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Background Animal experiments have been performed to predict toxicity in humans in many fields, in-cluding toxicology, medicine, and pharmacology, and have contributed to increasing life ex-pectancy. However, animal testing has been a controversial issue for over 100 years due to ethical concerns, and inter-species differences pose limitations for understanding human re-sponses to toxicity. In recent years, many researchers have developed in vitro and in silico al-ternatives to using animals (e.g., 3-dimensional [3D] organoid culture, organs-on-a-chip, and advanced computer modeling). In this study, we generated 3D alveolar organoids (AOs) for pulmonary toxicity testing following exposure to chemicals, instead of animal models or two-dimensional culture of a single cell type. Methods After human induced pluripotent stem cells were cultured with differentiation medium corresponding to each step for 14 days in 6-well plates, AOs were generated by forced aggregation and cultured with differentiation medium. The AOs were exposed to acrolein and sodium chromate for 24, 72, and 120 hours, and we determined the cytotoxicity of these chemicals using the MTT assay. Results Exposure to acrolein and sodium chromate for 24 hours decreased proliferation, but the organoid size did not change considerably. However, long-term exposure to acrolein and sodium chromate significantly decreased the organoid size. Conclusion These findings suggest that AOs could facilitate acute toxicity assessments based on measurements of cell viability in AOs, as well as sub-chronic toxicity assessments based on measurements of both size and viability.