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Iron is typically the dominant metal in the ultrafine fraction of airborne particulate matter. Various studies have investigated the toxicity of inhaled nano-sized iron oxide particles (FeO_xNPs) but their results have been contradictory, with some indicating no or minor effects and others finding effects including oxidative stress and inflammation. Most studies, however, did not use materials reflecting the characteristics of FeO_xNPs present in the environment. We, therefore, analysed the potential toxicity of FeO_xNPs of different forms (Fe₃O₄, α-Fe₂O₃ and γ-Fe₂O₃) reflecting the characteristics of high iron content nano-sized particles sampled from the environment, both individually and in a mixture (FeO_x-mix). A preliminary in vitro study indicated Fe₃O₄ and FeO_x-mix were more cytotoxic than either form of Fe₂O₃ in human bronchial epithelial cells (BEAS-2B). Follow-up in vitro (0.003, 0.03, 0.3 µg/mL, 24 h) and in vivo (Sprague–Dawley rats, nose-only exposure, 50 µg/m³ and 500 µg/m³, 3 h/d × 3 d) studies therefore focused on these materials. Experiments in vitro explored responses at the molecular level via multi-omics analyses at concentrations below those at which significant cytotoxicity was evident to avoid detection of responses secondary to toxicity. Inhalation experiments used aerosol concentrations chosen to produce similar levels of particle deposition on the airway surface as were delivered in vitro. These were markedly higher than environmental concentrations. No clinical signs of toxicity were seen nor effects on BALF cell counts or LDH levels. There were also no significant changes in transcriptomic or metabolomic responses in lung or BEAS-2B cells to suggest adverse effects.