Review of in vitro approaches for assessing the toxicity of chemical stressors across diverse physiological systems

Abstract The widespread presence of chemicals in the environment poses significant health risks to humans and wildlife. Existing traditional in vivo approaches, while informative, are limited by considerable ethical, economic, and logistical challenges that prevent timely and effective chemical testing and management strategies. To overcome these challenges, new approach methodologies (NAMs) offer promising alternatives for high-throughput, mechanistic, and ethically conscious chemical toxicity testing. The key to testing chemicals for potential adverse effects is the selection of appropriate bioassays that cover the range of biological or physiological pathways likely targeted by the contaminant of interest. In other words, chemical testing strategies need to cover a broad range of physiological systems to account for the mechanistic specificity of contaminants. Researchers and regulators thus face the challenge of navigating this interdisciplinary and rapidly growing space. This in-depth review attempts to address this important challenge by identifying and describing existing in vitro bioassays that can be used to assess functional impacts of chemical contaminants across vital physiological systems: endocrine, immune, cardiovascular, neurological, and hepatic. By summarizing the current tools and methodologies, this article provides a comprehensive guide for researchers and regulators to help identify relevant NAMs for use in modern chemical toxicity testing strategies.