ATotally Animal-free Model for Acute Chemicl Toxicity Testing

描述（由申请人提供）：Draize眼睛刺激试验自1944年以来一直在使用。一只有意识的兔子的一只眼睛被注入了测试物质，而另一只眼睛是对照组。然后在一小时到三周内评估角膜、结膜和虹膜的反应。由于该测试在道德和科学上的缺陷，人们一直在积极寻求替代方法，但目前还没有找到。今天，Draize测试仍然是唯一一种可以将物质分为四类眼睛刺激：腐蚀性/严重、中度、轻度和非刺激性。具体来说，没有任何一种体外方法可以识别中度和轻度刺激物，因此Draize测试继续进行。没有一种体外方法解决眼部炎症的问题，这是Draize眼睛刺激试验的一个组成部分，是由试验物质注入后死亡的细胞引起的。我们假设，一种具有终点的体外方法可以预测受伤眼睛的炎症潜力，除了腐蚀性/严重和非刺激性外，还可以识别中度和轻度刺激物。使用未用于移植的人类供体角膜，我们的初步研究表明，在完全不含动物（和动物产品）的系统中，通过茜素红S染色显示角膜内皮损伤水平，并通过角膜内皮全景图像分析中的红色染色定量，以100%的准确度预测腐蚀性/重度，中度，轻度和非刺激性类别的18种测试物质的刺激潜力。我们的目标是证明我们的方法可以完全取代Draize眼睛刺激测试。具体目的是：1)利用我们的方法对具有公开的历史Draize试验数据的试验物质进行分类；2)研究经试验物质处理的人供体角膜中受损角膜内皮中VIP的状态。

DESCRIPTION (provided by applicant): The Draize eye irritation test has been in use since 1944. One eye of a conscious rabbit is instilled with the test substance, while the other eye is the control. The reactions of the cornea, conjunctiva, and iris are then evaluated in a period of one hour to three weeks. Because of the test's moral and scientific short comings, a replacement has been actively sought, but there is still none in the horizon. Today, the Draize test remains the only one that can classify a substance into one of the four categories of eye irritation: corrosive/severe, moderate, mild, and non- irritants. Specifically, none of the in viro methods can identify the moderate and mild irritants and the Draize test continues. None of the in vitro method addresses the issue of ocular inflammation, an integral part of the Draize eye irritation test brought upon by dying cells following test substance instillation. We hypothesize that an in vitro method with an end point that can predict the inflammatory potential of the injured eye can identify moderate and mild irritants, in addition to the corrosive/severe and non- irritants. Using human donor corneas not used for transplantation, our preliminary studies demonstrated in a totally animal (and animal product)-free system that the levels of corneal endothelial damage, revealed by alizarin red S staining and quantified by image analysis of the red stain in the panoramic view of the corneal endothelium, predicted with 100% accuracy the irritation potential of 18 test substances in the corrosive/severe, moderate, mild, and non- irritants categories. Our goal is to demonstrate that our method can fully replace the Draize eye irritation test. The specific aims are to 1) use our method to classify test substances that have historical Draize test data available in public, 2) study the status of VIP in the damaged corneal endothelium in the test substance-treated human donor corneas.