ToxPoint: the case study for respiratory sensitization assessment of methyl methacrylate using new approach methodologies

Assessing chemicals for their respiratory sensitization potential remains a significant scientific and regulatory challenge. For regulatory purposes and risk management in particular, the ability to robustly identify and distinguish respiratory sensitizers from respiratory irritants and skin sensitizers is critical, yet validated predictive models have been lacking (Hargitai et al. 2024). Recent advances in mechanistic understanding and non-animal test systems enable a more evidence-based human-relevant assessment (Sullivan et al. 2017, OECD 2025). The adverse outcome pathway for respiratory sensitization describes key events (KEs) from covalent protein binding (KE1) through inflammatory signaling (KE2), dendritic cell activation (KE3), Th2 cell activation (KE4), to allergic respiratory hypersensitivity (KE5) (Sullivan et al. 2017, Hargitai et al. 2024). New approach methodologies (NAMs) targeting some of these KEs have emerged, including the ALIsens model, a 3D human-relevant in vitro system that evaluates respiratory sensitization potential by measuring dendritic cell surface markers and cytokine release, and an in chemico assay assessing peptide reactivity using glycine-para-nitroanilide (Gly-pNA) as a model nucleophile (OECD 2025, Simoneit et al. 2025, Gutleb et al. 2026). Methyl methacrylate (MMA) is a low molecular weight monomer used in plastics, adhesives, and medical devices. While it is a well-known respiratory irritant, its classification as a respiratory sensitizer has been debated primarily based on limited and non-guidance-conforming human challenge data (Pemberton and Kimber 2022, ECHA Chem 2025). In 2024, the Risk Assessment Committee (RAC) of the European Chemicals Agency (ECHA) proposed to classify MMA as a Respiratory Sensitizer Category 1 under the Classification, Labelling and Packaging (CLP) Regulation, in accordance with CLP Guidance, pending legal adoption in the European Union (EU). This proposal is based on a precautionary interpretation of human data of questionable reliability, despite the overall weight of evidence from other human clinical data and mechanistic assays providing inconsistent or negative results for respiratory sensitization (Pemberton and Kimber 2022). In contrast to the recent RAC/ECHA proposal to classify MMA as a respiratory sensitizer, Simoneit et al. (2025) demonstrated that MMA exhibits negligible peptide reactivity in the Gly-pNA chemoassay developed for identification of respiratory sensitizers, supporting a lack of protein binding potential (KE1) relevant for respiratory sensitization (Simoneit et al. 2025). While covalent protein binding (haptenation, KE1) is considered to be the necessary molecular initiating event for most chemical respiratory sensitizers, exceptions may exist, particularly for some metals and other non-electrophilic agents that might use different mechanisms (like inducing danger signals). However, for low molecular weight organic chemicals, lack of protein binding is generally interpreted as strong evidence against a respiratory sensitization potential (Hargitai et al. 2024).