In vitro toxicological evaluation of aerosols generated by a 4th generation vaping device using nicotine salts in an air-liquid interface system

Abstract

Background Electronic cigarettes (EC) have gained popularity, especially among young people, with the introduction of fourth-generation devices based on e-liquids containing nicotine salts that promise a smoother vaping experience than freebase nicotine. However, the toxicological effects of nicotine salts are still largely unknown, and the chemical diversity of e-liquids limits the comparison between different studies to determine the contribution of each compound to the cytotoxicity of EC aerosols. Therefore, the aim of this study was to evaluate the toxicological profile of controlled composition e-liquid aerosols to accurately determine the effects of each ingredient based on exposure at the air-liquid interface. Methods Human lung epithelial cells (A549) were exposed to undiluted aerosols of controlled composition e-liquids containing various ratios of propylene glycol (PG)/vegetable glycerin (VG) solvents, freebase nicotine, organic acids, nicotine salts, and flavoured commercial e-liquids. Exposure of 20 puffs was performed at the air-liquid interface following a standard vaping regimen. Toxicological outcomes, including cytotoxicity, inflammation, and oxidative stress, were assessed 24 h after exposure. Results PG/VG aerosols elicited a strong cytotoxic response characterised by a 50% decrease in cell viability and a 200% increase in lactate dehydrogenase (LDH) production, but had no effects on inflammation and oxidative stress. These effects occurred only at a ratio of 70/30 PG/VG, suggesting that PG is the major contributor to aerosol cytotoxicity. Both freebase nicotine and organic acids had no greater effect on cell viability and LDH release than at a 70/30 PG/VG ratio, but significantly increased inflammation and oxidative stress. Interestingly, the protonated form of nicotine in salt showed a stronger proinflammatory effect than the freebase nicotine form, while benzoic acid-based nicotine salts also induced significant oxidative stress. Flavoured commercial e-liquids was found to be cytotoxic at a threshold dose of ≈ 330 µg/cm². Conclusion Our results showed that aerosols of e-liquids consisting only of PG/VG solvents can cause severe cytotoxicity depending on the concentration of PG, while nicotine salts elicit a stronger pro-inflammatory response than freebase nicotine. Overall, aerosols from fourth-generation devices can cause different toxicological effects, the nature of which depends on the chemical composition of the e-liquid.

Quantification of Free Radicals from Vaping Electronic Cigarettes Containing Nicotine Salt Solutions with Different Organic Acid Types and Concentrations

“…The highest radical yields were observed for BA and CA e-liquids, which was determined to result from a combination of radical production and scavenging processes. These spin-trapped ROS measurement data are consistent with recent in vitro results for vaping aerosols from BA and SLA nicotine salts from JUUL devices, wherein BA but not SLA salts were found to induce oxidative stress . Under the assumption that Fenton-like chemistry occurs in the vaping process, the results from CA and TA are expected, as these are well-studied ligands for Fenton-like reactions, but the observation that BA produced the highest radical yield is unexpected and novel.…”

“…These spin-trapped ROS measurement data are consistent with recent in vitro results for vaping aerosols from BA and SLA nicotine salts from JUUL devices, wherein BA but not SLA salts were found to induce oxidative stress. 46 Under the assumption that Fenton-like chemistry occurs in the vaping process, the results from CA and TA are expected, as these are well-studied ligands for Fenton-like reactions, but the observation that BA produced the highest radical yield is unexpected and novel. Given that BA and LA are the two most common organic acid additives in nicotine salt e-liquids in the market currently, the results here have implications for their safe usage in commercial e-cigarette vape solutions due to the negligible radical production for LA nicotine salts and the significant radical production from BA nicotine salts.…”

“…Consistent with our spin-trapped ROS measurement results, a recent in vitro study also found that BA nicotine salt vaping aerosol from JUUL devices was the only tested formulation that produced significant oxidative stress response, whereas SLA nicotine salts, freebase nicotine salts, and the organic acids themselves did not. 46 BA has not been studied for radical production but has been studied for its radical scavenging abilities. 47,48 BA has relatively low scavenging capabilities; 47 whereas benzoic acids with additional methoxy and hydroxy groups have significantly higher radical scavenging capabilities.…”