The EpiAirway™-FT (full thickness) tissues (Mattek Corporation, Ashland, USA) contain tight junctions, ciliated and other bronchial cell types making it an ideal model for testing aerosolised substances in vitro. Toxicity is not a single endpoint but a combination of events that lead to tissue remodelling. By analysing cilia beat frequency (CBF), trans-epithelial electrical resistance (TEER), post-treatment tissue depth in conjunction with goblet cell hyperplasia (GCH) per tissue, we investigated this multi-endpoint approach for understanding different mechanisms of toxicity in vitro. We treated EpiAirway™ tissues for 6 days (basolaterally) with several chemicals and recorded data for CBF, TEER, tissue depth and GCH. IL-4, IL-17 and IL-13 resulted in up to 4-fold increases in GCH. Reduction in TEER was observed with Platelet Activation Factor (PAF) at 0.001 µM and above. Treatment with staurosporine appeared to reduce tissue depth and affect CBF (which was also affected by treatment with PAF at >0.001 µM). We also exposed tissues to Adenosine triphosphate (ATP), Procaterol Hydrochloride (USAN), Hydrogen Peroxide (H2O2) or Benzalkonium Chloride (BC) or a pH range (4 to 10) and analysed after 0.5 and 2 hours. Untreated tissues had an average CBF of 17.62 Hz (n=40). ATP (0.01 mM) and USAN (0.01 mM) increased CBF (as expected) to 19.68 Hz and 20.42 Hz respectively and BC (3 mM) decreased CBF (as expected) to 2.5 Hz. H2O2 (0.1 mM) unexpectedly increased CBF (19.55 Hz), even at 10 mM (19.37 Hz). Only pH4 adversely affected CBF yielding ciliostasis. When exposed to 3R4F cigarettes (Health Canada Intense regime / 88 minutes / Vitrocell^® VC10^® machine [VitroCell GmbH, Germany]), CBF was slightly elevated at 2 L/min compared to the air control and was undeterminable at higher concentrations. These data show that toxicity is a complex process but by analysing multiple endpoints, elucidation of the underlying mechanism could be possible using this 3D tissue model.