*4.1. Discussion on BTEX Results*

HS-SPME-GC-MS analysis of 97 e-liquids highlighted BTEX contamination. Experimental data obtained suggest that, during the period 2013–2015, contaminated e-liquids were commercially available on the EU market, particularly e-liquids imported into EU member states and manufactured in China. Taking into account all of the data obtained, no correlation was found between BTEX contamination levels and nicotine content, nor nicotine presence. The variability observed in BTEX contamination levels from one brand to another one is therefore likely to be related to the variability in contamination level of the basic components (i.e., propylene glycol and glycerol) and/or the flavoring additives included. In addition, the variability in BTEX contamination levels observed within the same brand is likely to be related to the flavoring additives used, and in the specific case of samples 10, 11 and 12 C, given the same flavor and nicotine content, to the contamination of basic components used in the production process of different batches. According to Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of substances and mixtures (CLP), benzene, toluene, ethylbenzene and m,o,p-xylenes are included in Annex VI, Table 3. Benzene is classified as carcinogenic for humans (Carc. 1A, H350: May cause cancer by inhalation), mutagenic (Muta. 1B, H340: May cause genetic defects), and represents a hazard when inhaled (Asp. Tox 1, H304: May be fatal if swallowed and enters airways; STOT RE 1, H372: causes damage to organs through prolonged and repeated exposure) [31]. Toluene is classified as reprotoxic (Repr. 2, H361d: Suspected of damaging the unborn child) and represents a hazard when inhaled (Asp.Tox 1, H304: May be fatal if swallowed and enters airways). Ethylbenzene and xylenes are both classified as follows: Acute tox. 4, H332: harmful if inhaled. Given all the information on toxicity classification reported above, more attention has necessarily to be paid to benzene, a human mutagenic and genotoxic carcinogen, detected in some e-liquids at high concentration levels. Therefore, an in-depth analysis of potential health effects due to inhalation exposure to benzene is due. Epidemiological studies over the years have provided evidence of a causal relationship between chronic inhalation exposure to benzene and serious adverse health effects and diseases, from non-cancer health effects (i.e., hematologic diseases and/or functional aberrations of immune, nervous, endocrine systems) to cancer (i.e., myeloid leukemia, non-Hodgkins lymphoma) [46]. Numerous studies have demonstrated that benzene metabolites, especially p-benzoquinone, are involved in the progression from cytotoxicity to carcinogenicity, as they activate oxygenated radical species able to cause DNA damage [47]. It has been estimated that approximately 50% of the quantity of inhaled benzene is adsorbed into the human body. Once introduced into the human body through the respiratory apparatus, benzene is preferentially adsorbed in fat-rich tissues (i.e., fat and bone marrow), owing to its lipophilic nature. Great concern about potential health hazards has been historically linked to occupational exposure (where higher benzene concentrations than in general environments are likely to be encountered) but knowledge on the issue, acquired over the years, has led the scientists and epidemiologists to be more and more focused on health effects induced by long term exposure of the general population to low concentrations of benzene. Although benzene is recognized as a 'non-threshold carcinogen' on the basis of the assumption that any exposure may result in some increase of risk, in the present study the carcinogenic risk related to the inhalation exposure to benzene resulting from the consumption of e-liquids affected by the highest contamination (brand A) has been estimated.

As reported in the results section, across all 97 e-liquids tested, benzene concentration levels ranged from 2.7 μg/L (in samples 3-B and 6-D, both produced in Italy) to 30,200.0 μg/L (sample 3-A produced in China). This means that, if we consider the daily average consumption of e-liquids by a regular vaper approximately equal to 3 ml per day [48], the total amount of benzene potentially inhaled by the vaper within one day would have ranged from 0.0081 μg to 90.6 μg. For the most contaminated Chinese brand (brand A) the total amount of daily inhaled benzene with 3 ml e-liquid consumption would have varied in the range 21.6–90.6 μg. Taking into account a regular vaper represented by an adult person with an average body weight of 60 kg, the daily consumption of brand A e-liquids would

result in benzene exposure of 0.00036–0.00151 mg/kg/day. A carcinogenic risk assessment for benzene may be performed comparing the estimated exposure with derived minimal effect level (DMEL) value, representing the level of exposure expressed as mg/kg/day below which the risk level of cancer is considered tolerable/acceptable (indicative tolerable risk level is 10-5 associated with a life-time risk for cancer of 1 per 100000 exposed individuals). The DMEL value for benzene, derived from reference values reported on Integrated Risk Information System (IRIS) website of United States Environmental Protection Agency (USEPA), is 0.0000182 mg/kg/day [49]. The comparison exposure-DMEL allows to point out that the daily consumption of Chinese e-liquids belonging to brand A would have resulted in a serious inhalation exposure scenario for active users with a risk level of cancer that is not acceptable. These results are of particular concern, also in light of the World Health Organization (WHO) guidelines for indoor air quality, published in 2010, where it is clearly stated that 'no safe level of exposure to benzene can be recommended' and that 'from a practical standpoint, it is expedient to reduce exposure levels to as low as possible' reducing or eliminating activities and materials that may release it [50].
