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.
This SBIR 2B is focused on the qualification and interlaboratory validation of a novel medium throughput genotoxicity assay in a metabolically competent human cell line HepaRG? integrated with a ?high throughput? innovative 96 well single-cell array platforms (CometChip) that provides automated analysis of DNA damage. This assay referred to as the HepaRG? CometChip Assay, will be aimed directly as an in vitro alternative to the in vivo Comet assay in rodent liver. The Comet assay is part of the required genetic toxicology test battery for pharmaceuticals, is used as a follow up for positive results from in vitro genotoxicity testing by the EU Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) program, and uses up 40 rodents per study. HepaRG? CometChip Assay is being developed for use in screening of pharmaceuticals and chemicals in safety assessment as a component of a genetic toxicology test battery. This Phase 2B is aimed at providing the needed validations trials to meet regulatory compliance and a submission to OECD for consideration as a Performance Based Test Guideline reducing the need for animal testing.
