A synthetic coolant (WS-23) in disposable electronic cigarettes impairs cytoskeletal function in EpiAirway microtissues exposed at the air liquid interface.

The design of popular disposable electronic cigarettes (ECs) was analyzed, and the concentrations of WS-23, a synthetic coolant, in EC fluids were determined for 22 devices from 4 different brands. All products contained WS-23 in concentrations that ranged from 1.0 to 40.1 mg/mL (mean = 21.4 ± 9.2 mg/mL). To determine the effects of WS-23 on human bronchial epithelium in isolation of other chemicals, we exposed EpiAirway 3-D microtissues to WS-23 at the air liquid interface (ALI) using a cloud chamber that generated aerosols without heating. Proteomics analysis of exposed tissues revealed that the cytoskeleton was a major target of WS-23. BEAS-2B cells were exposed to WS-23 in submerged culture to validate the main results from proteomics. F-actin, which was visualized with phalloidin, decreased concentration dependently in WS-23 treated BEAS-2B cells, and cells became immotile in concentrations above 1.5 mg/mL. Gap closure, which depends on both cell proliferation and migration, was inhibited by 0.45 mg/mL of WS-23. These data show that WS-23 is being added to popular EC fluids at concentrations that can impair processes dependent on the actin cytoskeleton and disturb homeostasis of the bronchial epithelium. The unregulated use of WS-23 in EC products may harm human health.

Ethyl maltol, vanillin, corylone and other conventional confectionery-related flavour chemicals dominate in some e-cigarette liquids labelled 'tobacco' flavoured.

BACKGROUND: The increased popularity of electronic cigarettes (e-cigarettes) has been linked to the abundance of flavoured products that are attractive to adolescents and young adults. In the last decade, e-cigarette designs have evolved through four generations that include modifications in battery power, e-cigarette liquid (e-liquid) reservoirs and atomiser units. E-liquids have likewise evolved in terms of solvent use/ratios, concentration and number of flavour chemicals, use of nicotine salts and acids, the recent increased use of synthetic cooling agents and the introduction of synthetic nicotine. Our current objective was to evaluate and compare the evolving composition of tobacco-flavoured e-liquids over the last 10 years. METHODS: Our extensive database of flavour chemicals in e-liquids was used to identify trends and changes in flavour chemical composition and concentrations. RESULTS: Tobacco-flavoured products purchased in 2010 and 2011 generally had very few flavour chemicals, and their concentrations were generally very low. In tobacco-flavoured refill fluids purchased in 2019 and Puff Bar Tobacco e-cigarettes, the total number and concentration of flavour chemicals were higher than expected. Products with total flavour chemicals >10 mg/mL contained one to five dominant flavour chemicals (>1 mg/mL). The most frequently used flavour chemicals in tobacco e-liquids were fruity and caramellic. CONCLUSIONS: There is a need for continuous surveillance of e-liquids, which are evolving in often subtle and harmful ways. Chemical constituents of tobacco flavours should be monitored as they clearly can be doctored by manufacturers to have a taste that would appeal to young users.

Disposable Puff Bar Electronic Cigarettes: Chemical Composition and Toxicity of E-liquids and a Synthetic Coolant.

The popularity of disposable fourth-generation electronic cigarettes (ECs) among young adults and adolescents has been increasing since the ban on flavored cartridge EC products such as JUUL. Although the constituents and toxicity of some cartridge-based fourth-generation ECs, such as JUUL, have been studied, limited data exist for other disposable ECs such as Puff. The purpose of this study was to determine flavor chemicals, synthetic coolants, and nicotine concentrations in 16 disposable Puff devices, evaluate the cytotoxicity of the different flavors from the Puff brand using in vitro assays, and investigate the health risks of synthetic coolants in EC products. Gas chromatography/mass spectrometry was used to identify and quantify chemicals in Puff EC fluids. One hundred and twenty-six flavor chemicals were identified in Puff fluids, and 16 were >1 mg/mL. WS-23 (2-isopropyl-N,2,3-trimethylbutyramide) was present in all products, and concentrations ranged from 0.8 to 45.1 mg/mL. WS-3 (N-ethyl-p-menthane-3-carboxamide) concentrations ranged from 1.5 to 16.4 mg/mL in 6/16 products. Nicotine concentrations ranged from 40.6 to 52.4 (average 44.8 mg/mL). All unvaped fluids were cytotoxic at dilutions between 0.1 and 10% in the MTT and neutral red uptake assays when tested with BEAS-2B lung epithelial cells. The cytotoxicity of Puff fluids was highly correlated with total chemical concentrations, nicotine, WS-23, both synthetic coolants, and synthetic coolants plus ethyl maltol. Lower concentrations of WS-23 than those in the fluids adversely affected cell growth and morphology. Concentrations of synthetic coolants exceeded levels used in consumer products. The margin of exposure data showed that WS-3 and WS-23 concentrations were high enough in Puff products to present a health hazard. Our study demonstrates that disposable Puff ECs have high levels of cytotoxic chemicals. The data support the regulation of flavor chemicals and synthetic coolants in ECs to limit potentially harmful health effects.

Flavour chemicals, synthetic coolants and pulegone in popular mint-flavoured and menthol-flavoured e-cigarettes.

BACKGROUND: The Food and Drug Administration (FDA) has recently banned flavours from pod-style electronic cigarettes (e-cigarettes), except for menthol and tobacco. JUUL customers have quickly discovered that flavoured disposable e-cigarettes from other manufacturers, such as Puff, are readily available. Our goal was to compare flavour chemicals, synthetic coolants and pulegone in mint-flavoured/menthol-flavoured e-cigarettes from JUUL and Puff, evaluate the cytotoxicity of the coolants and perform a cancer risk assessment for pulegone, which is present in both JUUL pods and disposable Puff products. METHODS: Identification and quantification of chemicals were performed using gas chromatography/mass spectrometry. Cytotoxicity of the coolants was evaluated with BEAS-2B cells using the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cancer risk of pulegone was calculated using the margin of exposure (MOE). RESULTS: Menthol was the dominant flavour chemical (>1 mg/mL) in all products from both manufacturers. Minor flavour chemicals (<1 mg/mL) differed in the JUUL and Puff fluids and may produce flavour accents. The concentrations of WS-3 and WS-23 were higher in Puff than in JUUL. WS-23 was cytotoxic in the MTT assay at concentrations 90 times lower than concentrations in Puff fluids. The risk of cancer (MOE<10 000) was greater for mint than for menthol products and greater for Puff than for JUUL. CONCLUSIONS: Switching from flavoured JUUL to Puff e-cigarettes may expose users to increased harm due to the higher levels of WS-23 and pulegone in Puff products. Cancer risk may be reduced in e-cigarettes by using pure menthol rather than mint oils to produce minty-flavoured e-cigarette products.

Adhesion and Removal of Thirdhand Smoke from Indoor Fabrics: A Method for Rapid Assessment and Identification of Chemical Repositories.

Thirdhand smoke (THS) is an environmental contaminant that may cause adverse health effects in smokers and nonsmokers. Currently, time-consuming analytical methods are necessary to assess chemicals in THS repositories, like upholstered furniture and clothing. Our goal was to develop a rapid, accessible method that can be used to measure THS contamination in common household fabrics and to evaluate remediation. Cotton, terry cloth, polyester, and wool were exposed to THS for various times in a controlled laboratory environment and then extracted in various media at room temperature or 60 °C to develop an autofluorescent method to quantify THS. Concentrations of nicotine and related alkaloids in the extracts were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high-performance liquid chromatography (HPLC). The autofluorescence of extracts was proportional to the time and amount of THS exposure received by cotton and terry cloth. Extracts of polyester and wool did not show autofluorescence unless heat was applied during extraction. Nicotine, nicotine alkaloids, and TSNA concentrations were higher in THS extracts from cotton and terry cloth than extracts of polyester and wool carpet, in agreement with the autofluorescence data. For fabrics spiked with 10 mg of nicotine, extraction efficiency was much higher from terry cloth (7 mg) than polyester (0.11 mg). In high relative humidity, nicotine recovery from both cotton and polyester was 80% (~8 mg). Our results provide a simple, rapid method to assess THS contaminants in household fabrics and further show that THS extraction is influenced by fabric type, heat, and humidity. Thus, remediation of THS environments may need to vary depending on the fabric reservoirs being treated. Understanding the dynamics of THS in fabrics can help set up appropriate remediation policies to protect humans from exposure.

Design features and elemental/metal analysis of the atomizers in pod-style electronic cigarettes.

BACKGROUND: The atomizers of electronic cigarettes (ECs) contain metals that transfer to the aerosol upon heating and may present health hazards. This study analyzed 4th-generation EC pod atomizer design features and characterized their elemental/metal composition. METHODS: Eleven EC pods from six brands/manufacturers were purchased at local shops and online. Pods were dissected and imaged using a Canon EOS Rebel SL2 camera. Elemental analysis and mapping of atomizer components was done using a scanning electron microscope coupled with an energy dispersive x-ray spectrometer. RESULTS: EC pods varied in size and design. The internal atomizer components were similar across brands except for variations occurring mainly in the wicks and filaments of some products. The filaments were either Elinvar (nickel, iron, and chromium) (36.4%), nichrome (36.4%), iron-chromium (18.2%), or nickel (9%). Thick wires present in 55% of the atomizers were mainly nickel and were joined to filaments by brazing. Wire-connector joints were Elinvar. Metal air tubes were made of Elinvar (50%), nickel, zinc, copper, and tin (37.5%), and nickel and copper (12.5%). Most of the wick components were silica, except for two pods (PHIX and Mico), which were mainly ceramic. Connectors contained gold-plated nickel, iron-chromium multiple alloys of nickel, zinc, gold, iron, and copper. Wick chambers were made of Elinvar. Outer casings were either nickel, copper-tin, or nickel-copper alloys. Magnets were nickel with minor iron, copper, and sulfur. Some frequently occurring elements were high in relative abundance in atomizer components. CONCLUSIONS: The atomizers of pods are similar to previous generations, with the introduction of ceramic wicks and magnets in the newer generations. The elements in EC atomizers may transfer into aerosols and adversely affect health and accumulate in the environment.

Electronic Cigarette Refill Fluids Sold Worldwide: Flavor Chemical Composition, Toxicity, and Hazard Analysis.

Flavor chemicals in electronic cigarette (EC) fluids, which may negatively impact human health, have been studied in a limited number of countries/locations. To gain an understanding of how the composition and concentrations of flavor chemicals in ECs are influenced by product sale location, we evaluated refill fluids manufactured by one company (Ritchy LTD) and purchased worldwide. Flavor chemicals were identified and quantified using gas chromatography/mass spectrometry (GC/MS). We then screened the fluids for their effects on cytotoxicity (MTT assay) and proliferation (live-cell imaging) and tested authentic standards of specific flavor chemicals to identify those that were cytotoxic at concentrations found in refill fluids. A total of 126 flavor chemicals were detected in 103 bottles of refill fluid, and their number per/bottle ranged from 1-50 based on our target list. Two products had none of the flavor chemicals on our target list, nor did they have any nontargeted flavor chemicals. A total of 28 flavor chemicals were present at concentrations ≥1 mg/mL in at least one product, and 6 of these were present at concentrations ≥10 mg/mL. The total flavor chemical concentration was ≥1 mg/mL in 70% of the refill fluids and ≥10 mg/mL in 26%. For sub-brand duplicate bottles purchased in different countries, flavor chemical concentrations were similar and induced similar responses in the in vitro assays (cytotoxicity and cell growth inhibition). The levels of furaneol, benzyl alcohol, ethyl maltol, ethyl vanillin, corylone, and vanillin were significantly correlated with cytotoxicity. The margin of exposure calculations showed that pulegone and estragole levels were high enough in some products to present a nontrivial calculated risk for cancer. Flavor chemical concentrations in refill fluids often exceeded concentrations permitted in other consumer products. These data support the regulation of flavor chemicals in EC products to reduce their potential for producing both cancer and noncancer toxicological effects.

Mitochondrial Stress Response in Neural Stem Cells Exposed to Electronic Cigarettes.

Stem cells provide a sensitive model to study exposure to toxicants, such as cigarette smoke. Electronic cigarettes (ECs) are popular nicotine delivery devices, often targeted to youth and pregnant mothers. However, little is known about how chemicals in ECs might affect neural stem cells, and in particular their mitochondria, organelles that maintain cell functionality and health. Here we show that the mechanism underlying EC-induced stem cell toxicity is stress-induced mitochondrial hyperfusion (SIMH), a transient survival response accompanied by increased mitochondrial oxidative stress. We identify SIMH as a survival response to nicotine, now widely available in EC refill fluids and in pure form for do-it-yourself EC products. These observed mitochondrial alterations combined with autophagy dysfunction to clear damaged mitochondria could lead to faulty stem cell populations, accelerate cellular aging, and lead to acquired mitochondriopathies. Any nicotine-containing product may likewise stress stem cells with long-term repercussions for users and passively exposed individuals. VIDEO ABSTRACT.

High-Nicotine Electronic Cigarette Products: Toxicity of JUUL Fluids and Aerosols Correlates Strongly with Nicotine and Some Flavor Chemical Concentrations.

Whereas JUUL electronic cigarettes (ECs) have captured the majority of the EC market, with a large fraction of their sales going to adolescents, little is known about their cytotoxicity and potential effects on health. The purpose of this study was to determine flavor chemical and nicotine concentrations in the eight currently marketed prefilled JUUL EC cartridges ("pods") and to evaluate the cytotoxicity of the different variants (e.g., "Cool Mint" and "Crème Brulee") using in vitro assays. Nicotine and flavor chemicals were analyzed using gas chromatography-mass spectrometry in pod fluid before and after vaping and in the corresponding aerosols. 59 flavor chemicals were identified in JUUL pod fluids, and 3 were >1 mg/mL. Duplicate pods were similar in flavor chemical composition and concentration. Nicotine concentrations (average 60.9 mg/mL) were significantly higher than those of any EC products we have previously analyzed. The transfer efficiency of individual flavor chemicals that were >1 mg/mL and nicotine from the pod fluid into aerosols was generally 35-80%. All pod fluids were cytotoxic at a 1:10 dilution (10%) in the MTT and neutral red uptake assays when tested with BEAS-2B lung epithelial cells. Most aerosols were cytotoxic in these assays at concentrations between 0.2 and 1.8%. The cytotoxicity of collected aerosol materials was highly correlated with nicotine and ethyl maltol concentrations and moderately to weakly correlated with total flavor chemical concentration and menthol concentration. Our study demonstrates that (1) some JUUL flavor pods have sufficiently high concentrations of flavor chemicals that may make them attractive to youth and (2) the concentrations of nicotine and some flavor chemicals (e.g., ethyl maltol) are high enough to be cytotoxic in acute in vitro assays, emphasizing the need to determine if JUUL products will lead to adverse health effects with chronic use.

Identification of Cytotoxic Flavor Chemicals in Top-Selling Electronic Cigarette Refill Fluids.

We identified the most popular electronic cigarette (EC) refill fluids using an Internet survey and local and online sales information, quantified their flavor chemicals, and evaluated cytotoxicities of the fluids and flavor chemicals. "Berries/Fruits/Citrus" was the most popular EC refill fluid flavor category. Twenty popular EC refill fluids were purchased from local shops, and the ingredient flavor chemicals were identified and quantified by gas chromatography-mass spectrometry. Total flavor chemical concentrations ranged from 0.6 to 27.9 mg/ml, and in 95% of the fluids, total flavor concentration was greater than nicotine concentration. The 20 most popular refill fluids contained 99 quantifiable flavor chemicals; each refill fluid contained 22 to 47 flavor chemicals, most being esters. Some chemicals were found frequently, and several were present in most products. At a 1% concentration, 80% of the refill fluids were cytotoxic in the MTT assay. Six pure standards of the flavor chemicals found at the highest concentrations in the two most cytotoxic refill fluids were effective in the MTT assay, and ethyl maltol, which was in over 50% of the products, was the most cytotoxic. These data show that the cytotoxicity of some popular refill fluids can be attributed to their high concentrations of flavor chemicals.

High concentrations of flavor chemicals are present in electronic cigarette refill fluids.

We characterized the flavor chemicals in a broad sample of commercially available electronic cigarette (EC) refill fluids that were purchased in four different countries. Flavor chemicals in 277 refill fluids were identified and quantified by gas chromatography-mass spectrometry, and two commonly used flavor chemicals were tested for cytotoxicity with the MTT assay using human lung fibroblasts and epithelial cells. About 85% of the refill fluids had total flavor concentrations >1 mg/ml, and 37% were >10 mg/ml (1% by weight). Of the 155 flavor chemicals identified in the 277 refill fluids, 50 were present at ≥1 mg/ml in at least one sample and 11 were ≥10 mg/ml in 54 of the refill fluids. Sixty-one% (170 out of 277) of the samples contained nicotine, and of these, 56% had a total flavor chemical/nicotine ratio >2. Four chemicals were present in 50% (menthol, triacetin, and cinnamaldehyde) to 80% (ethyl maltol) of the samples. Some products had concentrations of menthol ("Menthol Arctic") and ethyl maltol ("No. 64") that were 30 times (menthol) and 100 times (ethyl maltol) their cytotoxic concentration. One refill fluid contained cinnamaldehyde at ~34% (343 mg/ml), more than 100,000 times its cytotoxic level. High concentrations of some flavor chemicals in EC refill fluids are potentially harmful to users, and continued absence of any regulations regarding flavor chemicals in EC fluids will likely be detrimental to human health.

Counterfeit Electronic Cigarette Products with Mislabeled Nicotine Concentrations.

OBJECTIVES: We compared nicotine concentrations in one brand of refill fluids that were purchased in 4 countries and labeled 0 mg of nicotine/mL. We then identified counterfeit e-cigarette products from these countries. METHODS: Overall, 125 e-cigarette refill fluids were purchased in Nigeria, the United States (US), England, and China. Nicotine concentrations were measured using high performance liquid chromatography and compared to labeled concentrations. Refill fluids were examined to identify physical differences and grouped into authentic and counterfeit products. RESULTS: Whereas nicotine was in 51.7% (15/29) of the Nigerian, 3.7% (1/27) of the Chinese and 1.6% (1/61) of the American refill fluids (range = 0.4 - 20.4 mg/mL), 8 British products did not contain nicotine. Products from China, the US, and Nigeria with trace amounts of nicotine (0.4 to 0.6 mg/mL) were authentic; however, all products from Nigeria with more than 3.7 mg/mL were counterfeit. CONCLUSIONS: We introduce 2 novel issues in the e-cigarette industry, the production of counterfeit refill fluids under a brandjacked label and inclusion of nicotine in 81.3% of the counterfeit products labeled 0 mg/mL. This study emphasizes the need for better control and monitoring of nicotine containing products and sales outlets.

Evaluating Cell Processes, Quality, and Biomarkers in Pluripotent Stem Cells Using Video Bioinformatics.

There is a foundational need for quality control tools in stem cell laboratories engaged in basic research, regenerative therapies, and toxicological studies. These tools require automated methods for evaluating cell processes and quality during in vitro passaging, expansion, maintenance, and differentiation. In this paper, an unbiased, automated high-content profiling toolkit, StemCellQC, is presented that non-invasively extracts information on cell quality and cellular processes from time-lapse phase-contrast videos. Twenty four (24) morphological and dynamic features were analyzed in healthy, unhealthy, and dying human embryonic stem cell (hESC) colonies to identify those features that were affected in each group. Multiple features differed in the healthy versus unhealthy/dying groups, and these features were linked to growth, motility, and death. Biomarkers were discovered that predicted cell processes before they were detectable by manual observation. StemCellQC distinguished healthy and unhealthy/dying hESC colonies with 96% accuracy by non-invasively measuring and tracking dynamic and morphological features over 48 hours. Changes in cellular processes can be monitored by StemCellQC and predictions can be made about the quality of pluripotent stem cell colonies. This toolkit reduced the time and resources required to track multiple pluripotent stem cell colonies and eliminated handling errors and false classifications due to human bias. StemCellQC provided both user-specified and classifier-determined analysis in cases where the affected features are not intuitive or anticipated. Video analysis algorithms allowed assessment of biological phenomena using automatic detection analysis, which can aid facilities where maintaining stem cell quality and/or monitoring changes in cellular processes are essential. In the future StemCellQC can be expanded to include other features, cell types, treatments, and differentiating cells.