Background: Traditional animal toxicity tests can be time and resource intensive, thereby limiting the number of chemicals that can be comprehensively tested for potential hazards to humans and/or to the environment. Objective: We compared several types of data to demonstrate how alternative models can be used to inform both human and ecological risk assessment. Methods: We reviewed and compared data derived from high throughput in vitro assays to fish reproductive tests for seven chemicals. We investigated whether human-focused assays can be predictive of chemical hazards in the environment. We examined how conserved pathways enable the use of nonmammalian models, such as fathead minnow, zebrafish, and Xenopus laevis, to understand modes of action and to screen for chemical risks to humans. Results: We examined how dose-dependent responses of zebrafish embryos exposed to flusilazole can be extrapolated, using pathway point of departure data and reverse toxicokinetics, to obtain human oral dose hazard values that are similar to published mammalian chronic toxicity values for the chemical. We also examined how development/safety data for human health can be used to help assess potential risks of pharmaceuticals to nontarget species in the environment. Discussion: Using several examples, we demonstrate that pathway-based analysis of chemical effects provides new opportunities to use alternative models (nonmammalian species, in vitro tests) to support decision making while reducing animal use and associated costs. Conclusions: These analyses and examples demonstrate how alternative models can be used to reduce cost and animal use while being protective of both human and ecological health.