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

CometChip：开发肝细胞高通量 DNA 损伤检测方法

基本信息

批准号：
9789880
负责人：
Bevin P. Engelward
金额：
$ 79.99万
依托单位：
INTEGRATED LABORATORY SYSTEMS, LLC
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-19 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9789880
关键词：
Address Affect Animal Testing Animals Biological Biological Assay Biological Models Cancer Etiology Carcinogens Cell Culture Techniques Cell Line Cells Chemicals Comet Assay Congenital Abnormality Cosmetics DNA Damage DNA lesion Databases Development Environment Enzymes Evaluation Gene Mutation Goals Guidelines Hepatocyte Human Human Cell Line In Vitro Laboratories Lead Literature Liver Measures Metabolic Methods Mutagenicity Tests Mutagens Mutation Peer Review Performance Pesticides Pharmacologic Substance Phase Poison Protocols documentation Publications Quality Control Review Literature Rodent Sales Scheme Small Business Innovation Research Grant System Technology Testing Time Tissues Toxicity Tests Toxicogenetics Toxicology Translating United States National Institutes of Health Validation Xenobiotics automated analysis base cost drug candidate environmental chemical follow-up genotoxicity high throughput screening in vitro testing in vivo innovation instrumentation novel novel therapeutics programs response safety assessment screening tool virtual

项目摘要

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.

估计有 10,000 只动物用于毒理学安全评估，以满足所有监管要求测试时间跨度为 5 至 10 年且超过 1000 万美元。需要减少对动物测试的依赖并开发高度预测的人类生物学替代传统的毒理学动物毒性测试模型。使用替代测试进行安全评估需要严格的验证才能接受监管机构要求的替代方法。全球监管机构，直接测量DNA损伤、基因突变、染色体细胞培养物和动物体内的彗星试验是啮齿类动物肝脏损伤的一个组成部分。所需的遗传毒理学测试电池可检测广泛的 DNA 损伤，并且被认为是区分基因毒性致癌物和非基因毒性致癌物的有力工具这种监管检测方法昂贵、效率低下，并且可能需要多达 45 只动物进行检测。我们开发了一个中等通量的 Comet 检测平台 (CometChip®)。使用人类肝细胞系 HepaRG™，可维持 CYP450 的活性、诱导谱我们通过测试 60 多种化合物来验证该测定。集成实验室系统和 MIT 用于评估 DNA 损伤，现已能够进行实验室间试验来验证该测定法作为体内方法的非动物替代品该 SBIR 2B 旨在验证 CometChip® 技术进行检测。使用具有代谢能力的 HepaRG™ 细胞和肝细胞作为 DNA 损伤体内彗星测定的体外替代方案这将通过四个具体目标来实现：目标 1 进行文献综述并建立体内彗星测定反应数据库在同行评审文献中发表；目标 2 最终确定 HepaRG™ CometChip® 检测特异性 SOP、质量控制标准、仪器验证和符合 GLP 的具体协议；目标 3 每年对 100-150 种化合物进行 HepaRG™ CometChip® 检测，持续 3 年 Tox 21 的生物活性；具体目标 4 制定总体验证计划并执行实验室间验证；此 SBIR 2B 应用程序和主验证的具体目的计划开发一种体外替代监管机构使用的体内彗星测定 (OECD 489) 该 SBIR 2B 应用旨在验证全球范围内的肝脏遗传毒性。并转化通过多个 NIH SBIR II 期开发的创新测试方法作为所需动物测试的替代方案，可以更好地预测化学品如何可能影响人类和环境并减少对动物的依赖。