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Introduction: Because of the rising popularity of novel tobacco products, there has been an increasing interest in the biological effects of these products. This study was performed to investigate the biological effects of vapor from a novel tobacco vapor product (NTV) in comparison with cigarette smoke (CS) from a 3R4F reference cigarette by using two types of in vitro model. Materials and Methods: A two-dimensional (2D) culture system composed of a submerged monolayer of BEAS-2B cells was used to detect cellular oxidative stress responses. A three-dimensional (3D) culture system composed of a pseudostratified air-liquid interface culture of normal human bronchial epithelial cells was used to detect tissue inflammation. In both culture systems, exposures were conducted with an aqueous extract (AqE) of NTV vapor or CS. Results: In the 2D culture system, exposure to CS AqE induced glutathione oxidization and antioxidant response element reporter gene activation. NTV vapor AqE also induced reporter gene activity, but the concentration required for activation was higher than that of CS AqE. In the 3D culture system, exposure to CS AqE increased the secretion of various inflammatory mediators. In contrast, the effect of NTV vapor AqE on tissue inflammation was limited, and only interferon-c showed a slight increase in expression. Conclusions: Overall, these results demonstrate that NTV vapor has fewer effects on cellular oxidative stress and tissue inflammatory responses as compared with CS in in vitro models using human cells.

Summary A new reference cigarette, 1R6F, produced by the Kentucky Tobacco Research and Development Center, has been manufactured as a substitute for the 3R4F reference cigarette because of a depletion of 3R4F stock. The purpose of the current study was to investigate the interchangeability of 1R6F and 3R4F by comparing the chemical and biological characteristics of the mainstream smoke and to assess the inter-laboratory reproducibility by comparing the results obtained in the current study with a previous report. We analyzed 45 priority chemicals required by Health Canada for regulatory reporting and assessed the toxicological effects of cigarette smoke using in vitro standard toxicological assays recommended by the Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) under the International Organization for Standardization (ISO) standard and intense smoking regimens. The results of the chemical analysis and standard toxicological assays showed a good inter-laboratory reproducibility for 1R6F as a reference cigarette, while there were some slight reproducible differences between 1R6F and 3R4F. In addition, we investigated the interchangeability of 1R6F with 3R4F in some additional toxicological assays that detect oxidative stress because oxidative stress is a principle endpoint used in tobacco research with next generation tobacco and nicotine delivery products (NGPs). Both 1R6F and 3R4F elicited comparable responses in the oxidative stress assays. Overall, our results showed inter-laboratory reproducibility in chemical and standard toxicological assessments of 1R6F; thus, suggesting the suitability of 1R6F as a reference cigarette. In addition, the results obtained in the oxidative stress assays provide insight into the interchangeability of 1R6F with 3R4F when used as a comparator for NGPs.