Phototoxicity Screening Assay in Reconstituted Skin

描述(由申请人提供)：我们已经开发并预验证了一种光毒性的体外筛选试验，即重组皮肤中的增强型光毒性试验(EPARS)，以取代目前的标准动物光毒性试验。在消费品行业，化学品、化妆品、膳食补充剂和药品的光毒性潜力是一个主要且越来越令人担忧的问题，但社会和政府要求减少实验动物的使用。EPARS比动物试验更客观、更快速、成本更低。到目前为止，还没有替代的光毒性测试在美国得到验证(通过ICCVAM)，尽管3T3中性红摄取(3T3NRU)测试在ECVAM验证后已被欧盟接受。EPARS明显优于3T3NRU生存能力测试，因为它克服了3T3NRU测试的几个局限性。具体而言：(1)EPARS基于与人类皮肤形态非常相似的分化组织模型，而不是成纤维细胞单层；(2)组织由三维培养系统中的原代角质形成细胞组成，这是一个比小鼠肿瘤细胞系更相关的模型；以及(3)可以直接应用测试物质，避免了将制剂溶解到培养介质中往往存在的问题。如果充分发挥其潜力，EPARS将是可用于识别人类潜在光毒剂的最准确的检测方法。我们建议通过测试已知光毒素的扩展列表来确认EPARS的重复性、敏感性、特异性和可预测性，并验证在第一阶段确定的最具预测性和准确性的组织存活终点。我们还将使用人类基因芯片来测量基因表达模式的变化，并确定可用作光毒性诱导预测因子的基因。这项第二阶段的工作将补充和加强EPARS的基础，为提交给美国监管机构作为动物光毒性测试的替代方案做准备。

DESCRIPTION (provided by applicant): We have developed and prevalidated an in vitro screening test for phototoxicity, the Enhanced Phototoxicity Assay in Reconstituted Skin (EPARS), to replace the current standard animal phototoxicity tests. The phototoxic potential of chemicals, cosmetics, dietary supplements and Pharmaceuticals are a major and growing concern in the consumer products industry, but society and the government have demanded decreased use of experimental animals. EPARS is more objective, faster, and less expensive than animal tests. To-date, no alternative phototoxicity test has been validated in the U.S. (via ICCVAM), although the 3T3 Neutral Red Uptake (3T3NRU) assay has been accepted in the E.U. after validation by ECVAM. EPARS is significantly superior to the 3T3NRU viability test because it overcomes several of the 3T3NRU test's limitations. Specifically: (1) EPARS is based upon a differentiated tissue model that closely parallels human skin morphology, instead of a fibroblast monolayer; (2) the tissues are composed of primary human keratinocytes in a 3D culture system, a more relevant model than a mouse tumor cell line; and (3) test substances can be applied directly, avoiding the often problematic solubilization of formulations into culture media. If developed to its full potential, EPARS would be the most accurate assay available to identify potential phototoxic agents in humans. We propose to confirm the reproducibility, sensitivity, specificity and predictability of EPARS by testing an expanded list of known phototoxins and validate the most predictive and accurate tissue viability endpoints identified in Phase I. We will also use human gene microarrays to measure changes in gene expression patterns and identify genes that can be used as predictors for induction of phototoxicity. This Phase II work will supplement and strengthen the foundation of EPARS in preparation for submission to U.S. regulatory agencies as an alternative to animal phototoxicity testing.