Cytotoxicity Assessment of Heated Tobacco Product and Combustible Cigarette Aerosols Utilizing Whole Aerosol Exposure in the Neutral Red Uptake Assay

March 19, 2023

SOT 62nd Annual Meeting and ToxExpo, March 19-23 2023, Nashville, Tennessee, USA

Robert Leverette1, Thomas Shutsky1, Rebecca Payne2, Kristen Jordan1

1Scientific & Regulatory Affairs, RAI Services Company, Winston-Salem, NC, USA;
2Labcorp Early Development Laboratories Ltd., Harrogate, UK

In vitro toxicological methods are used to assess the biological activities of combustible and next generation tobacco products (NGP), including Heated Tobacco Products (HTP). Historically, toxicological testing of combustible cigarettes (CC) involved pad-collected total particulate matter (TPM) and/or gas-vapor phase (GVP) samples prepared in liquid solvents and applied to cell cultures. Exposure to freshly generated unfractionated whole aerosol (WA) at the air liquid interface (ALI) eliminates the generation of separate particulate and gas phase preparations. The WA cytotoxicity from four HTP (glo™) styles, a marketed HTP comparator, two marketed combustible cigarettes (nonmenthol and menthol) and the 1R6F Kentucky Reference cigarette was assessed with the Neutral Red Uptake (NRU) assay. WA exposures utilized a Vitrocell® VC10® robot connected to a 6/48 exposure module. H292 cells seeded on Transwell® culture inserts (24mm) were exposed (ALI) to either combustible or HTP aerosols. Liquid traps within the exposure module allowed quantification of delivered WA nicotine and carbonyl constituents. The CCs delivered 24 – 54 µg nicotine per 24-minute exposure, the HTPs 620 – 2751 µg nicotine per 180-minute exposure. WA from the CCs was cytotoxic, with IC 50 values of 2.03 ± 0.51, 1.81 ± 0.17 and 1.68 ± 0.56 µg nicotine for the nonmenthol, menthol, and reference CC, respectively. HTP aerosols were cytotoxic; however, their IC50 values ranged from 26.88 ± 13.61 to 134.76 ± 97.12 µg nicotine, which were up to 80 times less cytotoxic, on a per nicotine basis, when compared to the CC. These results add to the weight of evidence from multiple studies on the harm reduction potential of HTPs when compared to CCs, further supporting the tobacco harm reduction paradigm of NGPs.

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