Pulmonary effects of e-liquid flavors: a systematic review

Figures & data

Figure 1. Different generations of EC devices and their main differences. The figure was produced with Biorender.com.

Table 1. Flavors in e-liquid and some of the main flavoring chemicals they are composed of.

Flavoring chemical	Common flavors with chemical agents	References
Cinnamaldehyde, 2-methoxycinnamaldehyde	Cinnamon	(Behar et al., 2014; Gerloff et al., 2017)
3,4-Dimethoxybenzaldehyde	Cherry	(Kavvalakis et al., 2015)
2,5-dimethylpyrazine	Chocolate	(Kavvalakis et al., 2015)
Maltol, ethyl maltol	Caramel, Vanilla	(Gerloff et al., 2017; Tierney et al.,2016)
Menthol, Menthone	Mint	(Berkelhamer et al., 2019)
p-Anisaldehyde	Anise	(Behar et al., 2018a; Berkelhamer et al., 2019; Noël et al. 2020b)
Benzaldehyde	Almond	(Behar et al., 2018a; Berkelhamer et al., 2019)
Vanillin, O-vanillin	Vanilla	(Behar et al., 2014; Gerloff et al., 2017; Muthumalage et al. 2018)
Hexyl acetate, 3-Hexen-1-ol	Apple	(Kerasioti et al., 2020; Kim et al., 2018)
Isoamyl acetate	Banana	(Fetterman et al., 2018)
Methyl cinnamate	Balsamic, Strawberry	(Berkelhamer et al., 2019; Leigh et al.,2016)
Methyl anthranilate	Berry	(Tierney et al. 2016)
Other flavoring agents less known: Damascenone (α or β), 3-methyl-1,2- cyclopentanedione, acetamide, linalool, terpineol, citral, corylon, anisaldehyde, trimethylpyrazine, eugenol, benzaldehyde, limonene	Miscellaneous	(Gerloff et al., 2017; Tierney et al., 2016)

Figure 2. PRISMA flowchart. Articles were retrieved from HDAS and Scopus databases. Primary research articles that met the selection criteria were included irrespective of the year of publication. The figure was created with Lucidchart.com.

Table 2. In vitro studies where toxicological effects of flavored e-liquids were investigated with primary epithelial cells alone or together with immortalized normal pulmonary cells.

Cell type	Flavors or flavoring chemicals investigated	Exposure (Time & flavor dose)	E-liquid Nicotine Dose	Nicotine Control	Main findings	Reference
Primary human bronchial epithelial (HBECs), immortalized bronchial epithelial cells (BEAS-2B)	Hot Cinnamon Candies, ‘Sinicide’, Kola (Each containing cinnamaldehyde) Cinnamaldehyde alone at 5 different concentrations (0.05, 0.25, 0.5 & 5 mM).	0, 20, 40, 60, 80, 100, 120 min exposure to aerosol at ALI	No nicotine	N/A	Since aerosol transiently suppressed ciliary frequency beat (CBF), and cinnamaldehyde recapitulated and other differential toxic effects in both cell lines following metabolic and glycolysis assays using a Seahorse XFe21 extracellular flux analyzer. Cinnamaldehyde impaired mitochondrial respiration and glycolysis dose-dependently in BEAS-2B. Reduction of Intracellular ATP levels dose-dependently in differentiated hBE and BEAS-2B cells.	(Clapp et al. 2019)
Human bronchial epithelial cells (hBE)	Apple (3 different variants)	Puff duration of 2.6 s, 50 × 3 s puffs with 5 s in between were bubbled through 10 mL of culture medium to create EC extract. Cells were exposed to 100% of this media.	0, 18 mg/ mL	Nicotine alone controls present	Each apple flavor increased necrosis and apoptosis because of flavor effects. Each apple flavor reduced the secretion of TNF-α, IL-6, IP-10, MIP-1α, and MIP-1β because of flavor-effects. Nicotine alone induced a decrease in pro-inflammatory cytokines (IP-10 & MIP-1β) similar to cigarette smoke extract (CSE) and increased % of macrophages efferocytosing hBE cells but had no effect when in combination with e-liquid.	(Ween et al. 2020a)
Primary human 24-h alveolar macrophages, neutrophils, and natural killer (NK) cells.	Kola Hot Cinnamon Candies Menthol Banana pudding Menthol tobacco Banana ‘Sinicide’	Adherent alveolar macrophages were challenged in triplicate with cell culture medium containing 1, 0.5, or 0.25% flavored e-liquid for 1 h	0, 0.6, 1.2, 1.8, and 2.4 mg/ml	No nicotine alone controls present	Hot Cinnamon Candies, and Kola, and each decreased phagocytosis and viability of macrophages, neutrophils, and NK cells. Hot Cinnamon Candies, and Kola mediated suppression of alveolar macrophage phagocytosis was reversed by coexposure of cinnamaldehyde. No nicotine dose-dependent effects were observed. No nicotine x flavor interaction observed.	(Clapp et al. 2017)
Primary human bronchial epithelial cells	Diacetyl (DA)	1-h ALI aerosol exposure to 12, 25, and 50 mM of diacetyl.	No nicotine	N/A	LDH increased significantly with 50 mM DA- compared to PBS, 12- and 25-mM DA-exposed cultures. Transient decrease in trans-epithelial electrical resistance in all DA-exposed cultures. At 25 and 50 mM, airway basal cell injury with keratin five ubiquitination and decreased p63 expression were observed. The ubiquitin-proteasome-pathway partially mediates airway basal cell repair after acute DA exposure.	(McGraw et al., 2020)
16HBE14o- airway epithelial cell line (16HBE), Normal human bronchial epithelial cells (NHBE), THP-1 differentiated macrophages & primary alveolar macrophages (AM).	Cappuccino Chocolate Peppermint	E-cigarette vapor extracts (EVE) generated using 50 x 3s puffs and exposed to cells for 24-h	18 mg/mL (No nicotine-free e-liquids)	Nicotine alone controls present	Cappuccino, Chocolate and Peppermint caused cellular cytotoxicity by inducing morphological changes and cell shedding in 16HBE. All 10 flavors induced significant LDH release (chocolate highest and nicotine alone significantly higher) in 16HBE. Chocolate > cappuccino >peppermint > banana induced apoptosis. Nicotine and PG:VG did not induce apoptosis in 16HBE and primary HBE, the other cells were not specified). Chocolate flavor most cytotoxic following an LDH assay in NHBE in inducing necrosis and apoptosis. Nicotine showed significant toxicity but lower than chocolate, whereas PG:VG showed no effect. Mango, Tobacco, Banana and Chocolate caused decreased macrophage phagocytic capacity. PG:VG and nicotine alone showed a significant effect on efferocytosis. Chocolate > Banana > nicotine induced increase in IL-8 secretion by NHBE. Decrease in IL-1β by all flavors as well as nicotine. Effects are linked to concentration and benzene-ring containing chemicals.	(Ween et al. 2020b)
Human bronchial epithelial cells of COPD models, immortalized 16HBE cells	Virginia Tobacco (Flavor from tobacco extract) Menthol	Intense puffing regime of 55 mL over 4 s, with 30s rest interval) for 30 min at ALI	5 mg/ml	No nicotine alone control	Virginia tobacco-induced significantly higher LDH levels compared to menthol in the COPD cells Menthol in 16HBE induced a significantly lower LDH release when compared to air-only control. No difference in DNA damage in either cell line in Virginia tobacco or menthol when compared to cigarette controls. In COPD cells, cigarette, and Virginia tobacco-induced a significant decrease in IL-8 production. No alteration in IL-6 release compared to air-only controls. In 16HBE, IL-8 production was significantly higher in all groups compared to air-only controls. Tobacco significantly decreased IL-6 release compared to cigarette.	(O’Farrell et al. 2021)

Table 3. In vitro studies where toxicological effects of flavored e-liquids were carried out on immortalized pulmonary normal epithelial cells alone or together with carcinoma pulmonary cells.

Cell type	Flavors or flavoring chemicals investigated	Exposure (Time & flavor dose)	E-liquid Nicotine Dose	Nicotine Control	Main findings	Reference
Human bronchial epithelial cell line (16HBE14o)	2,5-dimethylpyrazine (chocolate, nutty flavor) Damascenone (apple, citrus, wine-like) Linalool (floral, spice) α-ionone (fruity, raspberry) Ethyl maltol (caramel) Furaneol (strawberry, sweet) Vanillin (vanilla)	24-h direct exposure (flavoring chemical)	No nicotine	N/A	2,5-Dimethylpyrazine reduces the physiological response to signaling molecules important in aspects of airway epithelial cell innate immunity. 2,5-Dimethylpyrazine increases airway epithelial ion conductance. 2,5-Dimethylpyrazine activates apical ion efflux via cystic fibrosis transmembrane conductance regulator (CFTR). Vanillin and 2,5-Dimethylpyrazine significantly altered airway epithelial cellular physiology indicative of cellular signaling events. Linalool, α-ionone, damascenone, ethyl maltol, and furaneol, no significant effects?	(Sherwood and Boitano, 2016)
Immortalized human bronchial epithelial cells (16-HBE, BEAS-2B)	Cool Cucumber Mango Strawberry coconut Classic Menthol Caffe Latte	66 puffs during 22 minutes with a three-second puff duration at 1.6 L/min flow rate and an inter puff interval of approximately 17s.	50 mg/mL nicotine	No nicotine control	Cool cucumber, classic menthol, caffe latte induced pro-inflammatory biomarkers (IL-1ß, IFN-γ, IL-17, etc.) depending on the cell line as 16-HBE and BEAS-2B present specific physiological differences. It is unclear whether these effects are in part influenced by nicotine content due to the lack of nicotine control samples. Classic Menthol induced the most significant mitochondrial ROS production in 16-HBE. Prostaglandin production was induced in BEAS-2B, not in 16-HBE, by strawberry coconut. IL-8 production was induced in 16-HBE, but not in BEAS-2B, by cool cucumber, classic menthol, strawberry coconut, and caffe latte.	(Muthumalage et al. 2019)
Normal human bronchial epithelial (NHBE) cells	Flavoring chemicals diacetyl and 2,3-pentanedione (Concentrations of diacetyl and 2,3-pentanedione were not specified)	24-h chemical exposure after media dilution in an ALI model	No nicotine	N/A	Cytoskeletal and cilia processes among common genes (142 genes) were perturbed by both diacetyl and 2,3-pentanedione. The expression of multiple genes involved in cilia biogenesis was significantly downregulated by diacetyl and 2,3-pentanedione. The number of ciliated cells significantly decreased.	(Park et al., 2019)
BEAS-2B and human pulmonary fibroblast (HPF)	Ethyl maltol Menthol	48-h chemical (diluted liquid) direct exposure	No nicotine	N/A	Both flavor chemicals were highly cytotoxic following a MTT assay at concentrations 30 (menthol) and 100 times (ethyl maltol) lower than the highest concentrations in the refill fluids. BEAS-2B cells (IC50 = 0.15 mg/ml) were more sensitive to ethyl maltol than hPF (IC50 = 0.28 mg/ml). No difference in sensitivity to menthol between HPF and BEAS-2B.	(Omaiye et al. 2019)
BEAS-2B	Menthol Virginia Tobacco	30 min x 3 (Each one after 12-h) aerosol exposure	5% nicotine (Specific mg/ml of nicotine not specified)	No nicotine control	Menthol- mitochondrial effects: proton leakage, a decrease in coupling efficiency, and a reduction of complex I, II, and IV 24-h post-menthol exposure reduction of basal respiration, maximal respiration, spare capacity, and complex I. Tobacco had no significant effects on mitochondrial respiration, but immediately post final exposure increased complex I, IV, and V. Whether this effect is synergistic with nicotine was unclear.	(Lamb, Muthumalage, and Rahman 2020)
Human pulmonary fibroblasts (HPF)	Cinnamon Ceylon	48-h e-liquid direct exposure to 1% and 0.3% doses.	Different concentrations of nicotine, but not specified.	No nicotine control	Nicotine concentration did not correlate with cytotoxicity in MTT assays. Cinnamon Ceylon cytotoxicity effect following MTT assay was dose-dependent. Cinnamaldehyde and 2-methoxycinnamaldehyde (2-MOCA) were the major components of cinnamon flavors. Cinnamaldehyde and 2-MOCA standards were highly cytotoxic in MTT assay.	(Behar et al. 2014b)
Human lung fibroblasts (HPF)	Bubblegum Butterscotch Caramel Butterfinger Menthol Artic Wisconsin frost Domestic JC Original French vanilla Vanilla Tahiti Tennessee cured Island Valencia Mint chocolate Swiss Dark Caramel Espresso Mercado Simply strawberry Arctic Menthol Summer Peach Black Cherry Chocolate truffle Cinnamon Ceylon	48-h direct e-liquid exposure to 0.001%, 0.01%, 0.03%, 0.1%, 0.3%, and 1% v/v.	0 to 24 mg/ml	No nicotine alone control	Most significant cytotoxicity following MTT assay was observed with cinnamon and menthol flavors which did not contain nicotine. Levels of nicotine were not correlated with the degree of MTT cytotoxicity. The humectants vegetable glycerin (VG) and propylene glycerin (PG) were non-cytotoxic for the hPF.	(Bahl et al. 2012)
Human airway epithelial cells (H292) & human lung fibroblast cells (HFL-1 cell line)	Tobacco Cinnamon roll Grape	30 s interval with a 4 s pulse for different time durations 5, 10, and 15 minutes at ALI	0, 6, 16, 18 and 24 mg	No nicotine alone control present	Tobacco and cinnamon roll-flavored e-liquid increased the secretion of inflammatory cytokines IL-6 and IL-8 in H292 and HFL-1. The addition of nicotine gave a striking, dose-dependent increase only in IL-8 secretion in HFL-1.	(Lerner et al. 2015)
Normal human bronchial epithelial (NHBE) cells	Menthol Tobacco Vanilla Blueberry	24-h direct e-liquid exposure	Different dilutions (0.03125%, 0.0625%, 0.125%, 0.25%, 0.5%, 1%, 2% & 3% of 24 mg/ml e-liquids.	No Nicotine control	Only menthol elicited dose-dependent cytotoxic effects on activating G0/G1cycle arrest, decreasing glutathione concentration, activation of caspase 3/7 activity, increasing nuclear factor-kB (NF-kB), & increased mitochondrial mass. Tobacco, vanilla, and menthol decreased mitochondrial membrane potential dose-dependently. Glutathione content decreased after exposure to tobacco, blueberry, and vanilla, but only above 1%. No nicotine-free e-liquids were tested. Therefore, it is unclear whether these effects are due to flavors alone or a combination of flavor plus nicotine. Base liquids (PG/VG), with or without nicotine, and commercial, flavored, nicotine-containing e-liquids (CFs), had little or no effect on cell viability, cell count, mitochondrial mass, and potential and oxidative stress.	(Czekala et al. 2019)
Pulmonary fibroblasts (HFL-1)	A mixture of tobacco, coconut, vanilla, and cookie	24-h direct e-liquid exposure 0.1%, 0.25%, 0.5% & 1%.	3 mg/ml (No nicotine-free group included)	Nicotine alone control present at 0.25% and 0.5%.	A dose-dependent decrease in cell viability using the mixture of e-liquids. An increase in IL-8 release induced only by 0.25% flavor mix. Elevated senescence-associated beta-galactosidase (SA-β-gal) activity in a dose-response fashion. Transforming growth factor-β1 (TGF-β1) induced myofibroblast differentiation was inhibited by flavor mix, decreasing α-smooth muscle actin and fibronectin protein levels at only one concentration 0.5%. No significant nicotine effect was observed.	(Lucas et al. 2020)
Human bronchial epithelial cell lines (BEAS-2B, H292)	Crème brûlée-favor	5s duration every 30s for 1-h to aerosol at ALI	50 mg/mL	No nicotine control	Aerosol decreased cell viability (≥50%) of BEAS-2B cells alone and increased nitric oxide (NO) production (≥30%), as well as iNOS gene expression. In H292 cells, aerosol increased the production of reactive oxygen species (ROS). Aerosol dysregulated expression of several genes related to biotransformation, inflammation, and airway remodeling, including CYP1A1, IL-6, and MMP12 in both cell lines. Because nicotine-free e-liquids were not investigated, it is unclear whether nicotine has any synergistic effects with Crème brûlée-flavor.	(Pinkston et al. 2020)
BEAS-2B, Human cystic fibrosis cell line (IB3-1), and Calu3	Apple, Cherry, Tobacco, Strawberry	24-h exposure to condensates (50%, 25%, and 12.5% diluted with media)	0, 6, 12 and 18 mg/mL	No nicotine alone control	Cherry- similarly cytotoxic to the C38 and BEAS-2B In BEAS-2B, tobacco- also cytotoxic following cell viability at all dilutions tested. Strawberry reduced cell viability in BEAS-2B cells was significantly greater than apple, cherry, and tobacco CALU-3 showed no cytotoxic response to any flavor. There was no nicotine concentration-dependent effect.	(Leslie et al. 2017)
Human pulmonary fibroblasts (HPF), alveolar epithelial cells (A549)	Vanilla Mint/Menthol Butterscotch Caramel Butterfinger	E-fluid (0.001%, 0.01%, 0.03%, 0.1%, 0.3%, 1.0%) and aerosol exposures. For each batch of aerosol, 24 puffs were collected into 4 mL of culture medium	18 mg/ml	No nicotine control	Vanilla (creamy/buttery) and Butterscotch (creamy/buttery) were cytotoxic following MTT assay both as fluid and as aerosol. Mint/Menthol was cytotoxic following MTT assay only as e-liquid. Butterfinger (creamy/buttery) and caramel aerosol were the most toxic in a dose-dependent fashion of all flavors following MTT assays. It is unclear whether the effects observed are due to nicotine plus as there are no nicotine controls.	(Behar et al., 2018)

Table 4. In vitro studies where toxicological effects of flavored e-liquids were investigated in pulmonary carcinoma cells alone or together with primary pulmonary cells.

Cell type	Flavors or flavoring chemicals investigated	Exposure (Time & flavor dose)	E-liquid Nicotine Dose	Nicotine Control	Main findings	Reference
Human bronchial epithelial cells (H292)	Strawberry Tobacco Pina colada Menthol Coffee	3 s puff duration, every 30s, with a 55 mL puff volume. A period of 30 min was used as this was the minimum exposure at ALI.	0, 6, 12, 18, or 24 mg/mL of nicotine	Nicotine alone control present.	Strawberry flavor was most cytotoxic following neutral red assay. Exposure to each aerosol decreased metabolic activity and cell viability. Coffee increased the release of IL-6, CXCL1, and CXCL2. Strawberry increased the release of IL-1ß IL-10, CYCL1, CXCL2, and CXCL10. No significant differences were observed between the different concentrations of nicotine and air control for both metabolic activity and viability. Unflavored e-liquids significantly decreased in IL-1β, CXCL1, and CXCL2 in response to 18 mg/mL nicotine aerosol versus air controls. Unflavored e-liquids significantly increased IL-6 in response to 24 mg/mL nicotine aerosol versus air control. Metabolic activity was significantly decreased in cells exposed to PG/VG and VG-only, but not PG-only aerosols compared to air controls.	(Leigh et al. 2016)
Primary bronchial epithelial cells and human distal lung epithelial cells (NCI-H441)	Mix-1: raspberry, orange, lemon, and lime Mix-2: ripe strawberry, sweet apples, and tart kiwi	40 ml/puff, 3s puff duration, 30s puff interval, 10 puffs/ session at ALI	0, 3 mg/ml	No nicotine alone control present	Exposure to mix-1 (nicotine-free) significantly increased transcript expression of pro-inflammatory cytokines CXCL8, IL6, NFKB1, TNF (not specified whether α or β ) by more than three-fold and IL-1ß by 44-fold. Mix-2 (nicotine-free) PI3 transcript was reduced two-fold. The presence of nicotine in mix-1 significantly reduced the release of inflammatory markers of IL-1ß, 1 L-10, IL-13, and IL-6. The presence of nicotine in mix-2 increased the transcript of TNF (not specified whether α or β)	(Ganguly et al. 2020)
H292 cells	Cinnamon Butter	2-h/day for 1 or 3 consecutive days aerosol exposure. Using sub-ohm EC	mg/mL	No nicotine control	1 Day of exposure is cytotoxic, alters gene expression, and increases reactive oxygen species (cinnamon> butter). After 3 days of exposure, cinnamon flavor is more cytotoxic following LDH assay and causes oxidative damage to a greater extent than butter-flavored e‑cig aerosol. Cinnamon-flavored aerosol induced pro-oxidant effects, e.g., increased 8-hydroxy-2-deoxyguanosine levels. It is unclear whether the effects observed are due to nicotine plus as there are no nicotine controls.	(Noël et al. 2020b)
A549 (3D) co-culture of alveolar and H441 cells	Menthol Cinnamon Tobacco	24-h exposure to condensed aerosol (puff regime was set to 55 mL puff volume, 3 s draw, 60s 200 puff)	0 mg/ mL & 18 mg/ mL	No Nicotine control present	Cell viability significantly decreased in mono- and 3D culture by mint and cinnamon. Significant IL-8 release only in monoculture after Cinnamon exposure. No increase in MCP-1 release E-liquids containing nicotine did not alter cytotoxicity or pro-inflammatory effects relative to nicotine-free.	(Bengalli et al. 2017)
CALU-3 airway epithelial cells	Banana Pudding (Southern Style) Kola Hot Cinnamon Candies Menthol Tobacco Solid Menthol Captain black cigar Peanut butter cookies Popcorn Black Licorice Vanilla Tobacco	24-h direct e-liquid exposure (0%, 0.1%, 0.3%, 1%, 3.0%, 6.0%, 10% (v/v))	0 mg/mL and 12 mg/mL of nicotine	Nicotine alone control present	Hot cinnamon candies and menthol tobacco show cytotoxicity (cell and mitochondria viability) in the absence of nicotine. Nicotine control decreases cell proliferation and viability dose-dependently and is cytotoxic compared to the PG/VG controls. Nicotine (12 mg/mL) containing captain black cigar e-liquid showed more cytotoxicity (MTT assay) compared to captain black cigar alone. Nicotine-free e-liquids inhibit cell proliferation and viability in a dose-dependent manner. Nicotine-free kola, hot cinnamon candies, and menthol tobacco significantly decrease viability (calcein assay) than vehicle control.	(Rowell et al. 2017)
Human adenocarcinoma alveolar basal epithelial cells (A549 cells)	Balsamic flavor (with and without nicotine)	50 min aerosol exposure followed by analysis at 2-h, 6-h, 24-h.	Nicotine concentration not specified	No nicotine alone control present	Nicotine-free balsamic aerosol caused a significant, progressive, time-dependent loss of viability and increased cytotoxicity following LDH assay. Nicotine-contained e-liquids compared to flavor alone caused a significant loss in viability and enhanced LDH release. Quantitative and qualitative responses are superimposable to that of cigarette smoke exposure. Humectants alone did not cause cytotoxicity even 24 hours after treatment.	(Cervellati et al., 2014)
Human airway epithelial cells (H292) AND human lung fibroblast cells (HFL-1)	Tobacco Cinnamon roll Grape	30 s interval with a 4 s pulse for different time durations 5, 10, and 15 minutes	0, 6, 16, 18 and 24 mg	No nicotine alone control present	Nicotine-free tobacco and cinnamon roll-flavored e-liquid increased the secretion of inflammatory cytokines IL-6 and IL-8 in H292 and HFL-1. Nicotine-contained tobacco flavored e-liquid had a significant increase in IL-8 secretion in a dose-dependent manner in HFL-1 but not on other markers.	(Lerner et al., 2015)
Human adenocarcinoma alveolar basal epithelial cells (A549)	Tobacco Menthol	Six puffs were dissolved per 1 mL of A549 culture medium, referred to as 6 total-puff- equivalents (TPE) of aerosol, and grown in control or treatment medium for 3–8 days.	48 mg/ml (No nicotine free e-liquids present)	No nicotine control	Both EC liquids and aerosols induced epithelial-to-mesenchymal transition (EMT) Both tobacco and Menthol flavors alter cell-to-cell junctions induced an EMT, internalization of E-cadherin, increased motility, and upregulation of EMT markers. The EMT was concurrent with the plasma membrane to nuclear translocation of active β-catenin. Due to the lack of nicotine controls, it is unclear whether the effects observed are a result of an interaction between flavor and nicotine or as a flavor effect. No cytotoxicity was measured.	(Zahedi et al. 2018)
Primary human epithelial cells (HBEC), cancerous human bronchial epithelial cells CALU-3	Banana Pudding (BP)	5–25 puffs	0 and 12 mg/ml nicotine	No nicotine alone control, but 0 mg/mL BP	BP acutely elevates cytosolic Ca^2+ in both pulmonary cell types in no nicotine samples. No adverse effects induced by PG:VG in HBEC and CALU-3. Nicotine increased ER Ca^2+ in CALU-3. Effect of nicotine on HBEC was not characterized.	(Rowell et al., 2020)

Table 5. Chemical composition analyses of flavored EC.

E-liquid flavor	Experimental approach	E-liquid Nicotine Dose	Main findings	Reference
Menthol, Vanillin, Maltol	Particle size distribution	18 mg/ mL of nicotine	Vanillin and nicotine increase e-cigarette particle size distribution. Increasing the glycerol (VG) proportion in e-liquid increases EC particle size.	(Lechasseur et al., 2019)
Menthol	GC−MS Analysis of Liquid, Vapor, and Particulate Phases, FTIR Spectrometry	18 mg nicotine/mL	EC emissions contained acetaldehyde, acrolein, benzaldehyde, benzene, toluene, ethylbenzene, and xylene The concentrations of these chemicals increased with increasing VG composition. Benzaldehyde, naphthalene, diphenyl ether, menthol, and nicotine were also detected in the aerosol condensates.	(Ooi et al., 2019)
Strawberry, Cinnamon, Sweet cream	Hydroxyl radicals in reaction mixes were measured based on the formation of 2-hydroxyterephthalic acid (2OHTA)	3, 12, 24 and 36 mg/mL	VG e-liquids generated higher •OH levels than PG e-liquids, as did flavored e-liquids relative to nonflavored e-liquids (cinnamon>sweet cream>strawberry>non-flavored). E-vapor, combined with ascorbic acid, can also induce •OH formation. The levels of •OH in 3 and 12 mg/ml nicotine solutions were 19.6% and 10.0% lower than that in the equivalent nicotine-containing-vapor solutions. Conversely, 24 and 36 mg/ml nicotine solution induced 40% more •OH than the equivalent e-vapor solutions.	(Son et al., 2019)
51 flavors	Mass spectrometry	5% nicotine	At least one flavoring chemical was detected in 47 of 51 unique flavors tested. Diacetyl was detected above the laboratory limit of detection in 39 of the 51 flavors tested, ranging from below the limit of quantification to 239 μg/EC. 2,3-Pentanedione and acetoin were detected in 23 and 46 of the 51 flavors tested at concentrations up to 64 and 529 μg/EC, respectively.	(Allen et al., 2016)
Mango, Banana, Tobacco, Cappuccino, Chocolate, Bubblegum, Peppermint, Cinnamon, Cherry, Apple	GC-MS		Vanillin was the most frequently detected flavoring chemical. It was found in banana, tobacco, cappuccino, chocolate, bubblegum, and cinnamon. Chocolate and cinnamon e-liquids had the most complex flavoring profile, with 14 and 15 flavoring peaks, respectively. Tobacco flavor had the least complex flavoring profile with only 2 peaks.	(Ween et al. 2020b)

Table 6. In vivo studies of toxicological effects of EC flavors.

Strain/sex	Exposure	E-liquid Nicotine Dose	Flavor(s) investigated.	Main findings	Reference
Adult Balb/c/ (male and female)	30 min twice daily, 6 days/week exposed for 18 days (aerosol exposure)	0 mg/mL &12 mg/mL	Black licorice (BL) Kola Banana Pudding Cinnacide	House dust mite (HDM) challenged mice exposed to nicotine-free cinnacide reduced airway inflammation and increased peripheral airway hyperresponsiveness compared to air-only controls. HDM-challenged mice exposed to flavored EC without nicotine had significant but heterogeneous effects on features of allergic airways disease compared to air controls. Nicotine-free BL increased macrophage, eosinophils, and macrophages count in bronchoalveolar lavage (BAL). Nicotine in all flavors induced an increase in macrophages and eosinophils in BAL compared to nicotine-free. Sex-difference in toxicity were not explored.	(Chapman et al. 2019)
C57BL/6 /female	2 h a day, 7 days a week, for 6 weeks	No nicotine	Vanilla	Exposures to VG/PG + vanilla increased lung tidal and minute volumes and tissue damping. Increased number of dendritic cells, CD4 + T cells, and CD19 + B cells in the lungs in the VG/PG-exposed group compared to air, irrespective of the presence of vanilla. A > 3-fold increase of 2-arachidonoylglycerol (2-AG), an anti-inflammatory mediator, and a 2-fold increase of 12-hydroxyeicosatetraenoic acid (12-HETE), an inflammatory mediator, following VG/PG exposure, with or without vanilla The PG/VG EC aerosol dysregulated genes related to biotransformation (Aldh8a1), transcription factors expressed in pulmonary surfactant (F2), synuclein-alpha (snca), which interacts with phospholipids and proteins, as well as IL-6 compared air controls.	(Szafran et al. 2020)
Balb/c/Pregnant and offspring	14–31 days exposure. Vaping topography profile of 3-s puff duration and a 55-mL puff volume every 30s.	36 mg/ml Nic (No 0 mg/mL available)	Cinnamon-flavor	Preconception and prenatal exposures to EC aerosols significantly decreased the offspring birth weight and body length compared to non-exposed controls Preconception exposure caused downregulation of 7 inflammation-related genes. Four genes were common to both dams and fetuses. Preconception EC exposure led to offspring with significantly increased lung tissue fraction at birth compared to unexposed controls. Increase in nicotinic receptors gene expression (α7nAChR) in the lungs.	(Noël et al. 2020a)
C57BL6/male	Whole-body exposure for 3 days or 4 weeks. Puff volume was 20 ml. Mice were exposed to CS or EC 4 times a day with 30-min smoke-free intervals.	18 mg/ml (No 0 mg/mL control)	Tobacco flavor	Increased BAL cellularity, MUC5AC production, and oxidative stress markers in BAL and lung compared to unexposed controls. In many cases, increases were more than for CS. BAL markers increased significantly in PG/VG + tobacco flavor + nicotine compared to PG/VG + nicotine.	(Glynos et al. 2018)
Crl:CD (Sprague-Dowley) rats/male and female	90-day nose-only inhalation to 3.2, 9.6 and 32 mg/kg/day of aerosol, which are referred to as low, mid, and high, respectively.	20 mg Nic	PG:VG control (77% PG: 23% VG) Formulation 1 (22.5% VG, 75.5% PG, 20 mg Nic) Formulation 2 (18.1% VG, 62.3% PG, 20 mg NIC and 17.6% proprietary flavor)	The inflammatory effects (increase in neutrophils, lymphocytes) in a dose-related manner were observed in the PG:VG control in both males and female groups. No flavor-dependent effect was observed for pulmonary toxicity. No nicotine-related pulmonary effects were observed.	(Werley et al. 2016)

Figure 3. The effects of flavors in e-liquids reported in different experimental models. The figure was produced with Biorender.com.

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Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article.