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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.

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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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TABLE D-1 Summary of Exposure, Comparison, and Control Conditions and Cell or Tissue Type Used in In Vitro Studies of E-Cigarettes Assessing Cytotoxicity

Reference	Exposure: Aerosol (A), Extracts (X), E-liquid (L)	Humectant Only as an Exposure	Nicotine Only as an Exposure	Combustible Tobacco Cigarette Smoke as a Control	Compares Flavors	Cell or Tissue Type Used and Comments
Aufderheide and Emura, 2017	A			10004		Immortalized primary NHBE cell line (CL-1548) Study used 3D constructs of cells.
Bahl et al., 2012	L	✔	✔		✔	hESC mNSC 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 al., 2016	X		✔	✔		HUVEC
Behar et al., 2014	L	✔	✔		✔	hPF hESC Primary cells (embryonic and adult) tested for aerosol effect using cinnamon Ceylon.

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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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Behar et al., 2016	L and A	✔		✔	hPF Human lung epithelial carcinoma cells (A549) hESC Combination of primary cell line and embryonic cells to test cinnamonaldehyde cytotoxicity by exposure to aerosols made from refill fluids.
Bharadwaja et al., 2017	L and A				Stress-specific recombinant bacterial cells: E. coli-RecA, E. coli-SodA, E. coli-CopA, and E. coli-DMO1 (as biosensors) Not a primary or mammalian-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 al., 2014	A	✔	✔		Immortalized human keratinocytes (HaCaT) Human lung epithelial carcinoma cells (A549)

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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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Reference	Exposure: Aerosol (A), Extracts (X), E-liquid (L)	Humectant Only as an Exposure	Nicotine Only as an Exposure	Combustible Tobacco Cigarette Smoke as a Control	Compares Flavors	Cell or Tissue Type Used and Comments
Farsalinos et al., 2013	A	✔	✔	✔	✔	Monolayer-cultured cardiomyoblast cells (H9c2) Reason provided for cell selection is the better culture stability and reproducibility than human cardiomyocytes.
Husari et al., 2016	X			✔		Human lung epithelial carcinoma cells (A549)
Leigh et al., 2016	A	✔	✔	✔	✔	Human lung mucoepidermoid cells (NCI-H292 cell line)
Lerner et al., 2015	A	✔	✔	✔	✔	Human bronchial airway epithelial cells (H292) HFL1
Lerner et al., 2016	A					HFL1
Misra et al., 2014	X		✔	✔		Human lung epithelial carcinoma cells (A549)
Neilson et al., 2015	A			✔		EpiAirway™: a human 3D airway tissue model Fully differentiated in vitro reconstructs of primary human tracheobronchial epithelium. Cultures express mucus-producing

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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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					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., 2013	L		✔	✔	Mouse BALB/3T3 fibroblasts
Sancilio et al., 2016	L	✔			HGF Cells were 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 al., 2017	L	✔			HGF
Scheffler et al., 2015a	A	✔	✔		Primary NHBE cells Human lung epithelial carcinoma cells (A549) Immortalized primary NHBE cell line (CL-1548)

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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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Reference	Exposure: Aerosol (A), Extracts (X), E-liquid (L)	Humectant Only as an Exposure	Nicotine Only as an Exposure	Combustible Tobacco Cigarette Smoke as a Control	Compares Flavors	Cell or Tissue Type Used and Comments
Scheffler et al., 2015b	A	✔	✔	✔		Primary NHBE cells 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., 2016	L				✔	Spheroidal cultures of oropharyngeal mucosa Freshly isolated specimens were 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 et al., 2014	L	✔	✔		✔	Clonetics® HPdLF Fibroblasts are considered to be mesenchymal stem cells because of their self-renewing and multipotent character.

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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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Wu et al., 2014	L		✔			Normal hTBE cells from young, healthy, non-smoking organ donors
Yu et al., 2016	X		✔			Spontaneously transformed immortal keratinocyte (HaCaT) HNSCC cell lines: HN30 and UMSCC10B The HN30 and UMSCC10B cell 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.

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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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TABLE D-2 Summary of Test Agents, Cell or Tissue Type Used, and Assays Employed in In Vitro Studies of E-Cigarettes Assessing Cytotoxicity

Reference	Test Agent(s)	Cell or Tissue Type Used	Dose and Time Course	Assay Employed
Aufderheide and Emura, 2017	Mainstream combustible tobacco cigarette smoke from reference 3R4F cigarettes (University of Kentucky) E-liquid aerosol (Tennessee cured flavor, no nicotine, Johnsons Creek, Hartland, WI)	Immortalized primary NHBE cell line CL-1548	Samples taken after 0, 4, 6, and 8 smoke/aerosol exposure repetitions and analyzed microscopically after histopathological preparation of the cultures.	Histopathology
Bahl et al., 2012	35 different flavors	hESC mNSC hPF	6 concentrations: 0.001%, 0.01%, 0.03%, 0.1%, 0.3%, and 1% Incubation at 37ºC, 5% CO₂, and 95% relative humidity for 48 hours	MTT assay
Barber et al., 2016	Combustible tobacco cigarette smoke extracts from Marlboro 100 cigarettes (16 mg tar, 1.2 mg/ml nicotine, Philip Morris) E-cigarette aerosol extract from NJoy OneJoy device, traditional flavor with 1.2% (12 mg/ml) or 1.8% (18 mg/ml) nicotine and eGo (OKC Vapes), desert sands flavor with 0, 12, or 18 mg/ml nicotine	HUVECs	48-hour exposure to the extracts	Endothelial cell viability, density and metabolic activity after exposure to mainstream and sidestream tobacco smoke extracts, e-aerosol extracts, and pure nicotine Activation/deposition of complement proteins onto endothelial cells was quantified as a means to monitor the progression of innate immune responses

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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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	E-cigarette aerosol extract from NJoy OneJoy device, traditional flavor with 1.2% (12 mg/ml) or 1.8% (18 mg/ml) nicotine and eGo (OKC Vapes), desert sands flavor with 0, 12, or 18 mg/ml nicotine			Activation/deposition of complement proteins onto endothelial cells was quantified as a means to monitor the progression of innate immune responses
Behar et al., 2014	10 cinnamon-flavored e-cigarette refill liquids from online vendors; various concentrations of nicotine, cinnamon flavoring, and percentages of PG and/or glycerol Cinnamaldehyde and 2-MOCA	hPF hESC	48 hours	MTT assay

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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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Reference	Test Agent(s)	Cell or Tissue Type Used	Dose and Time Course	Assay Employed
Behar et al., 2016	39 e-cigarette refill fluids purchased from online vendors Laboratory-made refill fluids Aerosols produced from the refill fluids (produced with unused cartomizer or tank using smoking machine) Cinnamon Ceylon aerosol produced from cartomizer-style e-cigarette	hPF Human lung epithelial carcinoma cells (A549) hESC	The Vea cartomizer device and unfilled cartomizers (Johnson Creek, Hartland, Wisconsin) operated at 2.9 V, 2.1 Ω, and 4 W. An Innokin iTaste MVP 3.0 battery with variable voltage and wattage and Innokin iClear 16D bottom dual-coil clearomizers (tanks) were operated at 3 V, 2.1 Ω, and 4.2 W or at 5 V, 2.1 Ω, and 11.9 W. 2 ml of fluid for each sample. Puff duration was 4.3 seconds. Time course varied by assay and cell type.	GC/MS MTT assay Nuclei stained with DAPI Live cell imaging assay Alkaline comet assay
Bharadwaja et al., 2017	E-cigarette liquid (NJOY brand containing glycerol, PG, 10 mg/ml nicotine, flavoring chemicals) Soluble e-liquid aerosol produced from the e-cigarette liquid	Stress-specific recombinant bacterial cells: E. coli-RecA, E. coli-SodA, E. coli-CopA, and E. coli-DMO1 (as biosensors)	Cells were exposed to various concentrations of e-liquid and soluble e-liquid aerosol.	UV-Vis spectroscopy Bioluminescence assay DNA fragmentation assay

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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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	Soluble e-liquid aerosol produced from the e-cigarette liquid
Cervellati et al., 2014	E-cigarette aerosol (e-cigarette Mini Touch T-Fumo T-TEX with e-liquid in balsamic flavors with or without nicotine, Cloudsmoke, Terna Trade) E-cigarette aerosol with humectants only (no additives such as flavors or nicotine). Combustible tobacco cigarette smoke (United Kingdom research cigarette, 12 mg tar, 1.1 mg nicotine)	Immortalized human keratinocytes (HaCaT) Human lung epithelial carcinoma cells (A549)	HaCaT cells were exposed to fresh combustible tobacco cigarette smoke in an exposure system that generated smoke by burning one UK research cigarette, and to e-cigarette mixtures using a vacuum pump to draw air through the cigarette and leading the smoke stream over the cell cultures for 50 minutes.	Ultrastructural morphology Trypan Blue exclusion test LDH assay Pro-inflammatory cytokines were measured in culture medium by the Bio-Plex cytokine assay kit

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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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Reference	Test Agent(s)	Cell or Tissue Type Used	Dose and Time Course	Assay Employed
Farsalinos et al., 2013	Combustible tobacco cigarette with 0.8 mg nicotine, 10 mg tar, and 10 mg carbon monoxide yields (Marlboro, Philip Morris Italia S.r.l., Rome, Italy) 20 commercially available e-liquids (17 tobacco flavored, 3 sweet or fruit flavored), with 6–24 mg/ml nicotine, manufactured or distributed by 5 different companies	Monolayer-cultured cardiomyoblast cells (H9c2)	Two sets of experiments were performed; one using regular voltage and a second using higher voltage, for e-cigarette aerosol production. The medium was aspirated and replaced by medium containing the combustible tobacco smoke and e-cigarette liquid extracts in one undiluted (100%) and 4 diluted samples (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%.	MTT assay

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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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Husari et al., 2016

E-cigarette aerosol was generated using pre-filled V4L CoolCart cartomizer cartridges (strawberry flavor, 3.5 Ω, 18 mg/ml labeled nicotine) and 4.2-V battery (Vapor Titan Soft Touch)
Reference 3R4F combustible tobacco cigarettes (University of Kentucky, 9.4 mg tar, 0.726 mg nicotine per cigarette)

Human lung epithelial carcinoma cells (A549)

Exposure to e-cigarette aerosol or combustible tobacco cigarette smoke extract was initiated 24 hours post-seeding by diluting smoke extract in complete media to the desired final concentration (e.g., 0.5, 1, 2, 4, 8 mg/ml). Images were taken 24 hours post-treatment.

Trypan blue exclusion assay

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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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Reference	Test Agent(s)	Cell or Tissue Type Used	Dose and Time Course	Assay Employed
Leigh et al., 2016	6 types of commercially available e-cigarettes (purchased from gas stations, convenience stores, online retailers, and local vape shops in Buffalo, New York, Daly City, California, and online) eGo tank system (Vision Spinner) e-cigarette device with battery output voltage fixed at 3.3 V and refill solutions in tobacco, piña colada, menthol, coffee, and strawberry flavors (purchased from a local vape shop in Buffalo, New York) Reference 3R4F combustible tobacco cigarettes (University of Kentucky)	Human lung mucoepidermoid cells (NCI-H292 cell line)	Air–liquid interface (ALI) exposure Health Canada Intense method, using the following conditions: 3-second puff duration, every 30 seconds, with a 55-ml puff volume, implemented continuously for 30 minutes, and resulting in a total of 55 puffs. Air exposures (control) generated using smoking machines were run during each experiment. Reference 3R4F combustible tobacco cigarettes (comparison) were smoked using the same method as for the e-cigarette products.	Neutral red uptake assay Trypan blue assay Cytokine release was measured as an indicator of cell inflammatory response

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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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5 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.

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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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Reference	Test Agent(s)	Cell or Tissue Type Used	Dose and Time Course	Assay Employed
Lerner et al., 2015	Refillable pen-style e-cigarette device (eGo Vision Spinner, China) and compatible clearomizer chamber (Anyvape, China) with 2.2-Ω heating element blu e-cigarettes (classic tobacco flavor containing 16 mg nicotine) CSE from a research-grade combustible tobacco cigarette	Human bronchial airway epithelial cells (H292) HFL1	H292: blu e-cigarette aerosol using a CSM-SSM machine (CH-Technologies Inc.) was drawn into the chamber every 30 seconds with a 4-second pulse for different time durations of 5, 10, and 15 minutes. HFL1 was treated with the following e-liquids: PG, glycerol, Vape Dudes (classic 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.	HFL1: Violet B 405-nm laser and 440/40 bandpass filter to detect increases in cellular fluorescence FlowJo V.10 for data compilation

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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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Lerner et al., 2016	blu classic tobacco e-cigarette with 16 mg nicotine (Lorillard, Greensboro, NC)	HFL1	E-cigarette puffs were regulated with 4-second puffs every 30 seconds for various sessions (5, 10, 15, or 20 minutes).	Mitochondria superoxide staining Mitochondria membrane potential staining DNA fragmentation assay IL-8 and IL-6 cytokine secretion
Misra et al., 2014	blu e-cigarettes containing glycerol-based e-liquids, with and without nicotine and two market flavors (classic tobacco and magnificent menthol) Reference 3R4F, 1R5F, and Marlboro gold combustible tobacco cigarettes	Human lung epithelial carcinoma cells (A549)	0–20 mg/ml	Neutral red uptake IL-8 release

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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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Reference	Test Agent(s)	Cell or Tissue Type Used	Dose and Time Course	Assay Employed
Neilson et al., 2015	NJOY bold (4.5% labeled nicotine) and NJOY menthol (3.0% labeled nicotine) Reference 3R4F combustible tobacco cigarettes	EpiAirway™: a human 3D airway tissue model	A VITROCELL VC 01 Smoking Robot (VC1/110613) and a 12/6 CF stainless-steel exposure module (VITROCELL Systems GmbH) Reference 3R4F cigarettes were 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.	MTT assay Integrity of the airway epithelium tight junctions was measured by TEER8 conducted according to the MatTek Corporation’s standard protocol.
Romagna et al., 2013	Combustible tobacco cigarette smoke extract 21 different e-cigarette liquids. Composition of e-liquids was (w/w) 46.17% PG USP, 44.92% glycerol USP, 8.11% water, 0.8% nicotine USP, and < 0.5% flavorings	Mouse BALB/3T3 fibroblasts	E-cigarette aerosol and combustible tobacco cigarette smoke extracts simulating e-cigarette use added to culture medium. 100%, 50%, 25%, 12.5%, 6.25%, 3.12% for 24 hours at 37ºC	MTT assay

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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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	glycerol USP, 8.11% water, 0.8% nicotine USP, and < 0.5% flavorings		3.12% for 24 hours at 37ºC
Sancilio et al., 2016	Two different cartridge solutions (nicotine content [w/v] 0 and 24 mg/ml) from Halo Company (Pompton Plains, NJ, USA) containing PG, glycerol, and natural and artificial flavorings (concentrations not provided by the manufacturer), diluted from 4.8 to 48 times	HGF	HGFs treated with pre-warmed fluids with or without nicotine. Cell medium was replaced every 24 hours. In the vaped samples, 1.5 ml of the cartridge solution was put in the cartomizer, warmed for 1 minute before the dilution and then harvested with a syringe from the cartomizer to a vial.	MTT assay Apoptosis Increase in green fluorescence for reactive oxygen species production Bax expression (pro-apoptotic protein)

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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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Reference	Test Agent(s)	Cell or Tissue Type Used	Dose and Time Course	Assay Employed
Sancilio et al., 2017	Two different cartridge solutions (nicotine content [w/v] 0 and 24 mg/ml) containing PG, glycerol, and natural and artificial flavorings (concentrations not provided by the manufacturer), diluted 24 times with DMEM	HGF	HGFs treated with 1 mg/ml nicotine (obtained by diluting 24 times the 24 mg/ml nicotine-containing fluid), warmed and not warmed before administration HGFs treated with the fluid without nicotine diluted 24 times, warmed and not warmed before administration HGFs also left untreated	TEM LDH assay Lysosome compartment analysis Human collagen type I concentration in supernatants was assayed using an ELISA Western blot for LC3 expression in HGF
Scheffler et al., 2015a	Reevo Mini-S e-cigarette (In-Smoke, Winnenden, Germany) with a 3.3-V/900-mAh battery and 2.2-Ω resistance with e-liquids purchased from Johnsons Creek (Hartland, WI, USA) in Tennessee cured flavor (75% PG USP, 25%	Primary NHBE cells Human lung epithelial carcinoma cells (A549) Immortalized primary NHBE cell line (CL-1548)	The e-cigarette was connected to the piston pump of a smoking robot and 200 puffs were taken with a puff volume of 35 ml, puff duration of 2 seconds, blow-out time of 7 seconds, and an interpuff interval of 10 seconds.	ROS-Glo™ H₂O₂ Assay (Promega, Madison, WI, USA) for oxidative stress CellTiter-Blue® Assay (Promega, Madison, WI, USA) for cell viability

Page 723 Cite

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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glycerol USP, 0.0% and 2.4% nicotine USP). Other ingredients listed on the bottle include deionized water, natural flavors, artificial flavor, and USP-grade citric acid (as a preservative).
Reference 3R4F combustible tobacco cigarettes (Kentucky) with a standard cellulose acetate filter tip

For combustible tobacco cigarette smoke exposure, 10 K3R4F cigarettes were each puffed by the smoking robot using the same parameters as described for the e-cigarette.

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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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Reference	Test Agent(s)	Cell or Tissue Type Used	Dose and Time Course	Assay Employed
Scheffler et al., 2015b	Aerosols from two e-cigarette liquids purchased from Johnsons Creek (Hartland, WI, USA) in Tennessee cured flavor (0% and 2.4% nicotine). Liquids contained USP-grade PG, USP-grade glycerol, deionized water, natural and artificial flavors, USP-grade nicotine, and USP-grade citric acid. Aerosols from humectants (glycerol and PG) obtained from Alfa Aesar (Karlsruhe, Germany), with a purity of 99.5%. Combustible tobacco cigarette smoke from 10 reference K3R4F combustible tobacco cigarettes (Lexington, Kentucky) with a standard cellulose acetate filter tip.	Primary NHBE cells	The e-cigarette was connected to the piston pump of a smoking robot and 200 puffs were taken with a puff volume of 35 ml, puff duration of 2 seconds, blow-out time of 7 seconds. For combustible tobacco cigarette smoke exposure, 10 K3R4F cigarettes were smoked by the smoking robot using the same parameters as described for the e-cigarette. Each cigarette was puffed 6 times. Cultures were analyzed 24 hours after exposure.	ROS-Glo™ H₂O₂ Assay (Promega, Madison, WI, USA) for oxidative stress CellTiter-Blue® Assay (Promega, Madison, WI, USA) for cell viability

Page 725 Cite

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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Welz et al., 2016	E-liquids in three flavors (apple, cherry, and tobacco) with a base mixture of 80% PG, 10% glycerol, and 10% water and 12 mg/ml nicotine	Spheroidal cultures of oropharyngeal mucosa	24-hour one-time incubation 2.5-hour incubation on 5 sequential days	MTT assay
Willershausen et al., 2014	E-liquids (eSmokerShop, GmbH, Hannover, Germany) in hazelnut, lime, and menthol flavors with 20–22 mg/ml nicotine in a PG-base	Clonetics® HPdLF	Up to 96-hour incubation depending on assay	PrestoBlue Cell Viability Assay ATP detection Cell visualization Migration assay
Wu et al., 2014	Tobacco-flavored e-liquid at various concentrations (0, 0.01, 0.1, 0.3% v/v) without nicotine or with 18 mg/ml of nicotine (InnoVapor LLC., Boise, ID)	Normal hTBE cells from young, healthy, non-smoking organ donors	24- and 48-hour exposures. The final nicotine concentrations were within the serum nicotine range of e-cigarette users.	Pro-inflammatory cytokines HRV-16 infection in e-liquid-–exposed normal hTBE cells LDH IL-6 levels by ELISA Taqman quantitative real-time RT-PCR to detect HRV RNA and human SPLUNC1 mRNA

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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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Reference	Test Agent(s)	Cell or Tissue Type Used	Dose and Time Course	Assay Employed
Yu et al., 2016	V2 (red American tobacco flavor) and VaporFi (classic tobacco flavor) e-cigarette brands in a 70% PG/30% glycerol base with 0.0% and 1.2% nicotine	Spontaneously transformed immortal keratinocyte (HaCaT) HNSCC cell lines: HN30 and UMSCC10B	HaCaT cells were treated for 8 weeks with 1% v/v extract UMSCC10B and HN30 were each treated for 1 week with 1% v/v extract. HaCaT cells were treated for 10 days at 0.5%, 1.0%, and 2.0% v/v aerosolized e-cigarette liquid. HaCaT cells were treated for 10 days, and UMSCC10B and HN30 for 12 days prior to colony counting.	Neutral comet assay γ-H2AX immunostaining Cell cycle changes by flow cytometry Trypan Blue staining Clonogenic survival Annexin V apoptotic assay

NOTE: 2-MOCA = 2-methoxycinnamaldehyde; CSE = cigarette smoke extract; DAPI = 4’,6-diamidino-2-phenylindole; GC/MS = gas chromatography/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.

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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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TABLE D-3 Summary of Results from In Vitro Studies of E-Cigarettes Assessing Cytotoxicity

Reference	Results and Observations
Aufderheide and Emura, 2017	Cultures exposed to both mainstream combustible tobacco cigarette smoke and e-liquid aerosol showed a clear reduction in mucus production and cilia bearing, but the effect was weaker for the aerosol than for the smoke.
Bahl et al., 2012	The MTT assay showed effects of refill solutions on cell survival that 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., 2016	Most of the exposure conditions resulted in significant effects on cell 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.
Barber et al., 2016	Results showed significant increase in the deposition of C1q and C5b9, and in C3b to a lesser extent. There were no changes in C4d.
Behar et al., 2014	The study established NOAELs of 0.03% for hESC and 0.01% for hPF; hESC was more sensitive than hPF.
Behar et al., 2014	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.

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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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Reference	Results and Observations
Behar et al., 2016	In the 48-hour MTT assay, hESC (embryonic stem cells) were more 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).
Behar et al., 2016	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 al., 2017	Following exposure to e-liquids and e-cigarette aerosol at various 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.
Bharadwaja et al., 2017	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.

Page 729 Cite

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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Reference	Results and Observations
Cervellati et al., 2014	Exposure to e-cigarette aerosol with humectants only (no flavorings 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 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 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.

Page 730 Cite

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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Reference	Results and Observations
Farsalinos et al., 2013	Of 20 samples tested, 4 samples exhibited a cytotoxic effect in the 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%–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 rates 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., 2016	Combustible tobacco cigarette smoke total particulate matter extract 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.

Page 731 Cite

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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Reference	Results and Observations
Leigh et al., 2016	Effects of e-cigarette aerosols on toxicity to bronchial epithelial cells 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-1β, CXCL1, and CXCL2, while exposure to aerosol with 24 mg/ml nicotine resulted in significantly increased IL-6. IL-1β 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-1β, 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.

Page 732 Cite

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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Reference	Results and Observations
Lerner et al., 2015	Fibroblasts cultured with e-liquid or combustible tobacco CSE 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 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 (% viability in means ± SD; 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, 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 ± 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 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.

Page 733 Cite

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.

×

Reference	Results and Observations
Lerner et al., 2016	Results showed a small but significant reduction in the amount of 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., 2014	No cytotoxicity was observed for any of the e-liquids tested up to their respective highest sample doses.
Misra et al., 2014	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.

Page 734 Cite

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.

×

Reference	Results and Observations
Neilson et al., 2015	Tissue cell viability following combustible tobacco cigarette smoke 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 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 controls.
Neilson et al., 2015	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 al., 2013	From the 21 samples examined, only the coffee-flavored e-liquid exhibited a cytotoxic effect, and this only at the highest 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.

Page 735 Cite

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.

×

Reference	Results and Observations
Sancilio et al., 2016	E-liquid exposure resulted in reduced metabolic activity in a time- 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-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 onefold.
Sancilio et al., 2017	E-liquids with nicotine exerted cytotoxicity as demonstrated by the 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.

Page 736 Cite

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.

×

Reference	Results and Observations
Scheffler et al., 2015a	Primary NHBE48 cells were the most sensitive, responding to 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 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 exposure to nicotine-containing and nicotine-free aerosol.
Scheffler et al., 2015a	The oxidative stress level is elevated in CL-1548 cells compared to A549 cells, but lower than those of primary NHBE48 cells.
Scheffler et al., 2015b	The authors found toxicological effects of e-cigarette aerosol and the humectant-only substances, whereas the nicotine concentration did not have an effect on 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., 2016	Both fruit- and tobacco-flavored extracts were cytotoxic to 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.

Page 737 Cite

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.

×

Reference	Results and Observations
Willershausen et al., 2014	Starting at 24 hours, the highest reduction in the proliferation was 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., 2014	Within the physiological nicotine range, e-liquid exposure did not 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.

Page 738 Cite

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.

×

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-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NHBE = normal human bronchial epithelial; NOAEL = no observed adverse effect level; PG = propylene glycol.

Page 739 Cite

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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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(Supplement 2):ii94–ii102. http://tobaccocontrol.bmj.com/content/25/Suppl_2/ii94 (accessed February 5, 2018).

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(Supplement 2):ii81–ii87. http://tobaccocontrol.bmj.com/content/25/Suppl_2/ii81 (accessed February 5, 2018).

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. https://doi.org/10.1371/journal.pone.0116732 (accessed February 5, 2018).

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.

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 replacement therapy products: E-liquids, extracts and collected aerosols. International Journal of Environmental Research & Public Health 11(11):11325–11347.

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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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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. https://doi.org/10.1186/1746-160X-10-39 (accessed February 5, 2018).

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. https://doi.org/10.1371/journal.pone.0108342 (accessed February 5, 2018).

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.