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)