Phototoxicity Screening Assay in Reconstituted Skin

描述（由申请人提供）：我们已经开发并预先验证了一种体外光毒性筛选试验，即重建皮肤增强光毒性试验（EPARS），以取代目前的标准动物光毒性试验。化学品、化妆品、膳食补充剂和药品的潜在光毒性是消费品行业日益关注的一个主要问题，但社会和政府已经要求减少实验动物的使用。与动物实验相比，EPARS更客观、更快、更便宜。迄今为止，尽管3T3中性红色摄取（3T3NRU）测定法在ECVAM验证后已被欧盟接受，但美国还没有其他光毒性测试（通过icvam）得到验证。EPARS明显优于3T3NRU生存能力测试，因为它克服了3T3NRU测试的一些局限性。具体来说：(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)在三维培养系统中，组织由原代人角质形成细胞组成，这是一种比小鼠肿瘤细胞系更相关的模型；(3)试验物质可以直接应用，避免了配方在培养基中溶解的问题。如果发展到其全部潜力，EPARS将是最准确的测定方法，可用于识别潜在的光毒性物质在人类。我们建议通过测试已知光毒素的扩展列表来确认EPARS的可重复性、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最准确的组织活力终点。我们还将使用人类基因微阵列来测量基因表达模式的变化，并识别可用作诱导光毒性预测因子的基因。第二阶段的工作将补充和加强EPARS的基础，准备提交给美国监管机构作为动物光毒性测试的替代方案。

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.