On the extension of the use of a standard operating procedure for nicotine, glycerol and propylene glycol analysis in e-liquids using mass spectrometry.

INTRODUCTION: Standard operating procedures (SOP), accessible to several laboratories, are essential for product verification. EU-JATC (European-Joint Action on Tobacco Control) SOP and the WHO TobLabNet (World Health Organization Tobacco Laboratories Network) SOP (SOP11) are available standard methodologies to measure nicotine, glycerol, and propylene glycol, and propose mass spectrometer (MS) as an alternative method to flame ionization detector (FID). This study conducted a comparison between FID and MS concentration results, following the MS method described in SOP11. METHODS: In May 2020, five test e-liquids in replicates (A-E) were prepared at the Istituto di Ricerche Farmacologiche Mario Negri and sent, with SOP 11, validation document and results datasheet to 32 different laboratories all over the world from WHO TobLabNet and EU-JATC (18 from JATC, ten from WHO TobLabNet and four academic laboratories). Among thirty-two independent laboratories that participated in the study, results were received from 30 laboratories. RESULTS: The e-liquids analyses, using the two approaches, were compared. Of the 30 laboratories surveyed, 21 utilized the FID approach exclusively, 7 opted for MS detection, and 2 employed both methods. The findings demonstrated that the gas chromatography-mass spectrometry (GC-MS) method offers comparable analytical capabilities regarding accuracy and precision for nicotine, glycerol, and propylene glycol to the FID approach. Through Pearson's correlation test with r≃1 showing a positive correlation between GC-FID and GC-MS data, and the Student's t-test, no significant differences between the two approaches were revealed, showing p>0.005 for almost all three analytes in all samples. CONCLUSIONS: This study indicates that it is possible to apply the available EU-JATC SOP and the WHO TobLabNet SOP11 even in laboratories that do not have access to an FID, for example, to analyze flavors, trace compounds or carcinogenic, mutagenic, or toxic for reproduction (CMR) in electronic cigarette liquids.

Alterations in the molecular regulation of mitochondrial metabolism in human alveolar epithelial cells in response to cigarette- and heated tobacco product emissions.

Mitochondrial abnormalities in lung epithelial cells have been associated with chronic obstructive pulmonary disease (COPD) pathogenesis. Cigarette smoke (CS) can induce alterations in the molecular pathways regulating mitochondrial function in lung epithelial cells. Recently, heated tobacco products (HTPs) have been marketed as harm reduction products compared with regular cigarettes. However, the effects of HTP emissions on human alveolar epithelial cell metabolism and on the molecular mechanisms regulating mitochondrial content and function are unclear. In this study, human alveolar epithelial cells (A549) were exposed to cigarette or HTP emissions in the form of liquid extracts. The oxygen consumption rate of differently exposed cells was measured, and mRNA and protein abundancy of key molecules involved in the molecular regulation of mitochondrial metabolism were assessed. Furthermore, we used a mitophagy detection probe to visualize mitochondrial breakdown over time in response to the extracts. Both types of extracts induced increases in basal-, maximal- and spare respiratory capacity, as well as in cellular ATP production. Moreover, we observed alterations in the abundancy of regulatory molecules controlling mitochondrial biogenesis and mitophagy. Mitophagy was not significantly altered in response to the extracts, as no significant differences compared to vehicle-treated cells were observed.

Coolants, organic acids, flavourings and other additives that facilitate inhalation of tobacco and nicotine products: implications for regulation.

To inform regulatory policy, this article summarises findings on inhalation facilitation from the ninth report of the WHO Study Group on Tobacco Product Regulation. Some additives counteract the harshness and bitterness of tobacco and nicotine product aerosols, making them easier to inhale. Additives that promote inhalability may perpetuate and increase the use of inhaled tobacco and nicotine products, especially by young people. Thus, as a class, additives that facilitate inhalation are an important regulatory target to prevent tobacco and nicotine product uptake. We defined inhalation facilitation as modifications to products during manufacturing that enhance the sensory experience and (potentially) behaviours associated with inhalation (eg, deeper puffs, faster inhalation, larger puff volume, shorter intervals in between puffs and use episodes). Evidence review showed that: (a) menthol and synthetic coolants decrease irritation caused by aerosol constituents by activating sensory perception receptors (eg, cooling receptors) and may promote dependence in inexperienced users; (b) acid additives and sugars, which lower the pH of aerosols and shift nicotine from free-base to protonated salt forms, reduce harshness and increase blood nicotine yield; (c) e-cigarette flavourings perceived as sweet or fruity reduce subjective bitterness, increase attractiveness and may escalate use, although their effects on perceived harshness are inconclusive; (d) sugars in tobacco impart sweet sensations, but limited industry-independent data preclude strong conclusions for sugars' roles in inhalation facilitation. Given these findings, WHO policy recommendations suggest that regulators might consider banning ingredients that facilitate inhalation in all commercial inhaled tobacco and nicotine products.

Determination of Nicotine Protonation State in E-Liquids by Low-Resolution Benchtop NMR Spectroscopy.

Over several years, e-liquids with "nicotine salts" have gained considerable popularity. These e-liquids have a low pH, at which nicotine occurs mostly in its monoprotonated form. Manufacturers usually accomplish this by the addition of an organic acid, such as levulinic acid, benzoic acid, or lactic acid. Nicotine in its protonated form can be more easily inhaled, enhancing the addictiveness and attractiveness of products. Several techniques have been described for measuring the protonation state of nicotine in e-liquids. However, nuclear magnetic resonance (NMR) spectroscopy is particularly suited for this purpose because it can be performed on unaltered e-liquids. In this article, we demonstrate the suitability of a benchtop NMR (60 MHz) instrument for determining the protonation state of nicotine in e-liquids. The method is subsequently applied to measure the protonation state of 33 commercially available e-liquids and to investigate whether the vaping process alters the protonation state of nicotine. For this purpose, the protonation state in the condensed aerosol obtained by automated vaping of different e-liquids was compared with that of the original e-liquids. Two distinct populations were observed in the protonation state of nicotine in commercial e-liquids: free-base (fraction of free-base nicotine αfb > 0.80) and protonated (αfb < 0.40). For 30 e-liquids out of 33, the information on the packaging regarding the presence of nicotine salt was in agreement with the observed protonation state. Three e-liquids contained nicotine salt, even though this was not stated on the packaging. Measuring the protonation state of nicotine before and after (machine) vaping revealed that the protonation state of e-liquids is not affected by vaping. In conclusion, it is possible to determine the nicotine protonation state with the described method. Two clusters can be distinguished in the protonation state of commercial e-liquids, and the protonation state of nicotine remains unchanged after vaping.

Across the world availability of flavour accessories for tobacco products.

OBJECTIVE: This study aimed to provide an inventory of different types of flavour accessories for combustible tobacco products in eight countries varying in their approaches to flavour legislation and cultural aspects, including tobacco use. METHODS: A standardised search protocol was developed and shared with local informants to acquire information on the availability and marketing of flavour accessories in web shops accessible from Brazil, India, Italy, Singapore, South Africa, Switzerland, the UK and the USA. Characteristics of the products and web shops were reported, and flavours were categorised in a flavour wheel. RESULTS: Flavour accessories were available in all participating countries. Reported types are flavour capsules, cards, filter tips and tubes for make-your-own cigarettes, drops, sprays, rolling paper, aroma markers, a flavour stone and a flavour powder. In total, 118 unique flavours were reported, which were mostly fruity and sweet. Marketing of these products was often associated with (menthol) flavour bans. CONCLUSIONS: The wide availability and variety of flavour accessories raise significant public health concerns, as they have attractive flavours, and thus hinder the regulatory aim of flavour bans. Flavour accessories are not tobacco products and thus not regulated as such. Therefore, it is recommended that policymakers include these products in comprehensive flavour bans, to close this loophole in existing tobacco control measures.

Menthol and related compounds in waterpipe products.

INTRODUCTION: The addition of cooling substances, such as menthol, might be attractive for youth to start smoking waterpipe by reducing the harshness of the smoke, thereby facilitating inhalation. These compounds simultaneously increase the addictiveness of tobacco and related products by stimulating nicotine uptake. Some menthol-like compounds also increase attractiveness by imparting a menthol/mint flavor. We provide an overview of the frequency and quantities of use of menthol-like substances in waterpipe tobacco, herbal molasses and steam stones. METHODS: The primary data source of this study was the European Common Entry Gate (EU-CEG). Product names and ingredients were obtained for 282 waterpipe tobacco products notified to The Netherlands in 2020. Subsequently, gas-chromatography-mass spectrometry (GC-MS) analysis was used to quantify seven menthol-like substances and nicotine in waterpipe tobacco (n=5), herbal molasses (n=1) and steam stones (n=12). RESULTS: Of the 282 EU-CEG-notified products, 39% have a menthol/mint declared flavor. GC-MS showed that 15 of the 18 investigated waterpipe products contained one or more menthol-like ingredients. GC-MS analysis showed that products termed 'freeze', 'ice' or 'mint' contained higher median menthol concentrations than products without these terms. CONCLUSIONS: Nearly all investigated waterpipe products contained menthol-like compounds, irrespective of their flavor. Such compounds are known to provide flavoring or cooling effects, and some are known to be carcinogenic. Our results can support the regulation of these substances in waterpipe products. Regulators should screen all waterpipe products, not only those with menthol or a similar indicator in product names.

An ingredient co-occurrence network gives insight into e-liquid flavor complexity.

INTRODUCTION: Part of the appeal of e-cigarettes lies in their available flavors. To achieve attractive flavors, e-liquids contain many different flavoring agents, which allow many flavoring combinations. To advance our knowledge of e-liquid flavors and compositions and to evaluate the effect of legislation, we determined whether there are ingredient combinations that are frequently used together. METHODS: We used e-cigarette ingredient data from the European Common Entry Gate system (EU-CEG) as available on 31 December 2022. RESULTS: In e-liquids, we found 214 ingredient pairs with a co-occurrence odds ratio greater than 10. Together, these consisted of 62 unique ingredients. Network analysis revealed that ingredients were grouped together based on their flavor and/or chemical structure. We identified two densely connected regions (clusters) in the network. One consisted of six ingredients with sweet-vanilla-creamy flavors. The second cluster consisted of 13 ingredients. While some of these have fruity flavors, others, such as alkyl carboxylic acids and dimethyl sulfide, are known to have unpleasant flavors. Additional data and literature analyses indicated that alkyl carboxylic acids can contribute to a creamy and sweet-fruity taste, whereas dimethyl sulfide can contribute to a more refined fruity taste. CONCLUSIONS: These results exemplify that the flavor of e-liquids is not just the sum of its parts. Big data analyses on product data can be used to detect such patterns, but expert knowledge and additional data are needed for further interpretation. Monitoring of e-liquid flavors as well as ingredients will remain important to regulate e-liquid product attractiveness.

Context matters: Neural processing of food-flavored e-cigarettes and the influence of smoking.

E-cigarettes are harmful, addictive, and popular. In e-cigarettes, nicotine is often paired with food-flavors. How this pairing of nicotine and food cues influences neural processing warrants investigation, as in smokers, both types of cues activate similar brain regions. Additionally, while most e-cigarettes are sweet, savory e-cigarettes are seemingly absent, although savory flavors are commonly liked in food. To understand how smoking status and type of flavor modulate reactions to food-flavored e-cigarettes, in comparison to actual food, neural and subjective responses to food odors were measured in a 2 (sweet vs. savory odor) x2 (food vs. e-cigarette context) x2 (smokers vs. non-smokers) design in 22 occasional/light smokers and 25 non-smokers. During fMRI scanning, participants were exposed to sweet and savory odors and pictures creating the two contexts. Liking and wanting were repeatedly measured on a 100-unit visual-analogue-scale. Results show that sweet e-cigarettes were liked (Δ = 14.2 ± 1.7) and wanted (Δ = 39.5 ± 3.1) more than savory e-cigarettes, and their cues activated the anterior cingulate more (cluster-level qFDR = 0.003). Further, we observed context-dependent variations in insula response to odors (cluster-level qFDR = 0.023, and = 0.030). Savory odors in an e-cigarette context were wanted less than the same odors in a food-context (Δ = 32.8 ± 3.1). Smokers and non-smokers reacted similarly to flavored product cues. Our results indicate that the principles of flavor preference in food cannot directly be applied to e-cigarettes and that it is challenging to design sweet and savory e-cigarettes to appeal to smokers only.

Reducing attractiveness of e-liquids: proposal for a restrictive list of tobacco-related flavourings.

OBJECTIVE: Electronic cigarettes are addictive and harmful, and flavour is a key factor determining their abuse liability. Both adult smokers and young non-smokers like sweet and fruity flavours in particular. In order to discourage e-cigarette use among youth, the Dutch government announced in 2020 to only allow tobacco flavours in e-liquids. We propose a restrictive list of flavourings that will only enable the production of e-liquids with a tobacco flavour. METHODS: We used e-liquid ingredient data notified via the European Common Entry Gate system before the government's announcement. First, we classified all e-liquids into flavour categories, and continued with the set of flavourings present in tobacco e-liquids. Five selection criteria related to prevalence of use, chemical composition, flavour description and health effects were defined to compile a restrictive list of tobacco flavourings. RESULTS: E-liquids marketed as having tobacco flavour contained 503 different flavourings, some with tobacco flavour, but also other (such as sweet) flavours. We excluded (1) 330 flavourings used in <0.5% of e-liquids, (2) 77 used less frequently in tobacco than in all e-liquids, (3) 13 plant extracts, (4) 60 that are sweet or not associated with a tobacco flavour and (5) 7 flavourings with hazardous properties. This resulted in a final list of 16 flavourings. CONCLUSIONS: Implementing this restrictive list will likely discourage e-cigarette use among youth, but could also make e-cigarettes less attractive as smoking cessation aid.

Circumventing cigarette regulation: Product characteristics of cigarette-like cigarillos on the Dutch market.

INTRODUCTION: Cigarillos have been reported to provide an alternative to cigarettes with a characterizing flavor, which are banned in the European Union. Additionally, they are cheaper than cigarettes. To better inform policy making, we wanted to gain better insight into the market situation for cigarillos in the Netherlands. METHODS: We analyzed product data notified by manufacturers via the European Common Entry Gate system (EU-CEG), as extracted on the 1 June 2022. First, we identified parameters that allowed classifying cigarillos into cigar-like, cigarette-like and intermediate-type cigarillos. Next, we compared product characteristics for these groups. RESULTS: We identified five parameters that allowed classifying cigarillos into subtypes: product weight, filter presence, leaf tobacco percentage in the product, flue-cured tobacco percentage in the product, and the number of flavorings. Almost a quarter (71/285) of the cigarillos on the Dutch market were classified as cigarette-like. Compared to other cigarillo types, these have a high number of flavorings (average n=43), including many sweet and fruity flavorings. The package types of cigarette-like cigarillos resemble those of cigarettes. However, 85% of the cigarette-like cigarillos are available in smaller pack sizes than allowed for cigarettes. When comparing data over the period 2019-2022, we found a decrease in the number of cigar-like cigarillos and an increase in the number of cigarette-like cigarillos, which hints at a shift in the market composition. CONCLUSIONS: Cigarette-like cigarillos can provide a way to evade cigarette regulation. Moreover, their characteristics make them attractive for consumers, including young people. Regulators should consider amending regulations to close the regulatory loopholes that allow evading tobacco legislation.

Sensory appeal and puffing intensity of e-cigarette use: Influence of nicotine salts versus free-base nicotine in e-liquids.

BACKGROUND: In the US, nicotine salts (with protonated nicotine instead of free-based nicotine) have been reported to lower the harshness and bitterness of e-cigarette aerosols, making it easier to inhale high levels of nicotine. This study aimed to determine whether nicotine salts also increase sensory appeal at lower concentrations (< 20mg/mL). Moreover, and novel, inhalation intensity of both types of e-liquids was compared. METHODS: In a randomized, double-blinded, within-participants design, healthy adults who use e-cigarettes (n=68) vaped tobacco-flavored e-liquids containing 12mg/mL of free-based nicotine or nicotine salt ad libitum, using their own device, during two online sessions (June-July 2021, Utrecht, The Netherlands). The sensory parameters perceived liking, nicotine intensity, harshness, and pleasantness were rated on a 100-unit visual analog scale. The intensity of use was determined by the recorded puff number, duration and interval. RESULTS: Test scores on appeal, harshness and puffing behavior parameters showed no significant differences between the nicotine salt and the free-base condition. The average inhalation time was 2.5seconds. Additional analyses found no significant effect of liquid order, age, gender, smoking status, vaping frequency and familiarity with nicotine salts. Significant positive correlations were found between the sensory parameters except for harshness. CONCLUSIONS: Contrary to a previous study that used higher nicotine concentrations and standardized puffing conditions in a laboratory setting, we did not observe the effects of nicotine salts on sensory appeal in our real-life study paradigm. Moreover, we did not see effects on study parameters related to puffing intensity.

Sensory methods to evaluate perception of flavours in tobacco and other nicotine-containing products: a review.

OBJECTIVES: Sensory methods use human senses to evaluate product attributes. This review provides an overview of the types of sensory methods used to evaluate the perception of flavour in tobacco and other nicotine-containing (ToNic) products and to discuss how sensory data could inform flavoured ToNic product policy. DATA SOURCES: PubMed, Embase and Web of Science. STUDY SELECTION: All peer-reviewed studies evaluating ToNic products using a sensory method published before 23 May 2020. DATA EXTRACTION: Two independent coders completed title/abstract and full-text screening to choose articles for inclusion (Cohen's kappa=0.85, strong agreement). Each coder completed data extraction on half the articles, recording relevant information (eg, sensory methods used, results). The coders categorised sensory methods and generated overarching themes. DATA SYNTHESIS: Of 110 articles identified, we included 29 articles containing 35 studies that used sensory methods to investigate ToNic products. The sensory methods included analytic methods such as discrimination and descriptive tests and hedonic methods such as liking tests. Six themes emerged regarding how sensory methods can be used to understand consumer perception and liking of ToNic products and to inform ToNic product policy. CONCLUSIONS: The identified studies highlight that sensory data can inform ToNic product policy. Analytic and sensory hedonic ratings can be used to assess a ToNic product's ability to promote addiction in the user (ie, abuse liability). Lastly, hedonic ratings can provide information to assess potential use behaviours.

Exploring the gateway hypothesis of e-cigarettes and tobacco: a prospective replication study among adolescents in the Netherlands and Flanders.

BACKGROUND: Studies demonstrated that adolescent e-cigarette use is associated with subsequent tobacco smoking, commonly referred to as the gateway effect. However, most studies only investigated gateways from e-cigarettes to tobacco smoking. This study replicates a cornerstone study revealing a positive association between both adolescent e-cigarette use and subsequent tobacco use; and tobacco and subsequent e-cigarette use in the Netherlands and Flanders. DESIGN: The longitudinal design included baseline (n=2839) and 6-month (n=1276) and 12-month (n=1025) follow-up surveys among a school-based cohort (mean age: 13.62). Ten high schools were recruited as a convenience sample. The analyses involved (1) associations of baseline e-cigarette use and subsequent tobacco smoking among never smokers; (2) associations of e-cigarette use frequency at baseline and tobacco smoking frequency at follow-up; and (3) the association of baseline tobacco smoking and subsequent e-cigarette use among non-users of e-cigarettes. FINDINGS: Consistent with prior findings, baseline e-cigarette use was associated with higher odds of tobacco smoking at 6-month (OR=1.89; 95% CI 1.05 to 3.37) and 12-month (OR=5.63; 95% CI 3.04 to 10.42) follow-ups. More frequent use of e-cigarettes at baseline was associated with more frequent smoking at follow-ups. Baseline tobacco smoking was associated with subsequent e-cigarette use (OR=3.10; 95% CI 1.58 to 6.06 at both follow-ups). CONCLUSION: Our study replicated the positive relation between e-cigarette use and tobacco smoking in both directions for adolescents. This may mean that the gateway works in two directions, that e-cigarette and tobacco use share common risk factors, or that both mechanisms apply.

Flavours and flavourings in waterpipe products: a comparison between tobacco, herbal molasses and steam stones.

ObjectivesFlavoured products are especially appealing to youth and contribute to the onset of waterpipe smoking and continued use of waterpipe tobacco. The goal of database and chemical analysis was to provide a clear overview of commonly used flavours and flavourings in tobacco and related waterpipe products, that is, herbal molasses and steam stones. METHODS: In 2019, 249 waterpipe tobacco products were registered in the European Common Entry Gate by manufacturers to be marketed in The Netherlands. Flavour categories were assigned to the registered products based on their brand names and product descriptions. Nicotine and eleven 1111 flavourings were identified and quantified in waterpipe tobacco (n=8), herbal molasses (n=7) and steam stones (n=4) by extraction and gas chromatography-mass spectrometry (GC-MS) analysis. RESULTS: Flavour categories could be assigned to 237 of 249 registered waterpipe tobacco products. Eight flavour main categories and 48 unique subcategories were identified and presented in a flavour wheel. All registered waterpipe tobacco products were flavoured, and the majority (78%) was fruit flavoured. Herbal molasses contained similar median flavouring levels, and steam stones contained lower median levels compared with waterpipe tobacco. Flavourings in waterpipe products were almost exclusively fruity and sweet, often in combination with menthol/mint flavourings. CONCLUSIONS: This study is the first to present a waterpipe tobacco flavour wheel, providing a quick overview of waterpipe tobacco flavours and thereby aiding communication among experts around the globe. GC-MS analysis revealed that the most prevalent flavourings are present in similar levels in herbal and tobacco waterpipe products. Banning flavourings in all waterpipe products would be a good strategy to reduce waterpipe smoking among youth.

Comprehensive Dutch market data analysis shows that e-liquids with nicotine salts have both higher nicotine and flavour concentrations than those with free-base nicotine.

OBJECTIVES: Recent years have seen an increase in e-liquids containing nicotine salts. Nicotine salts are less harsh and bitter than free-base nicotine and therefore can facilitate inhalation. Because inhalation-facilitating ingredients are banned in the European Union, we assessed the occurrence and characteristics of nicotine salt-containing e-liquids notified for the Netherlands. METHODS: We analysed data for 39 030 products, submitted by manufacturers in the European Union Common Entry Gate system, as extracted on 30 June 2020. RESULTS: Nicotine salts were present in 13% of e-liquids, especially in pod-related e-liquids (73%) and e-liquids registered from 2018 onwards (over 25%). We found six nicotine salt ingredients (NSIs): nicotine lactate, salicylate, benzoate, levulinate, ditartrate and malate. Nicotine salts also occurred as nicotine-organic acid ingredient combination (NAIC), like nicotine and benzoic acid. Nicotine concentrations were twofold higher in e-liquids with NSI (median 14 mg/mL) and NAIC (11 mg/mL) than for free-base nicotine (6 mg/mL). E-liquids with NSI contained a fourfold higher number (median n=17) and concentration (median 31.0 mg/mL) of flavour ingredients than e-liquids with free-base nicotine (n=4, 7.4 mg/mL). In NAIC-containing e-liquids, these were threefold higher (n=12, 21.5 mg/mL). E-liquids with nicotine salts were less often tobacco flavoured but more often had fruity or sweet flavours. CONCLUSIONS: A substantial and increasing share of e-liquids in the Netherlands contains nicotine salts. Their characteristics can make such e-liquids more addictive and more attractive, especially to young and beginning users. Policymakers are advised to consider regulating products containing nicotine salts.

Variations in cigarette brand characteristics: can consumers tell the difference?

OBJECTIVES: Sensory experience is an important determinant of smoking initiation, brand choice and harm perception, but little is known about how cigarette design shapes sensory experience. This study reports which variations in tobacco blend and design characteristics available on the market are likely to be perceived as different by consumers. METHODS: Truth Tobacco Industry Documents was reviewed for studies showing noticeable sensory differences resulting from variations in tobacco blend and design characteristics. These differences were compared with tobacco product data as available in the Dutch section of the European Common Entry Gate (EU-CEG) system on 30 April 2020. RESULTS: Industry documents identified discrimination thresholds for ventilation, pressure drop, tobacco weight, filter length, and tar and nicotine levels in smoke while evidence for other design characteristics was less conclusive. In the 103 different cigarette varieties in the EU-CEG database, five main types of cigarettes could be identified by principal component analysis, differing in (combinations of) design characteristics. The most significant differences between brand varieties were tar, nicotine and carbon monoxide emissions and associated parameters filter ventilation, filter length, cigarette length and tobacco weight. CONCLUSIONS: While some clusters of brand varieties provided a noticeably different product for consumers, in many cases design differences within these clusters did not exceed the expected discrimination threshold. This indicates that many products on the market are not discernibly different for consumers, and that proliferation of brand varieties has a non-sensory purpose, such as marketing. Policy makers should consider limiting available brand varieties and regulating design characteristics to reduce product appeal.

Neuromodulatory and neurotoxic effects of e-cigarette vapor using a realistic exposure method.

The most direct effects of inhaled harmful constituents are the effects on the airways. However, inhaled compounds can be rapidly absorbed and subsequently result in systemic effects. For example, e-cigarette vapor has been shown to evoke local effects in the lung, although little is known about subsequent effects in secondary target organs such as the brain. Traditionally, such effects are tested using in vivo models. As an alternative, we have combined two in vitro systems, which are Air-Liquid-Interface (ALI) cultured alveolar cells (A549) and rat primary cortical cultures grown on multi-well microelectrode arrays. This allows us to assess the neurological effects of inhaled compounds. We have used exposure to e-cigarette vapor, containing nicotine, menthol, or vanillin to test the model. Our results show that ALI cultured A549 cells respond to the exposure with the production of cytokines (IL8 and GROalpha). Furthermore, nicotine, menthol, and vanillin were found on the basolateral side of the cell culture, which indicates their translocation. Upon transfer of the basolateral medium to the primary cortical culture, exposure-related changes in spontaneous electrical activity were observed correlating with the presence of e-liquid components in the medium. These clear neuromodulatory effects demonstrate the feasibility of combining continuous exposure of ALI cultured cells with subsequent exposure of neuronal cells to assess neurotoxicity. Although further optimization steps are needed, such a combination of methods is important to assess the neurotoxic effects of inhaled compounds realistically. As such, an approach like this could play a role in future mechanism-based risk assessment strategies.

Challenges in Predicting the Change in the Cumulative Exposure of New Tobacco and Related Products Based on Emissions and Toxicity Dose-Response Data.

Many novel tobacco products have been developed in recent years. Although many may emit lower levels of several toxicants, their risk in the long term remains unclear. We previously published a method for the exposure assessment of mixtures that can be used to compare the changes in cumulative exposure to carcinogens among tobacco products. While further developing this method by including more carcinogens or to explore its application to non-cancer endpoints, we encountered a lack of data that are required for better-substantiated conclusions regarding differences in exposure between products. In this special communication, we argue the case for more data on adverse health effects, as well as more data on the composition of the emissions from tobacco products. Such information can be used to identify significant changes in relevance to health using the cumulative exposure method with different products and to substantiate regulatory decisions.