According to the director of ICCVAM, "advances in structure-activity relationship models, assessment of protein and peptide bonding potential, and the evaluation of responses in mechanism based cellular systems," as well as "integration of the responses from a battery of test models," have the potential to eventually replace animals in the prediction of sensitization potential (Stokes, 2007).
Industry scientists have developed methods to identify skin sensitizers based on changes in the gene expression profile of dendritic cells isolated from peripheral blood fractions, and specifically to distinguish chemical sensitizers from chemicals that are skin irritants (Ryan, et al., 2004; Gildea, et al., 2006). A set of target genes have been identified that are up- or down-regulated in cells following exposures to known allergens. The goal of these scientists is to be able to use computational and cell-based assays in a test battery that will be predictive in identifying human skin sensitizers without the use of animals.
Dentritic cells in the skin [Langerhans cells (LC)] have a role in the induction of allergic contact dermatitis and undergo a number of phenotypic and functional changes following skin exposure to an allergen. Some of these cellular alterations have been used as the basis for chemical allergen detection in cell-based assays (Ryan, et al., 2005). Chemical-induced migration of LC out of a human organotypic skin explant culture has also been used as a measure of sensitization potential (Jacobs, et al., 2004). Skin irritants can also induce LC migration and phenotype changes, so test chemicals must be evaluated at non-irritant concentrations. A 2006 workshop entitled Dendritic Cells and Skin Sensitization: Biological Roles and Uses in Hazard Identification examined dendritic cell-based approaches for assessing the skin sensitization potential of chemicals (Ryan, et al., 2007).
An in vitro assay for evaluating the skin sensitization potential of chemicals was developed using THP-1 cells (a human monocytic leukemia cell line) (Ashikaga, et al., 2006; Sakaguchi, et al., 2006). In this assay, the human Cell Line Activation Test (h-CLAT), chemical allergens are predicted by the up-regulation of CD86 and CD54 expression when cells are exposed to subtoxic concentrations of chemicals. The h-CLAT may also be able to classify allergen potency (Sakaguchi, et al., 2007).
Several labs have developed peptide reactivity assays to screen for chemical allergens (Aptula, et al., 2006; Gerberick, et al., 2004; 2007; Natsch, et al., 2007). This method is based on an early step in the mechanism of induction of skin sensitization - the binding of the chemical allergen to skin proteins and/or peptides. Most known chemical allergens are electophilic (electron seeking; usually positively charged) and react with peptides or protein amino acids that are nucleophiles (electron donors). The ability of known chemical allergens to bind with nucleophilic amino acids has been shown to correlate to the skin sensitization potential of a chemical.
(Quantitative) structure-activity relationship [(Q)SAR] methods are another approach to predicting a chemical's potential to cause skin sensitization. (Q)SAR modeling can be conducted using commercial software such as DEREK for Windows (Barratt & Langowski, 1999; Langton, et al., 2006) and TOPKAT, or a model can be constructed such as the logistic regression model of Fedorowicz, et al. (2005). Structure-activity relationships based on the electrophilic reaction mechanism were found useful in classifying a set of chemicals already evaluated in the Local Lymph Node Assay (LLNA) (Ashby, et al., 1995; Roberts, et al., 2007). A recent case study by the OECD is Derek for Windows Model for Skin Sensitisation (OECD, 2007, p.101-107). Patlewicz, et al. (2007) evaluated the performance of three models (TOPKAT, Derek for Windows, and TOPS-MODE) with the same dataset and concluded that "none performed sufficiently well to act as a standalone tool for hazard identification." (Q)SARs are discussed in greater detail in (Q)SAR: Emerging Technologies & Approaches.
The development and use of various screening methods have generated support that in vitro and/or in silico methods conducted as a test battery or tiered test scheme could completely replace the use of animals for skin sensitization testing. Stokes (2007) explained that new integrated approaches for skin sensitization testing would require "validation of each individual model as well as the entire test battery with the same set of reference substances for which there is high quality data from the accepted reference test method." ICCVAM has yet to establish the detailed processes and guidelines that would facilitate validation of a test battery, so this issue will become more of a priority as the in vitro and in silico methods for a skin sensitization test battery become feasible.
The European Commission is supporting the Sens-it-iv project with the goal of developing in vitro alternatives to animal tests currently used for the risk assessment of potential skin or lung sensitizers (Rovida, et al., 2007). The Sens-it-iv research is being performed by 28 European Union university and industry labs. The most recent study results can be obtained in the Sens-it-iv newsletters. A summary of the accomplishments of the first 12 months of this program (Oct. 2005-Oct. 2006) included the characterization and optimization of culture conditions for various human cell lines; generation of gene expression profiles for several types of cells; and development of a 3D epidermal (skin) model for studying epitehlial-dendritic cell interactions. Methods and cellular models that incorporate T-cells are being developed, including proteome (protein) expression profiles of all three cell types and methods to investigate hapten formation in vitro.
Current ICCVAM activities include assessing the validation status of various applications and related protocols of the LLNA assay as a result of a nomination for their review by the US Consumer Product Safety Commission (CPSC) in January 2007 (ICCVAM, 2007b). The CPSC claimed that proposed uses of the LLNA extend beyond the scope of the original ICCVAM review and therefore nominated the LLNA for further evaluation of its use "for classification purposes, the validation status of non-radioactive LLNA protocols, the LLNA limit test, the use of the LLNA to test mixtures, aqueous solutions and metals, and the applicability domain for which the LLNA has been validated" (ICCVAM, 2007b). ICCVAM is assembling a peer review panel to assess and comment on these LLNA protocols and applications in 2008.
ECVAM recently validated the reduced Local Lymph Node Assay (rLLNA), which uses fewer animals than the LLNA (ECVAM, 2007). This assay will be especially important in reducing animal use in large testing programs such as REACH. The status of ICCVAM's review of the rLLNA has not been reported.
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