The objective of this proposal is to focus on evaluating materials for their ability to cause photogenotoxicity. Photosensitivity is a harmful reaction that occurs when drugs or chemicals in the skin or eyes cause undesirable cellular damage when exposed to UV or visible light. Photogenotoxicity is used as a means to screen a material for its photocarcinogenic potential. Genotoxicity is one of the possible outcomes caused by phototoxins. Although non-photo genotoxicity has standardized testing methods, currently there are no regulatory approved photogenotoxicity assays. Applying the testing procedures used in genotoxicity testing towards photogenotoxicity is not a straightforward approach. In a review of photoclastogenic (UV induced chromosome disruption) compounds, more than 75% of compounds that were classified as photoclastogenic in mammalian assays were negative in the standard 3T3 Neutral Red Update (NRU) phototoxicity assay, demonstrating the high occurrence of pseudophotoclastogenicity. A limitation of many of these assays is the extensive processing of cells to analyze DNA damage, which can require hypotonic cell treatment or cell lysis, DNA fixation or unwinding, staining or electrophoresis, then visual counting and scoring. Cell lines have historically been used in most laboratory studies for genotoxicity assays. Nonetheless, the development of 3D tissue models have allowed for a much greater representation of living tissues. We propose to evaluate in vitro models consisting of hanging drop 3D liver cell culture and 3D reconstructed human epidermal (RHE) tissues. 3D Multi-cell human liver microtissues (InSphero) and 3D differentiated model of the human epidermis (EpiDerm - MatTek Corp) will be evaluated for their ability to predict photogenotoxins. Evaluation of phosphorylation of histone H2AX will be quantitatively measured as an indicator of DNA damage. DNA damage induces phosphorylation of Ser139 of the carboxy terminus of histone H2AX (γ-H2AX). Detection of γ-H2AX can be detected by both flow cytometry and immunofluorescence. Monitoring a mechanistic endpoint of DNA repair will allow for a greater accuracy of evaluating Photogenotoxicity by allowing for a more quantitative endpoint.
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 irritants 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 also 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.
Ocular irritation testing is extremely relevant to assuring adequate safety levels of public health as new formulations of chemicals and products are introduced. In most cases, these safety assessments are performed using the Draize Rabbit Eye test, resulting in thousands of rabbits used in testing every year. Alternatives have been discussed since the 80s without any appreciable acceptance from regulators. As more governmental agencies mandate the replacement of using live animals in regulatory safety assessment of consumer products, chemicals and cosmetics, the need for a complete alternative solution to ocular irritation testing grows. Currently, there is no single or set of tests that have been validated and accepted by regulatory agencies as a full replacement for rabbit ocular irritation testing.
The aim of this project is to evaluate the applicability of the Integrated In Vitro/Alternative Ocular (IIVAO) pronounced -/eye vay o/ Toxicology Testing Strategy to categorize potential ocular irritants into regulatory toxicity classes using four integrated in vitro and alternative ocular irritation assays. IIVAO will demonstrate it is possible to effectively replace mandated acute ocular irritation testing using the Draize Rabbit Eye Test with non-animal test methods. For the purpose of this project, we have focused on assessing the performance of IIVAO using Environmental Protection Agency (EPA) testing classifications.
IIVAO, an integrated testing strategy (ITS), comprises in vitro and alternative tests that have been chosen for their ability to effectively detect specific ranges of ocular irritation, which correspond to regulatory classifications of ocular irritation/corrosivity - the Chorioallantoic Membrane Vascular Assay (CAMVA), the Bovine Cornea Opacity/Permeability test (BCOP), the Porcine Cornea Opacity/Reversibility Assay (PorCORA), the MatTek EpiOcular(EO)-3D human tissue construct and the Porcine Confocal Assay (PorFocal). This grant project will confirm and validate a comprehensive testing protocol that allows for a thorough evaluation and categorization of test materials for ocular irritancy potential ranging from non-irritating to ocular corrosion without using live animals.
We intend to perform validations of each assay and collectively as an integrated system using a test set of chemicals that are well characterized for specific categories of ocular irritancy. Additionally, we propose to utilize the finalized integrated testing protocol to evaluate a number of 'Off-The-Shelf' commercial products for those categories of ocular irritancy to demonstrate the effectiveness of the IIVAO testing strategy with mixtures and final product formulations. The long-term project goal is to submit the IIVAO testing strategy to the EPA for consideration as a standalone alternative to the Draize Rabbit Eye test. Validation and acceptance of IIVAO will significantly reduce the number of rabbits used in the toxicological assessment of consumer products, chemicals and raw materials by replacing rabbits with an ITS of in vitro and alternative assays.