1. Introduction
Combustible cigarettes (CCs) are the number one cause of preventable death in the world [1]. Researchers, therefore, have worked for decades to characterize CCs and the harmfulness of smoking [2]. Resultantly, countries or regions (e.g., the European Union) have passed laws providing regulatory agencies with the authority to enact regulations and programs that mandate reporting and reducing harmful CC smoke constituents in the interest of protecting public health [3,4,5,6,7,8,9].
Electronic nicotine delivery systems (ENDSs), also known as e-cigarettes, have risen in popularity over the past 15 years as an alternative to CCs, and therefore, regulatory authorities have a concomitant interest in evaluating the potential toxicity of ENDS emissions relative to CC smoking. The harmful or potentially harmful constituents (HPHCs) in CC smoke are well documented to be linked to a number of tobacco-related diseases such as cancer and cardiovascular and respiratory diseases [1]. In contrast to cigarettes, ENDSs have been designed under the concept of delivering nicotine without combustion, as the byproducts of tobacco combustion, rather than nicotine itself, have been shown to be responsible for smoking-related diseases (cancer, cardiovascular and respiratory diseases, and reproductive effects) [10,11,12].
As a technology, ENDSs most often contain a battery, a heating element (comprising a wick and coil), and a nicotine-containing solution, commonly known as e-liquid [13]. The heating of the e-liquid produces an aerosol that is then inhaled by the consumer. Product characteristics (i.e., coil temperature, wicking, etc.) and formulation composition (i.e., ingredients, ingredient amount, etc.) can impact both chemical composition and biological activity [14,15,16]. Unlike tobacco smoke, which is a highly complex mixture (>5000 constituents), ENDS aerosols have been reported to exhibit significant reductions in both the number of measurable analytes and their abundance due to the absence of tobacco leaf and combustion byproducts [2,17,18,19,20,21]. A growing body of literature on ENDS products reports lower levels of specific aerosol toxicants such as tobacco-specific nitrosamines, carbonyls, and volatile organic compounds [13,15,22,23,24,25,26,27,28,29,30,31,32,33]. Correspondingly, depending on the device and formulation, e-liquids and ENDS aerosols have exhibited lower in vitro and in vivo biological activity as compared to CCs [16,34,35,36,37,38,39,40,41,42].
As new products continue to emerge with variations in device design, performance parameters, and formulation composition, regulatory agencies continue to advance science-based regulatory frameworks. Within the U.S., in 2016, a Deeming Rule was finalized granting the U.S. Food and Drug Administration (US FDA) the authority to regulate ENDSs [43]. In 2019, the US FDA announced a comprehensive plan outlining a continuum of risk for nicotine products, with CCs at the highest end of the spectrum and nicotine replacement therapies at the lowest end of the spectrum [44]. Later in 2019, the US FDA issued a proposed rule and further published industry guidance on obtaining market authorization for ENDS products, which included a selection of relevant HPHCs and other chemicals to measure in ENDS aerosol [45,46,47,48]. Globally, government authorities and standardization bodies such as the European Committee for Standardization (CEN), European Union (EU), British Standard Institute (BSI), French Standardization Association (AFNOR), Health Canada (HC), and National Standard of the Peoples Republic of China have established or adopted ENDS-pertinent regulations relative to product manufacturing, e-liquid constituents, emission characterization, or constituent limits [7,8,9,49,50,51,52,53,54,55,56,57,58,59,60,61,62]. To assist in compliance with the growing body of standards and established regulatory frameworks and to support the evaluation of potential health-related claims, we propose in this paper a simplified framework to guide manufacturers on the characterization and assessment of new/modified products before introduction to the market. While other product evaluation frameworks have been proposed, these frameworks may include recommendations such as clinical trials, long-term surveillance, and population modeling studies or involve comprehensive systems toxicology assessments [63,64,65,66]. These frameworks add valuable analyses but are less suited for early assessments of novel tobacco products and do not emphasize the thorough chemical analysis required for their evaluation.
Importantly, the recommendations presented within this manuscript are not intended as a substitute for the legal standards that regulatory agencies are authorized or required to apply.
2. A Proposed Framework: Chemical and Toxicological Evaluation
There are three primary aspects of novel ENDS product evaluation, the e-liquid, the device (battery and pod components), and the aerosolized emissions. Presented in Figure 1, this framework proposes a three-step process to cover all aspects within this evaluation: (1) a preliminary toxicological assessment of product components, ingredients, additives, and aerosol properties; (2) characterization of the product aerosol via analytical chemistry and in vitro toxicity studies; and (3) qualitative risk assessment against CC smoke (or relevant comparator product) to evaluate the harm reduction potential of the novel ENDS product [46]. Fundamentally, the primary objective of this framework is to provide a practical approach to novel ENDS product characterization followed by a qualitative risk assessment for the determination of reduced risk potential. This paper applies the proposed framework to six test articles as a case study providing insights into how to conduct chemical and toxicological studies of this kind.
Prior to the chemical and toxicological evaluation of aerosols, the framework begins with a toxicological review of the novel ENDS device componentry/properties with a focus on components in the container closure system, components in the aerosol path, and toxicological evaluation of all ingredients used to make the e-liquid(s). The toxicological work on the ENDS product is usually performed in two parts. The first involves screening early in product development designed to avoid potentially carcinogenic, cytotoxic, mutagenic, reprotoxic, sensitizing, or inflammatory materials or additives that reduces the likelihood of potential downstream toxicological findings [64,67]. The second involves extractable and leachable studies on the container closure system and parts in the airpath. This evaluation could be similar to what is performed on medical devices or combination device/drug products [68,69,70,71,72,73,74,75,76,77]. Although depicted in Figure 1 as occurring prior to the chemical and toxicological evaluation of the product aerosol, these studies may be performed in parallel.
Some countries have proposed or finalized lists of allowed or prohibited ingredients for ENDS products (e.g., Canada, EU, China) [7,9,60]. Prohibited ingredients typically include those that are recognized as carcinogenic, mutagenic, or reproductive and developmental toxicants. Once regulatory requirements are satisfied, the preliminary toxicological assessment of ingredients uses well-established quantitative risk assessment techniques to evaluate the individual health risk of constituents from inhalation exposure [78,79,80,81,82,83]. This approach is consistent with the recent adoption of CEN EN 17648:2022 within the EU and builds upon a previously published approach to e-liquid risk assessment [55,67]. A key requirement of the ingredient quantitative risk assessment is to have an estimated daily product usage, which may change during product development.
In product aerosol characterization, differences in design and engineering play an important role in the variability of total aerosol yield, the level of aerosol constituents, and nicotine dose [15,84,85,86]. Differences in aerosol composition may result in key differences in toxicant reduction between ENDS products as compared to CCs [14,26,84]. Performing in vitro studies to assess cytotoxicity, mutagenicity, and genotoxicity provides information on key toxicological endpoints relevant to noncancer and cancer adverse effects associated with tobacco product use, and therefore, these studies are informative of the potential toxicity of new ENDS products. Concluding the evaluation, a qualitative risk assessment is performed on the chemical and toxicological studies with the focus of these assessments to establish that, compared to CCs or appropriate comparator products, these products have no new or increased hazards [87].
Case Study Background: Characterization of JUUL2 System with Six Novel Formulations
The JUUL2 product launched in the U.K. in September 2021 and is a temperature-regulated ENDS that consists of a closed (1.2 mL capacity) pod and technologically advanced device (i.e., smartphone-compatible with device locking, tracking, and usage analytic features). Utilizing the JUUL2 System, six pre-filled e-liquid formulations at an 18 mg/mL nicotine concentration were chosen for evaluation: Virginia Tobacco (VT), Crisp Menthol (CM), Polar Menthol (PM), Autumn Tobacco (AT), Ruby Menthol (RM), and Summer Menthol (SM). Following the proposed framework, the device componentry and ingredient disclosures were toxicologically assessed, and products were cleared for further chemical and toxicological analyses.
3. Materials and Methods
3.1. Targeted Analytical Chemistry Stability Study
For the analysis of chemical constituent levels in ENDS aerosol, stability studies were conducted in general accordance with relevant regulatory frameworks [9,46] and guidance documents [88,89]. One, commercially representative, pre-production lot of each formulation was manufactured, shipped, and placed in stability for 12 months with analytical evaluation performed at 0, 3, 6, 9, and 12 months under long-term (LT) storage conditions (25 ± 2 °C/60% RH ± 5%).
To determine selected aerosol constituents for evaluation, U.S., U.K., Asian, EU, and European Member State guidance documents were considered. A total of fifty-four aerosol constituents were measured across ten analytical methodologies (Table 1). Based on chemical classification, measured constituents were grouped into the following categories for discussion and interpretation: (1) Primary Constituents, (2) Carbonyls, (3) Glycidol, (4) Nicotine Degradants, (5) Tobacco-Specific Nitrosamines (TSNAs), (6) Metals, (7) Organic Acids, (8) Volatile Organic Compounds (VOCs), (9) Esters and Alcohols. In addition, aerosol (10) pH was also measured. Five replicates (n = 5) were utilized per analytical assay, per time point evaluation.
Aerosol constituent analysis was performed by Enthalpy Analytical, LLC (Durham, NC, USA; Richmond, VA, USA). All analytical methods were validated for the analysis of ENDS aerosol according to International Council for Harmonisation (ICH) guidance Q2 (R1) [90]. All analytical method validations were reviewed by an independent accreditation body as part of their International Organization for Standardization (ISO) 17025 accreditation process; for more information, see the Supplementary Materials (File S1) [91].
For the collection of ENDS aerosol, whole-pod collections, determined from end-of-life (EOL) determination experiments, were utilized following the approach outlined by Jameson et al. (2023) [92]. Batteries were charged before use and changed after each collection. Two collection regimes were used for the JUUL2 System (e.g., pod and device), the puffing regime described in ISO 20768:2018 (non-intense (NI)) of a 55 mL puff volume, 3 s puff duration, and 30 s inter-puff interval (55/3/30) [93], and an intense (INT) puffing regime of a 110 mL puff volume, 6 s puff duration, and 30 s inter-puff interval (110/6/30). Both puffing regimes utilized a square wave puffing profile. For the JUUL2 products, puff counts utilized in targeted analyses ranged from 100 to 150 puffs under non-intense puffing and 68 to 90 puffs under intense puffing with an average cumulative DML (device mass loss) target of 1085 mg per collection.
Aerosols were collected utilizing a linear 20-port e-cigarette puffing machine (Cerulean SM450e, Milton Keynes, U.K.); see the Supplementary Materials for an example of the collection apparatus (File S1, Figure S1). Primary Constituents, Nicotine Degradants, and TSNAs methodologies trapped aerosol on a conditioned 55 mm glass fiber filter pad (GFFP). Pads were placed in a vial containing extraction solution and agitated for analysis. Organic Acids, Glycidol, and pH methodologies effervesced aerosols through one or more impingers containing trapping solution followed by sample clean-up or derivatization. For Carbonyls, Esters, and Alcohols, an in-tandem (conditioned) GFFP and impinger setup was utilized. Pads were mixed with impinger trapping solution followed by agitation, sample clean-up, or derivatization. For VOCs, a sorbent tube was utilized to trap aerosols; tubes were removed, added to an extraction vial containing trapping solution, agitated, and aliquoted for analysis. For Metals analyses, to minimize potential background contamination introduced from the collection setup, samples were collected using an SM450e equipped with a Halder Process Solutions HPS-EP5 electrostatic precipitator (EP) system (Halder Process Solutions, Moseley, VA, USA). This system includes a quartz glass tube equipped with a tungsten electrode whereby high voltage is applied between the tube and electrode causing aerosol particles to acquire charge and deposit on the tube walls. Tubes were subsequently rinsed with semiconductor-grade methanol to extract and digest the collected aerosol for analysis.
Select methodologies and constituents such as VOCs, TSNAs, Esters, and Alcohols were measured at initial timepoints and removed from subsequent analysis based on a lack of presence, minimal detection levels, or the unlikelihood of formation within the aerosol. For analysis and interpretation, all values were reported on a per-collection basis (also referenced as “whole-pod collection”). These results were compared to combustible reference cigarettes smoked under non-intense (35:2:60, ISO 3308:2012) and intense (55:2:30 ISO 20778:2018) puffing regimes compiled from published literature or internally sponsored studies [94,95]. See the Supplementary Materials (File S1) for additional method and comparison details.
3.2. In Vitro Toxicity Assessment
The in vitro toxicological evaluation was based upon the testing recommended for tobacco products by the Cooperation Center for Scientific Research Relative to Tobacco (CORESTA) [96]. Accordingly, three established toxicological assays, testing for cytotoxicity, mutagenicity, and mammalian genotoxicity, were performed using ENDS aerosol and CC smoke condensates (Section 3.3) in accordance with the standardized Organization for Economic Co-operation and Development (OECD) guidelines. All assays were also performed in accordance with U.S. FDA Good Laboratory Practice (GLP) regulations. The neutral red uptake (NRU) assay was performed using murine 3T3 and human A549 cells to evaluate the potential cytotoxicity of ENDS product aerosols and reference cigarette (3R4F) smoke in accordance with the OECD 129 guidance [97]. The ISO 10993-5 criterion of 70% cell viability was used to determine cytotoxicity [98]. Mutagenicity was measured in the TA98, TA100, TA102, TA1535, and TA1537 strains of Salmonella typhimurium using the reverse mutation (Ames) assay with preincubation in accordance with the OECD 471 guidance, and genotoxicity was evaluated in human TK6 cells using the in vitro micronucleus (MN) assay in accordance with the OECD 478 guidance [99,100,101]. Details have been previously published and are also provided in the Supplementary Materials (File S6) [102].
3.3. Aerosol and Smoke Generation and Chemical Characterization of Condensates Prepared for Toxicological Assays
Condensate production from JUUL2 ENDS pods was initiated alongside T = 0 month targeted analytical chemical stability studies and was performed using the same two puffing regimens and in the same manner as used for the stability studies. Each condensate was collected from a single lot of JUUL2 pods. Condensates were generated by collecting JUUL2 System aerosol on a non-conditioned 55 mm GFFP, connected in series to a glass impinger containing 20 mL of United States Pharmacopeia (USP)-grade ethanol, chilled in an ice bath (~0 °C). Batteries were charged before use and changed after each 50-puff collection until the pod EOL puff count was reached. Aerosol was collected across multiple pods until a target 1200 ± 100 mg aerosol collected mass (ACM) was reached on the GFFP. Upon completion of the aerosol collection, the GFFP was extracted in the contents of the impinger, and the mixture was filtered through gauze to produce the final condensate sample which was aliquoted in vials and stored frozen (<−65 °C) pending chemical and biological analysis.
The 3R4F reference cigarettes were obtained by Enthalpy Analytical and originally sourced from the University of Kentucky. Conditioning of the reference cigarettes conformed to ISO 3402 [103]. Smoke was produced with a rotary smoking machine (Borgwalt RM20, Hamburg, Germany) using the ISO 20778 smoking regime (55 mL puff volume, 2 s puff duration, 30 s interval with 100% ventilation holes blocked) [95]. Mainstream cigarette smoke was collected using a 92 mm GFFP connected in a series to an impinger as described above and stored frozen (<−65 °C) pending chemical and biological analysis.
To demonstrate the stability of the collected condensates during in vitro testing, the concentrations of primary constituents were analyzed at 0, 4, and 8 weeks (5 replicates) after ENDS aerosol condensate collection; see the Supplementary Materials (File S6) [102]. The chemical analysis methods were the same as those used in the targeted analytical chemistry stability studies and were performed by the same contract research organization.
4. Results
4.1. Chemical Evaluation
The complete list of JUUL2 aerosol quantitative chemistry stability results, applied calculations, source literature, and analytical CC values can be found in the Supplementary Materials (Files S1–S5).
4.1.1. Aerosol Chemical Characterization
Following the proposed framework, over the 12-month study, quantifiable analyte stability trends were evaluated and whole-pod collection results were normalized on a mg/mg nicotine basis for CC comparison. Aerosols generated from the six JUUL2 formulations (VT, CM, PM, AT, RM, SM) were analyzed for fifty-four (54) measured constituents, including HPHCs (established and proposed) under non-intense and intense puffing regimes. The majority (34 out of 54) of the measured constituents were either below the limit of quantification (LOQ) or below the limit of detection (LOD) in the JUUL2 System aerosols. Twenty (20) constituents were present at levels greater than the LOQ: nicotine, glycerol, propylene glycol, water, menthol (in mentholated formulations), benzoic acid, acetaldehyde, acrolein, formaldehyde, glycidol, β-nicotyrine, cotinine, myosmine, nicotine-n-oxide, nornicotine, NNN, chromium, iron, selenium, and ethyl acetate; see the Supplementary Materials (Files S2–S5).
4.1.2. Comparison to Combustible Cigarettes
The JUUL2 device is designed for adult smokers of combustible tobacco products, and available data indicate that smokers switch to these products when used as intended [104,105]. Therefore, an HPHC and chemical comparison to combusted cigarettes as a comparator product was deemed appropriate for the evaluation of changes in potential user exposures and subsequent changes in health risks associated with switching from combusted cigarettes to the JUUL2 System. Due to product and collection differences, aerosol and smoke comparisons were conducted on a per mg nicotine basis. The maximum nicotine-normalized value across T0-T12 was utilized for JUUL2 aerosol comparison. For the CC smoke, available published literature and internally conducted studies on 1R6F and 3R4F Kentucky reference cigarettes were utilized; see the Supplementary Materials (File S4).
Comparing ISO non-intense [94] CC smoke to ISO non-intense [93] JUUL2 aerosol, thirty-three (33) HPHC and chemical comparisons were evaluated. Excluded comparator constituents (21) include myosmine, arsenic, beryllium, cobalt, gold, iron, selenium, silver, tin, zinc, benzoic acid, propionic acid, 1,3, butadiene, isoprene, 1-butanol, benzyl acetate, ethyl acetate, ethyl acetoacetate, isoamyl acetate, isobutyl acetate, methyl acetate. Comparing ISO intense [95] CC smoke to intense JUUL2 aerosol, thirty-four (34) HPHC and chemical comparisons were evaluated. Excluded comparator constituents (20) include arsenic, beryllium, cobalt, gold, iron, selenium, silver, tin, zinc, benzoic acid, propionic acid, 1,3 butadiene, isoprene, 1-butanol, benzyl acetate, ethyl acetate, ethyl acetoacetate, isoamyl acetate, isobutyl acetate, methyl acetate. For all product (i.e., device and formulation)-specific comparisons between JUUL2 aerosol and CC smoke, see the Supplementary Materials (File S5).
Figure 2 and Figure 3 demonstrate marked reductions in overall levels of select HPHCs and target constituents relative to CC smoke for non-intense and intense puffing regimes, respectively. Excluding primary components that form the base formulation (propylene glycol, glycerol, water, menthol (in mentholated formulations), and nicotine), all compared constituents were present at lower levels relative to the yields of CCs, and the total average HPHC and chemical reductions were 96% or greater across both non-intense and intense puff regimes (Table in Section S1.13; File S5). For a demonstration that these reductions are significant, see Supplementary Materials, Tables in Sections S1.14 and S1.15 (File S5) for a statistical analysis of JUUL2 VT compared to the certified reference values for the 1R6F cigarette.
Categorically, across non-intense and intense puff regimes, similar percent reductions in mean aerosol constituent levels compared to CCs were exhibited with the exception of glycidol. Increased levels of glycidol under intense puffing are likely attributed to the longer puff duration. Of the quantifiable analytes, only ethyl acetate and benzoic acid could not be compared due to a lack of CC source values. Mean levels of chromium were found to be quantifiable only under intense puffing, at one timepoint, in one sample (T0; VT). Given the lack of consistent quantifiable measurements across replicates, regimes, and timepoints, the chromium results were deemed equivocal and removed from comparison; see the Supplementary Materials (File S1) for additional details.
4.1.3. Qualitative Risk Assessment
A qualitative risk assessment using the results from the targeted analytical chemistry stability studies conducted for each product was performed. Targeted constituents were grouped into either carcinogens or other toxicants that included respiratory toxicants, cardiovascular toxicants, and reproductive or developmental toxicants (Table 1) [4,47,48]. For the targeted analytes that were detected above the level of quantification, the highest value measured in the targeted analytical chemistry stability studies was used for comparison as a worst-case comparison with values from mainstream reference cigarette smoke. For carcinogens, there were significant reductions in levels measured in each JUUL2 product regardless of puffing regimen. For respiratory, cardiovascular, and reproductive and developmental toxicants, except for the main ENDS ingredients (nicotine, propylene glycol, and glycerin), there were also significant reductions that ranged from ≥40.5% to ≥99% in each JUUL2 product regardless of puffing regimen compared to levels in mainstream reference cigarette smoke.
Eight (8) of thirteen (13) carcinogenic HPHCs (established or proposed) evaluated were not detected above the limit of quantification or detection in any JUUL2 product. Four of the five carcinogens detected above the limit of quantification (acetaldehyde, formaldehyde, glycidol, and NNN) ranged from 66.7% to ≥99% lower in JUUL2 System aerosols compared to mainstream reference cigarette smoke. We believe the significant decreases in the levels of these carcinogens in JUUL2 System aerosols compared to reference mainstream cigarette smoke support substantial reductions in subsequent exposures and associated cancer risks from the use of the JUUL2 System relative to cigarette smoking.
4.2. Toxicological Evaluation
The complete in vitro toxicological results of VT, CM, PM, AT, RM, and SM JUUL2 pod aerosol condensate testing and 3R4F reference cigarette smoke condensate testing can be found in the Supplementary Materials, File S6.
4.2.1. NRU Cytotoxicity Assay
Results obtained with the vehicle and positive controls demonstrated the validity and sensitivity of the test system in each assay. None of the VT, CM, PM, AT, RM, or SM JUUL2 System condensates were found to be cytotoxic in the NRU assay using either BALB/c 3T3 and A549 cells regardless of puffing regimen (Figure 4) under the assay conditions. In contrast, the 3R4F condensate was found to be cytotoxic in both cell lines under the assay conditions, which is consistent with previous work [102,106]. See the Supplementary Materials (File S6) for complete NRU results on a mg ACM basis for ENDSs and mg total particulate mass (TPM) basis for 3R4F cigarettes.
4.2.2. Ames Mutagenicity Assay
Results obtained with the vehicle and strain-specific positive controls demonstrated the validity and sensitivity of these test systems for detecting chemical-induced mutagenicity in each assay. None of the VT, CM, PM, AT, RM, or SM JUUL2 System condensates were found to be mutagenic in the Ames assay in any S. typhimurium strain regardless of puffing regimen or metabolic activation under the assay conditions (Table 2). In contrast, the S9-treated 3R4F condensate was found to be mutagenic in three S. typhimurium strains (TA98, TA100, and TA1537 all with metabolic activation) under the conditions of the assay (Table 2). Complete results for all Ames mutagenicity assays are in the Supplementary Materials (File S6).
4.2.3. Micronucleus Genotoxicity Assay
The results obtained with the vehicle and positive controls demonstrated the suitability of each assay. For the JUUL2 System condensates, neither precipitation nor cytotoxicity were observed in any treatment condition, and therefore, MN frequencies were evaluated up to the solvent concentration limit (1% v/v). None of the JUUL2 VT, CM, PM, AT, RM, or SM condensates were found to be genotoxic in any of the test conditions of the MN assay regardless of the puffing regimen (Table S2; Supplementary Materials, File S6). In contrast, the 3R4F non-intense and intense condensates were respectively found to be equivocal and positive for genotoxicity (Table S2; Supplementary Materials, File S6).
5. Discussion
Following the principles of the “Tobacco Harm Reduction” strategy, a practical framework to evaluate novel ENDS products was presented. The application of the proposed framework on the novel JUUL2 product demonstrates that, as an alternative to combustible cigarettes, the JUUL2 device paired with JUUL2 formulations (VT, CM, PM, AT, RM, and SM) generates a significantly reduced number of HPHCs and chemicals quantified and does not exhibit cytotoxicity, mutagenicity, or genotoxicity under both non-intense and intense puffing conditions. The qualitative risk assessment confirmed that there were substantial reductions in the level of carcinogens in every JUUL2 product compared to CCs. Additionally, the qualitative risk assessment confirmed that except for the main ENDS ingredients, there was also a substantial reduction in levels of respiratory toxicants, cardiovascular toxicants, and reproductive or developmental toxicants in each JUUL2 product compared to CCs.
The proposed evaluation framework can be adapted for continual assessment throughout the product development lifecycle and, where applicable, be utilized to generate data in support of regulatory filings. As novel ENDS product development continues at a rapid pace with advancements in the device (cigalike, disposable, open tank, rechargeable, etc.), container system (pod-based, cartridge-based, non-refillable, refillable, etc.), aerosolization mechanism (cotton wick, silica wick, ceramic coil, etc.), formulation (free base nicotine, nicotine salt, natural vs. synthetic ingredients, etc.), and associated technology (Bluetooth, access restrictions, counterfeit compatibility, etc.), evaluation remains a critical step for manufacturers to have a role in understanding or reducing potential health risks from ENDSs. Despite progress, much work remains in terms of having a clear and complete list of recommended methods, guidance documents, and approaches that satisfy the multitude of regulatory requirements or total weight of evidence substantiation [107]. Offered within the JUUL2 case study are insights on study design and conduct, data analysis and interpretation, and qualitative risk assessment for both a chemical and in vitro toxicological characterization.
5.1. Study Design and Conduct Considerations
Various regulatory requirements and guidance documents helped define a list of assays (neutral red uptake cytotoxicity assay, mutagenicity with the Ames assay, and mammalian genotoxicity with the micronucleus assay) and select constituents for identification within the emissions but do not specifically address the manner in which studies are conducted. Associations such as CORESTA have formed subgroups (E-Vapour (EVAP) and In Vitro Toxicity Testing) that seek to align and standardize testing approaches primarily by adapting established CC methods for use with ENDSs [96]. Focus on this area of scientific research and study conduct further assists in generating robust methodologies that characterize e-liquids, aerosol emissions, and device properties potentially leading to the optimization of system characteristics that can minimize exposure.
Outlined within the JUUL2 case study are some examples of study conduct advancements that support analyses of the complete product, such as the use of “EOL” for reporting on a “whole-pod” basis. These advancements can become important when evaluating cross-product comparisons (e.g., per-cig yield vs. per-pod yield, 1 mL pod yield vs. 2 mL pod yield). Furthermore, the “EOL” puff collections also satisfy a more robust approach to aerosol condensate production for toxicological assay dosing. The chemical composition and stability of aerosol condensates utilized in toxicological evaluations are often minimally characterized, and there is a lack of a standardized condensate production approach [108]. This has led to oversight relative to the impact of factors such as the number of puffs per pod taken to achieve the target concentration and the nature of the condensate solvent. Additional collection methodology improvements such as the use of electrostatic precipitation within chemical metal analysis were incorporated to reduce potential contamination from the collection apparatus. Lowering the level of background trace metals also supports a lower effective quantification limit; however, it does not eliminate the possibility of random, sporadic quantifiable replicates (Section 4.1.2).
Undoubtedly, chemical and toxicological methods, techniques, and modifications will continue to develop alongside the advancement of product technology; therefore, critical review of the study conduct remains essential prior to data analysis and interpretation.
5.2. Data Analysis and Interpretation Considerations
For the aerosol chemical constituent comparison of ENDSs to CCs, several methods have been used to analyze and interpret results (e.g., per puff, normalized to per mg nicotine, per estimated daily exposure) [13,18,19,109]. One study has discussed the calculational impacts such as comparisons made on a per-nicotine basis versus per-puff and the impact of potential errors brought about by using imputed values and concluded that comparing results on a per-nicotine or per-puff basis exhibited minimal differences between the two approaches largely due to the significant amount of non-quantifiable analytes in the ENDS product [19]. However, when comparing only the quantifiable analytes on a per-nicotine basis, products with lower nicotine concentrations provided greater estimated toxicant exposures despite the greater mass of aerosol generated by the comparator product(s). Furthermore, the handling of non-quantifiable value imputation (midpoint of LOD/LOQ approach, LOD/√2, predicted values from models, and use of sub-detection limit values presented by the analytical method) was deemed inconsequential based on the substantial differences between CC smoke and ENDS aerosol [19].
Within the JUUL2 product case study, stability trends were assessed (Supplementary Materials, File S1) and aerosol chemical constituent comparisons were nicotine-normalized (mg/mg) on a “whole-pod” basis, negating the effect of any variable constituent generation during pod life [92,110]. Given the nicotine concentrations and deliveries were consistent across the JUUL2 product formulations and similar substantial decreases in ENDS aerosol constituent concentrations relative to CCs were observed, comparison on a per-nicotine basis was deemed appropriate. Where aerosol constituent levels below the LOD and LOQ were observed, values were imputed as LOD/2 and (LOQ + LOD/2), respectively. Across stability study timepoints, the highest value from any timepoint was used when comparing the JUUL2 System to CCs. The percent difference was not comparable (NC) if both the JUUL2 System and CCs were BLOQ or BLOD, if the JUUL2 System aerosol yield was quantifiable and the CC LOQ was greater than the JUUL2 System quantifiable yield, or if the CC yield was quantifiable and the JUUL2 LOQ was greater than the CC quantifiable yield.
Similar to chemistry, several methodologies have been utilized for a toxicological cross-product comparison [111,112,113,114,115]. Forest et al. (2022) discussed the different metrics and dose ranges used in studies for toxicological assessment of ENDSs, which included number of puffs, puffs/mL, puffs/m2, concentration of a compound (i.e., flavor), and ratio % (corresponding to the dilution of e-liquid, aerosol, or smoke extracts) [116]. While there are many ways to express doses and different dose ranges may be utilized, the question to balance is understanding the mechanistic aspect of the toxicological process or response (where unrealistically high doses can be used without inherent objection) versus exposure doses that mimic the real-life conditions of e-cigarette use [116]. Within the JUUL2 study, nicotine was chosen to characterize dosimetry across in vitro assays. The utilization of nicotine as a marker facilitates cross-product comparison and extrapolation of preclinical data and consumer use studies to help further explore the reduced risk potential of ENDS products [116].
5.3. Qualitative Risk Assessment Considerations
For each carcinogen measured above the LOD and LOQ, there was a 66.7% to ≥99% reduction across all JUUL2 System aerosols compared to mainstream reference cigarette smoke. The other carcinogens were not found above the LOQ or LOD and therefore were more than 99% reduced in all JUUL2 System aerosols compared to mainstream reference cigarette smoke. The World Health Organization (WHO) Study Group on Tobacco Product Regulation [117] identified nine toxicants as “most hazardous” in cigarette smoke, including five of the carcinogens reduced in all JUUL2 products’ aerosol relative to cigarette smoke:
NNK and NNN (TSNAs);
Benzo[a]pyrene (representative PAHs);
VOCs, 1,3-butadiene, acrolein, acetaldehyde, formaldehyde, and benzene;
Carbon monoxide (CO).
Fowles and Dybing (2003) estimated that combined, aldehydes and other small organic byproducts of combustion can account for approximately 44.3% (using EPA cancer risk values) to 62.4% (using Cal/EPA cancer risk values) of the cancer risk from mainstream cigarette smoke [118]. Acetaldehyde, formaldehyde, benzene, NNK, and NNN were measured in the targeted analytical chemistry stability studies and were found to be lower by 96% to ≥99% in all JUUL2 System aerosols compared to levels in mainstream reference cigarette smoke.
Carcinogens are generally assumed to have a linear dose–response relationship, which implies that there is an excess cancer risk associated with exposure to the constituent at any level, and the cancer risk for most carcinogens, including carcinogenic HPHCs, increases proportionally with increased exposure [119,120]. Taken together, of the 12 carcinogenic HPHCs evaluated in JUUL2 System aerosols, levels were not detected or were found at significantly lower levels compared to mainstream reference cigarette smoke, indicating a likely substantial reduction in user exposures and associated cancer risk.
With the exception of the primary e-liquid ingredients (nicotine, propylene glycol, and glycerin), the levels of established and proposed HPHCs with known respiratory, cardiovascular, or reproductive or developmental toxicities were also reduced in aerosol, regardless of puffing regimen, in all JUUL2 products compared to reference mainstream cigarette smoke. Substantially lower levels of these HPHCs support significant reductions in overall toxicant exposures and associated noncancer hazard from the use of the JUUL2 products relative to cigarette smoking. These conclusions based upon a comparison of aerosol constituent levels from targeted analytical chemistry stability studies between JUUL2 products and mainstream cigarette smoke are consistent with the in vitro assay results. The in vitro assay results showed that all JUUL2 products were not cytotoxic, not mutagenic, and not genotoxic within the assay conditions, while mainstream cigarette smoke was cytotoxic, mutagenic, and genotoxic within the assay conditions.
5.4. Regulatory Compliance
Dependent on the intended marketplace, country/region-specific regulatory standards or guidance documents should be followed. For example, in the US, modifications to the product characterization may include additional factors such as the requirement to analyze three (3) separate manufactured batches (seven replicates per constituent, per batch), the inclusion of e-liquid analyses, the inclusion of non-targeted analyses (on e-liquid and aerosol), or a battery of complementary clinical, behavioral, and populational studies. Contrasting the extensive characterization requirements in the U.S., Europe and European Member State guidance documents tend to emphasize the evaluation of ingredient sourcing and toxicological assessments thereof with additional considerations on banned ingredients, emission limits for specific analytes, and nicotine dose uniformity characterizations [9,62]. Within Asia, compliance attributes trend similar to the European entities, with a focus on factors such as permissions of e-liquid additives or “pollution” limits on specific analytes [60].
5.5. Limitations
The primary intent of this proposed framework was geared towards internal evaluation during the product development lifecycle. As such, this proposed framework is more limited than some other proposed product evaluation frameworks (Bergman et al., 2015; Murphy et al., 2017; Camacho et al., 2021), but also more extensive than others (Costigan and Meredith, 2015; Iskandar et al., 2016) [63,64,65,67,121]. This proposed framework is also consistent with the recent framework (Dempsey et al., 2023) proposed for heat-not-burn tobacco products [87]. While this practical framework and case study example focus on a reduction in toxicity relative to cigarettes, and could be performed relative to heat-not-burn tobacco products or other ENDS products, this framework does not cover aspects such as populational effects, which additional clinical, behavioral, or modeling studies work to address [64,65,121].
Discussed but not wholly presented within the JUUL2 case study is the critical aspect that all materials, components, or ingredients utilized within the novel ENDS product passed the preliminary toxicological assessment. Complete aerosol chemical analysis (e.g., non-targeted chemical analysis) was not included in the proposed framework because it is not universally required by regulators, although results for several U.S. products have been published [20,21]. For the intense puffing regimen chosen, no standardized regimen has been published to date; therefore, a maximum device cut-off (6 s) was selected; however, other puffing conditions have been deemed suitable for other products (including alternative non-intense puffing regimens).
Regarding the specific in vitro product testing within the proposed framework, although all in vitro product testing programs have limitations, the use of standardized OECD assays [97,100,101], ISO 17025 accredited laboratories [91], and similar aerosol collection techniques for all tested products minimizes these limitations to provide an adequate and scientifically valid in vitro toxicological assessment of generated aerosols. Our proposed framework does not incorporate the systems toxicology recommendations for in vitro assays by Iskandar et al. (2016) due to the limited availability of these methods at contract research organizations and, to date, their limited demonstrated regulatory value (IQOS TPL, 29 April 2019; page 39) [63,122].
6. Conclusions
In this study, a proposed framework for chemical and toxicological characterization was applied to the novel ENDS product JUUL2. Respective JUUL2 formulation, Virginia Tobacco, Crisp Menthol, Polar Menthol, Autumn Tobacco, Ruby Menthol, and Summer Menthol 18 mg/mL, aerosols were evaluated for relevant HPHCs and chemicals over a 12-month stability study, and cytotoxic, mutagenic, and genotoxic endpoints were assessed. Following the framework, the qualitative comparison to CCs demonstrates the products’ harm reduction potential through the significant reductions (>96% on average) in toxicant emissions coupled with the lack of toxicologically relevant responses under both non-intense and intense regimes.
Conceptualization, D.K.C., I.G.G., M.J.O. and G.L.; formal analysis, D.K.C., M.J.O. and G.L.; data curation, J.W., A.B., S.U., C.S., G.L. and M.J.O.; writing—original draft preparation, D.K.C.; writing—review and editing, I.G.G., M.J.O., G.L., K.C., C.J. and G.D.; visualization, D.K.C. and C.S.; supervision, I.G.G.; project administration, D.K.C. All authors have read and agreed to the published version of the manuscript.
Not applicable.
Not applicable.
The data presented in this study are available within the
At the time this work was performed, all authors were employed by Juul Labs, Inc. All testing was performed at Enthalpy Analytical LLC and Charles River Laboratories and paid for in full by Juul Labs, Inc.
Footnotes
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
Enlarge this image.Figure 2. Percent reduction comparison of mean JUUL2 pod aerosol constituent aerosol levels and mean combustible cigarette smoke constituent levels under non-intense puffing regimes.
Enlarge this image.Figure 3. Percent reduction comparison of mean JUUL2 pod aerosol constituent aerosol levels and mean combustible cigarette smoke constituent levels under intense puffing regimes.
![View Image - Figure 4. BALB/c 3T3 and A549 cytotoxicity of intense and non-intense ENDS aerosol and 3R4F smoke condensates, measured in the NRU assay. The dotted line represents the 70% relative viability cut-off defining cytotoxicity [98].](https://media.proquest.com/media/hms/THUM/8/eCJBX?_a=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%3D%3D&_s=js3bPiieWTLnk33q6tX%2Fwu5ZuAw%3D "View Image - Figure 4. BALB/c 3T3 and A549 cytotoxicity of intense and non-intense ENDS aerosol and 3R4F smoke condensates, measured in the NRU assay. The dotted line represents the 70% relative viability cut-off defining cytotoxicity [98].")
Enlarge this image.Figure 4. BALB/c 3T3 and A549 cytotoxicity of intense and non-intense ENDS aerosol and 3R4F smoke condensates, measured in the NRU assay. The dotted line represents the 70% relative viability cut-off defining cytotoxicity [98].
Measured aerosol constituents and pH.
| Primary | Nicotine | Propylene Glycol | Water | Glycerol |
| Menthol | Diethylene Glycol (PC) | Ethylene Glycol (RT, RDT) | ||
| Carbonyls | Acetaldehyde | Acrolein | Diacetyl | Formaldehyde |
| Acetyl Propionyl (RT) | Butyraldehyde | Crotonaldehyde (CA) | Furfural | |
| Glycidol | Glycidol (CA) | |||
| Nicotine | β-Nicotyrine | Cotinine | Myosmine | Nicotine N Oxide |
| Nornicotine | Anabasine | Anatabine | ||
| TSNAs | NNN (CA) | NNK (CA) | ||
| Metals | Arsenic | Cobalt | Lead | Tin |
| Beryllium | Copper | Nickel | Selenium | |
| Chromium | Iron | Silver | Gold | |
| Zinc | Cadmium | |||
| Organic Acids | Benzoic Acid | Propionic Acid | ||
| VOCs | 1,3-Butadiene | Acrylonitrile | Propylene Oxide (CA, RT) | Isoprene |
| Toluene | Benzene | |||
| Esters and | 1-Butanol | Benzyl Acetate | Ethyl Acetate (RT) | Ethyl Acetoacetate (RT) |
| Isoamyl Acetate (RT) | Isobutyl Acetate | Methyl Acetate (RT) | ||
| pH | pH |
CA = carcinogen, RT = respiratory toxicant, CT = cardiovascular toxicant, RDT = reproductive or developmental toxicant, PC = poisonous chemical, AD = addictive.
Mutagenicity (Ames) and genotoxicity (MN) results of intense and non-intense ENDS aerosol and 3R4F smoke condensates.
| Formulation | Ames Assay | MN Assay | ||
|---|---|---|---|---|
| Non-Intense | Intense | Non-Intense | Intense | |
| Condensate | Condensate | Condensate | Condensate | |
| Autumn Tobacco | Negative | Negative | Negative | Negative |
| Virginia Tobacco | Negative | Negative | Negative | Negative |
| Crisp Menthol | Negative | Negative | Negative | Negative |
| Polar Menthol | Negative | Negative | Negative | Negative |
| Ruby Menthol | Negative | Negative | Negative | Negative |
| Summer Menthol | Negative | Negative | Negative | Negative |
| 3R4F | Positive | Positive | Equivocal | Positive |
Supplementary Materials
The following supporting information can be downloaded at
References
1. U.S. Department of Health and Human Services. Reports of the Surgeon General. The Health Consequences of Smoking: A Report of the Surgeon General; Department of Health and Human Services: Atlanta, GA, USA, 2004; 956.
2. Rodgman, A.; Perfetti, T.A. The Chemical Components of Tobacco and Tobacco Smoke; 2nd ed. CRC Press: Boca Raton, FL, USA, 2013; 2332.
3. U. S. Food and Drug Administration. FDA’s Youth Tobacco Prevention Plan; [Web Page] 14 September 2020. Available online: https://www.fda.gov/tobacco-products/youth-and-tobacco/fdas-youth-tobacco-prevention-plan (accessed on 20 December 2023).
4. National Center for Chronic Disease Prevention and Health Promotion (US) Office on Smoking and Health. Preventing Tobacco Use among Youth and Young Adults: A Report of the Surgeon General; Centers for Disease Control and Prevention: Atlanta, GA, USA, 2012.
5. H.R. 1256–111th Congress (2009–2010). Family Smoking Prevention and Tobacco Control Act, in 21 U.S.C. § 301; Congress.gov, Library of Congress: Washington, DC, USA, 2009; 21.
6. Massachussetts Department of Public Health. 105 CMR 660.00: Cigarette and Smokeless Tobacco Products: Reports of Nicotine Ratings; Mass.gov, Massachussetts Department of Public Health. Massachussetts Court System: Boston, MA, USA, 2018.
7. Health Canada. Tobacco Reporting Regulations; Health Canada: Ottawa, ON, Canada, 2019; pp. 1-40.
8. Agência Nacional de Vigilância Sanitária. Resolution-RDC Nº 14, of March 15, 2012; National Health Surveillance Agency: Brasília, Brazil, 2012.
9. European Parliament and the Council of the European Union. Directive 2014/40/EU of the European Parliament and of the Council of 3 April 2014, on the Approximation of the Laws, Regulations and Administrative Provisions of the Member States Concerning the Manufacture, Presentation and Sale of Tobacco and Related Products and Repealing Directive 2001/37/EC, 2014. Off. J. Eur. Union; 2014; 127, pp. 1-38.
10. Farsalinos, K.E.; Polosa, R. Safety Evaluation and Risk Assessment of Electronic Cigarettes as Tobacco Cigarette Substitutes: A Systematic Review. Ther. Adv. Drug Saf.; 2014; 5, pp. 67-86. [DOI: https://dx.doi.org/10.1177/2042098614524430] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25083263]
11. National Academies of Sciences Engineering Medicine. Public Health Consequences of E-Cigarettes; National Academies Press: Washington, DC, USA, 2018; pp. 1-774.
12. U.S. Department of Health and Human Services. The Health Consequences of Smoking—50 Years of Progress: A Report of the Surgeon General; U.S. Department of Health and Human Services: Centers for Disease Control and Prevention: National Center for Chronic Disease Prevention and Health Promotion: Office on Smoking and Health: Atlanta, GA, USA, 2014; pp. 1-1081.
13. Tayyarah, R.; Long, G.A. Comparison of select analytes in aerosol from e-cigarettes with smoke from conventional cigarettes and with ambient air. Regul. Toxicol. Pharmacol.; 2014; 70, pp. 704-710. [DOI: https://dx.doi.org/10.1016/j.yrtph.2014.10.010] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25444997]
14. Talih, S.; Salman, R.; Karaoghlanian, N.; El-Hellani, A.; Shihadeh, A. Carbonyl Emissions and Heating Temperatures across 75 Nominally Identical Electronic Nicotine Delivery System Products: Do Manufacturing Variations Drive Pulmonary Toxicant Exposure?. Chem. Res. Toxicol.; 2023; 36, pp. 342-346. [DOI: https://dx.doi.org/10.1021/acs.chemrestox.2c00391]
15. El-Hellani, A.; Salman, R.; El-Hage, R.; Talih, S.; Malek, N.; Baalbaki, R.; Karaoghlanian, N.; Nakkash, R.; Shihadeh, A.; Saliba, N.A. Nicotine and Carbonyl Emissions From Popular Electronic Cigarette Products: Correlation to Liquid Composition and Design Characteristics. Nicotine Tob. Res.; 2016; 20, pp. 215-223. [DOI: https://dx.doi.org/10.1093/ntr/ntw280]
16. Wieczorek, R.; Phillips, G.; Czekala, L.; Sticken, E.T.; O’Connell, G.; Simms, L.; Rudd, K.; Stevenson, M.; Walele, T. A comparative in vitro toxicity assessment of electronic vaping product e-liquids and aerosols with tobacco cigarette smoke. Toxicol. In Vitro; 2020; 66, 104866. [DOI: https://dx.doi.org/10.1016/j.tiv.2020.104866]
17. U.S. Department of Health and Human Services. How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease: A Report of the Surgeon General; Centers for Disease Control and Prevention: National Center for Chronic Disease Prevention and Health Promotion: Office on Smoking and Health Communication: Atlanta, GA, USA, 2010.
18. Margham, J.; McAdam, K.; Forster, M.; Liu, C.; Wright, C.; Mariner, D.; Proctor, C. Chemical Composition of Aerosol from an E-Cigarette: A Quantitative Comparison with Cigarette Smoke. Chem. Res. Toxicol.; 2016; 29, pp. 1662-1678. [DOI: https://dx.doi.org/10.1021/acs.chemrestox.6b00188]
19. Cunningham, A.; McAdam, K.; Thissen, J.; Digard, H. The Evolving E-cigarette: Comparative Chemical Analyses of E-cigarette Vapor and Cigarette Smoke. Front. Toxicol.; 2020; 2, 586674. [DOI: https://dx.doi.org/10.3389/ftox.2020.586674]
20. Crosswhite, M.R.; Bailey, P.C.; Jeong, L.N.; Lioubomirov, A.; Yang, C.; Ozvald, A.; Jameson, J.B.; Gillman, I.G. Non-Targeted Chemical Characterization of JUUL Virginia Tobacco Flavored Aerosols Using Liquid and Gas Chromatography. Separations; 2022; 8, 130. [DOI: https://dx.doi.org/10.3390/separations8090130]
21. Crosswhite, M.R.; Jeong, L.N.; Bailey, P.C.; Jameson, J.B.; Lioubomirov, A.; Cook, D.; Yang, C.; Ozvald, A.; Lyndon, M.; Gillman, I.G. Non-Targeted Chemical Characterization of JUUL-Menthol-Flavored Aerosols Using Liquid and Gas Chromatography. Separations; 2022; 9, 367. [DOI: https://dx.doi.org/10.3390/separations9110367]
22. Farsalinos, K.E.; Gillman, I.G.; Melvin, M.S.; Paolantonio, A.R.; Gardow, W.J.; Humphries, K.E.; Brown, S.E.; Poulas, K.; Voudris, V. Nicotine Levels and Presence of Selected Tobacco-Derived Toxins in Tobacco Flavoured Electronic Cigarette Refill Liquids. Int. J. Environ. Res. Public Health; 2015; 12, pp. 3439-3452. [DOI: https://dx.doi.org/10.3390/ijerph120403439] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25811768]
23. Kim, H.-J.; Shin, H.-S. Determination of tobacco-specific nitrosamines in replacement liquids of electronic cigarettes by liquid chromatography–tandem mass spectrometry. J. Chromatogr. A; 2013; 1291, pp. 48-55. [DOI: https://dx.doi.org/10.1016/j.chroma.2013.03.035] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23602640]
24. Long, G.A. Comparison of Select Analytes in Exhaled Aerosol from E-Cigarettes with Exhaled Smoke from a Conventional Cigarette and Exhaled Breaths. Int. J. Environ. Res. Public Health; 2014; 11, pp. 11177-11191. [DOI: https://dx.doi.org/10.3390/ijerph111111177] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25350011]
25. Laugesen, M. Nicotine and toxicant yield ratings of electronic cigarette brands in New Zealand. N. Z. Med. J.; 2015; 128, pp. 77-82.
26. Goniewicz, M.L.; Knysak, J.; Gawron, M.; Kosmider, L.; Sobczak, A.; Kurek, J.; Prokopowicz, A.; Jablonska-Czapla, M.; Rosik-Dulewska, C.; Havel, C. et al. Levels of selected carcinogens and toxicants in vapour from electronic cigarettes. Tob. Control; 2013; 23, pp. 133-139. [DOI: https://dx.doi.org/10.1136/tobaccocontrol-2012-050859] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23467656]
27. Flora, J.W.; Meruva, N.; Huang, C.B.; Wilkinson, C.T.; Ballentine, R.; Smith, D.C.; Werley, M.S.; McKinney, W.J. Characterization of potential impurities and degradation products in electronic cigarette formulations and aerosols. Regul. Toxicol. Pharmacol.; 2016; 74, pp. 1-11. [DOI: https://dx.doi.org/10.1016/j.yrtph.2015.11.009] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26617410]
28. Farsalinos, K.E.; Kistler, K.A.; Pennington, A.; Spyrou, A.; Kouretas, D.; Gillman, G. Aldehyde levels in e-cigarette aerosol: Findings from a replication study and from use of a new-generation device. Food Chem. Toxicol.; 2018; 111, pp. 64-70. [DOI: https://dx.doi.org/10.1016/j.fct.2017.11.002]
29. Talih, S.; Salman, R.; El-Hage, R.; Karam, E.; Karaoghlanian, N.; El-Hellani, A.; Saliba, N.; Shihadeh, A. Characteristics and toxicant emissions of JUUL electronic cigarettes. Tob. Control; 2019; 28, pp. 678-680. [DOI: https://dx.doi.org/10.1136/tobaccocontrol-2018-054616]
30. Pellegrino, R.M.; Tinghino, B.; Mangiaracina, G.; Marani, A.; Vitali, M.; Protano, C.; Osborn, J.F.; Cattaruzza, M.S. Electronic cigarettes: An evaluation of exposure to chemicals and fine particulate matter (PM). Ann. Ig.; 2012; 24, pp. 279-288.
31. Marco, E.; Grimalt, J.O. A rapid method for the chromatographic analysis of volatile organic compounds in exhaled breath of tobacco cigarette and electronic cigarette smokers. J. Chromatogr. A; 2015; 1410, pp. 51-59. [DOI: https://dx.doi.org/10.1016/j.chroma.2015.07.094]
32. Lerner, C.A.; Sundar, I.K.; Watson, R.M.; Elder, A.; Jones, R.; Done, D.; Kurtzman, R.; Ossip, D.J.; Robinson, R.; McIntosh, S. et al. Environmental health hazards of e-cigarettes and their components: Oxidants and copper in e-cigarette aerosols. Environ. Pollut.; 2015; 198, pp. 100-107. [DOI: https://dx.doi.org/10.1016/j.envpol.2014.12.033] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25577651]
33. Chen, X.; Bailey, P.C.; Yang, C.; Hiraki, B.; Oldham, M.J.; Gillman, I.G. Targeted Characterization of the Chemical Composition of JUUL Systems Aerosol and Comparison with 3R4F Reference Cigarettes and IQOS Heat Sticks. Separations; 2021; 8, 168. [DOI: https://dx.doi.org/10.3390/separations8100168]
34. Breheny, D.; Thorne, D.; Baxter, A.; Bozhilova, S.; Jaunky, T.; Santopietro, S.; Taylor, M.; Terry, A.; Gaça, M. The in vitro assessment of a novel vaping technology. Toxicol. Rep.; 2020; 7, pp. 1145-1156. [DOI: https://dx.doi.org/10.1016/j.toxrep.2020.08.016] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32983902]
35. Rudd, K.; Stevenson, M.; Wieczorek, R.; Pani, J.; Trelles-Sticken, E.; Dethloff, O.; Czekala, L.; Simms, L.; Buchanan, F.; O’Connell, G. et al. Chemical Composition and In Vitro Toxicity Profile of a Pod-Based E-Cigarette Aerosol Compared to Cigarette Smoke. Appl. In Vitro Toxicol.; 2020; 6, pp. 11-41. [DOI: https://dx.doi.org/10.1089/aivt.2019.0015]
36. Platel, A.; Dusautoir, R.; Kervoaze, G.; Dourdin, G.; Gateau, E.; Talahari, S.; Huot, L.; Simar, S.; Ollivier, A.; Laine, W. et al. Comparison of the in vivo genotoxicity of electronic and conventional cigarettes aerosols after subacute, subchronic and chronic exposures. J. Hazard. Mater.; 2022; 423, 127246. [DOI: https://dx.doi.org/10.1016/j.jhazmat.2021.127246] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34844363]
37. Peitsch, M.C.; Hoeng, J. Toxicological Evaluation of Electronic Nicotine Delivery Products; Academic Press: Cambridge, MA, USA, 2021.
38. Scheffler, S.; Dieken, H.; Krischenowski, O.; Förster, C.; Branscheid, D.; Aufderheide, M. Evaluation of E-Cigarette Liquid Vapor and Mainstream Cigarette Smoke after Direct Exposure of Primary Human Bronchial Epithelial Cells. Int. J. Environ. Res. Public Health; 2015; 12, pp. 3915-3925. [DOI: https://dx.doi.org/10.3390/ijerph120403915] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25856554]
39. Husari, A.; Shihadeh, A.; Talih, S.; Hashem, Y.; El Sabban, M.; Zaatari, G. Acute Exposure to Electronic and Combustible Cigarette Aerosols: Effects in an Animal Model and in Human Alveolar Cells. Nicotine Tob. Res.; 2015; 18, pp. 613-619. [DOI: https://dx.doi.org/10.1093/ntr/ntv169]
40. Desai, R.W.; Demir, K.; Tsolakos, N.; Moir-Savitz, T.R.; Gaworski, C.L.; Weil, R.; Oldham, M.J.; Lalonde, G. Comparison of the toxicological potential of two JUUL ENDS products to reference cigarette 3R4F and filtered air in a 90-day nose-only inhalation toxicity study. Food Chem. Toxicol.; 2023; 179, 113917. [DOI: https://dx.doi.org/10.1016/j.fct.2023.113917]
41. Romagna, G.; Allifranchini, E.; Bocchietto, E.; Todeschi, S.; Esposito, M.; Farsalinos, K.E. Cytotoxicity evaluation of electronic cigarette vapor extract on cultured mammalian fibroblasts (ClearStream-LIFE): Comparison with tobacco cigarette smoke extract. Inhal. Toxicol.; 2013; 25, pp. 354-361. [DOI: https://dx.doi.org/10.3109/08958378.2013.793439]
42. Farsalinos, K.E.; Romagna, G.; Allifranchini, E.; Ripamonti, E.; Bocchietto, E.; Todeschi, S.; Tsiapras, D.; Kyrzopoulos, S.; Voudris, V. Comparison of the Cytotoxic Potential of Cigarette Smoke and Electronic Cigarette Vapour Extract on Cultured Myocardial Cells. Int. J. Environ. Res. Public Health; 2013; 10, pp. 5146-5162. [DOI: https://dx.doi.org/10.3390/ijerph10105146]
43. U.S. Food and Drug Administration. Deeming Tobacco Products To Be Subject to the Federal Food, Drug, and Cosmetic Act, as Amended by the Family Smoking Prevention and Tobacco Control Act; Restrictions on the Sale and Distribution of Tobacco Products and Required Warning Statements for Tobacco Products. Final Rule United States Government: Washington, DC, USA, 2016; pp. 28973-29106.
44. U.S. Food and Drug Administration. FDA’s Comprehensive Plan for Tobacco and Nicotine Regulation. [Web Page]. 31 July 2019; Available online: https://www.fda.gov/tobacco-products/ctp-newsroom/fdas-comprehensive-plan-tobacco-and-nicotine-regulation (accessed on 1 April 2021).
45. U.S. Food and Drug Administration. Premarket Tobacco Product Applications and Recordkeeping Requirements. Proposed Rule; U.S. Food and Drug Administration: Silver Spring, MD, USA, 2019; pp. 50566-50658.
46. U.S. Food and Drug Administration. Premarket Tobacco Product Applications for Electronic Nicotine Delivery Systems-Final Guidance; U.S. Department of Health and Human Services: U.S. Food and Drug Administration: Center for Tobacoo Products: Washington, DC, USA, 2019.
47. U.S. Food and Drug Administration. Reporting Harmful and Potentially Harmful Constituents in Tobacco Products and Tobacco Smoke Under Section 904(a)(3) of the Federal Food, Drug, and Cosmetic Act; Center of Tobacco Products; U.S. Department of Health and Human Services: Silver Spring, MD, USA, 2012.
48. U.S. Food and Drug Administration. Harmful and Potentially Harmful Constituents in Tobacco Products; Established List; Proposed Additions Department of Health and Human Services: Atlanta, GA, USA, 2019; Volume 84.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62. Dutch Ministry of Public Health, Welfare, and Sport. Tobacco and Smoking Products Regulations, Reference 966398-150196-WJZ; Ministry of Public Health, Welfare and Sport: Hague, The Netherlands, 2023.
63. Iskandar, A.R.; Gonzalez-Suarez, I.; Majeed, S.; Marescotti, D.; Sewer, A.; Xiang, Y.; Leroy, P.; Guedj, E.; Mathis, C.; Schaller, J.P. et al. A framework for in vitro systems toxicology assessment of e-liquids. Toxicol. Mech. Methods; 2016; 26, pp. 392-416. [DOI: https://dx.doi.org/10.3109/15376516.2016.1170251] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27117495]
64. Camacho, O.M.; Ebajemito, J.K.; Coburn, S.; Prasad, K.; Costigan, S.; Murphy, J.J. Evidence From the Scientific Assessment of Electronic Cigarettes and Their Role in Tobacco Harm Reduction. Contrib. Tob. Nicotine Res.; 2021; 30, pp. 63-108. [DOI: https://dx.doi.org/10.2478/cttr-2021-0007]
65. Murphy, J.; Gaca, M.; Lowe, F.; Minet, E.; Breheny, D.; Prasad, K.; Camacho, O.; Fearon, I.M.; Liu, C.; Wright, C. et al. Assessing modified risk tobacco and nicotine products: Description of the scientific framework and assessment of a closed modular electronic cigarette. Regul. Toxicol. Pharmacol.; 2017; 90, pp. 342-357. [DOI: https://dx.doi.org/10.1016/j.yrtph.2017.09.008] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28954704]
66. Berman, M.L.; Connolly, G.; Cummings, M.K.; Djordjevic, M.V.; Hatsukami, D.K.; Henningfield, J.E.; Myers, M.; O’Connor, R.J.; Parascandola, M.; Rees, V. et al. Providing a Science Base for the Evaluation of Tobacco Products. Tob. Regul. Sci.; 2015; 1, pp. 76-93. [DOI: https://dx.doi.org/10.18001/TRS.1.1.8]
67. Costigan, S.; Meredith, C. An approach to ingredient screening and toxicological risk assessment of flavours in e-liquids. Regul. Toxicol. Pharmacol.; 2015; 72, pp. 361-369. [DOI: https://dx.doi.org/10.1016/j.yrtph.2015.05.018]
68.
69. Smith, C.; Lyndon, M.; Jeong, L.; Lehman, D.; Jameson, J.B.; Chevva, H.; Ayala-Fierro, F.; Cook, D.; Carter, K.; Oldham, M. et al. Analytical approaches for the evaluation of data deficient simulated leachable compounds in ENDS products: A case study. Front. Chem.; 2023; 11, 1212744. [DOI: https://dx.doi.org/10.3389/fchem.2023.1212744]
70. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. M7(R1) Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk. ICH Harmonised Guideline; ICH Working Group: Geneva, Switzerland, 2017.
71. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. ICH harmonised guideline. Guideline for Elemental Impurities Q3D(R1); International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use: Geneva, Switzerland, 2019.
72.
73.
74. U.S. Food and Drug Administration. Memorandum: Approaches to the Evaluation of Extractables and Leachables in Tobacco Product Application Review; U.S. Department of Health and Human Services: Atlanta, GA, USA, 2020.
75. U.S. Food and Drug Administration. Q3D(R1) Elemental Impurities: Guidance for Industry; U.S. Department of Health and Human Services: Atlanta, GA, USA, 2020.
76. United States Pharmacopeia. General Chapter, ⟨1664⟩ Assessment of Drug Product Leachables Associated with Pharmaceutical Packaging/Delivery Systems; USP-NF: Rockville, MD, USA, 2020; Volume 39, [DOI: https://dx.doi.org/10.31003/USPNF_M7127_03_01]
77. United States Pharmacopeia. General Chapter ⟨1663⟩ Assessment of Extractables Associated with Pharmaceutical Packaging/Delivery Systems; USP-NF: Rockville, MD, USA, 2020; [DOI: https://dx.doi.org/10.31003/USPNF_M7126_03_01]
78. National Research Council. Science and Decisions: Advancing Risk Assessment; The National Academies Press: Washington, DC, USA, 2009.
79. U.S. Environmental Protection Agency. Risk Assessment Guidance for Superfund: Volume I: Human Health Evaluation Manual (Part A)-Interim Final; U.S. Environmental Protection Agency: Washington, DC, USA, 1989.
80. U.S. Environmental Protection Agency. Risk Assessment Guidance for Superfund (RAGS), Volume I: Human Health Evaluation Manual, Part F, Supplemental Guidance for Inhalation Risk Assessment-Final; U.S. Enivronmental Protection Agency: Washington, DC, USA, 2009.
81. U.S. Food and Drug Administration. Applying Human Factors and Usability Engineering to Medical Devices: Guidance for Industry and Food and Drug Administration Staff; U.S. Department of Health & Human Services: Atlanta, GA, USA, 2016.
82. U.S. Food and Drug Administration. M7(R1) Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk; US Department of Health and Human Services: Atlanta, GA, USA, 2018.
83. U.S. Food and Drug Administration. Memorandum: Use of Reference Values in the Toxicological Evaluation of Inhaled Tobacco Products; Center of Tobacco Products: Rockville, MD, USA, 2019.
84. U.S. Department of Health and Human Services. E-Cigarette Use Among Youth and Young Adults; U.S. Department of Health and Human Services: Atlanta, GA, USA, 2016.
85. Soulet, S.; Sussman, R.A. A Critical Review of Recent Literature on Metal Contents in E-Cigarette Aerosol. Toxics; 2022; 10, 510. [DOI: https://dx.doi.org/10.3390/toxics10090510]
86. Soulet, S.; Sussman, R.A. Critical Review of the Recent Literature on Organic Byproducts in E-Cigarette Aerosol Emissions. Toxics; 2022; 10, 714. [DOI: https://dx.doi.org/10.3390/toxics10120714]
87. Dempsey, R.; Rodrigo, G.; Vonmoos, F.; Gunduz, I.; Belushkin, M.; Esposito, M. Preliminary toxicological assessment of heated tobacco products: A review of the literature and proposed strategy. Toxicol. Rep.; 2023; 10, pp. 195-205. [DOI: https://dx.doi.org/10.1016/j.toxrep.2023.01.008]
88. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Q1A(R2) Stability Testing of New Drug Substances and Products; ICH Expert Working Group: Geneva, Switzerland, 2003.
89. Cooperation Centre for Scientific Research Relative to Tobacco. CORESTA Guide No. 26: Technical Guide for Designing E-Vapour Product Stability Studies; Cooperation Centre for Scientific Research Relative to Tobacco: Paris, France, 2019.
90. International Conference on Harmonisation. ICH Harmonised Tripartite Guideline. Validation of Analytical Procedures: Text and Methodology Q2(R1); ICH Expert Working Group: Geneva, Switzerland, 2005; Step 4
91. ISO/IEC 17025:2017. General Requirements for the Competence of Testing and Calibration Laboratories; International Organization for Standardization: Geneva, Switzerland, 2017.
92. Jameson, J.B.; Wang, J.; Bailey, P.C.; Oldham, M.J.; Smith, C.R.; Jeong, L.N.; Cook, D.K.; Bates, A.L.; Ullah, S.; Pennington, A.S.C. et al. Determination of chemical constituent yields in e-cigarette aerosol using partial and whole pod collections, a comparative analysis. Front. Chem.; 2023; 11, 1223967. [DOI: https://dx.doi.org/10.3389/fchem.2023.1223967]
93.
94.
95.
96. Jordan, K.; Wieczorek, R.; Moennikes, O.; Crooks, I.; Hashizume, T.; Miller, J.; Weber, E.; Yoshino, K. The Rationale and Strategy for Conducting In Vitro Toxicology Testing of Tobacco Smoke; Cooperation Centre for Scientific Research Relative to Tobacco: Paris, France, 2019.
97. Organisation for Economic Co-operation and Development. Guidance Document No. 129: On Using Cytotoxicity Tests to Estimate Starting Doses for Acute Oral Systemic Toxicity Tests. OECD Environment, Health and Safety Publications; OECD Publishing: Paris, France, 2010.
98.
99. Organisation for Economic Co-Operation and Development. Test No. 471 Bacterial Reverse Mutation (withdrawn). OECD Guidelines for the Testing of Chemicals; Organisation for Economic Co-Operation and Development: Paris, France, 1997.
100. Organisation for Economic Co-operation and Development. Test No. 487: In Vitro Mammalian Cell Micronucleus Test, OECD Guidelines for the Testing of Chemicals, Section 4. OECD Guidelines for the Testing of Chemicals; OECD Publishing: Paris, France, 2016.
101. Organisation for Economic Co-operation and Development. Test No. 471: Bacterial Reverse Mutation Test. OECD Guidelines for the Testing of Chemicals; OECD Publishing: Paris, France, 2020.
102. Lalonde, G.; Demir, K.; Yao, J.; Wolz, R.L.; Kosachevsky, P.; Gillman, I.G.; Oldham, M.J. Characterization of a rapid condensate collection apparatus for in vitro assays of electronic nicotine delivery systems. Toxicol. In Vitro; 2022; 84, 105434. [DOI: https://dx.doi.org/10.1016/j.tiv.2022.105434] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35820568]
103.
104. Carpenter, M.J.; Wahlquist, A.E.; Dahne, J.; Gray, K.M.; Cummings, K.M.; Warren, G.; Wagener, T.L.; Goniewicz, M.L.; Smith, T.T. Effect of unguided e-cigarette provision on uptake, use, and smoking cessation among adults who smoke in the USA: A naturalistic, randomised, controlled clinical trial. eClinicalMedicine; 2023; 63, 102142. [DOI: https://dx.doi.org/10.1016/j.eclinm.2023.102142] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37753443]
105. Shiffman, S.; Goldenson, N.I.; Black, R.A. Changes in Dependence over One Year among Adult Smokers Who Switched Completely or Partially to Use of the JUUL System [Poster Presentation]. Proceedings of the Vermont Behavioral Health Conference; Burlington, VT, USA, 7–8 October 2021.
106. Coffa, B.G.; Doshi, U.; Zhang, J.; Desai, P.; McKinney, W.J.; Lee, K.M. Neutral Red Uptake Cytotoxicity Assay in A549 Cells Under Different Culture Conditions: Part of Assay Evaluation for Potential Use in Direct Aerosol In Vitro Testing [Poster Presentation]. Proceedings of the Tobacco Science Research Confernce; Bonita Springs, FL, USA, 28 November–1 December 2017.
107. Ward, A.M.; Yaman, R.; Ebbert, J.O. Electronic nicotine delivery system design and aerosol toxicants: A systematic review. PLoS ONE; 2020; 15, e0234189. [DOI: https://dx.doi.org/10.1371/journal.pone.0234189] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32497139]
108. Smart, D.J.; Phillips, G. Collecting e-cigarette aerosols for in vitro applications: A survey of the biomedical literature and opportunities to increase the value of submerged cell culture-based assessments. J. Appl. Toxicol.; 2021; 41, pp. 161-174. [DOI: https://dx.doi.org/10.1002/jat.4064] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33015847]
109. Dawkins, L.; Cox, S.; Goniewicz, M.; McRobbie, H.; Kimber, C.; Doig, M.; Kośmider, L. ‘Real-world’ compensatory behaviour with low nicotine concentration e-liquid: Subjective effects and nicotine, acrolein and formaldehyde exposure. Addiction; 2018; 113, pp. 1874-1882. [DOI: https://dx.doi.org/10.1111/add.14271]
110. Belushkin, M.; Djoko, D.T.; Esposito, M.; Korneliou, A.; Jeannet, C.; Lazzerini, M.; Jaccard, G. Selected Harmful and Potentially Harmful Constituents Levels in Commercial e-Cigarettes. Chem. Res. Toxicol.; 2020; 33, pp. 657-668. [DOI: https://dx.doi.org/10.1021/acs.chemrestox.9b00470] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31859484]
111. Herr, C.; Tsitouras, K.; Niederstraßer, J.; Backes, C.; Beisswenger, C.; Dong, L.; Guillot, L.; Keller, A.; Bals, R. Cigarette smoke and electronic cigarettes differentially activate bronchial epithelial cells. Respir. Res.; 2020; 21, 67. [DOI: https://dx.doi.org/10.1186/s12931-020-1317-2]
112. Bishop, E.; Terry, A.; East, N.; Breheny, D.; Gaça, M.; Thorne, D. A 3D in vitro comparison of two undiluted e-cigarette aerosol generating systems. Toxicol. Lett.; 2022; 358, pp. 69-79. [DOI: https://dx.doi.org/10.1016/j.toxlet.2022.01.002]
113. Czekala, L.; Simms, L.; Stevenson, M.; Tschierske, N.; Maione, A.G.; Walele, T. Toxicological comparison of cigarette smoke and e-cigarette aerosol using a 3D in vitro human respiratory model. Regul. Toxicol. Pharmacol.; 2019; 103, pp. 314-324. [DOI: https://dx.doi.org/10.1016/j.yrtph.2019.01.036]
114. Behar, R.Z.; Wang, Y.; Talbot, P. Comparing the cytotoxicity of electronic cigarette fluids, aerosols and solvents. Tob. Control; 2018; 27, pp. 325-333. [DOI: https://dx.doi.org/10.1136/tobaccocontrol-2016-053472]
115. Hua, M.; Omaiye, E.E.; Luo, W.; McWhirter, K.J.; Pankow, J.F.; Talbot, P. Identification of Cytotoxic Flavor Chemicals in Top-Selling Electronic Cigarette Refill Fluids. Sci. Rep.; 2019; 9, 2782. [DOI: https://dx.doi.org/10.1038/s41598-019-38978-w] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30808901]
116. Forest, V.; Mercier, C.; Pourchez, J. Considerations on dosimetry for in vitro assessment of e-cigarette toxicity. Respir. Res.; 2022; 23, 358. [DOI: https://dx.doi.org/10.1186/s12931-022-02286-1]
117. World Health Organization. The Scientific Basis of Tobacco Product Regulation: Second Report of a WHO Study Group. Tobacco Free Initiative; World Health Organization: Geneva, Switzerland, 2008.
118. Fowles, J.; Dybing, E. Application of toxicological risk assessment principles to the chemical constituents of cigarette smoke. Tob. Control; 2003; 12, pp. 424-430. [DOI: https://dx.doi.org/10.1136/tc.12.4.424] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/14660781]
119. U.S. Environmental Protection Agency. Guidelines for Carcinogen Risk Assessment; Risk Assessment Forum: Washington DC, USA, 2005.
120. U.S. Food and Drug Administration. Memorandum-SE Review: Evaluating Carcinogenic HPHC Increases and Assumption of Linearity for Low-Dose Extrapolation; U.S. Food and Drug Administration (FDA): Silver Spring, MD, USA, 2017.
121. Bergman, Å.; Becher, G.; Blumberg, B.; Bjerregaard, P.; Bornman, R.; Brandt, I.; Casey, S.C.; Frouin, H.; Giudice, L.C.; Heindel, J.J. et al. Manufacturing doubt about endocrine disrupter science—A rebuttal of industry-sponsored critical comments on the UNEP/WHO report “State of the Science of Endocrine Disrupting Chemicals 2012”. Regul. Toxicol. Pharmacol.; 2015; 73, pp. 1007-1017. [DOI: https://dx.doi.org/10.1016/j.yrtph.2015.07.026] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26239693]
122. U.S. Food and Drug Administration. Technical Project Lead Review of IQOS, PMTA: PM0000424, PM0000425, PM0000426, and PM0000479. Center of Tobacco Products; Food and Drug Administration (FDA): Silver Spring, MD, USA, 2019.
© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.
Details
; Wang, Jiaming; Bates, Austin; Ullah, Sifat; Sulaiman, Christina; Carter, Karen; Jongsma, Candice; Dull, Gary; Gillman, I Gene