*4.2. Discussion of Flavoring Additives Results*

Among the flavoring additives identified, diacetyl is certainly worthy of an in-depth analysis. Diacetyl is a volatile α-diketone and is a natural constituent of many regularly consumed foods (i.e., dairy products, fruits, coffee). Due to its flavor characteristics, it is widely used in the food manufacturing industry as a flavoring additive. It is added to a wide selection of foods and beverages to mainly impart butter and caramel taste and smell, depending on the concentration used. Its use in the food manufacturing industry is approved by competent governmental bodies such as U.S. Food and Drug Administration (U.S. FDA) and the National Institute for Occupational Safety and Health (NIOSH) and is currently authorized in EU member states according to EU Regulation No 872/2012. The potential risks for consumers health associated with the dietary exposure have been deeply evaluated over the years. As a result of safety evaluations, diacetyl has been determined to be 'generally recognized as safe' (e.g., GRAS) by the FEMA Expert Panel, and has been included in the FEMA GRAS list of authorized flavoring substances [51]. The European Food Safety Authority was also asked to take a position on the issue and the final opinion was that, on the basis of the safety evaluations carried out so far, the use of diacetyl in food is of no safety concern for humans. In this regard, however, it is important to point out that toxicological evaluations used to approve and support diacetyl as a flavoring additive in foods are related to ingestion, and therefore do not provide assurance of safety when other routes of exposure are involved, such as inhalation. In the early 2000s, concerns were raised with respect to potential toxicity for humans associated with inhalation exposure to diacetyl following the reported cases of a severe obstructive lung disease in diacetyl-exposed workers at microwave popcorn manufacturing plants in USA [52]. Preliminary evidence of an association between the occupational exposure to diacetyl and adverse effects on human respiratory apparatus has been reported by Kreiss et al., from a decline in respiratory function to development of a rare irreversible lung disease characterized by fixed airflow obstruction, called bronchiolitis obliterans [52]. Extensive scientific research on diacetyl has been carried out from then both confirming preliminary hypothesis on exposure-occurrence of lung disease association and adding new relevant scientific data [53]. Recently published papers have highlighted both neurotoxicity and impairment of cilia function in human airway epithelium [54,55]. Therefore, in light of the knowledge progressively acquired, the inclusion of diacetyl as flavoring additive in the manufacturing process of liquid formulations for e-cigs has rapidly become a much-debated issue in the scientific community due to foreseeable toxicological implications from direct inhalation exposure. In reaction to this, a prompt response came from e-liquids manufacturers with the replacement of diacetyl with 2,3-pentanedione (acetylpropionyl), an α-diketone showing similar flavor properties, but this option was soon revealed to be unsuccessful when scientific data on acetylpropionyl toxicity started to be published [56]. Our findings, although related to a limited number of samples, are in line with the results obtained in previous investigations highlighting the presence of diacetyl in e-liquids commercially available in EU member states in the

pre-TPD implementation period and with characteristic flavors appealing to teenagers and young adults [19,20,30]. Farsalinos et al., 2015 analyzed both liquid and aerosol matrices of a total number of 159 samples purchased from 36 manufacturers and retailers in 7 different countries. Diacetyl was found in 74% of the samples investigated and in a large proportion of sweet-flavored e-liquids, with similar concentrations in both liquid and aerosol. The simultaneous presence of acetylpropionyl also suggested that, instead of being used as a replacement, acetylpropionyl is often used in conjunction with diacetyl. Further, the authors highlighted that, for 47% of diacetyl-containing e-liquids, the daily exposure level (μg/day) for vapers could be higher than NIOSH-defined safety limits for occupational exposure. Barhdadi et al. investigated 12 flavored e-liquids by applying the HS/GC-MS method, properly developed for the screening and quantification of diacetyl and acetylpropionyl in e-liquids. The samples were provided by the Belgium Federal Agency for Medicinal and Health Products and collected either upon inspections in vaping shops or through seizure activity by Belgian authorities in the period 2013–2015, similar to the present study. The authors reported that only two sweet-flavored e-liquids contained measurable amounts of diacetyl and the determined concentrations were 6.04 μg/g and 98.84 μg/g. Finally, 42 e-liquids selected from among the 14 most popular brands dominating both the USA and EU markets in 2013 were investigated by Varlet et al. in terms of chemical and biological constituents. Diacetyl was detected in three e-liquids, two of them characterized by tobacco flavors and one by candy flavor. Similarly to Farsalinos et al., comparison with the NIOSH safety limit was made, revealing that one tobacco flavored e-liquid that resulted diacetyl-positive could lead to exposure higher the recommended limit. Although approximate for estimating risk for e-cig users, the use of occupational exposure limits is affected by several limitations [19,57]. This approach has raised some resistance, mainly because occupational safety limits for toxicants, for instance for diacetyl, have been set for workers not for the general population and are related to inhalation exposure scenarios not applicable to e-cigs users. According to the authors' knowledge, other two studies carried out by Allen et al. in 2017 and Omayie et al. in 2019 have raised concerns about diacetyl, confirming its inclusion as flavoring additive in refill liquids for e-cigs (diacetyl detected in 39 of 51 tested refills and in 150 of 277 samples, respectively), but in both cases the investigated samples were considered dominating the current extra-EU market and therefore are not representative of the EU market before the implementation of TPD. To summarize, our findings on diacetyl, although related to a limited number of e-liquids manufactured in China and commercially available in the EU during the period 2013-2015, are in line with the results obtained in other investigations made on larger sets of samples representative of the EU market at that time. The only discrepancy on diacetyl presence detectable among the studies performed before the TPD implementation was reported by Girvalaki et al. in 2018. The authors evaluated the chemical composition of 122 e-liquids selected among the most commonly sold brands in 9 EU member states in mid-2016 before the TPD implementation. The result of this comprehensive investigation was a list of 177 compounds detected (e.g., flavoring additives and other VOCs), the majority with associated Globally Harmonized System of Classification and labeling of Chemicals (GHS) health hazard statements. Diacetyl, however, was not detected in the samples tested, and therefore not included in the list. This discrepancy between Girvalaki et al. and the other abovementioned studies may be related or to the different period of e-liquids selection (2013–2015 versus 2016), although both periods were before TP -implementation, when the first actions aimed to the progressive replacement/elimination of diacetyl started to be made on a voluntary basis by some EU manufacturers and importers, or it simply reflects the potential heterogeneity due to the multitude of samples commercially available on the EU market in the period of reference. To date, following the implementation of TPD in most EU member states in 2016, both manufacturers and importers are obliged to submit a notification to competent authorities reporting detailed information on refill liquids (Article 20) [13]. The notification must report the list of all the ingredients (including flavoring additives) contained in e-liquid formulations for e-cigs available on the market and indication of related quantities as well. It must be noted, however, that according to TPD, the use of diacetyl is neither explicitly prohibited nor subjected to restriction. In addition, due to difficulty in defining a

typical inhalation exposure scenario fitting all vapers habits (high variability in daily e-liquid amount consumed), there is no scientific consensus on the maximum allowed level of diacetyl in e-liquids. Therefore, to date, diacetyl use as a flavoring additive in e-liquids remains an open issue, suggesting not only that quality controls remain necessary, even in e-liquids labelled as diacetyl-free, but also that the potential solution at the EU level to ensure that e-liquids supplied to consumers are safe is to follow the direction of some EU member states that proposed the ban of diacetyl and other flavoring additives of concern [58].
