A novel in vitro alveolar model (ALIsens®) for hazard assessment of methyl methacrylate: No evidence for respiratory sensitisation potential

This study aimed to assess the potential of methyl methacrylate (MMA) to induce respiratory sensitisation using ALIsens®, a three-dimensional (3D) human-relevant alveolar model cultured at the air-liquid interface (ALI). The system addresses Key Events 2 and 3 (KE2 and KE3) of the proposed Adverse Outcome Pathway (AOP #39) for respiratory sensitisation. To confirm model responsiveness, ALIsens® was exposed to lipopolysaccharide (LPS) and thymic stromal lymphopoietin (TSLP), with chloramine T trihydrate (Ch-T) as a reference respiratory sensitiser, lactic acid (LA) as a non-sensitiser, and 2-mercaptobenzothiazole (2-MBT) as a positive control for skin-sensitisation. Following dose-range finding experiments it was not possible to determine the exposure dose of MMA reducing cell viability of the test system ALIsens® by 25% (CV75). Thus, the highest achievable exposure dose (500 µg/cm²) and half that dose (250 µg/cm²), respectively were used. The ALIsens® model evaluates respiratory sensitisation potential by measuring two mechanistic endpoints aligned with key events in the adverse outcome pathway: Surface marker expression on dendritic-like THP-1 cells (KE3) — specifically, upregulation of the thymic stromal lymphopoietin receptor (TSLPr) to ≥150%. Cytokine and chemokine release profiles (KE2). MMA did not trigger biological responses associated with respiratory sensitisation in the ALIsens® model. TSLPr expression remained below the 150% threshold compared to vehicle controls, and no significant increase in cytokine or chemokine release was observed. KE1 (protein binding) was previously investigated by Simoneit et al. (2025), while KE2 and KE3 were examined in this study. Across all endpoints - whether previously published (KE1) or generated here (KE2 and KE3) - MMA consistently yielded negative results. This evidence supports the conclusion that MMA lacks respiratory sensitisation potential, corroborated by negative outcomes in Specific Inhalation Challenge (SIC) studies.