The use of electronic cigarettes (e-cigarettes) potentially offers a safer alternative to conventional tobacco products. The advance in molecular biology and computational sciences offers new perspective to assess adverse biological responses for product risk assessment by combining omics screens with knowledge-based biological pathways. Our aim was to compare transcriptomic perturbations in MucilAirÔ, a commercially available lung epithelial tissue, after short repeated exposure to cigarette smoke (3R4F) and e-cigarette (Vype ePen) aerosols. We performed deep RNA sequencing and secreted inflammatory cytokine profiling postexposure. One hundred twenty-three genes were differentially expressed at fold change (FC) >1.5 and p-false discovery rate (pFDR) <0.1 for 3R4F exposure and 0 genes for Vype ePen aerosol exposure. When a relaxed filter pFDR <0.5 and FC >1.5 was applied, 29 genes were identified with e-cigarette aerosol exposure and used for validation of potential candidates by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Gene enrichment analysis was conducted and predicted a response to 3R4F smoke exposure in biological processes involving inflammation and oxidative stress pathways. No enrichment could be performed for Vype ePen aerosol exposure due to the lack of regulated gene candidates at those exposure conditions even after qRT-PCR validation. Of a panel of 33 cytokines screened, 8 were upregulated (FC >1.5 p < 0.05) following 3R4F smoke exposure, which was in agreement with our enrichment analysis. In conclusion, aerosol from the tested e-cigarette caused limited perturbations in gene and inflammatory cytokine expression compared to conventional cigarette smoke, as assessed using next-generation sequencing-based systems biology approaches in 3D commercially available reconstituted lung epithelial tissues.

The e-cigarette category is evolving rapidly, providing consumers with a variety of formats, ranging from cig-alike products to larger, high-powered modular devices. When generating an in vitro assessment approach across such diverse products, dosimetry considerations are paramount. In this article, we have compared nicotine quantification techniques in two studies using a Vitrocell VC 10 Smoking Robot to generate aerosols from different ecigarettes. In Study 1, a 3R4F reference cigarette and four different commercially available e-cigarettes were compared: puff-by-puff nicotine concentration was quantified at the same e-cigarette puffing regime (CRM No81) or with different puff durations, (2 or 3 seconds), comparing 3R4F puff-by-puff yields following ISO and HCI smoking regimes. In Study 2, 3R4F and one e-cigarette were assessed for puff-by-puff nicotine concentration in different locations (China and United Kingdom) comparing different nicotine quantification methods with gas chromatography-mass spectrometry and UPLC-MS/MS used in the two laboratories. Study 1 showed that 3R4F cigarette delivers different nicotine concentrations across the different regimes and puff number, supporting the nicotine methodology; e-cigarettes tested generated different amounts of nicotine across the devices tested, but showed consistent puff-by-puff delivery per device. Study 2 showed positive agreement between results across two different laboratories utilizing different methods for nicotine quantification; statistical analysis, combining all interlaboratory variables, indicated that laboratory differences and the interaction of laboratory and puff number were not significant (p = 0.067 and 0.960, respectively). These studies will add further knowledge to support the in vitro assessment of novel nicotine products, providing reliability and assurance in the area of in vitro dosimetry.

The U.S. Food and Drug Administration has regulatory authority over tobacco products, including conventional cigarettes and next generation products (NGPs) such as e-cigarettes and tobacco heating products (THPs). There is a desire by the industry, regulator and animal, protection organizations to incorporate non-animal test methods for tobacco product and NGP assessment. When assessing respiratory effects in vitro, reliable exposure systems that deliver aerosols to cellular/tissue cultures (such as human reconstructed airways or lung slices) at the air-liquid interface are needed. Using nicotine dosimetry, we report the characterization of a Vitrocell VC1 in our laboratories (IIVS, USA). Nicotine, generated from a 3R4F reference cigarette or NGP (e-cigarette and THP) aerosols at source and the exposure interface (culture media), was assessed using ultra-high-performance liquid chromatographytandem mass spectrometry. These data were compared to published dosimetry data for the same products, generated at a different laboratory (BAT R&D, Southampton, UK), on different exposure systems (VC10 and Borgwaldt RM20S) to confirm repeatability. The nicotine content of 3R4F and NGP aerosols at VC1 source generation was established. Results demonstrated no statistical difference between laboratories (IIVS and BAT; p = 0.903) when comparing puff-by-puff nicotine concentrations from the three products. Culture media nicotine assessment demonstrated no significant difference between replicate wells in the exposure module ( p = 0.855), indicating uniform delivery. This study demonstrates successful Vitrocell VC1 aerosol generation and delivery across multiple nicotine product categories, as characterized using nicotine as a dosimetry marker. The data suggest the VC1 established in our laboratory can reproducibly generate and deliver tobacco product and NGP aerosols for future in vitro assessment and matches the performance of reported exposure systems.