New approach methodologies based prediction for pulmonary toxicity.

ABSTRACT: In recent years, New Approach Methodologies (NAMs) are rapidly emerging as a central paradigm for advancing human-relevant approaches in toxicology, biomedical research, and environmental health science. In respiratory toxicology, traditional two-dimensional cell cultures have limited capacity to reflect human physiological responses, structural complexity, and relevant inhalation exposure environments. Additionally, in vivo models not only present ethical challenges but also lack sufficient human relevance to reliably predict human responses. Recent advances in three-dimensional in vitro models, particularly organoids and microphysiological systems such as organ-on-chip platforms, offer promising alternatives that better recapitulate the structural, molecular, and functional features of the human respiratory system. Lung organoids derived from pluripotent or adult stem cells provide multicellular architecture, lineage diversity, and regenerative capacity suitable for evaluation of toxic mechanisms. Meanwhile, organ-on-chip systems enable dynamic exposure environments, including airflow, mechanical stretch, and air-liquid interface (ALI) conditions, improving the relevance of inhalation exposure modeling. This review summarizes current advances in lung organoid and organ-on-chip models as NAMs for respiratory toxicity testing, discusses their application potential and limitations, and outlines future directions to accelerate their translation into predictive, regulatory-relevant toxicology frameworks.