Areas of emerging skin research include: studies on skin regeneration and the mechanisms involved in skin irritation and wound healing; modifying 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 tiered test schemes or as animal replacement methods 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, the ESAC statement for the validation assessment of the EpiDerm model made the suggestion that the EpiDerm 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. Both 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-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. The use of both endpoints improved the sensitivity, specificity, and accuracy of the results. When MatTek scientists modified the EpiDerm protocol with extended exposure times they likewise "obtained a significant increase in sensitivity without decreasing the specificity of the method" (Kandárová, et al., 2007).
Another example of protocol optimization of an existing in vitro skin method was reported by Cotovio, et al. (2005) for EPISKIN. A two-tiered strategy 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 resulted in an increase in assay sensitivity and a decrease in false positives.
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 (in progress). MatTek provides a protocol for the use of EpiDerm tissues in percutaneous absorption testing.
New endpoints continue to be investigated for the assessment of skin irritation. Changes in gene expression following chemical exposure, or toxicogenomics, has been studied in several in vitro skin models. Differential gene expression has been reported between irritant and non-irritant exposures (Borlon, et al., 2007; Fletcher, et al., 2001), however, the amount of data is not sufficient to identify specific biomarkers at this time.
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, and skin sensitization.
An in vitro method for skin phototoxicity was endorsed as scientifically valid by ECVAM in 1997, however, this method uses 3T3 mouse fibroblast cells. 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.
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 yet validated, are also being used within industry.
Tiered testing strategies are also being used for regulatory applications. A recent analysis proposed a decision-tree integrated testing scheme 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).
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