Readiness of the in vitro alveolar macrophage assay: an interlaboratory comparison using 12 blinded particles

Inhaled particles can induce persistent pulmonary inflammation when macrophage-mediated clearance mechanisms are overwhelmed. Distinguishing particles that trigger biological activity through intrinsic toxicity from those acting primarily via particle overload represents a central challenge in inhalation toxicology and regulatory safety assessment. Mechanistically anchored new approach methodologies (NAMs) that capture key macrophage responses may therefore provide valuable tools for particle hazard identification while reducing reliance on animal testing. The alveolar macrophage assay (AMA) was developed as an in vitro method to assess particle-induced macrophage activation using four biological endpoints reflecting oxidative stress, lysosomal activation, cytotoxicity, and inflammatory signaling. Previous work demonstrated predictivity of the AMA for short-term inhalation outcomes of nanomaterials. However, broader application of the method requires evidence that the assay can be reliably transferred across laboratories and produce reproducible results with a wide variety of particles. In the present study, an interlaboratory ring-study was conducted to evaluate the transferability and reproducibility of the AMA. Twelve blinded particles were tested in three independent laboratories using standardized protocols and predefined acceptance criteria. Across laboratories, comparable dose-response patterns were observed for most endpoints, and seven of the twelve particles were consistently classified as either biologically active or passive according to the established data interpretation procedure (DIP). Sources of variability were identified, including differences in assay sensitivity, and limitations related to dose metrics and partially soluble particles. A main source of variability was the fetal bovine serum (FBS), highlighting the urgent need for transition to animal-component-free and chemically defined culture media to improve reproducibility of in vitro methods. Taken together, the results demonstrate that the AMA can be successfully transferred across laboratories and generate reproducible biological responses when strict standardization and quality criteria are applied, which underlines the importance of good in vitro method practice (GIVIMP). These findings represent an important step toward the validation of the AMA and support its use as a NAM for mechanism-based hazard assessment and grouping of particles in inhalation toxicology.