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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. 703
TABLE D-1â Summary of Exposure, Comparison, and Control Conditions and Cell or Tissue Type Used in In 704 Vitro Studies of E-Cigarettes Assessing Cytotoxicity Exposure: Humectant Nicotine Combustible Aerosol (A), Only Only Tobacco Extracts (X), as an as an Cigarette Smoke Compares Cell or Tissue Type Used and Reference E-liquid (L) Exposure Exposure as a Control Flavors Comments Aufderheide A Ã¼ â¢ mmortalized primary NHBE cell I and Emura, line (CL-1548) 2017 â¢ tudy used 3D constructs of cells. S Bahl et al., 2012 L Ã¼ Ã¼ Ã¼ â¢ hESC â¢ mNSC â¢ hPF â¢ lthough all are primary cells, A 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 al., X Ã¼ Ã¼ â¢ HUVEC 2016 Behar et al., L Ã¼ Ã¼ Ã¼ â¢ hPF 2014 â¢ hESC â¢ rimary cells (embryonic and P adult) tested for aerosol effect using cinnamon Ceylon.
Behar et al., L and A Ã¼ Ã¼ â¢ hPF 2016 â¢ uman lung epithelial carcinoma H cells (A549) â¢ hESC â¢ ombination of primary cell C line and embryonic cells to test cinnamonaldehyde cytotoxicity by exposure to aerosols made from refill fluids. Bharadwaja et L and A â¢ Stress-specific recombinant al., 2017 bacterial cells: E. coli-RecA, E. coli-SodA, E. coli-CopA, and E. coli- DMO1 (as biosensors) â¢ ot a primary or mammalian- N derived cell. These bioluminescent E. coli strains 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 et A Ã¼ Ã¼ â¢ mmortalized human keratinocytes I al., 2014 (HaCaT) â¢ uman lung epithelial carcinoma H cells (A549) continued 705
TABLE D-1 Continued 706 Exposure: Humectant Nicotine Combustible Aerosol (A), Only Only Tobacco Extracts (X), as an as an Cigarette Smoke Compares Cell or Tissue Type Used and Reference E-liquid (L) Exposure Exposure as a Control Flavors Comments Farsalinos et A Ã¼ Ã¼ Ã¼ Ã¼ â¢ Monolayer-cultured cardiomyoblast al., 2013 cells (H9c2) â¢ eason provided for cell selection R is the better culture stability and reproducibility than human cardiomyocytes. Husari et al., X Ã¼ â¢ uman lung epithelial carcinoma H 2016 cells (A549) Leigh et al., A Ã¼ Ã¼ Ã¼ Ã¼ â¢ uman lung mucoepidermoid cells H 2016 (NCI-H292 cell line) Lerner et al., A Ã¼ Ã¼ Ã¼ Ã¼ â¢ uman bronchial airway epithelial H 2015 cells (H292) â¢ HFL1 Lerner et al., A â¢ HFL1 2016 Misra et al., X Ã¼ Ã¼ â¢ uman lung epithelial carcinoma H 2014 cells (A549) Neilson et al., A Ã¼ â¢ EpiAirwayâ¢: a human 3D airway 2015 tissue model â¢ ully differentiated in vitro F 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 were obtained from a single donor and therefore may not be representative of responses from a heterogeneous population (e.g., polymorphisms, ethnicities, sex-related factors). Romagna et al., L Ã¼ Ã¼ â¢ ouse BALB/3T3 fibroblasts M 2013 Sancilio et al., L Ã¼ â¢ HGF 2016 â¢ ells were obtained from healthy C 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 al., L Ã¼ â¢ HGF 2017 Scheffler et al., A Ã¼ Ã¼ â¢ rimary NHBE cells P 2015a â¢ uman lung epithelial carcinoma H cells (A549) â¢ mmortalized primary NHBE cell I line (CL-1548) continued 707
TABLE D-1 Continued 708 Exposure: Humectant Nicotine Combustible Aerosol (A), Only Only Tobacco Extracts (X), as an as an Cigarette Smoke Compares Cell or Tissue Type Used and Reference E-liquid (L) Exposure Exposure as a Control Flavors Comments Scheffler et al., A Ã¼ Ã¼ Ã¼ â¢ rimary NHBE cells P 2015b â¢ rimary cells came from two P 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 Ã¼ â¢ pheroidal cultures of S 2016 oropharyngeal mucosa â¢ reshly isolated specimens were F cut 1 mm3 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. Willershausen L Ã¼ Ã¼ Ã¼ â¢ CloneticsÂ® HPdLF et al., 2014 â¢ ibroblasts are considered to be F mesenchymal stem cells because of their self-renewing and multipotent character.
Wu et al., 2014 L Ã¼ â¢ ormal hTBE cells from young, N healthy, non-smoking organ donors Yu et al., 2016 X Ã¼ â¢ Spontaneously transformed immortal keratinocyte (HaCaT) â¢ NSCC cell lines: HN30 and H UMSCC10B â¢ he HN30 and UMSCC10B cell T lines were originally derived from the oropharynx; HN30 was derived from primary laryngeal tumor and UMSCC10B was derived from metastatic lymph node. NOTE: hESC = human embryonic stem cell; HFL1 = human fetal lung fibroblast; HGF = human gingival fibroblast; HNSCC = head and neck squamous cell carcinoma; HPdLF = human periodontal ligament fibroblast; hPF = human pulmonary fibroblast; hTBE = human tracheobronchial epithelial; HUVEC = human umbilical vein endothelial cell; mNSC = mouse neural stem cell; NHBE = normal human bronchial epithelial. 709
TABLE D-2 Summary of Test Agents, Cell or Tissue Type Used, and Assays Employed in In Vitro Studies of 710 E-Cigarettes Assessing Cytotoxicity Cell or Tissue Type Reference Test Agent(s) Used Dose and Time Course Assay Employed Aufderheide and â¢ Mainstream â¢ Immortalized â¢ Samples taken â¢ Histopathology Emura, 2017 combustible tobacco primary NHBE cell after 0, 4, 6, and cigarette smoke line CL-1548 8 smoke/aerosol from reference 3R4F exposure repetitions cigarettes (University and analyzed of Kentucky) microscopically after â¢ E-liquid aerosol histopathological (Tennessee cured preparation of the flavor, no nicotine, cultures. Johnsons Creek, Hartland, WI) Bahl et al., 2012 â¢ 5 different flavors 3 â¢ hESC â¢ 6 concentrations: â¢ MTT assay â¢ mNSC 0.001%, 0.01%, 0.03%, â¢ hPF 0.1%, 0.3%, and 1% â¢ ncubation at 37ÂºC, 5% I CO2, and 95% relative humidity for 48 hours Barber et al., 2016 â¢ Combustible â¢ HUVECs â¢ 8-hour exposure to 4 â¢ Endothelial cell tobacco cigarette the extracts viability, density and smoke extracts metabolic activity from Marlboro 100 after exposure to cigarettes (16 mg tar, mainstream and 1.2 mg/ml nicotine, sidestream tobacco Philip Morris) smoke extracts, e-aerosol extracts, and pure nicotine
â¢ E-cigarette aerosol extract from NJoy â¢ Activation/deposition OneJoy device, of complement traditional flavor proteins onto with 1.2% (12 mg/ endothelial cells ml) or 1.8% (18 mg/ was quantified as a ml) nicotine and eGo means to monitor the (OKC Vapes), desert progression of innate sands flavor with immune responses 0, 12, or 18 mg/ml nicotine Behar et al., 2014 â¢ 10 cinnamon- â¢ hPF â¢ 48 hours â¢ MTT assay flavored e-cigarette â¢ hESC refill liquids from online vendors; various concentrations of nicotine, cinnamon flavoring, and percentages of PG and/or glycerol â¢ Cinnamaldehyde and 2-MOCA continued 711
TABLE D-2 Continued 712 Cell or Tissue Type Reference Test Agent(s) Used Dose and Time Course Assay Employed Behar et al., 2016 â¢ 9 e-cigarette refill 3 â¢ hPF â¢ The Vea cartomizer â¢ GC/MS fluids purchased â¢ Human lung device and unfilled â¢ MTT assay from online vendors epithelial carcinoma cartomizers (Johnson â¢ uclei stained with N â¢ Laboratory-made cells (A549) Creek, Hartland, DAPI refill fluids â¢ hESC Wisconsin) operated â¢ ive cell imaging assay L â¢ Aerosols produced at 2.9 V, 2.1 Î©, and 4 â¢ lkaline comet assay A from the refill fluids W. An Innokin iTaste (produced with MVP 3.0 battery with unused cartomizer or variable voltage and tank using smoking wattage and Innokin machine) iClear 16D bottom â¢ Cinnamon Ceylon dual-coil clearomizers aerosol produced (tanks) were operated from cartomizer-style at 3 V, 2.1 Î©, and 4.2 e-cigarette W or at 5 V, 2.1 Î©, and 11.9 W. 2 ml of fluid for each sample. Puff duration was 4.3 seconds. â¢ Time course varied by assay and cell type. Bharadwaja et al., 2017 â¢ E-cigarette liquid â¢ Stress-specific â¢ ells were exposed to C â¢ UV-Vis spectroscopy (NJOY brand recombinant bacterial various concentrations â¢ Bioluminescence assay containing glycerol, cells: E. coli-RecA, of e-liquid and soluble â¢ DNA fragmentation PG, 10 mg/ml E. coli-SodA, E. e-liquid aerosol. assay nicotine, flavoring coli-CopA, and chemicals) E. coli-DMO1 (as biosensors)
â¢ Soluble e-liquid aerosol produced from the e-cigarette liquid Cervellati et al., 2014 â¢ E-cigarette aerosol â¢ Immortalized â¢ aCaT cells were H â¢ Ultrastructural (e-cigarette Mini human keratinocytes exposed to fresh morphology Touch T-Fumo T-TEX (HaCaT) combustible tobacco â¢ rypan Blue exclusion T with e-liquid in â¢ Human lung cigarette smoke in test balsamic flavors with epithelial carcinoma an exposure system â¢ LDH assay or without nicotine, cells (A549) that generated smoke â¢ Pro-inflammatory Cloudsmoke, Terna by burning one UK cytokines were Trade) research cigarette, measured in culture â¢ E-cigarette aerosol and to e-cigarette medium by the Bio- with humectants mixtures using a Plex cytokine assay kit only (no additives vacuum pump to such as flavors or draw air through the nicotine). cigarette and leading â¢ Combustible tobacco the smoke stream over cigarette smoke the cell cultures for 50 (United Kingdom minutes. research cigarette, 12 mg tar, 1.1 mg nicotine) continued 713
TABLE D-2 Continued 714 Cell or Tissue Type Reference Test Agent(s) Used Dose and Time Course Assay Employed Farsalinos et al., 2013 â¢ Combustible tobacco â¢ Monolayer-cultured â¢ wo sets of T â¢ MTT assay cigarette with 0.8 mg cardiomyoblast cells experiments were nicotine, 10 mg tar, (H9c2) performed; one using and 10 mg carbon regular voltage and a monoxide yields second using higher (Marlboro, Philip voltage, for e-cigarette Morris Italia S.r.l., aerosol production. Rome, Italy) â¢ he medium was T â¢ 20 commercially aspirated and replaced available e-liquids by medium containing (17 tobacco flavored, the combustible 3 sweet or fruit tobacco smoke and flavored), with 6â24 e-cigarette liquid mg/ml nicotine, extracts in one manufactured or undiluted (100%) and distributed by 5 4 diluted samples different companies (50%, 25%, 12.5%, and 6.25%). For the e-cigarette extract, 100% e-cigarette extract is equal to an aerosol extract concentration of 1%.
Husari et al., 2016 â¢ E-cigarette aerosol â¢ Human lung â¢ xposure to e-cigarette E â¢ rypan blue exclusion T was generated epithelial carcinoma aerosol or combustible assay using pre-filled cells (A549) tobacco cigarette V4L CoolCart smoke extract was cartomizer cartridges initiated 24 hours (strawberry flavor, post-seeding by 3.5 Î©, 18 mg/ml diluting smoke extract labeled nicotine) and in complete media 4.2-V battery (Vapor to the desired final Titan Soft Touch) concentration (e.g., â¢ Reference 3R4F 0.5, 1, 2, 4, 8 mg/ml). combustible tobacco Images were taken 24 cigarettes (University hours post-treatment. of Kentucky, 9.4 mg tar, 0.726 mg nicotine per cigarette) continued 715
TABLE D-2 Continued 716 Cell or Tissue Type Reference Test Agent(s) Used Dose and Time Course Assay Employed Leigh et al., 2016 â¢ types of 6 â¢ Human lung â¢ Airâliquid interface â¢ eutral red uptake N commercially mucoepidermoid (ALI) exposure assay available e-cigarettes cells (NCI-H292 cell â¢ ealth Canada Intense H â¢ rypan blue assay T (purchased from line) method, using the â¢ Cytokine release gas stations, following conditions: was measured as convenience stores, 3-second puff duration, an indicator of cell online retailers, and every 30 seconds, inflammatory response local vape shops in with a 55-ml puff Buffalo, New York, volume, implemented Daly City, California, continuously for 30 and online) minutes, and resulting â¢ Go tank system e in a total of 55 puffs. (Vision Spinner) â¢ ir exposures (control) A e-cigarette device generated using with battery output smoking machines voltage fixed at 3.3 V were run during each and refill solutions in experiment. tobacco, piÃ±a colada, â¢ Reference 3R4F menthol, coffee, and combustible tobacco strawberry flavors cigarettes (comparison) (purchased from a were smoked using the local vape shop in same method as for the Buffalo, New York) e-cigarette products. â¢ Reference 3R4F combustible tobacco cigarettes (University of Kentucky)
â¢ 5 nicotine concentrations were examined: 0, 6, 12, 18, and 24 mg/ml. â¢ o study effects of T 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. â¢ hree battery output T voltage settings were tested: 3.3, 4.0, and 4.8 V. continued 717
TABLE D-2 Continued 718 Cell or Tissue Type Reference Test Agent(s) Used Dose and Time Course Assay Employed Lerner et al., 2015 â¢ Refillable pen- â¢ Human bronchial â¢ 292: blu e-cigarette H â¢ FL1: Violet B 405- H style e-cigarette airway epithelial cells aerosol using a nm laser and 440/40 device (eGo Vision (H292) CSM-SSM machine bandpass filter to Spinner, China) â¢ HFL1 (CH-Technologies detect increases in and compatible Inc.) was drawn into cellular fluorescence clearomizer chamber the chamber every â¢ lowJo V.10 for data F (Anyvape, China) 30 seconds with compilation with 2.2-Î© heating a 4-second pulse element for different time â¢ blu e-cigarettes durations of 5, 10, and (classic tobacco 15 minutes. flavor containing 16 â¢ FL1 was treated H mg nicotine) with the following â¢ SE from a research- C e-liquids: PG, glycerol, grade combustible Vape Dudes (classic tobacco cigarette tobacco with or without nicotine), Vape Dudes (cinnamon roll without nicotine), Vape Dudes (grape vape without nicotine), Ecto (American tobacco with or without nicotine) and other e-liquids for 24 hours and then examined for morphological changes by phase-contrast microscopy.
Lerner et al., 2016 â¢ lu classic tobacco b â¢ HFL1 â¢ E-cigarette puffs â¢ Mitochondria e-cigarette with were regulated with superoxide staining 16 mg nicotine 4-second puffs every â¢ Mitochondria (Lorillard, 30 seconds for various membrane potential Greensboro, NC) sessions (5, 10, 15, or staining 20 minutes). â¢ DNA fragmentation assay â¢ L-8 and IL-6 cytokine I secretion Misra et al., 2014 â¢ blu e-cigarettes â¢ Human lung â¢ 0â20 mg/ml â¢ Neutral red uptake containing glycerol- epithelial carcinoma â¢ IL-8 release based e-liquids, cells (A549) with and without nicotine and two market flavors (classic tobacco and magnificent menthol) â¢ Reference 3R4F, 1R5F, and Marlboro gold combustible tobacco cigarettes continued 719
TABLE D-2 Continued 720 Cell or Tissue Type Reference Test Agent(s) Used Dose and Time Course Assay Employed Neilson et al., 2015 â¢ JOY bold (4.5% N â¢ EpiAirwayâ¢: a â¢ VITROCELL VC A â¢ MTT assay labeled nicotine) and human 3D airway 01 Smoking Robot â¢ ntegrity of the I NJOY menthol (3.0% tissue model (VC1/110613) and airway epithelium labeled nicotine) a 12/6 CF stainless- tight junctions was â¢ Reference 3R4F steel exposure module measured by TEER8 combustible tobacco (VITROCELL Systems conducted according cigarettes GmbH) to the MatTek â¢ Reference 3R4F Corporationâs standard cigarettes were protocol. smoked to the ISO smoking regime: 8 puffs/cigarette. E-cigarettes were puffed for 30 minutes, equating to 60 puffs at an independent intense puffing regime, defined as an 80-ml puff drawn over 3 seconds with 30-second intervals. Romagna et al., 2013 â¢ Combustible tobacco â¢ Mouse BALB/3T3 â¢ E-cigarette aerosol and â¢ MTT assay cigarette smoke fibroblasts combustible tobacco extract cigarette smoke extracts â¢ 21 different simulating e-cigarette e-cigarette liquids. use added to culture Composition of medium. 100%, 50%, e-liquids was (w/w) 25%, 12.5%, 6.25%, 46.17% PG USP, 44.92%
glycerol USP, 8.11% 3.12% for 24 hours at water, 0.8% nicotine 37ÂºC USP, and < 0.5% flavorings Sancilio et al., 2016 â¢ Two different â¢ HGF â¢ GFs treated with H â¢ MTT assay cartridge solutions pre-warmed fluids â¢ Apoptosis (nicotine content with or without â¢ ncrease in green I [w/v] 0 and 24 nicotine. Cell medium fluorescence for mg/ml) from was replaced every 24 reactive oxygen species Halo Company hours. In the vaped production (Pompton Plains, samples, 1.5 ml of the â¢ ax expression (pro- B NJ, USA) containing cartridge solution was apoptotic protein) PG, glycerol, put in the cartomizer, and natural and warmed for 1 minute artificial flavorings before the dilution and (concentrations then harvested with not provided by a syringe from the the manufacturer), cartomizer to a vial. diluted from 4.8 to 48 times continued 721
TABLE D-2 Continued 722 Cell or Tissue Type Reference Test Agent(s) Used Dose and Time Course Assay Employed Sancilio et al., 2017 â¢ Two different â¢ HGF â¢ GFs treated with H â¢ TEM cartridge solutions 1 mg/ml nicotine â¢ LDH assay (nicotine content (obtained by diluting â¢ Lysosome [w/v] 0 and 24 24 times the 24 mg/ compartment analysis mg/ml) containing ml nicotine-containing â¢ Human collagen PG, glycerol, fluid), warmed and type I concentration and natural and not warmed before in supernatants was artificial flavorings administration assayed using an (concentrations â¢ GFs treated with H ELISA not provided by the fluid without â¢ estern blot for LC3 W the manufacturer), nicotine diluted 24 expression in HGF diluted 24 times with times, warmed and DMEM not warmed before administration â¢ GFs also left H untreated Scheffler et al., 2015a â¢ Reevo Mini-S â¢ rimary NHBE cells P â¢ he e-cigarette was T â¢ ROS-Gloâ¢ H2O2 Assay e-cigarette (In-Smoke, â¢ Human lung connected to the piston (Promega, Madison, Winnenden, Germany) epithelial carcinoma pump of a smoking WI, USA) for oxidative with a 3.3-V/900- cells (A549) robot and 200 puffs stress mAh battery and â¢ Immortalized were taken with a â¢ CellTiter-BlueÂ® Assay 2.2-Î© resistance with primary NHBE cell puff volume of 35 ml, (Promega, Madison, e-liquids purchased line (CL-1548) puff duration of 2 WI, USA) for cell from Johnsons Creek seconds, blow-out time viability (Hartland, WI, USA) of 7 seconds, and an in Tennessee cured interpuff interval of 10 flavor (75% PG USP, seconds. 25%
glycerol USP, 0.0% â¢ For combustible and 2.4% nicotine tobacco cigarette USP). Other smoke exposure, 10 ingredients listed on K3R4F cigarettes were the bottle include each puffed by the deionized water, smoking robot using natural flavors, the same parameters artificial flavor, and as described for the USP-grade citric acid e-cigarette. (as a preservative). â¢ Reference 3R4F combustible tobacco cigarettes (Kentucky) with a standard cellulose acetate filter tip continued 723
TABLE D-2 Continued 724 Cell or Tissue Type Reference Test Agent(s) Used Dose and Time Course Assay Employed Scheffler et al., 2015b â¢ Aerosols from two â¢ rimary NHBE cells P â¢ he e-cigarette was T â¢ ROS-Gloâ¢ H2O2 Assay e-cigarette liquids connected to the piston (Promega, Madison, purchased from pump of a smoking WI, USA) for oxidative Johnsons Creek robot and 200 puffs stress (Hartland, WI, USA) were taken with a puff â¢ CellTiter-BlueÂ® Assay in Tennessee cured volume of 35 ml, puff (Promega, Madison, flavor (0% and 2.4% duration of 2 seconds, WI, USA) for cell nicotine). Liquids blow-out time of 7 viability contained USP-grade seconds. PG, USP-grade â¢ For combustible glycerol, deionized tobacco cigarette water, natural and smoke exposure, 10 artificial flavors, USP- K3R4F cigarettes grade nicotine, and were smoked by the USP-grade citric acid. smoking robot using â¢ Aerosols from the same parameters humectants (glycerol as described for the and PG) obtained from e-cigarette. Each Alfa Aesar (Karlsruhe, cigarette was puffed 6 Germany), with a times. purity of 99.5%. â¢ Cultures were â¢ Combustible tobacco analyzed 24 hours cigarette smoke from after exposure. 10 reference K3R4F combustible tobacco cigarettes (Lexington, Kentucky) with a standard cellulose acetate filter tip.
Welz et al., 2016 â¢ -liquids in three E â¢ Spheroidal cultures â¢ 24-hour one-time â¢ MTT assay flavors (apple, of oropharyngeal incubation cherry, and tobacco) mucosa â¢ .5-hour incubation on 2 with a base mixture 5 sequential days of 80% PG, 10% glycerol, and 10% water and 12 mg/ml nicotine Willershausen et al., â¢ E-liquids â¢ CloneticsÂ® HPdLF â¢ p to 96-hour U â¢ PrestoBlue Cell 2014 (eSmokerShop, incubation depending Viability Assay GmbH, Hannover, on assay â¢ ATP detection Germany) in â¢ Cell visualization hazelnut, lime, and â¢ Migration assay menthol flavors with 20â22 mg/ml nicotine in a PG-base Wu et al., 2014 â¢ Tobacco-flavored â¢ ormal hTBE cells N â¢ 4- and 48-hour 2 â¢ Pro-inflammatory e-liquid at various from young, healthy, exposures. The final cytokines concentrations (0, non-smoking organ nicotine concentrations â¢ RV-16 infection in H 0.01, 0.1, 0.3% v/v) donors were within the serum e-liquid-âexposed without nicotine or nicotine range of normal hTBE cells with 18 mg/ml of e-cigarette users. â¢ LDH nicotine (InnoVapor â¢ L-6 levels by ELISA I LLC., Boise, ID) â¢ Taqman quantitative real-time RT-PCR to detect HRV RNA and human SPLUNC1 mRNA continued 725
TABLE D-2 Continued 726 Cell or Tissue Type Reference Test Agent(s) Used Dose and Time Course Assay Employed Yu et al., 2016 â¢ V2 (red American â¢ Spontaneously â¢ aCaT cells were H â¢ eutral comet assay N tobacco flavor) and transformed treated for 8 weeks â¢ -H2AX Î³ VaporFi (classic immortal with 1% v/v extract immunostaining tobacco flavor) keratinocyte (HaCaT) â¢ MSCC10B and HN30 U â¢ ell cycle changes by C e-cigarette brands â¢ HNSCC cell were each treated for flow cytometry in a 70% PG/30% lines: HN30 and 1 week with 1% v/v â¢ rypan Blue staining T glycerol base with UMSCC10B extract. â¢ Clonogenic survival 0.0% and 1.2% â¢ aCaT cells were H â¢ Annexin V apoptotic nicotine treated for 10 days assay at 0.5%, 1.0%, and 2.0% v/v aerosolized e-cigarette liquid. â¢ aCaT cells were H treated for 10 days, and UMSCC10B and HN30 for 12 days prior to colony counting. NOTE: 2-MOCA = 2-methoxycinnamaldehyde; CSE = cigarette smoke extract; DAPI = 4â,6-diamidino-2-phenylindole; GC/MS = gas chromatog- raphy/mass spectrometry; hESC = human embryonic stem cell; HFL1 = human fetal lung fibroblast; HGF = human gingival fibroblast; HNSCC = head and neck squamous cell carcinoma; HPdLF = human periodontal ligament fibroblast; hPF = human pulmonary fibroblast; HRV = human rhinovirus; hTBE = human tracheobronchial epithelial; HUVEC = human umbilical vein endothelial cell; LDH = lactate dehydrogenase; mNSC = mouse neural stem cell; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NHBE = normal human bronchial epithelial; PG = propylene glycol; TEM = transmission electron microscopy.
APPENDIX D 727 TABLE D-3 Summary of Results from In Vitro Studies of E-Cigarettes Assessing Cytotoxicity Reference Results and Observations Aufderheide Cultures exposed to both mainstream combustible tobacco cigarette and Emura, smoke and e-liquid aerosol showed a clear reduction in mucus 2017 production and cilia bearing, but the effect was weaker for the aerosol than for the smoke. Bahl et al., The MTT assay showed effects of refill solutions on cell survival that 2012 ranged from no evidence of cytotoxicity to high levels of 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 (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 2016 density. There was also a slight reduction in viability, 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; 2014 hESC was more sensitive than hPF. Of 4 chemical additives tested, CAD and 2-MOCA were the most cytotoxic, producing similar IC50 for both hESC and hPF cells. By contrast, dipropylene glycol and vanillin were the least cytotoxic, and their IC50 were higher than a user would likely experience. continued
728 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES TABLE D-3â Continued Reference Results and Observations Behar et al., In the 48-hour MTT assay, hESC (embryonic stem cells) were more 2016 sensitive to cinnamon Ceylon and cinnamaldehyde aerosols 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). Cytoskeletal structure disruption (e.g., depolymerization of actin microfilaments and microtubules) was observed for both hESC and hPF exposed to cinnamaldehyde at MTT NOAEL and IC50 concentrations. Bharadwaja et Following exposure to e-liquids and e-cigarette aerosol at various al., 2017 concentrations, bioluminescent recombinant bacterial cells (as 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.
APPENDIX D 729 TABLE D-3â Continued Reference Results and Observations Cervellati et Exposure to e-cigarette aerosol with humectants only (no flavorings al., 2014 or nicotine) resulted in no change in either cell viability or LDH release over 24 hours. 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 minutes) 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 hours) 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 smokeâinduced 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 cytokine 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. continued
730 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES TABLE D-3â Continued Reference Results and Observations Farsalinos et Of 20 samples tested, 4 samples exhibited a cytotoxic effect in the al., 2013 3.7-V experiments: 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 concentrations. The range of myocardial cell survival for all e-cigarette samples at 3.7 V was 89.7%â112.1% at 6.25%, 90.6%â115.3% at 12.5%, 81.0%â106.6% at 25%, 7.4%â106.8% at 50%, and 2.2%â110.8% at 100% extract concentration. The âbaseâ sample was not cytotoxic at any extract concentration. Combustible tobacco cigarette smoke extract was significantly cytotoxic at concentrations above 6.25%, with viability rates being 76.9 Â± 2.0% at 6.25%, 38.2 Â± 0.6% at 12.5%, 3.082 Â± 0.2% at 25%, 5.2 Â± 0.8% at 50%, and 3.9 Â± 0.2% at 100% extract concentration. The absolute mean difference in viability between 3.7-V and 4.5-V experiments was 7.1 Â± 4.1% at 6.25%, 5.0 Â± 5.3% at 12.5%, 4.2 Â± 4.8% at 25%, 5.0 Â± 3.8% at 50%, and 17.0 Â± 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 2016 at concentrations of 2 mg/ml and higher attenuated cellular growth and triggered cell death. Similar effects only occurred from exposure to e-cigarette total particulate matter extract at concentrations of 64 mg/ml.
APPENDIX D 731 TABLE D-3â Continued Reference Results and Observations Leigh et al., Effects of e-cigarette aerosols on toxicity to bronchial epithelial cells 2016 differed significantly. Flavors have a significant and 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. With respect to cytokine release, compared with air controls, exposure to aerosol with 18 mg/ml nicotine resulted in significant decreases in IL-1Î², CXCL1, and CXCL2, while exposure to aerosol with 24 mg/ml nicotine resulted in significantly increased IL-6. IL-1Î² 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-1Î², 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. continued
732 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES TABLE D-3â Continued Reference Results and Observations Lerner et al., Fibroblasts cultured with e-liquid or combustible tobacco CSE 2015 exhibited a reduction in the number of 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 changesâenlarged 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 (% viability in means Â± SD; control: 90.53 Â± 5.34, PG: 88.40 Â± 2.99, glycerol: 91.97 Â± 6.23, Ecto American tobacco flavor 0 mg nicotine: 92.7 Â± 2.55, Ecto American tobacco flavor 24 mg nicotine: 78.57 Â± 6.67, p > 0.05). Exposure to humectants only (PG, glycerol) elicited no significant increase in release of IL-8 compared with the control group (15.9 Â± 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 Â± 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 exposure. The IL-8 levels were all significantly increased in cells exposed to e-cigarette aerosol compared with the air controls. In cells exposed to e-cigarette aerosols, small but significant increases in fluorescence were observed.
APPENDIX D 733 TABLE D-3â Continued Reference Results and Observations Lerner et al., Results showed a small but significant reduction in the amount of 2016 mtROS present after 20 minutes of aerosol exposure compared to 10- or 15-minute exposures. The level of ARE-inducible Nqo1 expression increased for the 10- and 20-minute 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-2 (MTCO2) subunit in cell lysates collected 18 hours after aerosol exposure. A reduced level of Complex I NDUFB8 subunit in addition to reduced COX-2 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 2014 their respective highest sample doses. E-liquid exposure resulted in greater IL-8 release at high doses (6.9â 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. continued
734 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES TABLE D-3â Continued Reference Results and Observations Neilson et al., Tissue cell viability following combustible tobacco cigarette smoke 2015 exposure was reduced in a time- and dose-dependent manner from 100% to 12% viability after 6 hours of exposure, relative to untreated controls. Exposure of EpiAirwayâ¢ 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 controls. A dose-dependent decrease in cell viability was seen following incremental hourly exposures to cigarette smoke for up to 6 hours, 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 al., 2013 exhibited a cytotoxic effect, and this only at the highest extract concentration. For this sample, the viability rate was 114.5 Â± 2.0% at 3.125%, 112.2 Â± 3.6% at 6.25%, 101.5 Â± 3.1% at 12.5%, 92.0 Â± 8.9% at 25%, 85.9 Â± 11.8% at 50%, and 51.0 Â± 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.
APPENDIX D 735 TABLE D-3â Continued Reference Results and Observations Sancilio et al., E-liquid exposure resulted in reduced metabolic activity in a time- 2016 and dose-dependent manner in HGFs. For e-liquids both with 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-liquidâ 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 onefold. Sancilio et al., E-liquids with nicotine exerted cytotoxicity as demonstrated by the 2017 increased levels of LDH, in parallel to the presence of 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 was detected. continued
736 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES TABLE D-3â Continued Reference Results and Observations Scheffler et Primary NHBE48 cells were the most sensitive, responding to al., 2015a e-liquid aerosol exposure with a decrease in viability up to 60% and 52% compared to clean air-exposed cells. In comparison, combustible tobacco cigarette mainstream smokeâexposed cells showed only 7% viability of clean airâexposed 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 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 al., 2015b the humectant-only substances, whereas the nicotine concentration did not have an effect on cell viability. The viability of combustible tobacco cigarette mainstream smokeâexposed cells was 4.5â8 times lower and the oxidative stress levels 4.5â5 times higher than those of e-cigarette aerosolâexposed cells, depending on the donor. Welz et al., Both fruit- and tobacco-flavored extracts were cytotoxic to 2016 oropharyngeal tissue, but fruit-flavored liquids showed a higher 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.
APPENDIX D 737 TABLE D-3â Continued Reference Results and Observations Willershausen Starting at 24 hours, the highest reduction in the proliferation was et al., 2014 observed for the treatment with menthol-flavored liquids, 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 ATP detection. The untreated human periodontal ligament fibroblasts and those incubated for 24 hours with PG showed good proliferation. 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 2014 cause noticeable cytotoxicity at either 24 or 48 hours. 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.
738 PUBLIC HEALTH CONSEQUENCES OF E-CIGARETTES TABLE D-3â Continued Reference Results and Observations Yu et al., 2016 E-cigarette exposure without nicotine induced a 10-fold increase in cell death, while e-cigarette exposure with nicotine induced a 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 twofold decrease in survival was seen in all cell lines. NOTE: 2-MOCA = 2-methoxycinnamaldehyde; CSE = cigarette smoke extract; hESC = human embryonic stem cell; HFL1 = human fetal lung fibroblast; HGF = human gingival fibroblast; HNSCC = head and neck squamous cell carcinoma; HPdLF = human periodontal ligament fibroblast; hPF = human pulmonary fibroblast; HRV = human rhinovirus; hTBE = human tracheobronchial epithelial; HUVEC = human umbilical vein endothelial cell; LDH = lactate dehydrogenase; mNSC = mouse neural stem cell; MTT = 3-(4,5-Â imethylthiazol-2-yl d )-2,5-diphenyltetrazolium bromide; NHBE = normal human bronchial epithelial; NOAEL = Â no observed adverse effect level; PG = propylene glycol.
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