Harmful and potentially harmful constituents (HPHCs) in two novel nicotine pouch products in comparison with regular smokeless tobacco products and pharmaceutical nicotine replacement therapy products (NRTs).

BACKGROUND: Tobacco-free nicotine pouches is a novel category of oral nicotine-delivery products. Among current tobacco users such pouches may serve as a low-risk alternative to cigarettes or conventional, tobacco-based oral products e.g., snus and moist snuff. In the United States (U.S.), the market leading nicotine-pouch brand is ZYN®. However, no data on the chemical characteristics of ZYN have been published. METHODS: We screened for 43 compounds potentially present in tobacco products in seven oral nicotine-delivery products: ZYN (dry and moist), snus (General®), moist snuff (CRP2.1 and Grizzly Pouches Wintergreen), and two pharmaceutical, nicotine replacement therapy products (NRTs, Nicorette® lozenge and Nicotinell® gum). Thirty-six of the tested compounds are classified as harmful and potentially harmful constituents (HPHCs) by the Center for Tobacco Products at the U.S. Food and Drug Administration (FDA-CTP). Five additional compounds were included to cover the GOTHIATEK® product standard for Swedish snus and the last two compounds were chosen to include the four primary tobacco specific nitrosamines (TSNAs). RESULTS: The tested products contained nicotine at varying levels. The two ZYN products contained no nitrosamines or polycyclic aromatic hydrocarbons (PAHs) but low levels of ammonia, chromium, formaldehyde, and nickel. In the NRT products we quantified low levels of acetaldehyde, ammonia, cadmium, chromium, lead, nickel, uranium-235, and uranium-238. The largest number (27) and generally the highest levels of HPHCs were quantified in the moist snuff products. For example, they contained six out of seven tested PAHs, and seven out of ten nitrosamines (including NNN and NNK). A total of 19 compounds, none of which were PAHs, were quantified at low levels in the snus product. NNN and NNK levels were five to 12-fold lower in snus compared to the moist snuff products. CONCLUSIONS: No nitrosamines or PAHs were quantified in the ZYN and NRT products. Overall, the number of quantified HPHCs were similar between ZYN and NRT products and found at low levels.

Analysis of waterpipe aerosol constituents in accordance with the ISO standard 22486.

This study analyzed commercial waterpipe tobacco products in accordance with the newly developed ISO 22486 as well as with commercial waterpipes and charcoals using the ISO 22486 puffing regime for comparison. The aerosols from these products were analyzed for their nicotine, humectant, tobacco specific nitrosamine, carbonyl, benzo[a]pyrene, and metal yields. Significant differences were observed among the waterpipe tobacco products when analyzed in accordance with the ISO standard 22486 and with different commercial waterpipes and charcoals. The concentrations of CO and benzo[a]pyrene observed in the consumers' configuration using the ISO 22486 puffing regime (with lit charcoal) were higher than those obtained with the ISO standard using electrical heating, with the yields for carbonyl compounds being lower or higher. The use of the recently published ISO standard for generating water pipe tobacco aerosols should be complemented with analysis by using the consumers' configuration. The necessity for this was demonstrated by the differences in CO and benzo[a]pyrene yields in the present work. It appears that the temperature (280°C) selected for electrical heating of waterpipe tobacco products in ISO 22486 is somewhat lower than that obtained with commercial charcoals, resulting in a generally lower yield of nicotine and total collected matter. In addition, there is a need to evaluate the contribution of commercial charcoals to the concentration of constituents in waterpipe aerosols. This is particularly true for compounds resulting from charcoal combustion, such as CO and benzo[a]pyrene.

Comparing the preclinical risk profile of inhalable candidate and potential candidate modified risk tobacco products: A bridging use case.

Cigarette smoking causes major preventable diseases, morbidity, and mortality worldwide. Smoking cessation and prevention of smoking initiation are the preferred means for reducing these risks. Less harmful tobacco products, termed modified-risk tobacco products (MRTP), are being developed as a potential alternative for current adult smokers who would otherwise continue smoking. According to a regulatory framework issued by the US Food and Drug Administration, a manufacturer must provide comprehensive scientific evidence that the product significantly reduces harm and the risk of tobacco-related diseases, in order to obtain marketing authorization for a new MRTP. For new tobacco products similar to an already approved predicate product, the FDA has foreseen a simplified procedure for assessing "substantial equivalence". In this article, we present a use case that bridges the nonclinical evidence from previous studies demonstrating the relatively reduced harm potential of two heat-not-burn products based on different tobacco heating principles. The nonclinical evidence was collected along a "causal chain of events leading to disease" (CELSD) to systematically follow the consequences of reduced exposure to toxicants (relative to cigarette smoke) through increasing levels of biological complexity up to disease manifestation in animal models of human disease. This approach leverages the principles of systems biology and toxicology as a basis for further extrapolation to human studies. The experimental results demonstrate a similarly reduced impact of both products on apical and molecular endpoints, no novel effects not seen with cigarette smoke exposure, and an effect of switching from cigarettes to either MRTP that is comparable to that of complete smoking cessation. Ideally, a subset of representative assays from the presented sequence along the CELSD could be sufficient for predicting similarity or substantial equivalence in the nonclinical impact of novel products; this would require further validation, for which the present use case could serve as a starting point.

Biomarkers of Exposure and Potential Harm among Natural American Spirit Smokers.

OBJECTIVES: We compared biomarkers of exposure and potential harm in smokers of American Spirit (AS) to smokers of Marlboro, Newport, Camel, and Pall Mall. METHODS: We conducted secondary analysis on: (1) data from a randomized clinical trial (RCT); and (2) the Population Assessment of Tobacco Use and Health (PATH) Study. Biomarkers analyzed included: total nicotine equivalents (TNE); 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL); N'-nitrosonornicotine and its N-glucuronide (total NNN);3-hydroxypropylmercapturic acid(3-HPMA); 2-hydroxypropylmercapturic acid (2-HPMA), 3-hydroxy-1 methylpropylmercapturic acid (HMPMA); S-phenylmercapturic acid(SPMA); 2-cyanoethylmercapturic acid (CEMA); phenanthrene tetraol(PheT);1-hydroxypyrene (1-HOP);8-iso-PGF2α; white blood count(WBC); prostaglandin E metabolite(PGEM); and high sensitivity C-reactive protein(hsCRP). RESULTS: AS smokers did not differ in TNE but had higher TNE per cigarette compared to other brands. Total NNAL, total NNN, CEMA, and 3-HPMA were lower in AS smokers. All other biomarkers were no different in AS smokers compared to all or the majority of the other brands. CONCLUSIONS: Levels of total NNAL, total NNN, acrylonitrile, and acrolein were reduced in AS smokers; however, it is not known whether reductions in exposure to these toxicants contribute to reduced harm. Higher TNE per cigarette smoked in AS smokers suggests a greater addictive potential compared to other brands. Regulatory action to ensure that consumers are not misled about the risks of the AS brand are recommended.

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%.

Novel approach for selective reduction of NNN in cigarette tobacco filler and mainstream smoke.

Research conducted during past decades to reduce the level of the tobacco specific nitrosamine N-nitrosonornicotine (NNN) and its precursor nornicotine in tobacco yielded identification of three tobacco genes encoding for cytochrome P450 nicotine demethylases converting nicotine to nornicotine. We carried out trials to investigate the effect of using tobaccos containing three non-functional nicotine demethylase genes on the selective reduction of NNN in cigarette tobacco filler and mainstream smoke. Our results indicate that the presence of non-functional alleles of the three genes reduces the level of nornicotine and NNN in Burley tobacco by 70% compared to the level observed in currently available low converter (LC) Burley tobacco varieties. The new technology, named ZYVERT™, does not require a regular screening process, while a yearly selection process is needed to produce LC Burley tobacco seeds for NNN reduction. The reduction of NNN observed in smoke of blended prototype cigarettes is proportional to the inclusion level of tobacco having ZYVERT™ technology. Inclusion of Burley tobacco possessing the new trait into a typical American blend resulted in a selective reduction of NNN in cigarette smoke, while the levels of other Harmful and Potentially Harmful Constituents (HPHC) currently in the abbreviated list provided by the US Food and Drug Administration are statistically equivalent in comparison with the levels obtained in reference prototype cigarettes containing LC Burley.

The use of charcoal in modified cigarette filters for mainstream smoke carbonyl reduction.

Carbonyls are harmful and potentially harmful constituents (HPHCs) in mainstream cigarette smoke (MSS). Carbonyls, including formaldehyde and acrolein, are carcinogenic or mutagenic in a dose-dependent manner. Past studies demonstrate significant reduction of HPHCs by charcoal filtration. However, limits of charcoal filtration and cigarette design have not yet been investigated in a systematic manner. Objective data is needed concerning the feasibility of HPHC reduction in combustible filtered cigarettes. This systematic study evaluates the effect of charcoal filtration on carbonyl reduction in MSS. We modified filters of ten popular cigarette products with predetermined quantities (100-400 mg) of charcoal in a plug-space-plug configuration. MSS carbonyls, as well as total particulate matter, tar, nicotine, carbon monoxide (TNCO), and draw resistance were quantified. Significant carbonyl reductions were observed across all cigarette products as charcoal loading increased. At the highest charcoal loadings, carbonyls were reduced by nearly 99%. Tar and nicotine decreased modestly (<20%) compared to reductions in carbonyls. Increased draw resistance was significant at only the highest charcoal loadings. This work addresses information gaps in the science base that can inform the evaluation of charcoal filtration as an available technological adaptation to cigarette design which reduces levels of carbonyls in MSS.

Evaluation of the Tobacco Heating System 2.2. Part 2: Chemical composition, genotoxicity, cytotoxicity, and physical properties of the aerosol.

The chemical composition, in vitro genotoxicity, and cytotoxicity of the mainstream aerosol from the Tobacco Heating System 2.2 (THS2.2) were compared with those of the mainstream smoke from the 3R4F reference cigarette. In contrast to the 3R4F, the tobacco plug in the THS2.2 is not burnt. The low operating temperature of THS2.2 caused distinct shifts in the aerosol composition compared with 3R4F. This resulted in a reduction of more than 90% for the majority of the analyzed harmful and potentially harmful constituents (HPHCs), while the mass median aerodynamic diameter of the aerosol remained similar. A reduction of about 90% was also observed when comparing the cytotoxicity determined by the neutral red uptake assay and the mutagenic potency in the mouse lymphoma assay. The THS2.2 aerosol was not mutagenic in the Ames assay. The chemical composition of the THS2.2 aerosol was also evaluated under extreme climatic and puffing conditions. When generating the THS2.2 aerosol under "desert" or "tropical" conditions, the generation of HPHCs was not significantly modified. When using puffing regimens that were more intense than the standard Health Canada Intense (HCI) machine-smoking conditions, the HPHC yields remained lower than when smoking the 3R4F reference cigarette with the HCI regimen.

Evaluation of the Tobacco Heating System 2.2. Part 3: Influence of the tobacco blend on the formation of harmful and potentially harmful constituents of the Tobacco Heating System 2.2 aerosol.

The Tobacco Heating System (THS2.2), which uses "heat-not-burn" technology, generates an aerosol from tobacco heated to a lower temperature than occurs when smoking a combustible cigarette. The concentrations of harmful and potentially harmful constituents (HPHCs) are significantly lower in THS2.2 mainstream aerosol than in smoke produced by combustible cigarettes. Different tobacco types and 43 tobacco blends were investigated to determine how the blend impacted the overall reductions of HPHCs in the THS2.2 mainstream aerosol. The blend composition had minimal effects on the yields of most HPHCs in the aerosol. Blends containing high proportions of nitrogen-rich tobacco, e.g., air-cured, and some Oriental tobaccos, produced higher acetamide, acrylamide, ammonia, and nitrogen oxide yields than did other blends. Most HPHCs were found to be released mainly through the distillation of HPHCs present in the tobacco plug or after being produced in simple thermal reactions. HPHC concentrations in the THS2.2 aerosol may therefore be further minimized by limiting the use of flue- and fire-cured tobaccos which may be contaminated by HPHCs during the curing process and carefully selecting nitrogen rich tobaccos with low concentrations of endogenous HPHCs for use in the tobacco plug blend.

Characterization of potential impurities and degradation products in electronic cigarette formulations and aerosols.

E-cigarettes are gaining popularity in the U.S. as well as in other global markets. Currently, limited published analytical data characterizing e-cigarette formulations (e-liquids) and aerosols exist. While FDA has not published a harmful and potentially harmful constituent (HPHC) list for e-cigarettes, the HPHC list for currently regulated tobacco products may be useful to analytically characterize e-cigarette aerosols. For example, most e-cigarette formulations contain propylene glycol and glycerin, which may produce aldehydes when heated. In addition, nicotine-related chemicals have been previously reported as potential e-cigarette formulation impurities. This study determined e-liquid formulation impurities and potentially harmful chemicals in aerosols of select commercial MarkTen(®) e-cigarettes manufactured by NuMark LLC. The potential hazard of the identified formulation impurities and aerosol chemicals was also estimated. E-cigarettes were machine puffed (4-s duration, 55-mL volume, 30-s intervals) to battery exhaustion to maximize aerosol collection. Aerosols analyzed for carbonyls were collected in 20-puff increments to account for analyte instability. Tobacco specific nitrosamines were measured at levels observed in pharmaceutical grade nicotine. Nicotine-related impurities in the e-cigarette formulations were below the identification and qualification thresholds proposed in ICH Guideline Q3B(R2). Levels of potentially harmful chemicals detected in the aerosols were determined to be below published occupational exposure limits.

Comparison of select analytes in aerosol from e-cigarettes with smoke from conventional cigarettes and with ambient air.

Leading commercial electronic cigarettes were tested to determine bulk composition. The e-cigarettes and conventional cigarettes were evaluated using machine-puffing to compare nicotine delivery and relative yields of chemical constituents. The e-liquids tested were found to contain humectants, glycerin and/or propylene glycol, (⩾75% content); water (<20%); nicotine (approximately 2%); and flavor (<10%). The aerosol collected mass (ACM) of the e-cigarette samples was similar in composition to the e-liquids. Aerosol nicotine for the e-cigarette samples was 85% lower than nicotine yield for the conventional cigarettes. Analysis of the smoke from conventional cigarettes showed that the mainstream cigarette smoke delivered approximately 1500times more harmful and potentially harmful constituents (HPHCs) tested when compared to e-cigarette aerosol or to puffing room air. The deliveries of HPHCs tested for these e-cigarette products were similar to the study air blanks rather than to deliveries from conventional cigarettes; no significant contribution of cigarette smoke HPHCs from any of the compound classes tested was found for the e-cigarettes. Thus, the results of this study support previous researchers' discussion of e-cigarette products' potential for reduced exposure compared to cigarette smoke.