PROJECT SUMMARY Genomic instability, induced by DNA damage, plays a critical role in the pathogenesis of many major human diseases, such as neurodegeneration, cancer, and cardiovascular disease, together with aging. A critical need exists for automated assays that can assess DNA damage and thereby bring the measurement of such damage into greater routine use, which will help advance our understanding of disease and its environmental causes. The comet assay, a widely used and versatile means of measuring many types of DNA damage, has been integrated into various regulatory guidelines for screening chemicals and pharmaceuticals e.g., the Organization for Economic Cooperation and Development guidelines, the European Commission’s Registration, Evaluation and Authorization of Chemicals Program, and ICH S2(R1) guidance 16. Moreover, its use has far-reaching recommendations e.g., the FDA, the National Toxicology Program, the European Food Safety Authority; the International Conference on Harmonization of Technical Requirements for Human Use; and the European Medicines Agency. However, the comet assay is time-consuming and labor intensive (multiple steps and multiple sample slides processed individually by hand), downsides which can also introduce errors and variation and be problematic for users. These limitations can be addressed by assay automation, a workflow solution that is currently absent in the market. Our Phase I project resulted in a novel, prototype Automated High-Throughput Comet Assay Device (AHTP-CAD) that successfully automated the comet assay without the need for operator involvement. A Technology Niche Analysis subsequently confirmed the demand, and identified potential markets. In this Phase II application, we will refine, optimize, and test the AHTP-CAD for benchtop use and widespread adoption. We will develop manufacturing/assembly processes and procedures for scaling up to high volume production. We will also benchmark and assess the operation of the AHTP-CAD to support users in performing their assays in compliance with Good Laboratory Practice. At the project completion, we expect a commercialization-ready device, which will uniquely address the unmet need of providing users with a standalone, automated analysis of DNA damage. The device will be sufficiently developed for demonstration to potential commercial partners. The availability of such a self-contained benchtop device will accelerate biomedical discovery and address the NIH mission of seeking fundamental knowledge about the nature and behavior of living systems and the application of that knowledge to enhance health, lengthen life, and reduce illness and disability.