Strain background can strongly influence the outcome of toxicity tests in animals. Time and cost constraints preclude in vivo approaches to longstanding concerns about the lack of genetic diversity in animal models. An in vitro platform is vastly more efficient for understanding the role of genetic background in toxicology testing, and sacrifices no animals. In this project we propose to build a large panel of genetically diverse ES cells from the Diversity Outcross line of mice. The DO mice are an advanced intercross of the 8 founder strains used for the Collaborative Cross. The DO strains capture over 90% of all sequence variants present in laboratory strains and harbor . They are ideally suited for complex trait mapping. ES cells offer tremendous flexibililty, and is also ideal for an in vitro genetics platform. DO ES cell lines immortalize DO genomes, creating a renewable resource. In principle, ES cells can provide access to almost any cell type by directed differentiation. In this way, the DO ES panel is extremely versatile as a permanent, renewable resource. In Phase I we demonstrated feasibility by deriving a small set of 100 ES cell lines and identifying a cytotoxicity QTL by in vitro genetic analysis. Since then, we have derived an additional 1,002 DO ES cell lines, and in Phase II we will validate and define screening panels of male and female DO ES cell lines that are well powered for QTL mapping. We will biologically test candidate genes to identify at least 10 mouse QTL genes, and then determine the fraction of those genes that are also modifers of cytotoxicity in human IPS cells. The key challenge to broad adoption of our technology is a demostration of the relevance of mouse QTL genes to human toxicological response. The aims of this project are not primarily directed toward building the DO ES cell lines. They are directed to validating their utility toward understanding mechanisms underlying variable susceptibility to toxicants in human cells. While there are many cell types and toxicities that can be approached using our technology, we focused this project on cytotoxicity in pluripotent stem cells to efficiently generate a well powered dataset to validat our technology. The Phase II project will establish our in vitro genetics platform as an important and novel advance in understanding human toxicological response.