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)