify sensitizers, but to rank their potency. The EE potency assay they describe exhibited high transferability between labs, could be easily modified to other commercially available human skin models, and correlated even better with human skin data than with mouse data collected for the same chemicals.
  • A validation study of the SenCeeTox method introduced by Cyprotex/CeeTox and coordinated by PETA demonstrated that, by combining peptide reactivity with measured cellular markers in the 3-dimensional human tissue construct EpiDerm (approved by the OECD for skin irritation and skin corrosion tests), they were able to classify potential sensitizers by hazard and by potency. The SenCeeTox assay was tested in two outside laboratories and will be tested in a third before being submitted for validation by EURL ECVAM.

With Europe’s March 2013 Cosmetics Directive banning the sale of cosmetics that have been tested on animals in the EU, and with further implementation of REACH regulations causing increased pressure for information, some observers warned of an inhibitory effect on innovation in cosmetics. That remains to be seen; however, what is clear from the year’s developments in skin sensitization testing is that legislative “encouragement” is spurring significant innovation in the area of alternatives.

Read more information on skin sensitization elsewhere on AltTox.org: