Areas of emerging skin research include: skin regeneration and the mechanisms involved in skin irritation and wound healing; enhancements of existing in vitro methods to make them acceptable for a broader range of testing applications; and, development of new methods for in vitro culture of skin cells, including co-cultures (keratinocytes + another type of skin cell), miniaturization, and high throughput testing platforms. Areas of emerging policy include: validation issues; harmonization issues; and, the use of in vitro and in silico methods in integrated and tiered test schemes for regulatory applications.
Additional work continues on modifying existing in vitro methods to make them acceptable for a broader range of testing applications and regulatory acceptance. For example, a study by Mallampati, et al. (2010) indicates that the EpiDerm full thickness-300 model can be used to asses the in vivo irritation potential of aliphatic hydrocarbons.
The ESAC statement on the validation assessment of the EpiDerm model suggested that the protocol be modified to increase the method’s sensitivity. Kidd, et al. (2007) addressed this issue using a tiered testing approach so that the method could be used to predict both skin irritants and corrosives. Two assay endpoints, MTT cell viability and IL-1α release, were used to identify substances as corrosive/severe irritants. Other substances were then evaluated for mild to moderate skin irritation potential using three additional exposure times with the two endpoints. Substances found negative in both rounds of testing were considered non-corrosive/non-irritating. Kidd, et al. (2007) found the use of both endpoints improved the sensitivity, specificity, and accuracy of the results. However, when MatTek scientists modified the EpiDerm protocol with extended exposure times they obtained a significant increase in sensitivity without decreasing the specificity, and the additional assay for IL-1α release did not further improve the results (Kandárová, et al., 2009).
Other examples of protocol optimization of existing in vitro methods have been reported. Cotovio, et al. (2005) developed a two-tiered strategy using the EpiSkin model that increased assay sensitivity and resulted in a decrease in false positives where the MTT viability assay was first assessed followed by assays for the release of adenylate kinase (a marker for cell membrane damage) and the inflammatory cytokines IL-1α and IL-8. Various protocol adaptations in the SkinEthic RHE model for skin irritation testing were evaluated against the EpiSkin performance standards resulting in the identification of an optimized protocol with an overall accuracy of 85% when testing the 20 ECVAM reference test substances (Tornier, et al., 2010).
New skin models to assess skin irritation and corrosion continue to be developed. These include the Leiden reconstructed human epidermal model in the Netherlands (El Ghalbzouri, et al., 2008), and the LabCyte EPI-MODEL in Japan (Katoh, et al., 2009). New models can be validated using the performance standards and reference chemicals set forth by ECVAM and/or the OECD.
The barrier function of in vitro epidermal models has been challenged as not being equal to the barrier of the human skin (Gibbs, et al., 2002; Netzlaff, et al., 2005). Netzlaff, et al. (2007) compared the barrier function of EpiSkin to an ex vivo human skin preparation using a diffusion cell. EpiSkin was a poorer barrier than the human skin for the two compounds evaluated. The permeability of the same two chemicals was compared in three in vitro skin models (SkinEthic, EpiDerm, and EPISKIN) and three ex vivo skin preparations (human epidermis, bovine udder skin, and pig skin) (Schäfer-Korting, et al., 2006). The in vitro models overestimated the permeation of both chemicals compared to the human epidermis, however, the performance and reproducibility of the in vitro tissues were sufficient for organization of a validation study for their use in predicting percurtaneous penetration. The interlaboratory study conducted in Germany “demonstrated that MatTek’s EpiDerm in vitro human skin tissue equivalent is an appropriate alternative to human and pig skin for the in vitro assessment of the permeation and penetration of substances when applied as aqueous solutions” (Schäfer-Korting, et al., 2008). MatTek provides a protocol for the use of EpiDerm tissues in percutaneous absorption testing. Dermal penetration testing and research involving the metabolizing capacity of in vitro skin models are covered in greater detail on AltTox in Toxicity Endpoints & Tests: Dermal Penetration.
Another study described how skin irritant potency might be determined using in vitro epidermal models. Spiekstra, et al. (2009) tested 4 chemicals (dose-responses at 24 hours) using two different in vitro models that have different barrier properties. They assessed various endpoints 3 cytokines and cytotoxicity. They found variations in chemical penetration between the 2 models and variation in biomarker sensitivity between the different chemicals tested. The results, however, ranked the chemicals the same irrespective of the model or endpoint used.
New endpoints continue to be investigated for the assessment of skin irritation. Changes in gene expression following chemical exposure, or toxicogenomics, have been studied in several in vitro skin models (Borlon, et al., 2007; Fletcher, et al., 2001; Niwa, et al., 2009; Törmä, et al., 2006; Wei, et al., 2006). One study found changes in the expression of some proteins (proteomics) following surfactant exposure of EpiDerm cultures (Fletcher & Basketter, 2006). A 2009 review of biomarkers for assessing skin irritation potential noted the “limited number of genomic and proteomic studies” (Gibbs, 2009). Biomarkers/assay endpoints used in skin irritation models, reviewed by Gibbs (2009), included IL-1α, IL-6, IL-8, PGE2, SKALP, HSP70, kinases, MTT assay, and lactate dehydrogenase assay.
In vitro skin models originally developed for skin irritation testing are now being used for a variety of testing and research purposes. Some of the applications for the EpiDerm™ model reported by the manufacturer include: skin irritation, skin corrosion, dermal phototoxicity, skin inflammation, percutaneous absorption, transdermal drug delivery, psoriasis research, gene expression analysis, and use in high throughput screening configurations. Other testing applications of in vitro skin models include: genotoxicity, immunotoxicity, metabolism, skin sensitization, and mechanical loading.
An in vitro method for skin phototoxicity that uses 3T3 mouse fibroblast cells was endorsed as scientifically valid by ECVAM in 1997. Assays based on human skin cells are now being developed for phototoxicity testing (Jirová, et al., 2005; Lelièvre, et al., 2007).
Co-culture models where keratinocytes are cultured with another type of skin cell are being developed for specific testing applications. Two examples are keratinocyte/neuron co-cultures for assessing sensory responses and keratinocyte/dendritic cell co-cultures for skin sensitization testing.
Differential cytokine release using in vitro skin models has been extensively explored for the discrimination of skin irritants and skin sensitizers (Coquette, et al., 2003, and many others). When scaled up, these endpoints have not been found to be sufficiently predictive of irritants vs sensitizers. Newer approaches include assessing cell signalling pathways in keratocyte cultures activated by sensitizers vs irritants (Koeper, et al., 2007), and the use of co-culture models of keratinocytes and dendritic cells (Jacobs, et al., 2004; Ryan, et al., 2005; 2007). This topic is covered in greater detail on AltTox in Toxicity Endpoints & Tests: Skin Sensitization.
Basic research in skin cell biology is still revealing information useful to the development of in vitro skin models and to the understanding of the mechanisms of chemical-induced skin irritation. One example is the recent studies on stem cells in the epidermis. Populations of stem cells in the adult skin are maintained to regenerate skin epithelial cells and repair wounds (Fuchs, 2007). Asymmetric cell division has been proposed as the method used by stem cells in the renewal of the epidermis. Lechler & Fuchs (2005) showed that “basal epidermal cells use their polarity to divide asymmetrically, generating a committed suprabasal cell and a proliferative basal cell.” The proliferative basal cell remains attached to the epidermal basement membrane, and the suprabasal cell moves (over several weeks time) up through the keratinocyte layers of the epidermis as it differentiates and eventually becomes part of the skin barrier. A better understanding of the cellular and molecular processes involved in skin regeneration and wound healing will lead to the development of better in vitro models and endpoints for skin irritation testing.
A recent study of skin cell biology during chemical-induced skin irritation revealed that immune cells are involved in the skin’s response to chemical irritants. Ouwehand, et al. (2010) noted that “skin irritation is generally not considered to be an immunological event; however, alterations in the density of Langerhans cells (LC) in the epidermis do occur.” Using ex vivo intact human skin and epidermal sheets, they studied “the migration of LC out of the epidermis after exposure of the skin to contact irritants” and found that chemokines and dermal fibroblasts are involved in LC migration towards the dermis following irritant exposure. The mechanism differs from allergen exposure.
Tiered testing strategies using a combination of in vitro assays, in silico methods, and/or human patch testing for skin irritation and risk assessment are now being performed for many types of substances, especially cosmetics, where some manufacturers have circumvented animal testing of their products for many years (Robinson, et al., 2000; Robinson & Perkins, 2002). Methods for repeat and chronic exposure for skin irritation, while not specifically validated, are also being used within industry.
A report by the Fund for the Replacement of Animals in Medical Experiments (FRAME) reviewed integrated testing strategies (ITS) for skin corrosion and irritation, and proposed a decision-tree tiered testing scheme that could be used to eliminate animal use for skin testing in the EU REACH program (Grindon, et al., 2007).
ITS and tiered testing strategies are also being adapted for regulatory applications. A decision-tree integrated testing scheme was proposed for incorporating the maximal use of non-animal methods for skin irritation and corrosion testing for regulatory applications within the EU, and especially to address the testing needs of the REACH (Registration, Evaluation and Authorisation of Chemicals) program (Grindon, et al., 2007). Tiered/integrated strategies for assessing skin irritation potential have continued to be refined (Grindon, et al., 2008; Hoffmann, et al., 2008; Hulzebos, et al., 2010; Macfarlane, 2009).
An ECVAM assessment of ITS indicated the need for “further guidance on construction and multi-parameter evaluation” (Hoffmann, 2008). An analysis by COLIPA on a tiered approach using alternatives concluded that “the safety assessments for skin irritation/corrosion of new chemicals for use in cosmetics can be confidently accomplished using exclusively alternative methods” (Macfarlane, et al., 2009). “The Integrated Assessment Scheme (IAS) defines weight factors for each piece of toxicological information under REACH in an Integrated Testing Strategy” (Hulzebos, et al., 2010). Hulzebos, et al. (2010) demonstrated the usefulness of IAS in decision making for skin irritation classification and labeling, and concluded that “it adds to the ToxRTool and the ITS of Hoffmann et al. on the same endpoint and similar methods.”