A Practical Framework for Novel Electronic Nicotine Delivery System Evaluation: Chemical and Toxicological Characterization of JUUL2 Aerosol and Comparison with Reference Cigarettes.

Electronic nicotine delivery systems (ENDSs) are designed as a non-combustible alternative to cigarettes, aiming to deliver nicotine without the harmful byproducts of tobacco combustion. As the category evolves and new ENDS products emerge, it is important to continually assess the levels of toxicologically relevant chemicals in the aerosols and characterize any related toxicology. Herein, we present a proposed framework for characterizing novel ENDS products (i.e., devices and formulations) and determining the reduced risk potential utilizing analytical chemistry and in vitro toxicological studies with a qualitative risk assessment. To demonstrate this proposed framework, long-term stability studies (12 months) analyzing relevant toxicant emissions from six formulations of a next-generation product, JUUL2, were conducted and compared to reference combustible cigarette (CC) smoke under both non-intense and intense puffing regimes. In addition, in vitro cytotoxicity, mutagenicity, and genotoxicity assays were conducted on aerosol and smoke condensates. In all samples, relevant toxicants under both non-intense and intense puffing regimes were substantially lower than those observed in reference CC smoke. Furthermore, neither cytotoxicity, mutagenicity, nor genotoxicity was observed in aerosol condensates generated under both intense and non-intense puffing regimes, in contrast to results observed for reference cigarettes. Following the proposed framework, the results demonstrate that the ENDS products studied in this work generate significantly lower levels of toxicants relative to reference cigarettes and were not cytotoxic, mutagenic, or genotoxic under these in vitro assay conditions.

Corrigendum: Analytical approaches for the evaluation of data deficient simulated leachable compounds in ENDS products: a case study.

[This corrects the article DOI: 10.3389/fchem.2023.1212744.].

Determination of chemical constituent yields in e-cigarette aerosol using partial and whole pod collections, a comparative analysis.

Literature reports the chemical constituent yields of electronic nicotine delivery systems (ENDS) aerosol collected using a range of aerosol collection strategies. The number of puffs to deplete an ENDS product varies widely, but collections often consist of data from the first 50-100 puffs. However, it is not clear whether these discrete puff blocks are representative of constituent yields over the life of a pod. We aimed to assess the effect of differing aerosol collection strategies on reported yields for select chemical constituents in the aerosol of closed pod-based ENDS products. Constituents analyzed were chosen to reflect important classes of compounds from the Final Premarket Tobacco Product Application Guidance. Yields were normalized to total device mass loss (DML). Collection strategies that consisted of partial pod collection were valid for determining yields of constituents whose DML normalized yields were consistent for the duration of pod life. These included primary aerosol constituents, such as propylene glycol, glycerol, and nicotine, and whole pod yields could be determined from initial puff blocks. However, changes were observed in the yields of some metals, some carbonyl compounds, and glycidol over pod life in a chemical constituent and product dependent manner. These results suggest that collection strategies consisting of initial puff block collections require validation per chemical constituent/product and are not appropriate for chemical constituents with variable yields over pod life. Whole pod collection increased sensitivity and accuracy in determining metal, carbonyl, and glycidol yields compared to puff block-based collection methodologies for all products tested.

Analytical approaches for the evaluation of data deficient simulated leachable compounds in ENDS products: a case study.

Leachable investigations are routinely undertaken across a range of sectors (e.g., pharmaceuticals, medical devices, etc.) to determine whether chemicals from a container closure system transfer into a product under normal conditions of use. For Electronic Nicotine Delivery Systems (ENDS) the container closure system includes all materials in contact with the e-liquid that is aerosolized and subsequently inhaled by the user. Currently, there is no guidance for conducting leachable studies for ENDS products, however, there are relevant guidance documents for orally inhaled drug products that can be applied to an ENDS container closure system. We present a case study of the analytical investigation of two leachable compounds identified in simulated leachable studies using aged JUULpods filled with unflavored e-liquid (PG/VG/nicotine/benzoic acid). Both compounds had limited toxicological information and were considered data deficient. A qualitative analysis of the aerosol collected from aged commercial JUULpods (Virginia Tobacco and Menthol), using a similar analytical method (LC-MS/MS) used in the simulated leachable studies, showed no trace or detectable levels of either leachable compound. Therefore, this qualitative analysis did not provide semi-quantitative values for the data-deficient leachable compounds necessary to support toxicological risk assessment. Further, no commercial authentic standards or reasonable synthetic route were available due to the molecular size and structural complexity of the compounds. Instead, method limits were established using an alternative approach to standard ICH guidelines. The experimentally determined method limit of quantitation, using spiked samples of simulated leachable e-liquid, provided conservative semi-quantitative values for each data deficient leachable compound in the aerosol that enabled a transfer efficiency from e-liquid to aerosol to be estimated. The transfer efficiency of each leachable compound was experimentally determined to be less than 2% based on the limit of quantitation, which then could be used to define a relevant exposure limit for the toxicological risk assessment. This work details a novel analytical approach for determining the transfer efficiency of data deficient leachable compounds from ENDS container closure systems into the ENDS aerosol to support toxicological health risk assessments.

An Approach to Flavor Chemical Thermal Degradation Analysis.

Toxicological evaluations of flavor chemicals for use in inhalation products that utilize heat for aerosol generation are complicated because of the potential effect heat may have on the flavor chemical. The objective was to develop a thermal degradation technique to screen flavor chemicals as part of a toxicological testing program for their potential use in ENDS formulations. Based upon published data for acetaldehyde, acrolein, and glycidol from ENDS products (common thermal degradants of propylene glycol and glycerin), the pyrolizer temperature was adjusted until a similar ratio of acetaldehyde, acrolein, and glycidol was obtained from a 60/40 ratio (v/v) of glycerin/propylene glycol via GC/MS analysis. For each of 90 flavor chemicals, quantitative measurements of acetaldehyde, acrolein, and glycidol, in addition to semiquantitative non-targeted analysis tentatively identifying chemicals from thermal degradation, were obtained. Twenty flavor chemicals transferred at greater than 99% intact, another 26 transferred at greater than 95% intact, and another 15 flavor chemicals transferred at greater than 90% intact. Most flavor chemicals resulted in fewer than 10-12 tentatively identified thermal degradants. The practical approach to the thermal degradation of flavor chemicals provided useful information as part of the toxicological evaluation of flavor chemicals for potential use in ENDS formulations.

Characterization of a rapid condensate collection apparatus for in vitro assays of electronic nicotine delivery systems.

In vitro testing of Electronic Nicotine Delivery System (ENDS) aerosol condensates is important in evaluating their potential toxicity. Collecting sufficient condensate for these tests is a time consuming and costly procedure. The "triple puff (TP)" is a novel system which collects the aerosol from three ENDS devices sequentially into a single filter pad and impinger. The TP substantially reduces condensate collection time relative to the conventional single ENDS, single puff (SP), device system. Both the TP and SP (using two puffing profiles) were used to generate condensates from JUUL ENDS e-liquid Mint 5.0% (nicotine by weight). Aerosols were collected using the filter pad and ethanol-containing impinger method. Condensates produced with the SP and TP were compared for concentrations of primary constituents and carbonyl compounds as well as for their cytotoxicity (OECD 129), mutagenicity (OECD 471) and genotoxicity (OECD 487). Condensates generated with the SP and TP, regardless of puffing regimen, were very similar chemically and equivalent in the biological assays tested (not cytotoxic, mutagenic, or genotoxic). The TP device significantly reduces production time of ENDS condensates relative to the standard SP method and thus may facilitate further research by reducing the time and effort required to collect ENDS condensates.

Determining the impact of flavored e-liquids on aldehyde production during Vaping.

1,2-Propylene glycol and glycerin, principal components of e-liquids, can thermally degrade to form acetaldehyde, acrolein, and formaldehyde when heated in electronic nicotine delivery systems (ENDS). Recently the flavors in e-liquids were suggested to be the major source of these aldehydes. We used the same 10 ENDS devices to test 5 e-liquid formulations (four flavored & one corresponding non-flavored) and measured device mass loss and levels of acetaldehyde, acrolein, and formaldehyde (30 replicate measurements per formulation). Despite finding reasonable variability in measurements of device mass loss, two out of 10 ENDS devices tested produced outlier values for aerosol levels acetaldehyde, acrolein, and formaldehyde. After removing these devices from further analysis, acceptable variability (≤20% RSD) in aerosol levels of acetaldehyde, and formaldehyde were found. The flavored formulations tested resulted in a consistent and selective increase of 150%-200% in acetaldehyde, no increase or decrease in acrolein and depending on the flavor formulation, an increase, a decrease or no change in formaldehyde levels. Comparison of our results to the literature illustrates the need for development of a standardized ENDS testing protocol. Our results further support that device variability must be fully characterized and considered before assessing the impact of e-liquid formulations.

Comparative levels of carbonyl delivery between mass-market cigars and cigarettes.

The recent 2016 deeming of cigars by the US Food and Drug Administration (FDA) has led to increased interest in cigar science, including ways to accurately measure the harmful and potentially harmful constituents (HPHCs) found within mainstream cigar smoke. At present, there are standardized methods for evaluating HPHCs in mainstream cigarette smoke but none specific to cigar analysis except for nicotine and carbon monoxide. This study sought to analyze carbonyl delivery in marketed cigars and cigarillos and compare them against levels found in cigarettes. To accomplish this the standard cigarette method, CORESTA recommended method 74 (CRM-74), was optimized for cigar smoking including an evaluation of the trapping efficiency and the stability of the carbonyl-hydrazone adducts due to the increased smoke time required for cigar collection. On a per product basis, carbonyl delivery from cigars smoked under CRM-64 conditions was found to yield similar levels of formaldehyde and greater levels of acetaldehyde, acrolein and crotonaldehyde than measured in mainstream cigarette smoke collected under conditions prescribed under ISO standard 3308. Furthermore, on a per product basis, cigarettes smoked under the ISO 20778 intense smoking regime delivered higher levels of formaldehyde, acrolein and crotonaldehyde as compared to cigars smoked under the CORESTA regime, while acetaldehyde was found to be higher in mainstream cigar smoke. Given the recent deeming, this work expands upon previously reported work, limited in scope by either number of products or analytes reported, through the analysis of carbonyl delivery found in the mainstream smoke for 12 brands of cigars and cigarillos.

Determination of Selected Chemical Levels in Room Air and on Surfaces after the Use of Cartridge- and Tank-Based E-Vapor Products or Conventional Cigarettes.

There is an ongoing debate regarding the potential of secondhand exposure of non-users to various chemicals from use of e-vapor products (EVPs). Room air levels of 34 chemicals (nicotine, propylene glycol (PG), glycerol, 15 carbonyl chemicals, 12 volatile organic chemicals (VOCs), and four selected trace elements) were measured where EVPs and cigarettes were used by n = 37 healthy adult tobacco users in an exposure chamber. The products used were MarkTen® 2.5% Classic (Group I), a Prototype GreenSmoke® 2.4% (Group II), Ego-T® Tank with subjects' own e-liquids (Group III) and subjects' own conventional cigarettes (Group IV). Products were used under controlled conditions and 4-h ad libitum use. Background (without subjects) and baseline levels (with subjects) were measured. Cumulative 4-h. levels of nicotine, PG and glycerol measured were several-fold below the time-weighted average limits used in workplace exposure evaluation. Most the other chemicals (>75%) were at or below the limit of quantification during EVP use. Significant levels of chemicals (17 out of 34) were observed in Group IV. Overall, our results indicate that under the study conditions with the products tested, cumulative room air levels of the selected chemicals measured over 4-h were relatively small and were several-fold below the current occupational regulatory and consensus limits.

Nicotine levels and presence of selected tobacco-derived toxins in tobacco flavoured electronic cigarette refill liquids.

BACKGROUND: Some electronic cigarette (EC) liquids of tobacco flavour contain extracts of cured tobacco leaves produced by a process of solvent extraction and steeping. These are commonly called Natural Extract of Tobacco (NET) liquids. The purpose of the study was to evaluate nicotine levels and the presence of tobacco-derived toxins in tobacco-flavoured conventional and NET liquids. METHODS: Twenty-one samples (10 conventional and 11 NET liquids) were obtained from the US and Greek market. Nicotine levels were measured and compared with labelled values. The levels of tobacco-derived chemicals were compared with literature data on tobacco products. RESULTS: Twelve samples had nicotine levels within 10% of the labelled value. Inconsistency ranged from -21% to 22.1%, with no difference observed between conventional and NET liquids. Tobacco-specific nitrosamines (TSNAs) were present in all samples at ng/mL levels. Nitrates were present almost exclusively in NET liquids. Acetaldehyde was present predominantly in conventional liquids while formaldehyde was detected in almost all EC liquids at trace levels. Phenols were present in trace amounts, mostly in NET liquids. Total TSNAs and nitrate, which are derived from the tobacco plant, were present at levels 200-300 times lower in 1 mL of NET liquids compared to 1 gram of tobacco products. CONCLUSIONS: NET liquids contained higher levels of phenols and nitrates, but lower levels of acetaldehyde compared to conventional EC liquids. The lower levels of tobacco-derived toxins found in NET liquids compared to tobacco products indicate that the extraction process used to make these products did not transfer a significant amount of toxins to the NET. Overall, all EC liquids contained far lower (by 2-3 orders of magnitude) levels of the tobacco-derived toxins compared to tobacco products.

Fluorescent detection of lipid peroxidation derived protein adducts upon in-vitro cigarette smoke exposure.

Oxidative stress in biological systems can result in radical-induced lipid peroxidation (LPO), which can lead to the production of secondary reactive by-products such as 4-hydroxy-2-nonenal (HNE), malondialdehyde (MDA), acrolein, and acetaldehyde. These deleterious compounds are known to react with and concomitantly modify nucleophilic amino acid residues on proteins. Oxidative stress induced by cigarette smoke (CS) has been put forth as a major mechanism for tobacco-induced pathologies. At present, there are few reliable biomarkers for measuring the extent of oxidatively-induced damage resulting from CS exposure in vivo. This study has utilized a previously reported CS exposure system to expose cultured cells in-vitro to whole CS and determine the extent of LPO resulting from CS exposure by quantifying the increase in HNE within the exposure media versus controls via gas chromatography mass spectrometry. Additionally, we obtained protein enriched cell lysate post-CS exposures and measured the fluorescent signal obtained via direct injection fluorescent analysis at 375 nm ex./415 nm em. This study determined that the fluorescent signal intensity was directly proportional to the quantity increase of HNE in CS exposed media. It further tested this correlation by performing HNE titration addition experiments to cultured cells and Western blot analysis on proteins obtained from cell lysates. Finally, the fluorescent signal increase from authentic BSA solutions incubated with increasing concentrations of HNE was measured. It is proposed that the fluorescent signal observed from the protein lysate of CS exposed cultured cells corresponds to the extent of biological damage resulting from secondary reactive by-products formed from LPO induced via CS exposure and represented by HNE. The fluorescent signals increased in intensity upon increasing CS dose up to 20 min and remained elevated over 24 h after cessation of CS exposure.

Development of a quantitative method for the analysis of tobacco-specific nitrosamines in mainstream cigarette smoke using isotope dilution liquid chromatography/electrospray ionization tandem mass spectrometry.

An improved method has been developed for the determination of the four major tobacco-specific nitrosamines (TSNA) in mainstream cigarette smoke. The new method offers decreased sample preparation and analysis time as compared to traditional methodologies. This method uses isotope dilution liquid chromatography coupled to a tandem mass spectrometer with electrospray ionization and is significantly more sensitive than traditional methods. It also shows no evidence of artifactual formation of TSNA. Sample concentrations were determined for four TSNA in mainstream smoke using two isotopically labeled TSNA analogues as internal standards. Mainstream smoke was collected on an industry standard 44-mm Cambridge filter pad, extracted with an aqueous buffer solution, and analyzed without further sample cleanup. This method has been validated through intra- and interlaboratory studies and has shown excellent recoveries, sensitivity, and repeatability. The limits of detection of each TSNA varied from 0.01 to 0.1 ng/mL, and the linear calibration range of the instrument in sample matrix spanned 0.5-200 ng/ mL, which allowed for the determination of the TSNA levels in cigarettes with a wide range of deliveries. Data are also reported from two commercially available industry reference cigarettes and show excellent agreement and reproducibility over a six-month time period (n > 50).