**5. Conclusions**

In the present paper, results from a study on the chemical characterization of levels of BTEX in 97 e-liquids, representative of the EU market between 2013 and 2015 prior the implementation of TPD in most EU member states, are reported. To our knowledge, there have been very few studies focused on BTEX analysis in refill fluids and cartridges for e-cigs commercially available on the EU market in the pre-TPD implementation period. Therefore, although the e-liquids investigated may not be representative of the current EU market, our findings may represent a useful reference for the ongoing evaluation on the effectiveness of e-liquid safety and quality requirements under the current legislative framework. Most of the e-liquids investigated were revealed to be affected, to a lesser or greater extent, by BTEX contamination. Few exceptions were observed (12 of 97 samples). High variability in BTEX total concentration level was observed from one brand to another, ranging from 2.7 μg/L to 32,151.1 μg/L. The contamination is likely to be related to the contamination of propylene glycol and glycerol, and/or the flavoring additives used. No correlation was found between BTEX concentration levels and nicotine content/presence. Moreover, it was estimated that an inhalation exposure of very high concern would have occurred for active users vaping the most contaminated e-liquids (brand A), characterized by high concentration levels (7,200–30,200 μg/L) of benzene, a known human carcinogen. Our findings, therefore, point out that higher quality ingredients should have been used and that quality control on the formulations should have been applied prior their introduction on the EU market in 2013–2015 period. Further investigations carried out on a limited number of e-liquids aimed at the identification of flavoring additives through GC-MS-O application confirmed, in the reference period of the present study, the use of diacetyl, a flavoring additive approved for foods but associated with the onset of a severe lung disease when inhaled. This finding is in line with results obtained by other investigations made in the same period on a larger number of e-liquids sold in EU, highlighting the use of diacetyl in the e-liquid manufacturing industry due to poor awareness of the potential harm to humans. There are now sufficient toxicological data on the potential adverse effects of diacetyl and other flavoring chemicals when directly inhaled into the human airways, and therefore harmonized regulation at EU level on flavoring additives use in e-liquids, resulting in ban or restriction, should be fully addressed, in order to ensure health protection.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4433/11/4/374/s1, Figure S1: Comparison of the Chromatograms of a blank sample and a sample spiked with the BTEX standard solution (calibration level 3).

**Author Contributions:** Conceptualization: J.P., R.D. and G.d.G.; methodology: J.P., C.A., M.F. and L.P.; investigation: J.P., C.A., M.F. and L.P.; data curation: J.P., C.A., M.F., A.D.G. and L.P.; writing—original draft preparation: J.P., A.D.G. and L.P.; supervision: R.D. and G.d.G. project administration: C.A., M.F., G.d.G. and R.D.; funding acquisition: G.d.G. and R.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** The work has been carried with the coordination of the Italian National Institute of Health in the framework of the National project entitled 'New articles and new health risks: the electronic cigarette' (project code: CCM 2013, date of approval: 12 December 2013), financially supported by the Ministry of Health of Italy.

**Conflicts of Interest:** The authors declare no conflict of interest.
