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Public Health Consequences of E-Cigarettes (2018)

Chapter: Appendix D Cytotoxicity Tables

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Suggested Citation:"Appendix D Cytotoxicity Tables." National Academies of Sciences, Engineering, and Medicine. 2018. Public Health Consequences of E-Cigarettes. Washington, DC: The National Academies Press. doi: 10.17226/24952.
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D Cytotoxicity Tables This appendix contains summary tables (Tables D-1, D-2, and D-3) of in vitro studies in which cytotoxicity is assessed. D-1 PREPUBLICATION COPY: UNCORRECTED PROOFS

D-2 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES TABLE D-1 Summary Table of Exposure, Comparison, and Control Conditions and Cell or Tissue Type Used in In Vitro Studies of E-cigarettes assessing Cytotoxicity Exposure: Combustible Aerosol Humecta Nicotine tobacco (A), extracts nt only as only as cigarette (X), an an smoke as a Compare Citation e-liquid (L) exposure exposure control s flavors Cell or Tissue Type Used and Comments Aufderhei A Immortalized primary normal human bronchial epithelial de and (NHBE) cell line CL-1548 Emura (2017)a Study used 3D constructs of cells. Bahl et al. L Human embryonic stem cells (hESC) (2012)b Mouse neuronal stem cells (mNSC) Human pulmonary fibroblasts (hPF) Although all are primary cells, consideration must be given to the low capacity of some embryonic cells to metabolize chemicals via Phase I and II enzymes and efflux processes. Barber et X Human umbilical vein endothelial cells (HUVEC) al. (2016)c Behar et L hPF al. (2014)d hESC Primary cells (embryonic and adult) tested for aerosol effect using Cinnamon Ceylon Behar et L and A hPF al. (2016)e Lung epithelial cells (A549) hESC Combination of primary cell line and embryonic cells to test PREPUBLICATION COPY: UNCORRECTED PROOFS

APPENDIX D D-3 Exposure: Combustible Aerosol Humecta Nicotine tobacco (A), extracts nt only as only as cigarette (X), an an smoke as a Compare Citation e-liquid (L) exposure exposure control s flavors Cell or Tissue Type Used and Comments cinnamonaldehyde cytotoxicity by exposure to aerosols made from refill fluids. Bharadwaj L and A Stress-specific Recombinant Bacterial Cells: E. coli-RecA, E. a et al. coli-SodA, E. coli-CopA, and E. coli-DMO1 (as biosensors) (2017)f Not a primary or mammalian-derived cell. These bioluminescent E. coli stains are engineered to serve as biosensors of DNA strand breaks (E. coli-RecA), reactive oxygen species generation (E. coli-SodA), presence of heavy metals such as copper (E. coli-CopA), and cell membrane damage (E. coli-DMO1). Cervellati A Immortalized human keratinocytes HaCaT et al. (2014)g A549 Farsalinos A Monolayer-cultured cardiomyoblast cells (H9c2) et al. (2013)h Reason provided for cell selection is the better culture stability and reproducibility than human cardiomyocytes. Husari et X A549 al. (2016)i Leigh et A NCI-H292 cell line (human lung mucoepidermoid cells) al. (2016)j Lerner et A Human bronchial airway epithelial cells (H292) al. (2015)k Human fetal lung fibroblasts (HFL1) Lerner et A Human lung fibroblast (HFL1) al. (2016)l Misra et X Human lung epithelial carcinoma cells (A549) al. (2014)m Neilson et A EpiAirway: a human 3D airway tissue model PREPUBLICATION COPY: UNCORRECTED PROOFS

D-4 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES Exposure: Combustible Aerosol Humecta Nicotine tobacco (A), extracts nt only as only as cigarette (X), an an smoke as a Compare Citation e-liquid (L) exposure exposure control s flavors Cell or Tissue Type Used and Comments al. (2015)n Fully differentiated in vitro reconstructs of primary human tracheobronchial epithelium. Cultures express mucus producing goblet cells, ciliated cells with actively beating cilia, basal cells, and club cells (Clara). However, cells obtained from a single donor and therefore may not be representative of responses from a heterogeneous population (e.g., polymorphisms, ethnicities, gender-related factors). Romagna L Mouse BALB/3T3 fibroblasts et al. (2013)o Sancilio et L Human gingival fibroblast (HGF) al. (2016)p Cells obtained from healthy gingival tissue taken from adult subjects during surgical dental extractions. However, fibroblasts are considered to be mesenchymal stem cells because of their self-renewing and multipotent character. Sancilio et L Human gingival fibroblast (HGF) al. (2017)q Scheffler A NHBE48 et al. (2015a)r A549 CL-1548 Scheffler A Normal human bronchial epithelial cells (NHBE) et al. (2015b)s Primary cells came from two donors (cells named NHBE48 and NHBE33). Responses and endpoints vary depending on the origin (donor) of the cells. In some instances, changes and differences are quite significant. Welz et al. L Spheroidal cultures of oropharyngeal mucosa PREPUBLICATION COPY: UNCORRECTED PROOFS

APPENDIX D D-5 Exposure: Combustible Aerosol Humecta Nicotine tobacco (A), extracts nt only as only as cigarette (X), an an smoke as a Compare Citation e-liquid (L) exposure exposure control s flavors Cell or Tissue Type Used and Comments (2016)t Freshly isolated specimens were cut 1mm3 mucosal cubes. Cultures became spheroidal in shape and recoated with interacting endogenous epithelium. In vitro system used in this study is much closer to actual in vivo situation than other in vitro systems tested for e- liquid toxicity. Willershau L Clonetics® human periodontal ligament fibroblasts (HPdLF) sen et al. (2014)u Fibroblasts are considered to be mesenchymal stem cells because of their self-renewing and multipotent character. Wu et al. L Normal human tracheobronchial epithelial (hTBE) cells from (2014)v young, healthy, non-smoking organ donors Yu et al. X Spontaneously transformed immortal keratinocyte (HaCaT) (2016)w Head and neck squamous cell carcinoma (HNSCC) cell lines: HN30 and UMSCC10B The HN30 and UMSCC10B cell lines were originally derived from the oropharynx; HN30 derived from primary laryngeal tumor and UMSCC10B derived from metastatic lymph node. SOURCES: a Aufderheide and Emura, 2017. b Bahl et al., 2012. c Barber et al., 2016. d Behar et al., 2014. e Behar et al., 2016. f Bharadwaj et al., 2017. g Cervellati et al., 2014. h Farsalinos et al., 2013. I Husari et al., 2016. PREPUBLICATION COPY: UNCORRECTED PROOFS

D-6 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES j Leigh et al., 2016. k Lerner et al., 2015. l Lerner et al., 2016. m Misra et al., 2014. n Neilson et al., 2015. o Romagna et al., 2013. p Sancilio et al., 2016. q Sancilio et al., 2017. r Scheffler et al., 2015a. s Scheffler et al., 2015b. t Welz et al., 2016. u Willershausen et al., 2014. v Wu et al., 2014. w Yu et al., 2016. PREPUBLICATION COPY: UNCORRECTED PROOFS

APPENDIX D D-7 TABLE D-2 Summary Table of Test Agents, Cell or Tissue Type Used, and Assays Employed in In Vitro Studies of E-cigarettes assessing Cytotoxicity Cell or Tissue Citation Test Agent(s) Type Used Dose and Time Course Assay Employed Aufderheide Mainstream combustible tobacco Immortalized Samples taken after 0, 4, 6 and 8 Histopathology and Emura cigarette smoke from reference 3R4F human smoke/aerosol exposure repetitions and (2017)a cigarettes (University of bronchial analyzed microscopically after Kentucky) epithelial cell histopathological preparation of the lines CL-1548 cultures. E-liquid aerosol (“Tennessee Cured” flavor, no nicotine, Johnsons Creek, Hartland, WI) Bahl et al. 35 different flavors Human 6 concentrations: 0.001%, 0.01%, 0.03%, MTT assay (2012)b Embryonic 0.1%, 0.3%, and 1%. Stem Cells (hESC); Incubation at 37ºC, 5% CO2 and 95% relative humidity for 48 h Mouse Neuronal Stem Cells (mNSC) Human Pulmonary Fibroblasts (hPF) Barber et al. Combustible tobacco cigarette Human 48-hour exposure to the extracts Endothelial cell viability, (2016)c smoke extracts from Marlboro 100 umbilical vein density and metabolic cigarettes, (16 mg tar, 1.2 mg/ml endothelial activity after exposure to nicotine, Philip Morris) cells mainstream and (HUVECs) sidestream tobacco smoke E-cigarette aerosol extract from extracts, e-aerosol extracts NJoy OneJoy device, Traditional and pure nicotine Flavor with 1.2% (12 mg/ml) or 1.8% (18 mg/ml) nicotine and eGo Activation/deposition of (OKC Vapes), “Desert Sands” flavor complement proteins onto with 0, 12 or 18 mg/ml nicotine endothelial cells was PREPUBLICATION COPY: UNCORRECTED PROOFS

D-8 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES Cell or Tissue Citation Test Agent(s) Type Used Dose and Time Course Assay Employed quantified, as a means to monitor the progression of innate immune responses Behar et al. 10 cinnamon-flavored e-cigarette Human 48 hours MTT assay (2014)d refill liquids from online vendors; embryonic various concentrations of nicotine, stem cells cinnamon flavoring, and percentages of propylene glycol (PG) and/or Lung glycerol fibroblasts Cinnamaldehyde and 2- methoxycinnamaldehyde (2-MOCA) Behar et al. 39 e-cigarette refill fluids purchased hPF The Vea cartomizer device and unfilled GC/MS (2016)e from online vendors cartomizers (Johnson Creek, Hartland, Lung epithelial Wisconsin) operated at 2.9 V, 2.1 and MTT assay Laboratory-made refill fluids cells (A549) 4W. An Innokin iTaste MVP 3.0 battery with variable voltage and wattage and Nuclei stained with DAPI Aerosols produced from the refill hESC Innokin iClear 16D bottom dual coil fluids (produced with unused clearomizers (tanks) were operated at 3 V, Live cell imaging assay cartomizer or tank using smoking 2.1 and 4.2 W or at 5 V, 2.1 and machine) 11.9W. 2 ml of fluid for each sample. Alkaline comet assay Puff duration was 4.3s. Cinnamon Ceylon aerosol produced from cartomizer-style e-cigarette Time course varied by assay and cell type. Bharadwaja E-cigarette liquid (NJOY brand Stress-specific Cells were exposed to various UV-Vis spectroscopy et al. (2017)f containing glycerin, propylene Recombinant concentrations of e-liquid and soluble e- glycol, 10 mg/ml nicotine, flavoring Bacterial Cells: liquid aerosol. Bioluminescence assay chemicals) E. coli-RecA, E. coli-SodA, DNA fragmentation assay Soluble e-liquid aerosol produced E. coli-CopA, from the e-cigarette liquid and E. coli- DMO1 (as biosensors) Cervellati et E-cigarette aerosol (e-cigarette Mini Immortalized HaCaT cells were exposed to fresh Ultrastructural PREPUBLICATION COPY: UNCORRECTED PROOFS

APPENDIX D D-9 Cell or Tissue Citation Test Agent(s) Type Used Dose and Time Course Assay Employed al. (2014)g Touch T-Fumo T-TEX with e-liquid human combustible tobacco cigarette smoke in morphology, Trypan Blue in balsamic flavors with or without keratinocytes an exposure system that generated smoke exclusion test and LDH nicotine, Cloudsmoke, Terna Trade) (HaCaT) by burning one UK research cigarette, and assay. to e-cigarette mixtures using a vacuum E-cigarette aerosol with humectants A549 pump to draw air through the cigarette Pro-inflammatory only (no additives such as flavors or (adenocarcino and leading the smoke stream over the cytokines were measured nicotine) mic human cell cultures for 50 minutes in culture medium by the alveolar basal Bio-Plex cytokine assay Combustible tobacco cigarette epithelial, lung) kit. smoke (United Kingdom research cells cigarette, 12 mg tar, 1.1 mg nicotine) Farsalinos et Combustible tobacco cigarette with Monolayer- Two sets of experiments were performed; MTT assay al. (2013)h 0.8 mg nicotine, 10 mg tar and 10 cultured H9c2 one using regular voltage and a second mg carbon monoxide yields cardiomyoblast using higher voltage fore-cigarette aerosol (Marlboro, Philip Morris Italia S.r.l., cells production Rome, Italy). The medium was aspirated and replaced by medium containing the combustible 20 commercially-available e-liquids tobacco smoke and e-cigarette liquid (17 tobacco flavored, 3 sweet or fruit extracts in one undiluted (100%) and 4 flavored), with 6 mg/ml to 24 mg/ml diluted samples (50%, 25%, 12.5% and nicotine, manufactured or distributed 6.25%). For the e-cigarette extract, 100% by 5 different companies e-cigarette extract is equal to an aerosol extract concentration of 1%. Husari et al. E-cigarette aerosol was generated A549 Exposure to e-cigarette aerosol or Trypan blue exclusion (2016)i using pre-filled V4L CoolCart combustible tobacco cigarette smoke assay for cell counting. cartomizer cartridges (strawberry extract was initiated 24 hours post- flavor, 3.5 Ohm, 18 mg/ml labeled seeding by diluting smoke extract in nicotine) and 4.2 V battery (Vapor complete media to the desired final Titan Soft Touch). concentration (e.g., 0.5, 1, 2, 4, 8mg/ml). Images were taken 24 hours post- Reference 3R4F combustible treatment tobacco cigarettes (University of Kentucky, 9.4 mg tar, 0.726 mg nicotine per cigarette). PREPUBLICATION COPY: UNCORRECTED PROOFS

D-10 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES Cell or Tissue Citation Test Agent(s) Type Used Dose and Time Course Assay Employed Leigh et al. 6 types of commercially available e- NCI-H292 cell Air-liquid interface (ALI) exposure Neutral red uptake assay (2016)j cigarettes (purchased from gas line for metabolic activity. stations, convenience stores, online Health Canada Intense method, using the retailers and local vape shops in following conditions: 3 s puff duration, Trypan blue assay for cell Buffalo, New York, Daly City, every 30 s, with a 55 ml puff volume, viability. California, and online. implemented continuously for 30 min, and resulting in a total of 55 puffs. Cytokine release was eGo tank system (Vision Spinner) e- measured as an indicator cigarette device with battery output Air exposures (control) generated using of cell inflammatory voltage fixed at 3.3 V and refill smoking machines were run during each response. solutions in tobacco, piña colada, experiment. menthol, coffee, and strawberry flavors (purchased from a local Reference 3R4F combustible tobacco ‘vape’ shop in Buffalo, New York). cigarettes (comparison) were smoked using the same method as for the e- Reference 3R4F combustible cigarette products. tobacco cigarettes (University of Kentucky) Five nicotine concentrations were examined: 0, 6, 12, 18 and 24 mg/ml To study effects of humectants, H292 cells were exposed at the ALI to aerosols generated from the e-GO device filled with unflavored liquids containing the same nicotine concentration of 24 mg/ml in (1) PG-only; (2) glycerol-only or (3) a 50/50 mixture of PG/glycerol Three battery output voltage settings were tested: 3.3, 4.0 and 4.8 V. Lerner et al. Refillable pen-style e-cigarette Human H292: Blu e-cigarette aerosol using a HFL1: Violet B 405 nm (2015b)k device (eGo Vision Spinner, China) bronchial CSM-SSM machine (CH-Technologies laser and 440/40 band-pass and compatible clearomizer chamber airway Inc.) was drawn into the chamber every filter to detect increases in (Anyvape, China) with 2.2 ohm epithelial cells 30 seconds with a 4 second pulse for cellular fluorescence. PREPUBLICATION COPY: UNCORRECTED PROOFS

APPENDIX D D-11 Cell or Tissue Citation Test Agent(s) Type Used Dose and Time Course Assay Employed heating element (H292) different time durations 5, 10, and 15 minutes respectively. FlowJo V. 10 for data Blu e-cigarettes (Classic tobacco Human fetal compilation flavor containing 16 mg nicotine) lung fibroblasts HFL-1 was treated with the following e- (HFL1) liquids: PG, glycerol, Vape Dudes Cigarette Smoke Extract (CSE) from (Classic tobacco with or without a research grade combustible nicotine), Vape Dudes (Cinnamon roll tobacco cigarette. without nicotine), Vape Dudes (Grape vape without nicotine), Ecto (American tobacco with or without nicotine) and other e-liquids for 24 hrs and then examined for morphological changes by phase-contrast microscopy Lerner et al. blu Classic Tobacco E-cigarette with Human lung E-cigarette puffs were regulated with 4 s Mitochondria superoxide (2016)l 16 mg nicotine (Lorillard, fibroblasts puffs every 30 s for various sessions (5 staining Greensboro, NC). (HFL-1) min, 10 min, 15 min, or 20 min) Mitochondria membrane potential staining DNA fragmentation assay IL-8 and IL-6 cytokine secretion Misra et al. blu e-cigarettes containing glycerol- A549 0-20 mg/ml Neutral red uptake (2014)m based e-liquids, with and without nicotine and two market flavors IL-8 release for (Classic Tobacco and Magnificent inflammation Menthol) Reference 3R4F, 1R5F, and Marlboro Gold combustible tobacco cigarettes PREPUBLICATION COPY: UNCORRECTED PROOFS

D-12 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES Cell or Tissue Citation Test Agent(s) Type Used Dose and Time Course Assay Employed Neilson et NJOY Bold (4.5% labeled nicotine) Commercially A VITROCELL VC 01 Smoking Robot MTT assay for cell al. (2015)n and NJOY Menthol (3.0% labeled available (VC1/110613) and a 12/6 CF stainless- viability nicotine) human 3D steel exposure module (VITROCELL airway tissue Systems GmbH). Integrity of the airway Reference 3R4F combustible model, epithelium tight junctions tobacco cigarettes EpiAirway™ Reference 3R4F cigarettes were smoked was measured by TEER8 to the ISO smoking regime. Cigarettes conducted according to the were smoked to eight puffs/cig. E- MatTek Corporation’s cigarettes were puffed for 30 min, standard protocol equating to 60 puffs at an independent intense puffing regime, defined as a 80 ml puff drawn over 3 s with 30 s intervals Romagna et Combustible tobacco cigarette 3T3 cells E-cigarette aerosol and combustible MTT assay al. (2013)o smoke extract. (murine tobacco cigarette smoke extracts fibroblasts) simulating e-cigarette use added to culture 21 different e-cigarette liquids. medium. 100%, 50%, 25%, 12.5%, Composition of e-liquids, was (w/w) 6.25%, 3.12% 46.17% PG USP, 44.92% glycerol for 24hrs at 37ºC. USP, 8.11% water, 0.8% nicotine USP, and < 0.5% flavorings. Sancilio et Two different cartridge solutions Human HGFs treated with pre-warmed fluids MTT assay for metabolic al. (2016)p (nicotine content w/v; 0 and 24 gingival with or without nicotine. Cell medium activity mg/ml, respectively) from Halo fibroblasts was replaced every 24 h. In the vaped Company (Pompton Plains, NJ, (HGFs) samples, 1.5 ml of the cartridge solution Apoptosis USA) containing PG, glycerol, and was put in the cartomizer, warmed for 1 natural artificial flavorings min before the dilution and then harvested Increase in green (concentrations not provided by the with a syringe from the cartomizer to a fluorescence for reactive manufacturer), diluted from 4.8 to 48 vial. oxygen species production times Bax expression (pro- apoptotic protein) Sancilio et Two different cartridge solutions HGF HGFs treated with 1 mg/ml nicotine Transmission Electron al. (2017)q (nicotine content w/v; 0 and 24 (obtained by diluting 24 times the 24 Microscopy (TEM) mg/ml, respectively) containing PG, mg/ml nicotine containing fluid), warmed PREPUBLICATION COPY: UNCORRECTED PROOFS

APPENDIX D D-13 Cell or Tissue Citation Test Agent(s) Type Used Dose and Time Course Assay Employed glycerol, and natural artificial and not warmed before administration Lactate dehydrogenase flavorings (concentrations not assay provided by the manufacturer), HGFs treated with the fluid without diluted 24 times with DMEM nicotine diluted 24 times, warmed and not Lysosome Compartment warmed before administration Analysis HGFs also left untreated Human collagen type I concentration in supernatants was assayed using an ELISA Western blot for LC3 expression in HGF Scheffler et Reevo Mini-S e-cigarette (In-Smoke, A549 The e-cigarette was connected to the ROS-Glo™ H2O2 Assay al. (2015a)r Winnenden, Germany) with a piston pump of a smoking robot and 200 (Promega, Madison, WI, 3.3V/900 mAh battery and 2.2 Ohm NHBE puffs were taken with a puff volume of 35 USA) for oxidative stress resistance with e-liquids purchased ml, puff duration of 2 s, blow-out time of from Johnsons Creek (Hartland, WI, Immortalized 7 s, and an interpuff interval of 10 s. CellTiter-Blue® Assay USA) in “Tennessee Cured” flavor CL-1548 cells (Promega) for cell (75% PG USP, 25% glycerol USP, For combustible tobacco cigarette smoke viability 0.0% and 2.4% nicotine USP). Other exposure, 10 K3R4F cigarettes were ingredients listed on the bottle smoked by the smoking robot using the include: deionized water, natural same parameters as described for the e- flavors, artificial flavor, and USP cigarette. Each cigarette was puffed six grade citric acid (as a preservative) times. Reference 3R4F combustible tobacco cigarettes (Kentucky) with a standard cellulose acetate filter tip Scheffler et Aerosols from two e-cigarette liquids NHBE The e-cigarette was connected to the ROS-Glo™ H2O2 Assay al. (2015b)s liquids purchased from Johnsons piston pump of a smoking robot and 200 (Promega, Madison, WI, Creek (Hartland, WI, USA) in puffs were taken with a puff volume of 35 USA) for oxidative stress “Tennessee Cured” flavor (0 and ml, puff duration of 2 s, blow-out time of 2.4% nicotine). Liquids contained 7 s. CellTiter-Blue® Assay PREPUBLICATION COPY: UNCORRECTED PROOFS

D-14 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES Cell or Tissue Citation Test Agent(s) Type Used Dose and Time Course Assay Employed USP-grade propylene glycol, USP- (Promega) for cell grade glycerol, deionized water, For combustible tobacco cigarette smoke viability natural and artificial flavors, USP- exposure, 10 K3R4F cigarettes were grade nicotine, and USP-grade citric smoked by the smoking robot using the acid. same parameters as described for the e- cigarette. Each cigarette was puffed six Aerosols from humectants (glycerol times. and propylene glycol) obtained from Alfa Aesar (Karlsruhe, Germany), Cultures analyzed 24 hours after with a purity of 99.5%. exposure. Combustible tobacco cigarette smoke from 10 reference K3R4F combustible tobacco cigarettes (Lexington, Kentucky) with a standard cellulose acetate filter tip Welz et al. E-liquids in three flavors (apple, Fresh tissue (1) 24 h one-time incubation MTT assay (2016)t cherry, and tobacco) with a base samples of mixture of 80% PG, 10% G, and healthy human (2) 2.5 h incubation on five sequential 10% water and 12 mg/ml nicotine oropharyngeal days mucosa as- sembled into mucosal tissue cultures (spheroidal in vitro model) Willershause E-liquids (eSmokerShop, GmbH, Clonetics® Up to 96 hour incubation depending on PrestoBlue Cell Viability n et al. Hannover, Germany) in hazelnut, HPdLF assay Assay (2014)u lime, and menthol flavors with 20-22 (Human mg/ml nicotine in a PG-base Periodontal ATP-Detection Ligament Fibroblasts) Cell visualization Migration assay PREPUBLICATION COPY: UNCORRECTED PROOFS

APPENDIX D D-15 Cell or Tissue Citation Test Agent(s) Type Used Dose and Time Course Assay Employed Wu et al. Tobacco-flavored e-liquid at various Normal human 24 and 48 h exposures. The final nicotine Pro-inflammatory (2014)v concentrations (0, 0.01, 0.1, 0.3% tracheobronchi concentrations were within the serum cytokines v/v) without nicotine or with 18 al epithelial nicotine range of e-cigarette users mg/ml of nicotine (InnoVapor LLC., (hTBE) cells HRV-16 infection in e- Boise, ID) liquid-exposed normal hTBE cells Lactate dehydrogenase IL-6 levels by ELISA Taqman quantitative real- time RT-PCR to detect HRV RNA and human SPLUNC1 mRNA Yu et al. V2 (Red American Tobacco flavor) HaCaT HaCaT cells were treated for 8 weeks Neutral comet assay (2016)w and VaporFi (Classic Tobacco with 1% v:v extract flavor) e-cigarette brands) in a 70% HNSCC cell -H2AX PG/30% glycerol base with 0.0% lines: HN30 UMSCC10B and HN30 were each treated immunostaining abd 1.2% nicotine. and for 1 week with 1% v:v extract. UMSCC10B Cell cycle changes by flow HaCaT cells were treated for 10 days at cytometry 0.5%, 1.0%, and 2.0% v:v aerosolized e- cigarette liquid. Trypan Blue staining to assess cytotoxic effects HaCaT cells were treated for 10 days, and UMSCC10B and HN30 for 12 days prior Clonogenic survival to colony counting. Annexin V apoptotic assay SOURCES: a Aufderheide and Emura, 2017. b Bahl et al., 2012. c Barber et al., 2016. d Behar et al., 2014. PREPUBLICATION COPY: UNCORRECTED PROOFS

D-16 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES e Behar et al., 2016. f Bharadwaj et al., 2017. g Cervellati et al., 2014. h Farsalinos et al., 2013. i Husari et al., 2016. j Leigh et al., 2016. k Lerner et al., 2015. l Lerner et al., 2016. m Misra et al., 2014. n Neilson et al., 2015. o Romagna et al., 2013. p Sancilio et al., 2016. q Sancilio et al., 2017. r Scheffler et al., 2015a. s Scheffler et al., 2015b. t Welz et al., 2016. u Willershausen et al., 2014. v Wu et al., 2014. w Yu et al., 2016. PREPUBLICATION COPY: UNCORRECTED PROOFS

APPENDIX D D-17 TABLE D-3 Summary of Results from In Vitro Studies of E-cigarettes assessing Cytotoxicity Citation Results and Observations Aufderheide Cultures exposed to both mainstream combustible tobacco cigarette smoke and e-liquid aerosol showed a clear reduction in and Emura mucus production and cilia bearing, but the effect was weaker for the aerosol than for the smoke. (2017)a Bahl et al. The MTT assay showed effects of refill solutions on cell survival that ranged from no evidence of cytotoxicity to high levels of (2012)b toxicity. Cinnamon Ceylon had the strongest effects and was the only sample that was cytotoxic for all three cell types. Fifteen refill samples were moderately cytotoxic to hESC, and in general, mNSC responded similarly to these samples. In general, hESC were more sensitive than hPF, but Freedom Smoke Menthol Arctic and Global Smoke Caramel produced stronger cytotoxic effects on hPF than on the other two cells. The humectants (propylene glycol [PG] and glycerol) were non-cytotoxic for all cell types. Five Butterscotch or Caramel flavored samples were also non-cytotoxic at the highest dose tested. The relevance of exposure to refill liquid (as compared with aerosols) in cytotoxicity studies is a concern. Barber et al. Most of the exposure conditions resulted in significant effects on cell density. There was also a slight reduction in viability, (2016)c independent of nicotine concentration or the exact formulation of the extract. Authors observed a significant decrease in metabolic activity for cells that were exposed to combustible tobacco cigarette smoke or e-cigarette aerosol extracts, independent of the formulation of the extract. Exposure to pure nicotine did not alter endothelial cell metabolic activity. Results showed significant increase in the deposition of C1q and C5b- 9, and in C3b to a lesser extent. There were no changes in C4d. Behar et al. The study established NOAELs of 0.03% for hESC and 0.01% for hPF. hESC was more sensitive than hPF. (2014)d Of 4 chemical additives tested, CAD and 2MOCA were the most cytotoxic, producing similar IC50s for both hESC and hPF cells. By contrast, dipropylene glycol and vanillin were the least cytotoxic, and their IC50s were higher than a user would likely experience. Behar et al. In the 48-hour MTT assay, hESC (embryonic stem cells) were more sensitive to Cinnamon Ceylon and cinnamaldehyde aerosols (2016)e than hPF and A549 (respiratory) cells. By contrast, hESC tolerated short-term exposure to cinnamaldehyde for a longer time (8 hours) than hPF (2 hours). PREPUBLICATION COPY: UNCORRECTED PROOFS

D-18 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES Citation Results and Observations Cytoskeletal structure disruption (e.g., depolymerisation of actin microfilaments and microtubules) was observed for both hESC and hPF exposed to cinnamaldehyde at MTT NOAEL and IC50 concentrations. Bharadwaja Following exposure to e-liquids and e-cigarette aerosol at various concentrations, bioluminescent recombinant bacterial cells (as et al. (2017)f biosensors) showed dose-dependent and stress-specific responses. Interestingly, cells exposed to e-liquid showed greater inhibition of bioluminescence at high concentrations, which declined dose-dependently with dilutions, whereas cells exposed to e-cigarette aerosols showed the opposite effect, with bioluminescence increasing in a dose-dependent manner with exposure to decreasing concentrations of e-cigarette aerosol. These changes in bioluminescence expression indicate potential cellular damage, such as DNA damage, oxidative stress, ion homeostasis, and membrane damage. Both e-liquid and aerosol exposure resulted in cellular damage, but e-cigarette aerosol exposure showed damage without significant growth inhibition. Results of the DNA fragmentation assay showed considerable DNA breaks at high doses of e-liquid exposure, compared with lower doses (which showed partial DNA fragmentation) and controls. Cervellati et Exposure to e-cigarette aerosol with humectants only (no flavorings or nicotine) resulted in no change in either cell viability or al. (2014)g LDH release over 24 h. Exposure to e-cigarette aerosol with flavoring caused significant progressive loss of viability and increased LDH release in both cell types. E-cigarette aerosol with both flavoring and nicotine caused rapid (50 min) and marked loss in viability and enhanced LDH release. This is similar to effects of combustible tobacco cigarette smoke exposure, which caused an early (6 h) and progressive decrease in cell viability and increased LDH release. The authors observed a similar trend during the different time points in both cell lines, but keratinocytes appeared more susceptible to combustible tobacco cigarette smoke-induced toxicity after 24 hours. The morphology of the cells exposed to combustible tobacco cigarette smoke shows clear signs of cellular damage and presence of vacuoles. By contrast, cells treated with e-cigarette aerosol with humectants only (no flavors or nicotine), remained intact with the same ultrastructural aspect of control cells, even 24 hours after treatment. In cells exposed to e-cigarette with flavors, an increase in vacuolization and alteration of cytoplasmic membrane was observed. The degeneration of intracellular organelles was more pronounced after exposure to e-cigarette aerosols with flavors and nicotine, especially in HaCaT cells, which showed a marked vacuolization consequent to the expansion of the mitochondria and the endoplasmic reticulum. Results suggest that e-liquid and/or aerosol components contain some pro-inflammatory stimuli leading to a change in the secretome pattern depending on the cells lines employed. Fluctuations in cytokines release after other e-cigarette and combustible tobacco cigarette smoke exposures were also observed, but interpreting these effects was possible due to subsequent cell death. Farsalinos et Of 20 samples tested, four samples exhibited a cytotoxic effect in the 3.7 V experiments: PREPUBLICATION COPY: UNCORRECTED PROOFS

APPENDIX D D-19 Citation Results and Observations al. (2013)h “Cinnamon-Cookies” flavor was slightly cytotoxic at the highest extract concentration, while both samples of “El Toro Cigarillos” and “El Toro Puros” were cytotoxic at both 100% and 50% extract concentration. The range of myocardial cell survival for all e-cigarette samples at 3.7 V was: 89.7%–112.1% at 6.25%, 90.6%–115.3% at 12.5%, 81.0%–106.6% at 25%, 7.4%–106.8% at 50% and 2.2%–110.8% at 100% extract concentration. The “base” sample was not cytotoxic at any extract concentration. Combustible tobacco cigarette smoke extract was significantly cytotoxic at concentrations above 6.25%, with viability rate being: 76.9 ± 2.0% at 6.25%, 38.2 ± 0.6% at 12.5%, 3.082 ± 0.2% at 25%, 5.2 ± 0.8% at 50% and 3.9 ± 0.2% at 100% extract concentration The absolute mean difference in viability between 3.7 V and 4.5 V experiments was: 7.1 ± 4.1% at 6.25%, 5.0 ± 5.3% at 12.5%, 4.2 ± 4.8% at 25%, 5.0 ± 3.8% at 50% and 17.0 ± 12.2% at 100% extract concentration. Only the difference at 6.25% extract concentration was statistically significant (p = 0.039). None of the 4 samples was considered cytotoxic. IC50 could be determined only for combustible tobacco cigarette smoke extract and for “El Toro Cigarrillos” and “El Toro Puros”, since for every other e-cigarette sample viability was higher than 50% at all extract concentrations. The lowest NOAEL and IC50 were observed in combustible tobacco cigarette smoke extract. Husari et al. Combustible tobacco cigarette smoke total particulate matter extract at concentrations of 2 mg/ml and higher attenuated cellular (2016)i growth and triggered cell death. Similar effects only occurred from exposure to e-cigarette total particulate matter extract at concentrations of 64 mg/ml. Leigh et al. Effects of e-cigarette aerosols on toxicity to bronchial epithelial cells differed significantly. Flavors have a significant and (2016)j differential effect on toxicity: e-cigarette aerosols with menthol, coffee and strawberry flavors significantly reduced cell viability and metabolic activity compared to air controls. E-cigarette aerosols with coffee and strawberry flavors also significantly increased cytokine levels compared to both air controls and reference combustible tobacco cigarettes. No significant differences (p < 0.05) in metabolic activity and cell viability were observed between the e-cigarette aerosols with various nicotine concentrations and the air control, or among the varying nicotine concentrations when compared against each other. However, significant differences (p < 0.05) were found between the various nicotine concentrations and combustible tobacco cigarette smoke. Of note, metabolic activity of exposed cells was measured by neutral red uptake assay, but the definition of this endpoint is not clear because neutral red assay is a quantitative estimation of the number of viable cells in culture. PREPUBLICATION COPY: UNCORRECTED PROOFS

D-20 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES Citation Results and Observations With respect to cytokine release, compared with air controls, exposure to aerosol with 18 mg/ml nicotine resulted in significant decreases in IL-1 , CXCL1, and CXCL2, while exposure to aerosol with 24 mg/ml nicotine resulted in significantly increased IL-6. IL-1 and CXCL2 levels were also significantly decreased between 18 mg/ml nicotine aerosol and the reference combustible tobacco cigarette. Significant differences were observed among aerosols with variable nicotine concentrations for IL-1 , IL-6, CXCL1, and CXCL2. Exposure of H292 cells to e-cigarette humectant-only aerosols significantly decreased cell viability (p < 0.05) compared to air controls, but toxic effects were significantly less than from exposure to combustible tobacco cigarette smoke. Effects of humectant aerosols on cell metabolic activity differed significantly, decreasing significantly in cells exposed to PG/glycerol and glycerol-only aerosols, but not to PG-only compared with air controls. With respect to cytokine release, all tested cytokines increased significantly except CXCL1 and CXCL10 in cells exposed to PG-only compared with air controls. Aerosol from the 4.0 V and 4.8 V devices significantly decreased (p < 0.05) metabolic activity and cell viability compared with the air control. Aerosol generated with the 3.3 V device was not different than air and significantly less toxic than combustible tobacco cigarette smoke (p < 0.05). Aerosol generated with the device at the highest (4.8 V) setting significantly increased all tested cytokines compared with air controls. Lerner et al. Fibroblasts cultured with e-liquid or combustible tobacco cigarette smoke extract (CSE) exhibited a reduction in the number of (2015)k cells per count area. Many of the treated cells were enlarged and vacuolarized, and this effect was greater in CSE treated cells and cells treated with 5% e-liquids. Compared to control cells, e-liquid and CSE treated cells showed morphological changes— enlarged cells and spindle formation. Morphological changes were similar in cells exposed to e-liquid without nicotine added to cells at 1% concentration and 1% PG. In contrast, fibroblasts cultured in 1% e-liquid with nicotine resulted in morphological changes that resemble cells treated with 1% CSE. Lung fibroblast viability following treatments with 2.5% PG, glycerol, or commercial e-liquids was not significantly different than control after 24 hours (control; 90.53 ± 5.34, PG; 88.40 ± 2.99, glycerol; 91.97 ± 6.23, Ecto American tobacco flavor 0 mg nicotine; 92.7 ± 2.55, Ecto American tobacco flavor 24 mg nicotine; 78.57 ± 6.67, % viability in means ± SD, p > 0.05). Exposure to humectants (PG, glycerol) only elicited no significant increase in release of IL-8 compared with the control group (15.9 ± 12.02 pg/ml) after 24 hour treatment. Of the four commercially available e-liquids, only “cinnamon roll”-flavored e- liquid stimulated a significant increase in IL-8 secretion (458.14 ± 26.20 pg/ml). IL-8 and IL-6 secretion at 16 hours post- exposure was significantly higher for cells exposed to e-cigarette aerosols than air controls for each exposure time period. The release of IL-6 into culture media was dose-dependent. IL-6 secretion was significantly higher after 10 minute exposure than 5 minute exposures. The IL-8 levels were all significantly increased in cells exposed to e-cigarette aerosol compared with the air PREPUBLICATION COPY: UNCORRECTED PROOFS

APPENDIX D D-21 Citation Results and Observations controls. In cells exposed to e-cigarette aerosols, small but significant increases in fluorescence were observed. Lerner et al. Results showed a small but significant reduction in the amount of mtROS present after 20 minutes of aerosol exposure compared (2016)l to 10- or 15-minute exposures. The level of ARE inducible Nqo1 expression increased for the 10 min and 20 min exposure sessions. Similarly, 10 minutes of exposure of HFL-1 to e-cigarette aerosol increased average Nqo1 levels when total cellular proteins were collected 18 hours following the exposures. After 24 hours, the level of mtROS in cells treated with the copper metal nanoparticles increased significantly. E-cigarette aerosol-exposed cells exhibited complex IV sensitivity as observed by decreased levels of COX II (MTCO2) subunit in cell lysates collected 18 hours after aerosol exposure. A reduced level of complex I NDUFB8 subunit in addition to reduced COX II was observed in cell lysates harvested 90 minutes after exposure. 5-minute aerosol exposure did not produce any difference in DNA fragmentation, whereas, 10 and 15 minute exposures resulted in significant increases in DNA fragmentation compared to air control groups. However, as the exposure duration increased, the likelihood for DNA damage increased in the air control group as well. 10-minute aerosol exposure resulted in increased IL-6 secretion (45.70 pg/ml) at 18 hours post-e-cigarette exposure, compared with IL-6 levels (7.34 pg/ml) from the air control group. IL-8 levels (28.02 pg/ml) also increased compared with the air control group (16.42 pg/ml). Misra et al. No cytotoxicity was observed for any of the e-liquids, tested up to their respective highest sample doses. (2014)m E-liquid exposure resulted in greater IL-8 release at high doses (6.9–13.8 mg/ml). Any IL-8 release from blu MM e-liquid treatments that were significant when compared with IL-8 release from exposure to combustible tobacco cigarettes occurred at doses approximately 42-fold higher than the combustible tobacco cigarettes. Neilson et al. Tissue cell viability following combustible tobacco cigarette smoke exposure was reduced in a time- and dose-dependent manner (2015)n from 100% to 12% viability after 6 hours of exposure, relative to untreated controls. Exposure of EpiAirway™ tissue to either variety of e-cigarette did not reduce tissue viability relative to untreated control tissues. Thus, an ET50 for e-cigarette aerosol could not be calculated. No statistical difference in viability was seen between NJOY Bold or NJOY Menthol and diluting air PREPUBLICATION COPY: UNCORRECTED PROOFS

D-22 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES Citation Results and Observations controls. A dose-dependent decrease in cell viability was seen following incremental hourly exposures to cigarette smoke for up to 6 h, resulting in reductions of around 90% at the highest dose. By contrast, the two e-cigarettes did not cause cytotoxic effects under any of the test conditions, despite a much larger puff volume and exposure frequency in the e-cigarette machine smoking regime. Romagna et From the 21 samples examined, only the “coffee” flavored e-liquid exhibited a cytotoxic effect, and this only at the highest al. (2013)o extract concentration. For this sample, the viability rate was 114.5 2.0% at 3.125%, 112.2 3.6% at 6.25%, 101.5 3.1% at 12.5%, 92.0 8.9% at 25%, 85.9 11.8% at 50% and 51.0 2.6% at 100% extract concentration. Combustible tobacco cigarette smoke extract exhibited significant cytotoxicity at extract concentrations greater than 12.5%. For the majority of e-liquids (13 of 21), viability was not statistically different between extract concentrations. Thus, NOAEL for these samples was defined as 100% concentration. None of the 12 tobacco-flavored e-cigarette liquids tested were associated with a statistically significant difference in fibroblast viability. Sancilio et al. E-liquid exposure resulted in reduced metabolic activity in a time-and dose-dependent manner in HGFs. For e-liquids both with (2016)p and without nicotine at 5 mg/ml and 2 mg/ml concentrations, the metabolic activity was reduced up to 20 % of the control. There were no significant changes in apoptosis in the treated HGFs compared with untreated cells. After 48 hours, cell viability decreased in all the experimental conditions (about 60 % versus about 85 % in the controls), with a higher range in the 1-N sample (35.85 % of viable cells) The reactive oxygen species production showed a peak after 24 hours of treatment compared with untreated controls (771.6 [nicotine], 798.6 [warmed nicotine], 458.9 [no nicotine], and 687.6 [warmed, no nicotine] versus 200 [untreated]). In the nicotine-free fluid-treated HGFs, the ROS production was lower than in the other experimental conditions. However, effects were seen after 48 hours (540.7 nicotine free versus 271.1 untreated), whereas the other samples showed no significant changes compared with the control after 48 hours. Bax protein expression did not appear to be affected after 6 hours of exposure, but after 24 hours, it was higher in the e-liquid exposed conditions than in the control sample (1.485-fold increase [nicotine], 1.605-fold increase [warmed nicotine], 1.490-fold increase [no nicotine], and 1.405-fold increase [warmed no nicotine] on the untreated samples). After 48 hours, Bax expression in the nicotine, warmed nicotine, and nicotine-free conditions remained higher than in the untreated HGFs (1.735, 1.695, and 1.385 fold increase on the untreated samples, respectively) while in the warmed e-liquid without nicotine, the increase was close to one-fold. Sancilio et al. E-liquids with nicotine exerted cytotoxicity as demonstrated by the increased levels of LDH, in parallel to the presence of PREPUBLICATION COPY: UNCORRECTED PROOFS

APPENDIX D D-23 Citation Results and Observations (2017)q numerous vacuoles in the cytoplasm, as well as a decrease in collagen I production and an augmented LC3 II expression. Autophagic vesicles and an increased number of pro-collagen I molecules were present in the cytoplasm of fibroblasts exposed to nicotine-free fluids. In the same samples, the time-dependent activation of the lysosomal compartment with no changes in LC3 expression were detected. Scheffler et Primary NHBE48 cells were the most sensitive, responding to e-liquid aerosol exposure with a decrease in viability up to 60% al. (2015a)r and 52% compared to clean air-exposed cells. In comparison, combustible tobacco cigarette mainstream smoke-exposed cells showed only 7% viability of clean air-exposed cells. Immortalized CL-1548 cells are less sensitive to e-liquid aerosol (75% and 70% viability) and combustible tobacco cigarette smoke exposure (10% viability) compared to primary NHBE48 cells, but are still significantly more sensitive than A549 cells (88% viability for e-liquid aerosol, 21% for mainstream smoke exposure). In all cell types, no significant differences were seen after the exposure to nicotine-containing and nicotine-free aerosol. The oxidative stress level is elevated in CL-1548 cells compared to A549 cells, but lower than those of primary NHBE48 cells. Scheffler et The authors found toxicological effects of e-cigarette aerosol and the humectant-only substances, whereas the nicotine al. (2015b)s concentration did not have an effect on the cell viability. The viability of combustible tobacco cigarette mainstream smoke- exposed cells was 4.5–8 times lower and the oxidative stress levels 4.5–5 times higher than those of e-cigarette aerosol exposed cells, depending on the donor. Welz et al. Both fruit- and tobacco-flavored extracts were cytotoxic to oropharyngeal tissue, but fruit-flavored liquids showed a higher (2016)t toxicity than tobacco-flavored ones. Additionally, incubation of mucosal tissue cultures with fruit-flavored extracts showed DNA fragmentation, but no serious DNA damage was seen in tissue cultures incubated in tobacco-flavored extracts. Willershausen Starting at 24 hours, the highest reduction in the proliferation was observed for the treatment with menthol-flavored liquids, et al. (2014)u which was the only statistically significant reduction as compared to control cells. After an incubation time of 48 hours with the menthol flavored liquid the difference in comparison both to the control cells and the nicotine-treated cells was highly statistically significant (p < 0.001). Hazelnut flavor or lime flavor only caused a slight not statistically significant reduction of the proliferation rates at 48 hours. After 96 hours of incubation this strong growth-reducing effect of the menthol-flavored liquids persisted and was still statistically significant In comparison to the untreated cells, incubation with hazelnut-flavored (p < 0.024), lime-flavored (p < 0.009) or menthol- flavored liquids (p < 0.001) led to a statistically significant reduction of the ATP detection. The untreated human periodontal ligament fibroblasts and those incubated for 24 hours with PG showed good proliferation. PREPUBLICATION COPY: UNCORRECTED PROOFS

D-24 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES Citation Results and Observations Those incubated with nicotine, hazelnut or lime-flavored liquids showed a slight growth reduction, while incubation with the menthol-flavored liquid produced a strong growth inhibition. The inhibitory effect of menthol flavor exposure on the fibroblast cells was especially noticeable in the migration assay. Only the menthol-flavored liquid caused a highly statistically significant reduction (p < 0.001) of cell migration after 72 hours in comparison to the control cells as well as to the cells treated with nicotine. Wu et al. Within the physiological nicotine range, e-liquid exposure did not cause noticeable cytotoxicity at either 24 or 48 hours. (2014)v Exposure to e-liquid without nicotine increased IL-6 protein levels in a dose-dependent manner at both 24 and 48 hours. Addition of nicotine to e-liquid only marginally enhanced the IL-6 levels. Cells exposed to tobacco-flavored e-liquid (without or with nicotine) had higher levels of HRV load than unexposed cells at both 6 and 24 hours. Compared with e-liquid without nicotine, the addition of nicotine into e-liquid either did not alter (at 6 hours) or slightly increased (at 24 hours, p = 0.05) HRV load. HRV infection significantly increased IL-6 production at both 6 and 24 hours in cells that were pre-exposed to the control (medium alone) or e-liquid with and without nicotine. Yu et al. E-cigarette exposure without nicotine induced a 10-fold increase in cell death, while e-cigarette exposure with nicotine induced a (2016)w 10-fold increase compared with controls. UMSCC10B showed a statistically significant increased accumulation of arrest in G1, and HN30 showed an increase in G2, both independently of e-cigarette nicotine content. A stepwise decrease in colony count and decreased survival was observed with increasing e-cigarette doses in both brands, independently of nicotine content. After exposure to 0.5% v:v nicotine-free e-cigarette aerosol, greater than a two-fold decrease in survival was seen in all cell lines. SOURCES: a Aufderheide and Emura, 2017. b Bahl et al., 2012. c Barber et al., 2016. d Behar et al., 2014. e Behar et al., 2016. f Bharadwaj et al., 2017. PREPUBLICATION COPY: UNCORRECTED PROOFS

APPENDIX D D-25 g Cervellati et al., 2014. h Farsalinos et al., 2013. i Husari et al., 2016. j Leigh et al., 2016. k Lerner et al., 2015. l Lerner et al., 2016. m Misra et al., 2014. n Neilson et al., 2015. o Romagna et al., 2013. p Sancilio et al., 2016. q Sancilio et al., 2017. r Scheffler et al., 2015a. s Scheffler et al., 2015b. t Welz et al., 2016. u Willershausen et al., 2014. v Wu et al., 2014. w Yu et al., 2016. PREPUBLICATION COPY: UNCORRECTED PROOFS

D-26 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES REFERENCES Aufderheide, M., and M. Emura. 2017. Phenotypical changes in a differentiating immortalized bronchial epithelial cell line after exposure to mainstream cigarette smoke and e-cigarette vapor. Experimental and Toxicologic Pathology 69(6):393-401. Bahl, V., S. Lin, N. Xu, B. Davis, Y. H. Wang, and P. Talbot. 2012. Comparison of electronic cigarette refill fluid cytotoxicity using embryonic and adult models. Reproductive Toxicology 34(4):529-537. Barber, K. E., B. Ghebrehiwet, W. Yin, and D. A. Rubenstein. 2016. Endothelial cell inflammatory reactions are altered in the presence of e-cigarette extracts of variable nicotine. Cellular and Molecular Bioengineering 10(1):124-133. Behar, R. Z., B. Davis, Y. Wang, V. Bahl, S. Lin, and P. Talbot. 2014. Identification of toxicants in cinnamon-flavored electronic cigarette refill fluids. Toxicology in Vitro 28(2):198-208. Behar, R. Z., W. T. Luo, S. C. Lin, Y. H. Wang, J. Valle, J. F. Pankow, and P. Talbot. 2016. Distribution, quantification and toxicity of cinnamaldehyde in electronic cigarette refill fluids and aerosols. Tobacco Control 25(Suppl 2):ii94-ii102. Bharadwaj, S., R. J. Mitchell, A. Qureshi, and J. H. Niazi. 2017. Toxicity evaluation of e-juice and its soluble aerosols generated by electronic cigarettes using recombinant bioluminescent bacteria responsive to specific cellular damages. Biosensors and Bioelectronics 90:53-60. Cervellati, F., X. M. Muresan, C. Sticozzi, R. Gambari, G. Montagner, H. J. Forman, C. Torricelli, E. Maioli, and G. Valacchi. 2014. Comparative effects between electronic and cigarette smoke in human keratinocytes and epithelial lung cells. Toxicology in Vitro 28(5):999-1005. Farsalinos, K. E., G. Romagna, E. Allifranchini, E. Ripamonti, E. Bocchietto, S. Todeschi, D. Tsiapras, S. Kyrzopoulos, and V. Voudris. 2013. Comparison of the cytotoxic potential of cigarette smoke and electronic cigarette vapour extract on cultured myocardial cells. International Journal of Environmental Research and Public Health 10(10):5146-5162. Husari, A., A. Shihadeh, S. Talih, Y. Hashem, M. El Sabban, and G. Zaatari. 2016. Acute exposure to electronic and combustible cigarette aerosols: Effects in an animal model and in human alveolar cells. Nicotine & Tobacco Research 18(5):613-619. Leigh, N. J., R. I. Lawton, P. A. Hershberger, and M. L. Goniewicz. 2016. Flavourings significantly affect inhalation toxicity of aerosol generated from electronic nicotine delivery systems (ENDS). Tobacco Control 25(Suppl 2):ii81-ii87. Lerner, C. A., P. Rutagarama, T. Ahmad, I. K. Sundar, A. Elder, and I. Rahman. 2016. Electronic cigarette aerosols and copper nanoparticles induce mitochondrial stress and promote DNA fragmentation in lung fibroblasts. Biochemical and Biophysical Research Communications 477(4):620-625. Lerner, C. A., I. K. Sundar, H. Yao, J. Gerloff, D. J. Ossip, S. McIntosh, R. Robinson, and I. Rahman. 2015. Vapors produced by electronic cigarettes and e-juices with flavorings induce toxicity, oxidative stress, and inflammatory response in lung epithelial cells and in mouse lung. PLoS ONE 10(2):e0116732. Misra, M., R. D. Leverette, B. T. Cooper, M. B. Bennett, and S. E. Brown. 2014. Comparative in vitro toxicity profile of electronic and tobacco cigarettes, smokeless tobacco and nicotine PREPUBLICATION COPY: UNCORRECTED PROOFS

APPENDIX D D-27 replacement therapy products: E-liquids, extracts and collected aerosols. International Journal of Environmental Research & Public Health 11(11):11325-11347. Neilson, L., C. Mankus, D. Thorne, G. Jackson, J. DeBay, and C. Meredith. 2015. Development of an in vitro cytotoxicity model for aerosol exposure using 3D reconstructed human airway tissue; application for assessment of e-cigarette aerosol. Toxicology in Vitro 29(7):1952-1962. Romagna, G., E. Allifranchini, E. Bocchietto, S. Todeschi, M. Esposito, and K. E. Farsalinos. 2013. Cytotoxicity evaluation of electronic cigarette vapor extract on cultured mammalian fibroblasts (ClearStream-LIFE): Comparison with tobacco cigarette smoke extract. Inhalation Toxicology 25(6):354-361. Sancilio, S., M. Gallorini, A. Cataldi, and V. di Giacomo. 2016. Cytotoxicity and apoptosis induction by e-cigarette fluids in human gingival fibroblasts. Clinical Oral Investigations 20(3):477-483. Sancilio, S., M. Gallorini, A. Cataldi, L. Sancillo, R. A. Rana, and V. di Giacomo. 2017. Modifications in human oral fibroblast ultrastructure, collagen production and lysosomal compartment in response to e-cigarette fluids. Journal of Periodontology 88(7):673-680. Scheffler, S., H. Dieken, O. Krischenowski, and M. Aufderheide. 2015a. Cytotoxic evaluation of e-liquid aerosol using different lung-derived cell models. International Journal of Environmental Research and Public Health 12(10):12466-12474. Scheffler, S., H. Dieken, O. Krischenowski, C. Forster, D. Branscheid, and M. Aufderheide. 2015b. Evaluation of e-cigarette liquid vapor and mainstream cigarette smoke after direct exposure of primary human bronchial epithelial cells. International Journal of Environmental Research and Public Health 12(4):3915-3925. Welz, C., M. Canis, S. Schwenk-Zieger, S. Becker, V. Stucke, F. Ihler, and P. Baumeister. 2016. Cytotoxic and genotoxic effects of electronic cigarette liquids on human mucosal tissue cultures of the oropharynx. Journal of Environmental Pathology, Toxicology and Oncology 35(4):343-354. Willershausen, I., T. Wolf, V. Weyer, R. Sader, S. Ghanaati, and B. Willershausen. 2014. Influence of e-smoking liquids on human periodontal ligament fibroblasts. Head & Face Medicine 10:39. Wu, Q., D. Jiang, M. Minor, and H. W. Chu. 2014. Electronic cigarette liquid increases inflammation and virus infection in primary human airway epithelial cells. PLoS ONE 9(9):e108342. Yu, V., M. Rahimy, A. Korrapati, Y. Xuan, A. E. Zou, A. R. Krishnan, T. Tsui, J. A. Aguilera, S. Advani, L. E. Crotty Alexander, K. T. Brumund, J. Wang-Rodriguez, and W. M. Ongkeko. 2016. Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines. Oral Oncology 52:58-65. PREPUBLICATION COPY: UNCORRECTED PROOFS

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Millions of Americans use e-cigarettes. Despite their popularity, little is known about their health effects. Some suggest that e-cigarettes likely confer lower risk compared to combustible tobacco cigarettes, because they do not expose users to toxicants produced through combustion. Proponents of e-cigarette use also tout the potential benefits of e-cigarettes as devices that could help combustible tobacco cigarette smokers to quit and thereby reduce tobacco-related health risks. Others are concerned about the exposure to potentially toxic substances contained in e-cigarette emissions, especially in individuals who have never used tobacco products such as youth and young adults. Given their relatively recent introduction, there has been little time for a scientific body of evidence to develop on the health effects of e-cigarettes.

Public Health Consequences of E-Cigarettes reviews and critically assesses the state of the emerging evidence about e-cigarettes and health. This report makes recommendations for the improvement of this research and highlights gaps that are a priority for future research.

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