**1. Introduction**

Electronic cigarette (e-cig) use has increased extremely quickly worldwide over the last decade due to an intense marketing campaign aiming to advertise them as an aid to reducing and/or eliminating addiction to tobacco cigarette smoke [1]. Emerging in 2006 in China, e-cigs became widely available on the market throughout the world in 2008–2009. EU Commission public opinion surveys focused on the smoking attitudes of European citizens across 27 European Union (EU) member states highlighted that

e-cig consumption increased from 7.2% to 11.6% between 2011 and 2014 and is expected to increase further [2]. Despite the claims of manufacturers and retailers advertising e-cigs as a healthier way to smoke nicotine and other chemicals in public places, to date reliable sociological data confirming the effectiveness of e-cigs use in changing smokers' behavior (e.g., smoking cessation and/or reduction) are not exhaustive enough to draw certain conclusions [3–5]. On one hand, public opinion surveys have provided data suggesting a relationship between e-cig consumption and quitting and significant reduction of traditional tobacco smoking [6]. On the other hand, however, scientific research still raises doubts regarding the role of e-cigs in smoking cessation and highlights the interchangeable and simultaneous use of e-cigs with tobacco cigarettes [7,8]. Moreover, a controversial debate is still ongoing within the scientific community on potential adverse effects on the health of both users and bystanders. Concerns about e-cig consumption, specifically related to e-liquids composition, are: (a) the potential inhalation exposure to chemicals of concern present in e-liquid formulations as contaminants of the main ingredients (i.e., aromatic hydrocarbons, aldehydes, PAHs, heavy metals); (b) the potential exposure to harmful by-products formed during the vaporization process; and (c) the unknown and unpredictable long-term health effects due to flavoring additive and main ingredient (i.e., glycerol and propylene glycol) inhalation exposure [9–12]. In view of the health-related concerns raised by the international scientific community and EU member states' competent authorities, specific provisions concerning e-cigs manufacture, labelling, and advertising were included in the EU Tobacco Products Directive 2014/40/EU (TPD), entered into force on May 2014 and fully implemented in EU countries between 2016 and 2018 [13]. E-liquids, available on the market in bottles or in replaceable cartridges, are basically a mixture of propylene glycol, glycerol, and water (the latter generally in smaller quantities). The inclusion of propylene glycol and glycerol in e-liquid formulations is common due to humectant and solvent properties, although the use of other chemicals, such as ethanol (EtOH), has been recently reported in literature [14]. This basic formulation may be enriched with nicotine (in variable and allowed quantities) and a wide selection of flavoring additives, in order to provide users a satisfying and enhanced sensory perception while vaping.

### *1.1. Flavoring Additives*

It is estimated that several hundred flavoring chemicals are currently used for e-liquid formulations, allowing consumers to choose on the market among several flavors belonging to menthol, tobacco, fruit (i.e., cherry, blueberry, strawberry, apple), sweets (i.e., caramel, vanilla, liquorice, chocolate) categories, to mention the most popular ones [15,16]. Scientific reports on addictive behaviors highlighted the key role of flavors in vaping initiation, especially among young adults, and the resulting addiction along to nicotine [17]. The inclusion of flavoring additives in e-liquids is one of the most debated issues. They are approved in foods, beverages, and cosmetics and included in the Generally Recognized As Safe (GRAS) list of the Flavors and Extracts Manufacturers' Association (FEMA); therefore their use is intended through ingestion and dermal contact routes, not for direct inhalation. As a result, both short- and long-term effects due to inhalation exposure cannot be predicted. Due to the lack of epidemiological data able to elucidate the issue and to be reliable foundations for human risk assessment, precautionary measures have to be taken. Moreover, besides this general precautionary principle, specific flavorings are worthy of further attention for their potential toxicity. For instance, 2,3-butanedione (usually named diacetyl) has been widely used in the past in microwave popcorn in the USA with the purpose to generate, depending on the concentration, buttery and caramel tastes. It is a chemical mentioned in the GRAS list and approved in certain limits for ingestion, therefore it is used as additive in foods [18]. Due to its flavoring properties it is also used in the manufacturing process of e-liquid formulations and its presence has been documented in previous investigations carried out in EU Member States, raising concerns in the scientific community regarding potential health implications [19,20]. In this regard, recently published scientific papers based on epidemiological data collected over recent decades have revealed that inhalation exposure to diacetyl is likely related to increased risk of a specific lung disease called bronchiolitis obliterans [21,22]. The use of flavoring

chemicals for e-liquid manufacture stimulated scientists to focus on safety and quality aspects of the formulations. As a result, the number of scientific publications on the chemical characterization of e-liquids in terms of flavoring additives has recently increasing. To cite the most recent studies, in 2017 Aszyk et al. carried out a comprehensive determination of flavoring additives on 25 e-liquid samples highlighting that limonene and benzyl acetate were the two most frequently detected [23]. In 2018 Girvalaki et al. reported findings from qualitative and quantitative analysis performed on 122 of the most commonly sold e-liquids in 9 EU member states. Among the 293 flavoring chemicals identified, menthol was the most frequently detected compound, regardless the overall e-liquid flavor [24]. Specific flavoring chemicals with known respiratory irritant properties or identified as inhalation toxicants were detected in other studies in relevant amounts, i.e., benzaldehyde by Kosmider et al., methyl cyclopentenolone and menthol by Vardavas et al., diacetyl and acetylpropionyl by Barhdadi et al. [19,25,26].
