Generation of Electronic Cigarette Aerosol by a Third-Generation Machine-Vaping Device: Application to Toxicological Studies

A major unanswered question is whether long-term exposure to e-cig aerosol results in pulmonary toxicity. In addition, the general safety of e-cigs regarding human health is still a matter of controversy. In August 2016, the U.S. FDA expanded its regulatory authority on all tobacco products, including e-cigs. E-cig research, however, is challenging and complex due mostly to 1) the absence of standardized assessments; 2) the wide variety of e-cig devices (~2,800 different models from 466 identified brands)²⁴; 3) over 7,700 unique e-liquid flavors²⁴; 4) the various possible combinations of humectant ratios. Given the complexity of the field, it is essential, in order to face the challenge and generate sound scientific evidence, that careful considerations to the experimental conditions and reproducible processes are employed. In the present study, the focus was put on the description of an e-cig aerosol generation technique that can enable investigators to obtain unique data sets related to realistic and comprehensive e-cig aerosol exposure-related effect continuums. These can be of timely relevance to address e-cig-related safety or toxicity questions for the establishment of regulations on e-cig design features that potentially can have a direct impact on public health policies.

In the present article, meaningful exposure environments were generated using a computer-controlled system able to integrate the latest generation of e-cig devices as well as allowing for predefined or user-defined automated puffing profiles and set operating conditions (e.g., constant power source, standard values of resistance, voltage, or temperature). These automated puffing profiles include the standard conditions: 55 mL puff volume, 3 s puff duration, 30 s puff interval, and square puff profile, from the "Routine analytical machine for e-cigarette aerosol generation and collection – Definitions and standard conditions" provided by the Coresta Recommended Method (CRM) N°81²⁵ (Table 2). Since the system used can generate various automated puffing profiles, it also complies with ISO 20768 (Vapour products – Routine analytical vaping machine – Definitions and standard conditions)²⁶ puffing regime requirements. As expected, e-cig puffing regime standard conditions contrast with the ones from ISO 3308²⁷, which defines the standard conditions for cigarette-smoking machines (35 mL puff volume, 2 s puff duration, 60 s puff interval, and bell puff profile). These differences between cigarette smoking patterns and e-cig vaping patterns among users are well established²⁸. In the present study, the examples and data provided show that aerosols generated from this system and a third-generation e-cig device with adjustable voltage produce high TPM concentrations, reaching up to 0.27 and 0.82 mg per 55 and 70 mL puff, respectively. E-cig aerosols at these concentrations were collected right after the exposure chamber (Table 1-2, Figure 5). The results also show that there is more than a 160-fold difference in the particulate mass per puff produced with voltages varying from 1.8 to 4.8 V (Table 1). This voltage range is characteristic of the operating settings of e-cig devices on the U.S. market, which allow for the application of voltage ranging from 2.9 to 5.2 V²⁹. The results are also consistent with previously published data^18,21 where high levels of TPM collected at the outlet of the e-cig generator were reported for similar topography profiles (1.4 to 5.8 mg/puff). Critical steps within the protocol include adding a few drops of e-liquid to the atomizer prior to each exposure session to ensure a) that no dry burn is produced; b) e-liquid is available in the tank during the entire duration of the exposure; and verify that the e-cig aerosol is generated as expected by taking regular readings on the real-time concentration measurement device. It is well established that e-cig users try to avoid dry puffs, which occur in dry burn conditions. This vaping condition is related to the formation of high levels of aldehydes, including formaldehyde, a known carcinogen and respiratory toxicant^13,30. Therefore, ensuring that this condition is avoided during the exposures is crucial. Finally, in terms of nicotine exposure, mice exposed to e-cig aerosol from a 36 mg/mL nicotine-containing e-liquid for 2 h per day for 28 days (levels of 0.12 mg/puff) presented serum cotinine concentrations of 91 ng/mL (Figure 8); a level similar to that of cigarette smokers (> 100 ng/mL)^31,32,33, which is even lower than that of regular e-cig users (median saliva cotinine of 252 ng/mL)³⁴. It was reported in a vaping topography study that 235 was the maximum number of puffs per day taken by e-cig users^35,36. This is very similar to our exposure profile producing 1 puff every 30-sec for 2-h per day (total of 240 puffs). Thus, this vaping topography profile models e-cig users daily puff consumption and behavior.

Over the past decade, e-cig devices evolved from first-generation, cigarette-like, single-use, low-powered devices, to second-generation removable and refillable tank style devices, and now to third-generation tank-style devices with customizable features²⁴ for 1) the atomizer's coil resistance: the element responsible for heating the e-liquid, and 2) the power controller, which a) can operate at various voltages, b) affects the temperature of the heating element and c) determines whether or not the boiling temperature of the solution is reached^24,37. During e-cig use, the e-liquid is typically heated at 200 °C or greater³⁸, and it is in the aerosol form that its constituents interact with biological matrices. Therefore, the characterization of e-cig aerosol is essential. E-liquids solvents differ in volatility such that solutions composed mainly of PG (70%), which are less viscous and evaporate at a lower temperature³⁷, produce aerosols with relatively smaller particles that increase the user's 'throat hit' experience²⁰. On the other hand, VG-based e-liquids aerosolize at higher temperatures³⁷ and produce aerosols with relatively larger particles which, from a user's experience, increases the flavor and the amount of vapor generated^5,17,39. Thus, it has previously been established that the PG/VG ratio of the e-liquid influences the size distribution of the particles present in the e-cig aerosol^19,20. As shown in Figure 5, using an e-liquid composed of a 50/50 PG/VG ratio, e-cig aerosols with median diameters of ~ 100 nm were obtained. These results are in the same range as those reported by Baassiri, et al.²⁰. This suggests that in addition to the e-liquid base, the exposure parameters, including the e-cig operating settings (resistance, voltage, and power) and puffing profile, may affect the physical characteristics of the aerosols produced. Moreover, the nicotine concentration and flavoring chemicals added to the e-liquid base also can potentially influence the e-cig aerosol physicochemical properties. It was previously shown that an e-liquid that is less viscous produces an aerosol composed of finer particles, resulting in a less dense vapor, yielding a lower TPM concentration¹⁷. Using the same PG/VG ratio for both e-liquids tested, the e-liquid containing 36 mg/mL of nicotine and cinnamon flavoring chemical, implying that it is more diluted than the e-liquid base only (PG/VG + nicotine + cinnamon flavor versus PG/VG alone), appeared less viscous than the e-liquid composed solely of PG and VG. The apparent difference in viscosity between the two e-liquids may explain the disparity in the mass per puff obtained under equal e-cig vaping settings (Table 2). However, lower TPM may not correlate with less harmful aerosol, since the particle size distribution and the chemical characterization of the aerosol must also be considered. Indeed, the thermal degradation of VG and the chemical interactions of the e-liquid components produce emissions of harmful aldehydes, including formaldehyde and acetaldehyde, known to be potent threats to human health^15,17,40. As noted in Table 3, the chemical analysis of the e-cig aerosol produced here revealed that it also contained acrolein, monochlorophenol, catechol and benzothiazole. All are known respiratory irritants, while catechol is additionally classified as possibly carcinogenic to humans (group 2B) according to the International Agency on Research on Cancer (IARC)^41,42,43. This adds to the effects related to the chemistry of the flavoring agent incorporated into the e-liquid. For example, cinnamaldehyde and diacetyl, two of the Flavor and Extract Manufacturers Association high-priority flavoring chemicals for respiratory hazard, when inhaled by workers, have been shown to impair lung function and cause irreversible lung damage (bronchiolitis obliterans, namely 'popcorn lung')⁴⁴. Cinnamaldehyde has been shown to be highly cytotoxic in vitro^45,46,47 and is very popular in e-liquids⁴⁸. In the current study, the presence of cinnamaldehyde was identified in the e-cig aerosol from the cinnamon flavored e-liquid (Table 3 and Figure 7). Overall, this demonstrates the need to analyze e-cig aerosols for both, physical and chemical characteristics.

As mentioned above, the exposure technique described here can be extremely versatile. It can allow for the modifications of the puffing regime (via the software), of the operating features of the e-cig device or even of the type of exposure chamber (nose-only and whole-body) (via the hardware). This provides the investigator with all the flexibility to adapt or adjust the experimental conditions to the need of each research project. Troubleshooting this technique includes ensuring that the connections between the e-cig condenser, tubes, pumps and chambers are adequately secured, and that all chambers are properly sealed (for more detailed information refer to user manual). As noted and as tested in this study, a variety of factors can influence e-cig aerosol production and composition²². These factors are associated with the ratios and constituents of the e-liquid formulation, which impact the chemical component of the aerosol, as well as the selected e-cig device characteristics and operation settings, which influence the heating conditions used to aerosolize the e-liquid, and thus the composition as well as the physical component of the aerosol. E-liquids are composed of GRAS food additives, however, their safety following heating and aerosolization has not been established. Most importantly, e-cig users inhale these aerosols and control the puffing profile as well as the choice of both e-liquid and the operating settings (resistance and voltage) of their e-cig devices. These are key factors which can significantly impact the e-cig aerosol emissions and should therefore be carefully controlled and reported in experimental research.

As most experimental methods, the present e-cig exposure technique has advantages and limitations. While versatile and well suited for toxicological studies, it is also known that mice are nose-breathers and that whole-body exposures may also allow for dermal and oral absorption in addition to the inhalation exposure route. The advantages and disadvantages of using whole-body and nose-only inhalation exposures have been described extensively elsewhere^49,50. While nose-only exposures more closely mimic the inspiration/expiration patterns that govern the transport and deposition of particles in the respiratory tract, this mode of exposure is more stressful to the animals and is not adequate for long-term inhalation studies using large number of animals⁴⁹. In addition, the studies which compared whole-body and nose-only exposures in rodents exposed by inhalation to the same toxicant under the same exposure conditions (TiO₂ nanoparticles, cigarette smoke) found no statistical difference between those two modes of exposure for lung particle deposition and lung responses^50,51. Since the effects induced by chronic exposures to e-cig aerosol are largely undocumented and under-investigated, the e-cig exposure system described in this manuscript is useful for bridging this knowledge gap. Also, the third-generation machine-vaping device used in this study is oriented in a horizontal configuration. There is a possibility that the orientation of the device could have an effect on aerosol production; however, to the best of our knowledge, for third-generation e-cig devices, the orientation variable has not been tested previously. The horizontal orientation is the preferred position for beginner users of e-cig. This helps promote better wicking and minimizes the risks of e-liquid leaking. Thus, the horizontal orientation is representative of vaping behaviors of populations of e-cig users and has been used by other research groups²¹. It is also important to note that the power displayed on the e-cig device may slightly differ from the actual power supplied to the device^22,52, and that therefore it also may be advisable to measure the power supply values externally or use a corded power supply for a steady supply of energy.

There is a substantial research and knowledge gap for biomarkers of toxicity associated with long-term exposure to e-cig aerosols. This exposure system represents a step forward in this field by allowing the investigators to determine the effects of long-term inhalation exposures of animals to aerosolized e-cig liquid. Other existing e-cig exposure methods also have the capability for investigating the impact of puffing regime and operating settings of e-cig devices on toxicological endpoints^19,20,22,53. These exposure systems will help provide scientific evidence for future regulations on new alternative tobacco products. Ultimately, well-conducted and suitable toxicological studies will help better inform the policymakers, healthcare providers and the 9 million Americans that are e-cig users⁴. Most importantly, exposure systems that do not reproduce real-life vaping scenarios should be avoided. E-liquids are typically heated at 200 °C or greater temperatures³⁸ in an e-cig device, therefore, scenarios where the e-liquid is simply nebulized, or warmed to 37 °C and then nebulized⁸, should not be considered as representative of e-cig users consumption. Currently, e-cig consumers may reach potentially harmful e-cig aerosol constituent levels by using design features of third-generation e-cig devices that allow for the adjustment of distinctive heating conditions via changes in the atomizer's coil resistance and the battery voltage. Therefore, more experimental studies are needed to determine the health effects related to chronic inhalation exposures to e-cig aerosols. This begins by establishing reproducible and standardized e-cig exposure systems^25,26. Thus, having a versatile e-cig exposure system that allows for a broad range of exposure scenarios, including automated representative vaping topography profiles, is an asset to the conduct of experimental studies.