Advances in Cytotoxicity Testing: From <em>In Vitro</em> Assays to <em>In Silico</em> Models

Cytotoxicity testing remains a cornerstone of modern toxicology, providing essential insight into how chemicals and drugs affect cell viability and function. Classical colourimetric assays such as MTT, LDH release, and neutral red uptake established the methodological basis of in vitro toxicology and continue to serve as regulatory benchmarks. However, their limited mechanistic depth and physiological relevance have prompted the field to evolve towards more predictive and human-centred approaches. Recent advances in high-content imaging, flow cytometry, and real-time impedance analysis have transformed cytotoxicity testing into a multiparametric discipline capable of detecting subtle, adaptive, and sub-lethal cellular responses. Large-scale initiatives such as Tox21 and ToxCast, supported by computational pipelines like tcpl, have standardised data interpretation and improved reproducibility. In parallel, three-dimensional organoid cultures, organ-on-chip platforms, and bioprinted constructs now replicate the architec-ture, perfusion, and metabolic activity of human tissues, enhancing translational accuracy. Stem cell–based models using human embryonic and induced pluripotent stem cells provide ethically sustainable systems for organ-specific and developmental toxicity testing, while in silico tools - QSAR, machine learning, and physiologically based pharmacokinetic (PBPK) modelling - enable quantitative in vitro–in vivo extrapolation (QIVIVE). Together, these developments underpin New Approach Methodologies (NAMs) and Integrated Approaches to Testing and Assessment (IATA), marking the transition from descriptive assays to predictive, mechanism-anchored frameworks that advance both biomedical research and regulatory science.