Multi-omic applications for understanding and enhancing tropical fruit flavour.

Consumer trends towards nutrient-rich foods are contributing to global increasing demand for tropical fruit. However, commercial cultivars in the breeding pipeline that are tailored to meet market demand are at risk of possessing reduced fruit flavour qualities. This stems from recurrent prioritised selection for superior agronomic traits and not fruit flavour, which may in turn reduce consumer satisfaction. There is realisation that fruit quality traits, inclusive of flavour, must be equally selected for; but currently, there are limited tools and resources available to select for fruit flavour traits, particularly in tropical fruit species. Although sugars, acids, and volatile organic compounds are known to define fruit flavour, the specific combinations of these, that result in defined consumer preferences, remain unknown for many tropical fruit species. To define and include fruit flavour preferences in selective breeding, it is vital to determine the metabolites that underpin them. Then, objective quantitative analysis may be implemented instead of solely relying on human sensory panels. This may lead to the development of selective genetic markers through integrated omics approaches that target biosynthetic pathways of flavour active compounds. In this review, we explore progress in the development of tools to be able to strategically define and select for consumer-preferred flavour profiles in the breeding of new cultivars of tropical fruit species.

Editing VvDXS1 for the creation of muscat flavour in Vitis vinifera cv. Scarlet Royal.

Muscat flavour represents a group of unique aromatic attributes in some grape varieties. Biochemically, grape berries with muscat flavour produce high levels of monoterpenes. Monoterpene biosynthesis is mainly through the DOXP/MEP pathway, and VvDXS1 encodes the first enzyme in this plastidial pathway of terpene biosynthesis in grapevine. A single-point mutation resulting in the substitution of a lysine with an asparagine at position 284 in the VvDXS1 protein has previously been identified as the major cause for producing muscat flavour in grapes. In this study, the same substitution in the VvDXS1 protein was successfully created through prime editing in the table grape Vitis vinifera cv. 'Scarlet Royal'. The targeted point mutation was detected in most of the transgenic vines, with varying editing efficiencies. No unintended mutations were detected in the edited alleles, either by PCR Sanger sequencing or by amplicon sequencing. More than a dozen edited vines were identified with an editing efficiency of more than 50%, indicating that these vines were likely derived from single cells in which one allele was edited. These vines had much higher levels of monoterpenes in their leaves than the control, similar to what was found in leaf samples between field-grown muscat and non-muscat grapes.

Unlocking lager's flavour palette by metabolic engineering of Saccharomyces pastorianus for enhanced ethyl ester production.

Despite being present in trace amounts, ethyl esters play a crucial role as flavour compounds in lager beer. In yeast, ethyl hexanoate, ethyl octanoate and ethyl decanoate, responsible for fruity and floral taste tones, are synthesized from the toxic medium chain acyl-CoA intermediates released by the fatty acid synthase complex during the fatty acid biosynthesis, as a protective mechanism. The aim of this study was to enhance the production of ethyl esters in the hybrid lager brewing yeast Saccharomyces pastorianus by improving the medium chain acyl-CoA precursor supply. Through CRISPR-Cas9-based genetic engineering, specific FAS1 and FAS2 genes harbouring mutations in domains of the fatty acid synthesis complex were overexpressed in a single and combinatorial approach. These mutations in the ScFAS genes led to specific overproduction of the respective ethyl esters: overexpression of ScFAS1I306A and ScFAS2G1250S significantly improved ethyl hexanoate production and ScFAS1R1834K boosted the ethyl octanoate production. Combinations of ScFAS1 mutant genes with ScFAS2G1250S greatly enhanced predictably the final ethyl ester concentrations in cultures grown on full malt wort, but also resulted in increased levels of free medium chain fatty acids causing alterations in flavour profiles. Finally, the elevated medium chain fatty acid pool was directed towards the ethyl esters by overexpressing the esterase ScEEB1. The genetically modified S. pastorianus strains were utilized in lager beer production, and the resulting beverage exhibited significantly altered flavour profiles, thereby greatly expanding the possibilities of the flavour palette of lager beers.

Microbial terroir: associations between soil microbiomes and the flavor chemistry of mustard (Brassica juncea).

Here, we characterized the independent role of soil microbiomes (bacterial and fungal communities) in determining the flavor chemistry of harvested mustard seed (Brassica juncea). Given the known impacts of soil microbial communities on various plant characteristics, we hypothesized that differences in rhizosphere microbiomes would result in differences in seed flavor chemistry (glucosinolate content). In a glasshouse study, we introduced distinct soil microbial communities to mustard plants growing in an otherwise consistent environment. At the end of the plant life cycle, we characterized the rhizosphere and root microbiomes and harvested produced mustard seeds for chemical characterization. Specifically, we measured the concentrations of glucosinolates, secondary metabolites known to create spicy and bitter flavors. We examined associations between rhizosphere microbial taxa or genes and seed flavor chemistry. We identified links between the rhizosphere microbial community composition and the concentration of the main glucosinolate, allyl, in seeds. We further identified specific rhizosphere taxa predictive of seed allyl concentration and identified bacterial functional genes, namely genes for sulfur metabolism, which could partly explain the observed associations. Together, this work offers insight into the potential influence of the belowground microbiome on the flavor of harvested crops.

In vitro toxicological evaluation of aerosols generated by a 4th generation vaping device using nicotine salts in an air-liquid interface system.

BACKGROUND: Electronic cigarettes (EC) have gained popularity, especially among young people, with the introduction of fourth-generation devices based on e-liquids containing nicotine salts that promise a smoother vaping experience than freebase nicotine. However, the toxicological effects of nicotine salts are still largely unknown, and the chemical diversity of e-liquids limits the comparison between different studies to determine the contribution of each compound to the cytotoxicity of EC aerosols. Therefore, the aim of this study was to evaluate the toxicological profile of controlled composition e-liquid aerosols to accurately determine the effects of each ingredient based on exposure at the air-liquid interface. METHODS: Human lung epithelial cells (A549) were exposed to undiluted aerosols of controlled composition e-liquids containing various ratios of propylene glycol (PG)/vegetable glycerin (VG) solvents, freebase nicotine, organic acids, nicotine salts, and flavoured commercial e-liquids. Exposure of 20 puffs was performed at the air-liquid interface following a standard vaping regimen. Toxicological outcomes, including cytotoxicity, inflammation, and oxidative stress, were assessed 24 h after exposure. RESULTS: PG/VG aerosols elicited a strong cytotoxic response characterised by a 50% decrease in cell viability and a 200% increase in lactate dehydrogenase (LDH) production, but had no effects on inflammation and oxidative stress. These effects occurred only at a ratio of 70/30 PG/VG, suggesting that PG is the major contributor to aerosol cytotoxicity. Both freebase nicotine and organic acids had no greater effect on cell viability and LDH release than at a 70/30 PG/VG ratio, but significantly increased inflammation and oxidative stress. Interestingly, the protonated form of nicotine in salt showed a stronger proinflammatory effect than the freebase nicotine form, while benzoic acid-based nicotine salts also induced significant oxidative stress. Flavoured commercial e-liquids was found to be cytotoxic at a threshold dose of ≈ 330 µg/cm². CONCLUSION: Our results showed that aerosols of e-liquids consisting only of PG/VG solvents can cause severe cytotoxicity depending on the concentration of PG, while nicotine salts elicit a stronger pro-inflammatory response than freebase nicotine. Overall, aerosols from fourth-generation devices can cause different toxicological effects, the nature of which depends on the chemical composition of the e-liquid.

Free Radicals in Little Cigar Mainstream Smoke and the Potential Influence of Flavoring Chemicals on Free Radical Production.

Implementation of the Tobacco Control Act in 2009 banned characterizing flavors in cigarettes (except menthol and tobacco), but substitution has occurred by the continued availability of alternative flavored products (i.e., flavored little cigars). Little is known about how flavorants in noncigarette tobacco products impact human health. Thus, we investigated the impact of flavorants on free radical production in the mainstream smoke of little cigars. Gas- and particulate-phase free radical yields in mainstream smoke generated from 12 commercial little cigar brands and research little cigars and cigarettes were measured via electron paramagnetic resonance spectroscopy using the International Organization of Standardization (ISO) smoking protocol. Flavorants were extracted from unsmoked little cigars and analyzed by gas chromatography-mass spectroscopy. Gas- and particulate-phase radical yields from little cigars ranged from 13.5 to 97.6 and 0.453-1.175 nmol/unit, respectively. Comparatively, research cigarettes yielded an average of 4.9 nmol gas-phase radicals/unit and 0.292 nmol particulate-phase radicals/unit. From the products, 66 flavorants were identified, with each brand containing 4-24 individual flavorants. The free radical content was strongly correlated with the number of flavorants present in each cigar (r = 0.74, p = 0.01), indicating that highly flavored little cigars may produce higher levels of toxic free radicals. The presence of the flavorant ethyl methylphenylglycidate (strawberry) was associated with >2-fold higher levels of GP radicals (p = 0.001). Our results show that free radical delivery from little cigars is greater than that from research cigarettes and provide empirical evidence for the harmfulness of flavored tobacco products. Additionally, it demonstrates that flavorants present in combustible tobacco products can influence the levels of free radicals produced. Therefore, future tobacco product standards should consider little cigars.

Ozonolysis of Terpene Flavor Additives in Vaping Emissions: Elevated Production of Reactive Oxygen Species and Oxidative Stress.

The production of e-cigarette aerosols through vaping processes is known to cause the formation of various free radicals and reactive oxygen species (ROS). Despite the well-known oxidative potential and cytotoxicity of fresh vaping emissions, the effects of chemical aging on exhaled vaping aerosols by indoor atmospheric oxidants are yet to be elucidated. Terpenes are commonly found in e-liquids as flavor additives. In the presence of indoor ozone (O3), e-cigarette aerosols that contain terpene flavorings can undergo chemical transformations, further producing ROS and reactive carbonyl species. Here, we simulated the aging process of the e-cigarette emissions in a 2 m3 FEP film chamber with 100 ppbv of O3 exposure for an hour. The aged vaping aerosols, along with fresh aerosols, were collected to detect the presence of ROS. The aged particles exhibited 2- to 11-fold greater oxidative potential, and further analysis showed that these particles formed a greater number of radicals in aqueous conditions. The aging process induced the formation of various alkyl hydroperoxides (ROOH), and through iodometric quantification, we saw that our aged vaping particles contained significantly greater amounts of these hydroperoxides than their fresh counterparts. Bronchial epithelial cells exposed to aged vaping aerosols exhibited an upregulation of the oxidative stress genes, HMOX-1 and GSTP1, indicating the potential for inhalation toxicity. This work highlights the indirect danger of vaping in environments with high ground-level O3, which can chemically transform e-cigarette aerosols into new particles that can induce greater oxidative damage than fresh e-cigarette aerosols. Given that the toxicological characteristics of e-cigarettes are mainly associated with the inhalation of fresh aerosols in current studies, our work may provide a perspective that characterizes vaping exposure under secondhand or thirdhand conditions as a significant health risk.

The potential for Scotch Malt Whisky flavour diversification by yeast.

Scotch Whisky, a product of high importance to Scotland, has gained global approval for its distinctive qualities derived from the traditional production process, which is defined in law. However, ongoing research continuously enhances Scotch Whisky production and is fostering a diversification of flavour profiles. To be classified as Scotch Whisky, the final spirit needs to retain the aroma and taste of 'Scotch'. While each production step contributes significantly to whisky flavour-from malt preparation and mashing to fermentation, distillation, and maturation-the impact of yeast during fermentation is crucially important. Not only does the yeast convert the sugar to alcohol, it also produces important volatile compounds, e.g. esters and higher alcohols, that contribute to the final flavour profile of whisky. The yeast chosen for whisky fermentations can significantly influence whisky flavour, so the yeast strain employed is of high importance. This review explores the role of yeast in Scotch Whisky production and its influence on flavour diversification. Furthermore, an extensive examination of nonconventional yeasts employed in brewing and winemaking is undertaken to assess their potential suitability for adoption as Scotch Whisky yeast strains, followed by a review of methods for evaluating new yeast strains.

Use patterns of flavored non-cigarette tobacco products among US adults, 2010-2019.

OBJECTIVE: Flavored non-cigarette tobacco product (NCTP) use is common among US adult tobacco users. To update the estimates of use patterns of flavored NCTPs, this study assessed current NCTP use among adults by flavor use and flavor categories from 2010 to 2019. METHODS: We analyzed data from the 2010-2019 Tobacco Use Supplement to the Current Population Survey to estimate the weighted proportion of adult NCTP users by flavor use across survey waves. Flavor use was defined as past 30-day use of any menthol/mint or fruit/other flavors. We used the 2018-2019 data to examine the differences in demographic characteristics and tobacco use patterns among users of menthol/mint or fruit/other flavors compared to exclusive users of tobacco flavor, by product type. RESULTS: Compared to 2014-2015, electronic nicotine delivery system (ENDS) users were more likely (79.0% vs. 66.6%, p < 0.001) to report flavor use in 2018-2019, whereas cigar (26.9% vs. 31.2%, p = 0.030) and pipe (56.3% vs. 65.5%, p = 0.015) smokers were less likely to report flavor use in 2018-2019. In 2018-2019, the most prevalent flavor categories were exclusive use of tobacco flavor among cigar (73.1%) and smokeless tobacco (48.3%) users, and use of fruit/other flavors among ENDS (64.9%) and pipe (48.4%) users. Flavored users were more likely to be young adults aged 18-24 years (cigars, ENDS, smokeless tobacco) and Non-Hispanic Black or Hispanic persons (cigars, ENDS, pipes) compared to tobacco-flavored users. CONCLUSIONS: Flavored product use increased among adult ENDS users but decreased among cigar and pipe smokers. These findings could inform tobacco regulatory efforts concerning flavored NCTPs.

Sex differences in appeal, reward, and sensory experience of E-cigarette flavors among adults who smoke cigarettes.

OBJECTIVE: Flavors enhance nicotine e-cigarette appeal by altering sensory experience. Females may be more sensitive to tobacco-associated cues and uniquely affected by flavor. The current study is an exploratory analysis to examine differences by sex on reward, appeal, and sensory experience of popular e-cigarette flavors. METHODS: Adults (N = 121) who use cigarettes (≥1 cigarette/day) were enrolled in a laboratory study in which they vaped four e-cigarette flavors (tobacco, cherry, menthol, vanilla; in separate lab sessions) in one of two freebase nicotine concentrations (6 mg/ml,18 mg/ml). Following exposures, participants rated e-cigarette reward using the Drug Effects Questionnaire (DEQ), flavor and overall e-cigarette appeal using Labeled Hedonic Scales (LHS), and intensity of flavor, irritation, coolness, fruitiness, and sweetness using Generalized Labeled Magnitude Scales (gLMS). Linear mixed models were conducted for outcomes to analyze effects of sex, flavor, and sex x flavor interaction. RESULTS: For DEQ ratings, there was a trend (p = .08) toward a sex x flavor interaction, in which menthol flavor produced more reward than other flavors for females, but not males. For LHS ratings, there was a significant sex x flavor interaction (p = .03) for overall e-cigarette experience with females but not males rating menthol higher than other flavors. All gLMS scales but irritation showed that females generally had greater differences between flavors compared to males (ps ≤ 0.03). CONCLUSION: Menthol in e-cigarettes may play an important role in mediating appeal and/or reward among females. This may be due in part to their ability to better detect sensory effects of e-cigarettes.

Non-tobacco nicotine E-cigarette use and flavored E-cigarette use among young adults in the United States.

BACKGROUND: E-cigarette flavors can create sensations of sweetness and coolness while masking the aversiveness of nicotine. Recently, non-tobacco nicotine (NTN) products were introduced to the market, but little is known about flavors in NTN e-cigarette use. We examined associations between flavors (i.e., sweet, mint/menthol) and susceptibility to and use of NTN e-cigarettes. METHODS: 1239 US young adults (18-25 years) completed an anonymous, online survey in Fall 2021. The analytic sample included 520 participants who had used e-cigarettes and heard of NTN. Multinomial logistic regression models analyzed associations of flavored e-cigarette use (sweet and mint/menthol) with NTN e-cigarette use status (i.e., current [past-month] use, past [ever but not current] use, susceptible to use, and non-susceptible to use [reference]). RESULTS: Overall, 46.2% of participants reported current NTN use, 14.8% reported past use, 16.7% were susceptible to use, and 22.3% reported no susceptibility. Participants reported dual-use of sweet and mint/menthol NTN e-cigarette flavors (56.5%), sweet flavors use (24.8%), and mint/menthol flavor use (1.7%). Ever dual use of sweet and mint/menthol flavors was associated with current (OR = 9.64, 95%CI: 3.21-28.98) and past NTN e-cigarette use (8.30, [2.10-32.80]). Ever sweet flavor use was associated with current NTN use (3.80, 95%CI: 1.44-10.03) and susceptibility to future use (4.25, [1.53-11.81]). Similar findings were observed for mint/menthol flavors (current: 5.03, [1.41-17.99]; susceptible: 5.65, [1.64-19.51]). CONCLUSION: The use of sweet and mint/menthol flavors was significantly associated with NTN e-cigarette use among US young adults, highlighting the need for ongoing surveillance of flavored NTN e-cigarettes and appropriate regulations to discourage use.

Cross-sectional patterns and longitudinal transitions of unflavored and flavored cigar use without and with cigarettes among United States adults.

INTRODUCTION: Over half of US adults who smoke cigars use flavored cigars, illustrating their broad appeal; however, their long-term impact on cigar and cigarette use is unknown. METHODS: Using restricted data from Waves 1-5 (2013-2019) of the Population Assessment of Tobacco and Health Study, we investigated cross-sectional patterns and longitudinal transition rates of unflavored and flavored cigar use with and without cigarettes among a nationally representative sample of US adults. RESULTS: Proportionally, more adults who used flavored cigars without or with cigarettes were younger and female. More adults with exclusive cigar use were non-Hispanic Black. More adults with dual use had lower educational attainment. The median number of cigars smoked daily and tobacco dependence was highest among adults who used flavored cigars with cigarettes. Only 14.6% of adults with exclusive flavored cigar use at Wave 1 continued their use to Wave 5, with most transitioning to non-current (46.4%) or exclusive cigarette use (22.9%). Likewise, 13.8% of adults with dual flavored cigar and cigarette use at Wave 1 continued their use to Wave 5, with 57.6% transitioning to exclusive cigarette use and 19.7% transitioning to non-current use. Comparatively, 72.9% of adults with exclusive cigarette use continued their use to Wave 5, while 23.6% transitioned to non-current use. CONCLUSION: Adult cigar use was less stable than cigarette use, particularly among those who use flavored cigars. Future research should investigate whether these transition patterns between flavored and unflavored cigar and cigarette use vary across sociodemographic groups and their potential long-term health implications.

Would banning menthol cigarettes, flavored cigars, and flavored e-cigarettes prompt users to seek illicit channels for obtaining them in the United States?

BACKGROUND: Banning flavors in tobacco and nicotine products may reduce youth initiation and prompt quit attempts but such bans may lead to illicit markets. We examined how likely current users would be to seek flavored products from illicit channels under various ban scenarios. METHODS: Cross-sectional surveys of 2552 current users of menthol cigarettes or flavored cigars and 2347 users of flavored e-cigarettes were conducted between 2021 and 2022 in the United States. For each ban scenario, respondents reported if they would have intentions to seek the banned flavored products from any illicit channels and identified the specific illicit channel they would consider. Logistic regressions were used to estimate how the likelihood of having intentions to seek illicit channels was associated with demographics, ban scenarios, and status of tobacco use. RESULTS: Under various ban scenarios, 24-30% of people who smoked said they would seek illicit channels to obtain the banned products compared with 21-41% of dual users and 35-39% of users of flavored e-cigarettes. Online retailers were favored by people who smoked while users of flavored e-cigarettes favored local retailers. Heavy users were more likely to say they would try illicit channels. Under bans restricting more types of flavored tobacco products, users would be less likely to try illegal channels. CONCLUSIONS: A significant proportion of users of flavored tobacco and nicotine products would not reject using illicit banned products. Tailored programs are needed to apply to the groups with a higher risk of seeking illicit channels for banned products.

Quality evaluation of natural monomer flavors for Chinese tobacco industry based on dual mode combined Fourier transform ion cyclotron resonance mass spectrometry and isotopic fine structures.

RATIONALE: Natural monomer flavors can modify the taste of cigarettes. However, no report was published to establish the quality control method for their chemical compositions. METHODS: In this study, licorice, a traditional natural monomer flavor used in tobacco aroma processing, was selected, and the fingerprint was developed by high-performance liquid chromatography (HPLC). Next, the chemical markers of samples from different places of origin were discovered by multivariate statistical analysis. Then, its chemical constituents were identified by combination of HPLC-Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), direct infusion FT-ICR-MS (DI-FT-ICR-MS), and the technology of isotopic fine structures (IFSs). Moreover, its characteristic constituents were quantitatively analyzed using HPLC. RESULTS: The 14 common peaks were assigned in the fingerprint, and 8 of them were considered as qualitative markers by multivariate statistical analysis. A total of 42 chemical constituents were detected using HPLC-FT-ICR-MS, and 13 of them were unambiguously identified by references. Meanwhile, the elemental compositions of other eight unknown chemical components were decisively determined using IFSs. Subsequently, the contents of five characteristic constituents in 11 batches of samples were determined. CONCLUSIONS: The integration strategy established here can discover and quantify the chemical markers for improving the quality control standard of natural monomer flavor of licorice. It is expected that the strategy will be valuable for further quality control of other natural monomer flavors in Chinese tobacco industry.

Exposure to Aldehyde Cherry e-Liquid Flavoring and Its Vaping Byproduct Disrupt Pulmonary Surfactant Biophysical Function.

Over the past decade, there has been a significant rise in the use of vaping devices, particularly among adolescents, raising concerns for effects on respiratory health. Pressingly, many recent vaping-related lung injuries are unexplained by current knowledge, and the overall implications of vaping for respiratory health are poorly understood. This study investigates the effect of hydrophobic vaping liquid chemicals on the pulmonary surfactant biophysical function. We focus on the commonly used flavoring benzaldehyde and its vaping byproduct, benzaldehyde propylene glycol acetal. The study involves rigorous testing of the surfactant biophysical function in Langmuir trough and constrained sessile drop surfactometer experiments with both protein-free synthetic surfactant and hydrophobic protein-containing clinical surfactant models. The study reveals that exposure to these vaping chemicals significantly interferes with the synthetic and clinical surfactant biophysical function. Further atomistic simulations reveal preferential interactions with SP-B and SP-C surfactant proteins. Additionally, data show surfactant lipid-vaping chemical interactions and suggest significant transfer of vaping chemicals to the experimental subphase, indicating a toxicological mechanism for the alveolar epithelium. Our study, therefore, reveals novel mechanisms for the inhalational toxicity of vaping. This highlights the need to reassess the safety of vaping liquids for respiratory health, particularly the use of aldehyde chemicals as vaping flavorings.

What Is (Un)Flavored? A Scoping Review of the Conceptualization of Flavored Cigarillos.

INTRODUCTION: To identify how flavor is conceptualized with respect to cigarillos to illuminate areas of concordance and discordance and provide a pathway for the harmonization of tobacco flavor measurement. AIMS AND METHODS: Three methods were used to identify research measuring flavors respective to cigarillo products including: (1) querying research databases using key words "cigarillo," "tobacco" and "flavor"/'flavour,' (2) sub-sampling of the eligible literature with backward and forward reference searching, and (3) a bibliographic search of key experts. Two reviewers independently screened titles, abstracts, and full texts. Inclusion criteria required manuscripts to be available in English, published prior to 3/1/2022, be original and peer-reviewed, and include a measure of flavor pertaining to cigarillo products. A total of 163 manuscripts met these criteria and were subsequently evaluated. RESULTS: Cigarillo flavor determinations were made based on the presence of flavoring agents, sensory characteristics, marketing or package characteristics, pre-defined lists, the absence of any of these, and/or were undefined. Individual flavors and their related classifications were not altogether mutually exclusive due to differences in conceptualization which has evolved over time. CONCLUSIONS: Flavor measures continue to evolve, likely in response to both tobacco control policies and industry shifts. There is a lack of standardization in flavor conceptualization that can be ameliorated through intentional integration of multiple forms of research such as identifying patterns of flavoring agents and/or marketing strategies that elicit a specific flavor profile. By doing this, we may more comprehensively evaluate the impact of flavors, their constituents, and related marketing strategies on broader population health. IMPLICATIONS: There are discrepancies in the conceptualization of flavor in tobacco and nicotine products. The lack of standardized, definitive language limits our ability to comprehensively evaluate the population-level health impact of flavors in addition to our ability to effectively inform, implement, and enforce policies restricting flavors in tobacco and nicotine products.

A Method for Amending Loose Smokeless Tobacco With Menthol for Administration in Clinical Studies.

INTRODUCTION: Menthol has long been incorporated as a flavor additive in tobacco products and can impact use behaviors. Despite its inclusion in some of the most popular flavored smokeless tobacco (ST) products (eg, "mint" flavored products), few studies have systematically investigated the impact of menthol on ST use behaviors in prospective empirical studies. Rigorous investigation of ST menthol content on behavioral and physiological outcomes requires ST products with stable and precise levels of menthol; however, commercial product composition variability prevents product comparisons when evaluating the effects of systematic changes in menthol content on clinical outcomes. AIMS AND METHODS: We developed amended loose moist snuff ST products by treating commercially available, unflavored loose ST with an ethanol-based menthol spiking solution or a nonmentholated ethanol control solution to develop test products with different levels of menthol: 0, 1, 3, and 5 mg menthol/g tobacco. We evaluated the stability of menthol content in these products over 24 months and evaluated menthol exposure associated with the products through pharmacokinetic analysis of plasma menthol-glucuronide in human participants (n = 22). RESULTS: Menthol content of the amended products was on target, homogenous, and stable for up to 24 months. Menthol exposure (menthol-glucuronide Cmax and AUC) significantly differed between each test product. CONCLUSIONS: These data suggest that stable products with nonoverlapping menthol content can be developed using a menthol spiking solution and can be subsequently administered for clinical assessments of mentholated loose ST. IMPLICATIONS: The results from this study suggest that a menthol spiking solution can be used to mentholate unflavored, loose ST to a target menthol content. With this method, the ST menthol content was stable for at least 24 months, and the products exposed users to menthol in a dose-dependent manner. This method yielded loose ST products with precise, stable levels of menthol to allow systematic evaluation of ST menthol content on clinical outcomes. The method may have applications for systematically evaluating changes in other tobacco product ingredients.

Changes in Sales of Vaping Products and Cigarettes Associated With the New York State Flavored Vaping Product Sales Restriction.

INTRODUCTION: New York (NY) implemented a statewide restriction on the retail sale of flavored vaping products to reduce availability of vaping products having youth-appealing flavors in 2020. We assessed the intended effects of the NY law on sales of flavored vaping products and explored whether policy implementation had unintended effects on consumer behavior by evaluating policy-associated changes in sales of combusted cigarettes, which could serve as more harmful substitute products for NY consumers of flavored vaping products. AIMS AND METHODS: We analyzed custom product-level weekly retail tobacco sales scanner data for NY and a comparison state (California [CA]) for convenience stores and other outlets from June 2018 through June 2021. We categorized flavor descriptors for vaping products as flavored or as tobacco or unflavored and categorized cigarettes as menthol or non-menthol. We used a difference-in-difference model to assess the effect of the sales restriction on unit sales of flavored and unflavored vaping products and menthol and non-menthol cigarettes in NY compared with CA. RESULTS: Following NY policy implementation, flavored and total vaping product sales decreased in NY relative to CA. Unflavored vaping product sales increased in NY, while menthol cigarette sales did not change significantly relative to CA sales. CONCLUSIONS: The NY flavored vaping product policy was associated with fewer sales of flavored and total vaping products. The increase in sales of unflavored vaping products did not suggest complete substitution, and sales data suggest that consumers did not turn to cigarettes after flavored vaping products became unavailable. IMPLICATIONS: This study provides evidence that NY's flavored vaping product policy is associated with reduced flavored vaping product access and sales. Our analyses of potential unintended consequences indicate that some consumers switched from flavored to unflavored vaping products, but that cigarette sales did not change concurrent with the policy which means that decreased availability of flavored vaping products did not result in vapers switching to cigarettes. NY's policy had its intended effect with limited unintended consequences.

The Impact of High or Low Doses of Nicotine in a Mouse Model of Vapor Self-Administration.

INTRODUCTION: A wide variety of nicotine concentrations and formulations are available to users of electronic nicotine delivery systems (ENDS). This is increasingly true when considering the many flavors available with ENDS products. To date, there have been few preclinical investigations into the impact of nicotine doses, with and without flavors, on vaping-related behaviors. This present study evaluated how nicotine concentrations relevant to tank-based and pod-based ENDS, with and without flavors, impact reinforcement-related behavior in a mouse model. AIMS AND METHODS: Adult male and female C57/BL6J mice were used in vapor-inhalation self-administration assays. Mice were assigned e-liquids containing 6 mg/mL or 60 mg/mL nicotine. Additional mice were assigned these nicotine doses with green apple or menthol flavorants. Mice were trained on fixed-ratio 1 for 10, 2-hour sessions, then five sessions at FR3, three progressive ratio sessions, and two FR3 sessions. RESULTS: We observed male mice exhibited higher reinforcement-related behavior to menthol-flavored 6 mg/mL nicotine when compared to female mice. Males were only observed to have a menthol-induced enhancement of self-administration at 6 mg/mL nicotine and not 60 mg/mL nicotine. However, female mice exhibited significant menthol-induced increases in reinforcement-related behaviors with 60 mg/mL nicotine. CONCLUSIONS: These data provide evidence that males and females exhibit different dose sensitivities to nicotine. These sex-dependent differences in nicotine sensitivity also indicate that flavor-induced enhancement in nicotine intake is dependent on the different doses for each sex. IMPLICATIONS: There has been much discussion recently regarding the impact of flavors on vaping-related behavior. Our current study may support prior investigations that suggest flavors enhance the palatability of nicotine-containing products. However, this current study provides evidence that males and females exhibit different sensitivities to nicotine.

The Effect of State and Local Flavored Cigar Sales Restrictions, on Retail Sales of Large Cigars, Cigarillos, and Little Cigars in Massachusetts, California, Illinois, and New York.

INTRODUCTION: In 2009, the Family Smoking Prevention and Tobacco Control Act prohibited flavored cigarettes but allowed for flavored cigars. Since, there has been a 34% increase in youth cigar use and widened racial disparities. State and local jurisdictions have increasingly enacted flavored tobacco product sales restrictions. As more jurisdictions consider implementing flavor restrictions, it is important to understand their effect on tobacco markets that have high flavor proliferation, including the cigar market. AIMS AND METHODS: This study uses data from Truth Initiative's flavor policy database and NielsenIQ retailer scanners for California, Illinois, Massachusetts, and New York. We use a three-way fixed-effect model to assess the impact of the percentage of the population covered by a flavored cigar sales restriction on per capita unit sales of cigars. RESULTS: We find that population coverage by cigar sales restrictions was significantly associated with decreases in per capita cigar sales. More specifically, a 25% increase in the percentage of the population covered by a flavored cigar sales restriction was associated with a decrease in per capita all cigar sales of 15%-19%, 4%-10% for large cigars, 17%-21% for cigarillos, and 2%-41% for little cigars. CONCLUSION: Flavored cigar sales restrictions are an effective policy to reduce per capita cigar sales. The Food and Drug Administration (FDA)'s proposed product standards would increase population covered by a flavored cigar sales restriction to 100%, leading to potential significant reductions in cigar sales, especially little cigar, and cigarillo sales. This may also substantially reduce youth cigar use and racial disparities in cigar use. IMPLICATIONS: In April 2022, the U.S. FDA published a proposed rule to prohibit characterizing flavors in all cigars and menthol cigarettes. Besides this proposed rule, there has been little federal action to date to reduce sales of flavored cigars. However, as of March 31, 2022, Massachusetts and 333 localities across 10 states have enacted policies that restrict the sale of flavored cigars and other tobacco products. We find that population coverage by cigar sales restrictions is significantly associated with decreases in per capita cigar sales.