Modelling Long-Term Repeat-Dose Toxicity – Challenges Faced

Modelling of repeat/chronic dose toxicity is a complex area of research, but will be restricted to consideration of epithelial barriers in this short paper. Papers from the group of Pfaller will deal with organ culture approaches (Balls et al., 2001). Chronic and/or repeat approaches to toxicity have been examined by a number of authors (e.g., Balls et al., 1982; Dierickx and Ekwall, 1992; Clothier and Samson, 1996; Clothier et al., 1997). Problems arise not only with respect to the choice of target cells but also regarding the medium composition (Wilkinson and Clothier, 2005), since defined in vitro functions must be retained, which depends on certain medium supplements (Rubin and Rice, 1986) and cell stability in vitro. The ability to grow epithelial cells in the undifferentiated state and then control differentiation is critical (Rheinwald and Green, 1975; Ward et al., 1997), as toxicity can affect differentiation triggers, the differentiation control process (Rubin and Rice, 1986; Gray et al., 1999), and/or maintenance of the differentiated state (Barker and Clothier, 1997). In addition, in vitro toxicity should include evaluation of recovery from injury (e.g. Clothier et al., 1997, 1999a), which is part of in vivo toxicity profiling. Monitoring endpoint assay methods should allow for repeat exposure of cultures, and assess both functional and toxicological endpoints. Many routinely employed and even validated assays require fixation and solubilisation of cultures, thus numerous replicate cultures must be performed (e.g. Dierickx and Ekwall, 1992; Spielmann et al., 1998). Hence, the FRAME Alternative Laboratory has been developing endpoint assays that allow repeat monitoring of the same cultures (e.g. Shaw et al., 1991; Clothier and Samson, 1996) with standardised protocols (e.g. Gray et al., 2004). New methods and imaging systems such as confocal microscopy (Garside et al., 1998) and Terahertz radiation (Arnone et al., 1999; Clothier and Bourne, 2003) are still required. The Alamar BlueTM assay is based on the conversion of resazurin to resorufin (O’Brien et al., 2000) at concentrations that do not apparently harm the differentiation capacity of keratinocytes or corneocytes (Clothier and Samson 1996; Clothier Modelling Long-Term Repeat-Dose Toxicity – Challenges Faced