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TSRC, Tob. Sci. Res. Conf., 2018, 72, abstr. 017

Development of a 3D human oral tissue model for oral permeation of smokeless moist snuff

KEYSER B.(1); ROWE J.(2); WEIDMAN R.(2); FIELDS W.(1)
(1) RAI Services Company, Winston-Salem, NC, USA; (2) R.J. Reynolds Tobacco Company, Winston-Salem, NC, USA

Tobacco product use is a risk factor in the development of oral cancer, although epidemiology studies show this risk is far less with smokeless tobacco product use than cigarette smoking. While smokeless tobacco contains harmful and potentially harmful constituents (HPHCs), the oral permeation of HPHCs is not completely understood. The goal of this study was to determine the suitability of a 3D human buccal oral tissue model, EpiOral™, to study this process by using an extract of CORESTA moist snuff smokeless tobacco reference (CRP2). CRP2 extract was prepared in complete artificial saliva (CAS) with a ratio of 300 mg of CRP2 to 1 mL of CAS, sterile filtered and stored at -80°C until time of exposure with the stock extract or a 150 mg/mL dilution. CRP2 extract (150 or 300 mg/mL), CAS alone (vehicle control) or caffeine/CAS solution (0.15%; positive control) were applied to the apical side of EpiOral™ tissues and permeation was measured for 3 hours with fresh receiver solution (basolateral compartment) replaced every 30 minutes. After the last time point, EpiOral™ tissues were rinsed and cytotoxicity (via 3-(4,5-d-2-yl)-2,5-diphenyltetrazolium bromide; MTT) was measured. Subsequently, tobacco-specific nitrosamines (TSNAs) and caffeine concentrations were measured in each receiver solution by liquid chromatography (LC). Application of CRP2 extract (150 or 300 mg/mL), CAS alone or caffeine/CAS solution (0.15%) to the EpiOral™ tissues did not produce cytotoxicity as measured via MTT. Permeation over time (i.e., flux) was observed to achieve a linear steady state for each analyte measured over the 3 hours. Collectively, these data suggest that the EpiOral™ model could increase the understanding of the permeation of different tobacco constituents from moist snuff smokeless tobacco into oral tissue.