One of the goals of nanotoxicology research is to identify the physicochemical properties of ENM that can lead to adverse heath effects.{1-4} This goal is not achievable by primarily relying on animals to perform safety est all the new materials that are emerging at the rate nanotechnology is developing.{5,6} In addition, in vivo screening is expensive. A complete set of regulatory tests for a single chemical, including for carcinogenicity, chronic, reproductive and development toxicity testing requires hundreds of animals and could cost millions of dollars.{2,5-7} To avoid a similar conundrum in nano safety testing, we need alternative ways to screen for ENM toxicity. This should be a multi-disciplinary approach that includes comprehensive physicochemical characterization of ENM, in vitro screening to identify the mechanisms of toxicity, in silico methods to establish property-activity relationships and hazard ranking that can be used to prioritize animal testing.'^ In vitro assays are an indispensible part in this effort because these techniques allow the identification of specific biological and mechanistic pathways that are required for knowledge generation and for introducing the robust science that is needed to establish a toxicological paradigm that replaces descriptive experiments in animals. Recent advances in developing standard mechanism-based cellular assays, imaging techniques and rapid throughput screening platforms enable large numbers of ENM to be tested under standardized conditions.{8-13} The accompanying large data volume and analytical information can be dealt with by bioinformatics, including computerized models that allow hazard ranking, building of property-activity relationships, and using the information on dose, kinetics, ENM physicochemical properties and quantifiable biological response outcomes to plan and execute in vivo experiments. This integrative approach is called a predictive toxicological approach, which is officially defined as the assessment of in vivo toxic potential of a material or substance based on in vitro and in silico methods (Fig. 1).^''The National Research Council of the U.S. National Academy of Sciences (NAS) recently opined that toxicological testing in the twenty-first century should undergo a paradigm shift from a predominant observational science in animals to a target-specific and predictive in vitro science that utilizes mechanisms of injury and toxicological pathways to guide the conductance of in vivo studies.{14-16} This opinion is also compatible with the increased public and regulatory demand to reduce animal use for toxicological screening, e.g., the recent enactment of European Union REACH legislation. This legislation requires the development of extensive toxicological testing of existing and new substances through the use of non-animal test methods.{17} All these developments call for substantial improvement and expansion of existing in vitro approaches to meet the challenge of performing hazard assessment for a rapidly expanding number of new ENM with novel physicochemical properties.