The current industry standard for predicting contact dermal sensitization is the murine Local Lymph Node Assay (LLNA). A significant limitation of the LLNA is the frequency of false positives, as well as the occurrence of false negatives. This limitation occurs as a result of the LLNA inability to always correctly distinguish between substances that are strongly irritating and those that are sensitizing. The LLNA assesses sensitization by evaluating a dose-response of proliferating lymphocytes within a test group compared to a vehicle control group. Sensitizing substances induce lymphocyte proliferation, but irritating substances can also induce lymphocyte proliferation, thus preventing an accurate classification. Even though over-prediction of substances as sensitizers sounds acceptable, in the context of regulatory guidelines for public safety, this recognized limitation of the LLNA results in a significant issue during testing. Current testing guidelines allow for strong irritant to be tested at lower concentrations to avoid systemic toxicity and excessive local skin irritation. Limiting the maximum test concentrations as a means to prevent excessive skin irritation allows for the introduction of false negatives results, especially when testing a strong irritant that is lso weakly sensitizing. Thus there is a need for an improved endpoint to achieve greater accuracy in the LLNA. Animal testing is currently the only accepted regulatory means for identifying sensitizing substances. The LLNA, which was adopted 10 years ago, is the preferred assay over the older guinea- pig maximization test (GPMT). The LLNA is currently seen as the gold standard of sensitization testing. Although animal-free assays are currently not accepted by regulatory agencies, there is a growing interest to develop animal-alternative assays. Since the LLNA is used as a standard of comparison when new alternative tests are developed, any false predictions in the LLNA will inevitably complicate analysis of other assays. Even though the ability to distinguish sensitizers from irritants can be accomplished by more extensive molecular testing, additional testing is rarely performed in toxicological studies. To this end, we will evaluate using cytokine IL-18 and IL-18 receptor expression as a sensitizer specific-marker to enhance the prediction capability of the LLNA. Significant evidence has demonstrated that IL-18 is an essential component of contact dermal sensitization (Antonopolous et al., 2008). To determine if IL-18 can be used as a potential supplementary endpoint in the LLNA, we will take the following approaches: benchmark chemicals consisting of known non-sensitizing irritants, known false-positive irritants and known sensitizers will be tested in the LLNA.
Aim 1 will measure IL-18 serum levels.
Aim 2 will determine if the IL-18 receptor is upregulated on T-lymphocytes in response to sensitizers.
Aim 3 will determine the effect of inhibiting IL-18 function during sensitization with the IL-18 binding protein. If IL-18 can correctly discriminate irritants from sensitizers, this will allow for improved accuracy of testing results, greatly increasing the safety of consumer products.
Allergic Contact Dermatitis (ACD), the clinical manifestation of contact sensitization, is a serious health concern caused by exposure to pharmaceuticals, industrial chemicals, and consumer products, causing physical, emotional and occupational issues for those affected. The murine Local Lymph Node Assay (LLNA) is the standard assay to predict dermal sensitizers. A significant limitation of the LLNA is the frequency of false positive, as well as the occurrence of false negatives. We will use various analyses of cytokine IL-18 as sensitizer-specific marker to enhance the accuracy and applicability of the LLNA.