title: Development, qualification, validation and application of the neutral red uptake assay in Chinese Hamster Ovary (CHO) cells using a VITROCELL® VC10® smoke exposure system.
abstract: 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 the NRU assay in Chinese Hamster Ovary (CHO) cells, following exposure to fresh whole smoke generated with the VITROCELL® VC10® system is 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.
  
  
  OBJECTIVE
  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 of whole smoke; exposure-related changes in in vitro cytotoxicity were observed with reproducible IC50values; comparative analysis showed that the heat-not-burn cigarette was significantly (P<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.
authors.affiliation: RAI Services Company, Scientific & Regulatory Affairs, 401 North Main Street, Winston-Salem, NC 27101, USA. Electronic address: fieldsw@rjrt.com.
authors.forename: Wanda
authors.initials: W
authors.lastname: Fields
authors.affiliation: RAI Services Company, Scientific & Regulatory Affairs, 401 North Main Street, Winston-Salem, NC 27101, USA.
authors.forename: Kathy
authors.initials: K
authors.lastname: Fowler
authors.affiliation: Covance Laboratories Ltd., Otley Road, Harrogate, North Yorkshire HG1 3PY, UK.
authors.forename: Victoria
authors.initials: V
authors.lastname: Hargreaves
authors.affiliation: Covance Laboratories Ltd., Otley Road, Harrogate, North Yorkshire HG1 3PY, UK.
authors.forename: Lesley
authors.initials: L
authors.lastname: Reeve
authors.affiliation: RAI Services Company, Scientific & Regulatory Affairs, 401 North Main Street, Winston-Salem, NC 27101, USA.
authors.forename: Betsy
authors.initials: B
authors.lastname: Bombick
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D018929:Cell Culture Techniques; D006224:Cricetinae; D003412:Cricetulus; D009499:Neutral Red / Q000378:metabolism; D052638:Particulate Matter / Q000633:toxicity*; D012906:Smoke / Q000009:adverse effects*
chemical_list: D052638:Particulate Matter; D012906:Smoke; D009499:Neutral Red
keywords: ALI; CHO cells; Eclipse; Neutral red uptake; Smoke exposure; VITROCELL®