title: Development of a BALB/c 3T3 neutral red uptake cytotoxicity test using a mainstream cigarette smoke exposure system.
abstract: BACKGROUND
  Tobacco smoke toxicity has traditionally been assessed using the particulate fraction under submerged culture conditions which omits the vapour phase elements from any subsequent analysis. Therefore, methodologies that assess the full interactions and complexities of tobacco smoke are required. Here we describe the adaption of a modified BALB/c 3T3 neutral red uptake (NRU) cytotoxicity test methodology, which is based on the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) protocol for in vitro acute toxicity testing. The methodology described takes into account the synergies of both the particulate and vapour phase of tobacco smoke. This is of particular importance as both phases have been independently shown to induce in vitro cellular cytotoxicity.
  
  
  RESULTS
  The findings from this study indicate that mainstream tobacco smoke and the gas vapour phase (GVP), generated using the Vitrocell® VC 10 smoke exposure system, have distinct and significantly different toxicity profiles. Within the system tested, mainstream tobacco smoke produced a dilution IC50 (dilution (L/min) at which 50% cytotoxicity is observed) of 6.02 L/min, whereas the GVP produced a dilution IC50 of 3.20 L/min. In addition, we also demonstrated significant dose-for-dose differences between mainstream cigarette smoke and the GVP fraction (P < 0.05). This demonstrates the importance of testing the entire tobacco smoke aerosol and not just the particulate fraction, as has been the historical preference.
  
  
  CONCLUSIONS
  We have adapted the NRU methodology based on the ICCVAM protocol to capture the full interactions and complexities of tobacco smoke. This methodology could also be used to assess the performance of traditional cigarettes, blend and filter technologies, tobacco smoke fractions and individual test aerosols.
authors.affiliation: British American Tobacco, Group R&D, Southampton, Hampshire SO15 8TL, UK. David_Thorne@bat.com.
authors.forename: David
authors.initials: D
authors.lastname: Thorne
authors.forename: Joanne
authors.initials: J
authors.lastname: Kilford
authors.forename: Rebecca
authors.initials: R
authors.lastname: Payne
authors.forename: Linsey
authors.initials: L
authors.lastname: Haswell
authors.forename: Annette
authors.initials: A
authors.lastname: Dalrymple
authors.forename: Clive
authors.initials: C
authors.lastname: Meredith
authors.forename: Deborah
authors.initials: D
authors.lastname: Dillon
mesh_terms: D016475:3T3 Cells; D000818:Animals; D051379:Mice; D008807:Mice, Inbred BALB C; D009499:Neutral Red / Q000378:metabolism*; D012906:Smoke
chemical_list: D012906:Smoke; D009499:Neutral Red