Project Summary Pharmaceuticals and various industrial chemicals undergo comprehensive toxicological assessment in order to safeguard human health. Genetic toxicology is a key component of this risk assessment. This testing evaluates chemicals for their potential to damage DNA, an integral factor in the development of cancer and other diseases. For decades, in vivo assays have guided genotoxic risk assessment due to their ability to mirror physiological responses in humans. Their endogenous metabolism provides the enzymatic activation necessary for generating genotoxic intermediates, many of which are difficult to detect with in vitro assays. Additionally, their complete organ systems provide the structure for identifying organ specific genotoxicity, especially effects in liver and blood. Leading in vivo assays include the mammalian micronucleus assay, comet assay, chromosomal aberration assays, and rodent-based mutational assays. While these have served as a pillar of genotoxic risk assessment, regulatory agencies worldwide have begun to restrict and even eliminate the use of animals in toxicology research. This significant shift in risk assessment has left the field of genetic toxicology in need of a non-animal, metabolically-active systems that effectively models genotoxic risk in humans. In vitro assays provide initial insight into genotoxic risk, but many lack endogenous metabolic activation or only focus on individual cell types/organs. We will address the pressing need for an effective in vitro genotoxicity assay through our development of a comprehensive hen’s egg model for genotoxic analysis. The hen’s egg is a unique system in that it is not considered an animal model due to deficiencies in brain activity and pain perception early in development. Yet, similar to in vivo models, the hen’s egg has endogenous metabolic activity and functioning hepatic and hemopoietic organ systems. We will leverage these features to develop an assay that simultaneously evaluates genotoxicity in the blood and liver. Our utilization of flow cytometry and high content imaging will automate micronuclei quantification in the erythrocytes and hepatocytes. Furthermore, we will incorporate the assessment of genotoxic and cytotoxic biomarkers, providing valuable insight into organ specific toxicity and genotoxic mode of action. The development, optimization and validation of our hen’s egg genotoxicity assay will meet the critical need for an accurate, metabolically-active in vitro genotoxicity assay, while fulfilling upcoming regulatory requirements.