Wanda Fields a, Kathy Fowler a, Victoria Hargreaves b, Lesley Reeve b, Betsy Bombick a
a RAI Services Company, Scientific & Regulatory Affairs, 401 North Main Street,Winston-Salem, NC 27101, USA
b Covance Laboratories Ltd., Otley Road, Harrogate, North Yorkshire HG1 3PY, UK
This study discribes the development, validation and application of the NRU assay in Chinese Hamster Ovary (CHO) cells, following exposure to fresh whole smoke generated with the VITROCELL®VC10®system. The VITROCELL® VC10® successfully provided consistent generation and delivery of whole smoke from a cigarette (3R4F) and a primarily “heat-not-burn” cigarette (Eclipse).
Cytotoxicity assessment of combustible tobacco products by neutral red uptake (NRU) has historically used total particulate matter (TPM) or solvent captured gas vapor phase (GVP), rather than fresh whole smoke. Here, the development, validation and application of theNRU assay in Chinese Hamster Ovary (CHO) cells, following exposure to freshwhole smoke generated with the VITROCELL®VC10®systemis described.Whole smoke exposure is particularly important as both particulate and vapor phases of tobacco smoke show cytotoxicity in vitro. The VITROCELL® VC10®system provides exposure at the air liquid interface (ALI) to mimic in vivo conditions for assessing the toxicological impact of smoke in vitro. Instrument and assay validations are crucial for comparative analyses. Goals of this study: 1) demonstrate functionality of the VITROCELL® VC10® system by installation, operational and performance qualification, 2) develop and validate a cellular system for assessing cytotoxicity following whole smoke exposure and 3) assess the whole smoke NRU assay sensitivity for statistical differentiation between a reference combustible cigarette (3R4F) and a primarily “heat-not-burn” cigarette (Eclipse). Results: The VITROCELL® VC10® provided consistent generation and delivery ofwhole smoke; exposure-related changes in in vitro cytotoxicity were observed with reproducible IC50 values; comparative analysis showed that the heat-not-burn cigarettewas significantly (P b 0.001) less cytotoxic than the 3R4F combustible cigarette, consistent with the lower levels of chemical constituents liberated by primarily-heating the cigarette versus burning.