CometChip: Development of a high throughput DNA damage assay in hepatocytes

估计有10，000只动物用于毒理学安全性评估，以满足所有监管要求。 测试时间跨度为5至10年，费用超过1000万美元。存在一个临界 需要减少对动物试验的依赖，并开发高度预测性的基于人类的生物技术， 安全评估模型。替代传统的动物毒性毒理学试验 采用替代试验进行的安全评估需要严格验证， 监管机构的替代方法。遗传毒理学测试组合要求， 全球监管机构，直接测量DNA损伤，基因突变，染色体 细胞培养和动物中的损伤。啮齿动物肝脏中的体内彗星试验是 所需的遗传毒理学测试电池，检测范围广泛的DNA损伤， 被认为是区分遗传毒性致癌物和非遗传毒性致癌物的有力工具 致癌物质。这种调节测定是昂贵的、低效的，并且可能需要多达45只动物来进行。 每种测试化合物。我们开发了一种中等通量的彗星分析平台（CometChip®） 使用人肝细胞系HepaRG™，其维持CYP 450的活性、诱导谱 和II期酶细胞。我们已经通过测试超过60种化合物来鉴定该测定法， 综合实验室系统和麻省理工学院用于评估DNA损伤， 能够进行实验室间试验，以验证该测定法作为非动物替代品， 体内彗星试验。SBIR 2B旨在验证CometChip®技术， 使用具有代谢能力的HepaRG™细胞和肝细胞作为研究对象， 体外替代体内彗星试验。这将通过四个具体目标来实现： 目的1进行文献综述并建立体内彗星试验响应数据库， 同行评审文献中的出版物;目标2最终确定HepaRG™ CometChip®检测特异性 SOP、质量控制标准、仪器验证和GLP合规性方案;特定 目标3每年对100-150种化合物进行HepaRG™ CometChip®检测，持续3年， 根据Tox 21的生物活性;具体目标4制定主确认计划并执行 实验室间确认;本SBIR 2B应用程序和主确认的特定目的 计划开发监管机构使用的体内彗星试验（OECD 489）的体外替代方法 用于评估肝脏遗传毒性。此SBIR 2B应用程序旨在验证 并将通过多个NIH SBIR第二阶段开发的创新测试方法转化为 计划作为替代所需的动物测试，将更好地预测化学品如何可能 影响人类和环境，减少对动物的依赖。

An estimated 10,000 animals are used in toxicology safety assessments to meet all regulatory requirements with testing spanning 5 to 10 yrs and exceeding $10 million dollars. There is a critical need to reduce reliance on animal testing and to develop highly predictive human-based biological models for safety assessments. Replacement of traditional animal toxicity tests for toxicology safety assessments with alternative tests requires rigorous validations for acceptance of an alternative method by regulatory agencies. The genetic toxicology test battery required by regulatory agencies worldwide, directly measures DNA damage, gene mutation, chromosomal damage in cell culture and in animals. The in vivo Comet assay in rodent liver is a component of the required genetic toxicology test battery that detects a broad range of DNA lesions and is considered a powerful tool to distinguish between genotoxic carcinogens and nongenotoxic carcinogens. This regulatory assay is expensive, inefficient and can require up to 45 animals for each test compound. We developed a medium throughput Comet assay platform (CometChip®) using a human liver cell line HepaRG™ that maintains active, inducible spectrum of CYP450s and Phase II enzyme cells. We have qualified this assay by testing more than 60 compounds at Integrated Laboratory systems and MIT for use in the assessment of DNA damage and are now able to conduct interlaboratory trials to validate this assay as a non-animal alternative to the in vivo Comet assay. This SBIR 2B is directed at validating CometChip® technology to detect DNA damage using metabolically competent HepaRG™ cells and hepatocytes as an in vitro alternative to the in vivo Comet assay. This will be accomplished in four Specific Aims: Aim 1 Conduct literature review and build database of in vivo Comet assay responses for publication in peer-reviewed literature; Aim 2 Finalize HepaRG™ CometChip® assay specific SOPs, quality control criteria, validation of instrumentation and GLP-compliant protocols; Specific Aim 3 Conduct HepaRG™ CometChip® assay on 100-150 compounds per year for 3 years based on biological activity from Tox 21; Specific Aim 4 Develop Master Validation Plan and Execute Interlaboratory Validation; The specific purpose of this SBIR 2B application and Master Validation Plan is to develop an in vitro alternative to the in vivo Comet assay (OECD 489) used by regulators worldwide for the assessment of genotoxicity in liver. This SBIR 2B application is aimed validating and translating an innovative test method developed through multiple NIH SBIR Phase II programs as an alternative to required animals testing that will better predict how chemicals may affect humans and the environment and reduce reliance on animals.