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Recently, Sustainable Aviation Fuel (SAF) blends and novel combustion technologies have been introduced to reduce aircraft engine emissions. However, there is limited knowledge about the impact of combustion technology and fuel composition on toxicity of primary Particulate Matter (PM) emissions, comparable to regulated non-volatile PM (nvPM). In this study, primary PM was collected on filters using a standardised approach, from both a Rich-Quench-Lean (RQL) combustion rig and a bespoke liquid fuelled Combustion Aerosol Standard (CAST) Generator burning 12 aviation fuels including conventional Jet-A, SAFs, and blends thereof. The fuels varied in aromatics (0–25.2%), sulphur (0–3000 ppm) and hydrogen (13.43–15.31%) contents. Toxicity of the collected primary PM was studied in vitro utilising Air-Liquid Interface (ALI) exposure of lung epithelial cells (Calu-3) in monoculture and co-culture with macrophages (differentiated THP-1 cells). Cells were exposed to PM extracted from filters and nebulised from suspensions using a cloud-based ALI exposure system. Toxicity readout parameters were analysed 24 h after exposure. Results showed presence of genotoxicity and changes in gene expression at dose levels which did not induce cytotoxicity. DNA damage was detected through Comet assay in cells exposed to CAST generated samples. Real-Time PCR performed to investigate the expression profile of genes involved in oxidative stress and DNA repair pathways showed different behaviours after exposure to the various PM samples. No differences were found in pro-inflammatory interleukin-8 secretion. This study indicates that primary PM toxicity is driven by wider factors than fuel composition, highlighting that further work is needed to substantiate the full toxicity of aircraft exhaust PM inclusive of secondary PM emanating from numerous engine technologies across the power range burning conventional Jet-A and SAF.