Influence of Nitrite on Formation of Tobacco-Specific Nitrosamines in Electronic Cigarette Liquids and Aerosols.

Tobacco-specific nitrosamine (TSNA) formation occurred during aerosol generation from select commercial cig-a-like e-cigarette products. To understand the drivers behind the potential formation of TSNAs in electronic cigarette (e-cigarette) aerosols and e-liquids, model e-liquid systems were generated in the lab to demonstrate that nitrite can react with nicotine and minor alkaloids to form TSNAs in e-liquids. In the presence of nitrite and nicotine, TSNA levels in e-liquids increased over time and the process was accelerated by elevated temperature. Additionally, TSNAs formed during aerosol generation when nitrite was present in the corresponding e-liquids. The commercial e-cigarette products that showed higher levels and formation of TSNAs were observed to contain nitrite and minor alkaloid impurities in the corresponding e-liquids. This study provides valuable information about drivers for TSNA formation in e-liquids and e-cigarette aerosols that may be applied to the evaluation and quality assurance of e-cigarette products.

Formation of Diacetyl and Other α-Dicarbonyl Compounds during the Generation of E-Vapor Product Aerosols.

Exposure to diacetyl (DA) has been linked to the respiratory condition bronchiolitis obliterans. Previous research has demonstrated that DA and other α-dicarbonyl compounds can be detected in both the e-liquids and aerosols of e-vapor products (EVPs). While some EVP manufacturers may add these compounds as flavor ingredients, the primary objective of this work was to determine the potential for the formation of α-dicarbonyl compounds during the generation of aerosols from EVPs where no DA or other α-dicarbonyl compounds are added to the e-liquid. A novel ultraperformance liquid chromatography-mass spectrometry-based analytical method for the determination of DA, acetyl propionyl, glyoxal, and methylglyoxal was developed and validated. Next, eight commercially available cig-a-like-type EVPs were evaluated for α-dicarbonyl formation. Increased levels of α-dicarbonyls were observed in the aerosols of all evaluated EVPs compared to their respective e-liquids. Mechanistic studies were conducted using a model microwave reaction system to identify key reaction precursors for DA generated from propylene glycol (PG) and carbon-13-labeled glycerin (GLY). These studies, along with the corresponding retrosynthetic analysis, resulted in the proposed formation pathway where hydroxyacetone is generated from PG and/or GLY. Hydroxyacetone then participates in an aldol condensation with formaldehyde where formaldehyde can also be generated from PG and/or GLY; the resultant product then dehydrates to form DA. This proposed pathway was further investigated through in situ synthetic organic experiments within the model microwave reaction system. This work establishes that DA is formed in the aerosol generation process of the EVPs tested though at levels below toxicological concern.

An evaluation of electronic cigarette formulations and aerosols for harmful and potentially harmful constituents (HPHCs) typically derived from combustion.

U.S. FDA draft guidance recommends reporting quantities of designated harmful and potentially harmful constituents (HPHCs) in e-cigarette e-liquids and aerosols. The HPHC list comprises potential matrix-related compounds, flavors, nicotine, tobacco-related impurities, leachables, thermal degradation products, and combustion-related compounds. E-cigarettes contain trace levels of many of these constituents due to tobacco-derived nicotine and thermal degradation. However, combustion-related HPHCs are not likely to be found due to the relatively low operating temperatures of most e-cigarettes. The purpose of this work was to use highly sensitive, selective, and validated analytical methods to determine if these combustion-related HPHCs (three aromatic amines, five volatile organic compounds, and the polycyclic aromatic hydrocarbon benzo[a]pyrene) are detectable in commercial refill e-liquids, reference e-cigarette e-liquids, and aerosols generated from rechargeable e-cigarettes with disposable cartridges (often referred to as "cig-a-likes"). In addition, the transfer efficiency of these constituents from e-liquid to aerosol was evaluated when these HPHCs were added to the e-liquids prior to aerosol formation. This work demonstrates that combustion-related HPHCs are not present at measurable levels in the commercial and reference e-liquids or e-cigarette aerosols tested. Additionally, when combustion-related HPHCs are added to the e-liquids, they transfer to the aerosol with transfer efficiencies ranging from 49% to 99%.

Determination of Selected Chemical Levels in Room Air and on Surfaces after the Use of Cartridge- and Tank-Based E-Vapor Products or Conventional Cigarettes.

There is an ongoing debate regarding the potential of secondhand exposure of non-users to various chemicals from use of e-vapor products (EVPs). Room air levels of 34 chemicals (nicotine, propylene glycol (PG), glycerol, 15 carbonyl chemicals, 12 volatile organic chemicals (VOCs), and four selected trace elements) were measured where EVPs and cigarettes were used by n = 37 healthy adult tobacco users in an exposure chamber. The products used were MarkTen® 2.5% Classic (Group I), a Prototype GreenSmoke® 2.4% (Group II), Ego-T® Tank with subjects' own e-liquids (Group III) and subjects' own conventional cigarettes (Group IV). Products were used under controlled conditions and 4-h ad libitum use. Background (without subjects) and baseline levels (with subjects) were measured. Cumulative 4-h. levels of nicotine, PG and glycerol measured were several-fold below the time-weighted average limits used in workplace exposure evaluation. Most the other chemicals (>75%) were at or below the limit of quantification during EVP use. Significant levels of chemicals (17 out of 34) were observed in Group IV. Overall, our results indicate that under the study conditions with the products tested, cumulative room air levels of the selected chemicals measured over 4-h were relatively small and were several-fold below the current occupational regulatory and consensus limits.

Gas Chromatography-Mass Spectrometry Method to Quantify Benzo[a]Pyrene in Tobacco Products.

The USA Food and Drug Administration (FDA) established benzo[a]pyrene (B[a]P) as a harmful and potentially harmful constituent (HPHC) found in tobacco products. Tobacco manufacturers are required to report HPHC quantities to the FDA; however, there is currently no standardized method for determination of B[a]P in smokeless tobacco products (STPs). This work details a sensitive, selective and rapid method for the determination of B[a]P in STPs, cigarette filler and tobacco. Tobacco is extracted using methanol followed by solid-phase extraction and concentration prior to analysis by gas chromatography/mass spectrometry in the selected ion monitoring mode. Cooperation Centre for Scientific Research Relative to Tobacco reference products and 3R4F Kentucky reference cigarette filler were used for method validation. All method validation requirements were met including linearity, accuracy, precision, robustness, limit of detection (LOD) and limit of quantitation (LOQ), and stability. Calibration range of 0.5-125 ng mL-1 was achieved with the coefficient of determination (R2) greater than 0.995. The method LOQ and LOD were 0.729 and 0.216 ng/g, respectively. Using standardized methods for the measurement of HPHCs in tobacco products will reduce variability and ensure accurate data for regulatory reporting.

Development/verification of methods for measurement of exhaled breath and environmental e-vapor product aerosol.

Concerns have been raised about the potential health effects of potential bystander exposure to exhaled aerosols from e-vapor products (EVPs). An exhaled breath collection system (EBS) was developed and analytical methods were verified for collection and analysis of exhaled breath from users of EVPs. Analytical methods were adapted and verified for collection of environmental air samples during EVP use in an exposure chamber. Analysis of constituents in exhaled breath focused on nicotine, propylene glycol, and glycerin (because these are reported as the major constituents in EVPs) and selected carbonyl compounds (acetaldehyde, acrolein, and formaldehyde). Analysis of environmental samples included nicotine, propylene glycol, glycerin, 12 volatile organic compounds (VOCs), 15 carbonyl compounds and 4 metals. The EBS and analytical methods used were found to be suitable for collection and analysis of the target constituents in exhaled breath. Environmental sampling for background levels of VOCs and carbonyl compounds found only acetone, acetaldehyde, benzene, ethylbenzene, formaldehyde, isoprene, methyl ethyl ketone, hexaldehyde, propionaldehyde, and toluene above the limit of quantification in some samples. None of the targeted metals were detected. Background levels of VOCs and carbonyl compounds were consistent with levels previously reported for ambient air.

A Well-Mixed Computational Model for Estimating Room Air Levels of Selected Constituents from E-Vapor Product Use.

Concerns have been raised in the literature for the potential of secondhand exposure from e-vapor product (EVP) use. It would be difficult to experimentally determine the impact of various factors on secondhand exposure including, but not limited to, room characteristics (indoor space size, ventilation rate), device specifications (aerosol mass delivery, e-liquid composition), and use behavior (number of users and usage frequency). Therefore, a well-mixed computational model was developed to estimate the indoor levels of constituents from EVPs under a variety of conditions. The model is based on physical and thermodynamic interactions between aerosol, vapor, and air, similar to indoor air models referred to by the Environmental Protection Agency. The model results agree well with measured indoor air levels of nicotine from two sources: smoking machine-generated aerosol and aerosol exhaled from EVP use. Sensitivity analysis indicated that increasing air exchange rate reduces room air level of constituents, as more material is carried away. The effect of the amount of aerosol released into the space due to variability in exhalation was also evaluated. The model can estimate the room air level of constituents as a function of time, which may be used to assess the level of non-user exposure over time.

Insights from analysis for harmful and potentially harmful constituents (HPHCs) in tobacco products.

A total of 20 commercial cigarette and 16 commercial smokeless tobacco products were assayed for 96 compounds listed as harmful and potentially harmful constituents (HPHCs) by the US Food and Drug Administration. For each product, a single lot was used for all testing. Both International Organization for Standardization and Health Canada smoking regimens were used for cigarette testing. For those HPHCs detected, measured levels were consistent with levels reported in the literature, however substantial assay variability (measured as average relative standard deviation) was found for most results. Using an abbreviated list of HPHCs, statistically significant differences for most of these HPHCs occurred when results were obtained 4-6months apart (i.e., temporal variability). The assay variability and temporal variability demonstrate the need for standardized analytical methods with defined repeatability and reproducibility for each HPHC using certified reference standards. Temporal variability also means that simple conventional comparisons, such as two-sample t-tests, are inappropriate for comparing products tested at different points in time from the same laboratory or from different laboratories. Until capable laboratories use standardized assays with established repeatability, reproducibility, and certified reference standards, the resulting HPHC data will be unreliable for product comparisons or other decision making in regulatory science.

Insights from a multi-year program designed to test the impact of ingredients on mainstream cigarette smoke toxicity.

CONTEXT: Cigarette tobacco ingredients may alter the distribution of chemical constituents present in smoke. When considering the toxicological relevance of potential ingredient-related effects on chemical and biological measurements assessing cigarette smoke toxicity, it is critical to understand the intrinsic variability of tobacco and cigarette smoke that is influenced by the environmental conditions during growing, agricultural practices during preparation, cigarette manufacturing tolerances, and stability of the assay methods. OBJECTIVE: To understand possible effects of ingredients on cigarette smoke toxicity, various chemical and biological endpoints were measured in smoke from experimental cigarettes (added ingredient) to the intrinsic variability of control cigarettes (no added ingredient). MATERIALS AND METHODS: Data were collected during a multi-year program testing a variety of cigarette ingredients from several chemical classes. Chemical analysis of mainstream cigarette smoke,and biological procedures (Salmonella mutagenicity, cytotoxicity, and smoke inhalation) were performed using validated and controlled laboratory methods. The within-study and temporal variation of control cigarettes manufactured in parallel with experimental cigarettes was calculated and used to measure intrinsic variability. RESULTS: The overwhelming majority of data generated from experimental cigarettes fell within the experiment variability represented by the pooled standard error of the entire multi-year dataset for the control cigarettes. CONCLUSION: The results of this evaluation add to a growing body of the literature regarding a weight of evidence assessment of cigarette ingredient toxicity. When assessed against the variability of assay methodology, natural agricultural change, and manufacturing control, the ingredients studied here demonstrated little relevant influence on the mainstream cigarette smoke toxicity endpoints measured.

A comprehensive evaluation of the toxicology of cigarette ingredients: carbohydrates and natural products.

CONTEXT: Eleven carbohydrates and natural product ingredients were added individually to experimental cigarettes. OBJECTIVE: A battery of tests was used to compare toxicity of mainstream smoke from these experimental cigarettes to matched control cigarettes without test ingredients. MATERIALS AND METHODS: Smoke fractions from each cigarette type were evaluated using analytical chemistry; in vitro cytotoxicity (neutral red uptake) and in vitro bacterial (Salmonella) mutagenicity (five strains) testing. For 10 ingredients (ß-cyclodextrin, cleargum, D-sorbitol, high fructose corn syrup, honey, invert sugar, maltodextrin, molasses, raisin juice concentrate, and sucrose), 90-day nose-only smoke inhalation studies using rats were also performed. RESULTS: In general, addition of each ingredient in experimental cigarettes resulted in minimal changes in smoke chemistry; the exceptions were D-sorbitol and sucrose, where reductions in amount of 60% to 80% of control values for some smoke constituents were noted. Additionally, each ingredient resulted in small increases in smoke formaldehyde concentrations. Except for a reduction in cytotoxicity by inclusion of maltodextrin and an increase by inclusion of plum juice concentrate, the cytotoxicity and mutagenicity results were unaffected by addition of the other ingredients in experimental cigarettes. There were also very few statistically significant differences within any of the 10 inhalation studies, and when present, the differences were largely sporadic and inconsistent between sexes. CONCLUSION: The carbohydrates and natural products tested here as ingredients in experimental cigarettes as a class increased formaldehyde, but resulted in minimal toxicological responses, even at high inclusion levels compared with the levels used in commercial cigarette products.

An evaluation of the toxicity of 95 ingredients added individually to experimental cigarettes: approach and methods.

CONTEXT: Ingredients have been used in modern cigarette manufacturing to facilitate tobacco processing, provide flavor, and preserve tobacco. Concern has been raised regarding the use of ingredients in cigarette manufacturing due to the possible generation of toxic chemicals resulting from their combustion when added to tobacco. OBJECTIVE: Investigate the impact of individual ingredients on cigarette smoke toxicity. MATERIALS AND METHODS: A total of 95 ingredients were tested individually through addition at different concentrations to the tobacco of experimental cigarettes. Mainstream cigarette smoke chemistry analysis, bacterial mutagenicity testing, and cytotoxicity testing were conducted. Additionally, 31 of the ingredients were tested in 90-day nose-only rat inhalation studies using mainstream cigarette smoke. Studies were designed following conventional toxicity testing methods employed for food additives and other consumer products. RESULTS: The studies reported here demonstrate that high levels of some ingredients can change the quantity of some smoke constituents, altering the smoke chemistry profile. From the panel of biological endpoints monitored, these added ingredients produced minimal changes in the overall toxicity profile of mainstream cigarette smoke. In some cases, the addition of high levels of an ingredient caused a small reduction in toxicity findings, probably due to displacement of burning tobacco. CONCLUSION: The battery of testing results presented here is a useful addition to the available scientific information for cigarette ingredients and extends the dataset which can be used for evaluating their appropriate use.

Development of a quantitative method for the analysis of tobacco-specific nitrosamines in mainstream cigarette smoke using isotope dilution liquid chromatography/electrospray ionization tandem mass spectrometry.

An improved method has been developed for the determination of the four major tobacco-specific nitrosamines (TSNA) in mainstream cigarette smoke. The new method offers decreased sample preparation and analysis time as compared to traditional methodologies. This method uses isotope dilution liquid chromatography coupled to a tandem mass spectrometer with electrospray ionization and is significantly more sensitive than traditional methods. It also shows no evidence of artifactual formation of TSNA. Sample concentrations were determined for four TSNA in mainstream smoke using two isotopically labeled TSNA analogues as internal standards. Mainstream smoke was collected on an industry standard 44-mm Cambridge filter pad, extracted with an aqueous buffer solution, and analyzed without further sample cleanup. This method has been validated through intra- and interlaboratory studies and has shown excellent recoveries, sensitivity, and repeatability. The limits of detection of each TSNA varied from 0.01 to 0.1 ng/mL, and the linear calibration range of the instrument in sample matrix spanned 0.5-200 ng/ mL, which allowed for the determination of the TSNA levels in cigarettes with a wide range of deliveries. Data are also reported from two commercially available industry reference cigarettes and show excellent agreement and reproducibility over a six-month time period (n > 50).

Determination of trace metals in drinking water using solid-phase extraction disks and X-ray fluorescence spectrometry.

A convenient method is described for monitoring Cd, Ni, Cu, and Pb at trace levels in drinking water samples. These metals are preconcentrated on a chelating solid-phase extraction disk and then determined by X-ray fluorescence spectrometry. The method tolerates a wide pH range (pH 6-14) and a large amount of alkaline and alkaline earth elements. The preconcentration factor is well over 1600, assuming a 1 L water sample volume. The limits of detection for Cd, Ni, Cu, and Pb are 3.8, 0.6, 0.4, and 0.3 ng/mL, respectively. These are well below the federal maximum contaminant level values, which are 5, 100, 1300, and 15 ng/mL, respectively. The proposed method has many advantages including ease of operation, multielement capability, nondestructiveness, high sensitivity, and relative cost efficiency. The solid-phase extraction step can be conducted in the field and then the disks can be mailed to a laboratory for the analysis, eliminating the cost of transporting large volumes of water samples. Furthermore, the color of the used extraction disk provides an initial estimate of the degree of contamination for some transition metals (for example, Ni and Cu). Thus, the overall cost for analysis of metals in drinking water can be minimized by implementing the method, and small water supply companies with limited budgets will be better able to comply with the Safe Drinking Water Act.