In vitro toxicological assessment of secondary organic aerosol precursors from road transport

Exposure to unabated organic aerosol emissions is known to induce pulmonary inflammation and exacerbate respiratory symptoms, primarily through oxidative stress and direct toxic injury. However, the mechanisms by which specific compounds within the primary emissions from road transport, that can also act as secondary organic aerosol precursors, impact health require further characterization. An in vitro safety assessment of selected intermediate-/semi-volatile organic compounds (I/SVOCs) was conducted using human alveolar epithelial A549 cells, murine macrophage RAW 264.7 cells, and rat alveolar macrophage NR8383 cells under both conventional submerged monolayer cultures and advanced air–liquid interface (ALI) exposure systems. The first stage of the biological evaluation comprised a series of experiments using selected I/SVOCs commonly found in road transport-derived aerosols, namely polycyclic aromatic hydrocarbons (e.g., naphthalene and pyrene) and alkanes (e.g., dodecane, tetradecane, and docosane). Furthermore, actual gasoline internal combustion engine exhaust was collected on polytetrafluoroethylene (PTFE) membranes and subsequently subjected to biological evaluation.

The in vitro assessment of the I/SVOCs demonstrated significant cytotoxicity, oxidative stress, and inflammatory responses in all tested cell lines. Traditional submerged cultures revealed concentration-dependent effects, including reactive oxygen species (ROS) generation, glutathione depletion, apoptosis, G₂/M cell cycle arrest, and increased levels of pro-inflammatory cytokines. Moreover, advanced human ALI organotypic airway tissue models exposed via the VITROCELL® Essentials ALI exposure system (VITROCELL SYSTEMS GmbH, Waldkirch, Germany) were employed to more accurately replicate real-world respiratory exposure conditions. The ALI model represents an advanced in vitro airway system in which differentiated primary airway cells, cultured on microporous membrane scaffolds, are directly exposed to aerosols and gases at the air–liquid interface. Unlike submerged monolayer cultures, these differentiated primary cells exhibit transcriptional profiles that more closely resemble the in vivo human airway epithelium. Consequently, ALI airway models more accurately reproduce in vivo airway architecture, including barrier integrity and metabolic activity, while enabling exposure scenarios that better reflect real-world human inhalation. In addition, the integration of the ALI exposure system with a volatile organic compound (VOC) generator and a real-time multi-gas analyser, connected via a bypass sampling line downstream of a mixing chamber, enhanced the exposure precision, flexibility, and physiological relevance. Collectively, the findings are expected to provide a robust basis for the classification and prioritization of I/SVOCs according to their potential health risks, thereby supporting informed decision-making in air quality regulation and public health protection.

Acknowledgement: This research was funded by the European Union’s Horizon Europe research and innovation programme within the AEROSOLS project under grant agreement No. 101096912 and UK Research and Innovation (UKRI) under the UK government’s Horizon Europe funding guarantee [grant numbers 10092043 and 10100997].