Robert Leverette1, Thomas Shutsky1, John Wertman1, Katarina Aleksa2, Dhatri Lakshmanan2, Rebecca Payne3, Kristen Jordan1
1Scientific & Regulatory Affairs, RAI Services Company, Winston-Salem, NC, USA;
2Labstat International Inc. Kitchener, ON, Canada;
3Labcorp Early Development Laboratories Ltd., Harrogate, UK
This poster shows whole aerosol exposures of combustible and next generation tobacco products, including Electronic Nicotine Delivery Systems. A Vitrocell® VC10® robot generated and delivered aerosols to the Mammalian 6/48 aerosol dilution and exposure system, with up to 7 concurrent doses plus a clean air control. Dosimetry module allowed the capture and quantification of deposited aerosol constituents (nicotine, glycerol and carbonyls). The study utilized the Neutral Red Uptake (NRU) assay in which mammalian cells were exposed to either combined TPM + GVP (submerged culture) or Whole Aerosol (ALI).
In vitro toxicological methods are being used to assess the biological activities of combustible and next generation tobacco products, including Electronic Nicotine Delivery Systems (ENDS). Historically, toxicological testing of combustible cigarettes involved pad-collected total particulate matter (TPM) and / or gas-vapor phase (GVP) samples extracted or trapped in solvents and applied to cell cultures, resulting in the fractionation of the aerosol phases. Exposure of cell cultures to whole aerosol (WA) at an air-liquid interface (ALI) prevents this separation of the particulate and gas phases. Two independent studies were conducted to determine the aerosol cytotoxicity from six ENDS (Vuse Alto®) and a marketed combustible cigarette (CC). Both studies utilized the Neutral Red Uptake (NRU) assay in which mammalian cells were exposed to either combined TPM + GVP (submerged culture) or WA (ALI). CHO cells seeded in 96-well plates were exposed to increasing concentrations of TPM + GVP for 24 hours. WA exposures utilized a Vitrocell® VC10® robot and 6/48 exposure module. H292 cells seeded on Transwell® culture inserts (24mm) were exposed (ALI) to either combustible or ENDS aerosols. Liquid traps within the exposure module allowed WA dosimetry via nicotine quantification. GVP and WA carbonyl constituents were also quantified. The combustible cigarette was cytotoxic, both TPM + GVP (IC50 = 6.2 μg nicotine / mL) and WA (IC50 = 2.4 μg nicotine), while an IC50 could not be calculated for the ENDS, even at the concentrations of delivered nicotine (up to ~120 μg/mL TPM + GVP, ~3 mg, WA). These studies were not designed for direct comparison, complicating attempts to relate the results of the submerged culture (TPM + GVP) and ALI (WA) exposures due to differences in cell types, culture methods (96-well plates versus 24mm culture inserts), exposures and doses utilized. However, both approaches demonstrated the ENDS aerosols were noncytotoxic, compared to the combustible cigarette, at the doses tested. The WA approach allowed direct exposure, at higher concentrations, of an aerosol more representative of that delivered to a product user, versus the fractionated TPM + GVP preparations.