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Background: Smokeless tobacco (SLT) is a tobacco-based product consumed without burning or smoking. Snuff, commonly known as naswar, is a widely used SLT product in Pakistan. This study is designed to investigate the concentrations of lead (Pb) and cadmium (Cd) in snuff and their associated health risks to consumers., Methods: Blood samples from snuff consumers and non-consumers were analyzed for Pb and Cd concentrations. Additionally, samples of drinking water and wheat flour were also analyzed for Pb and Cd. Health risk assessments were calculated using hazard quotient (HQ) and incremental lifetime cancer risk (ILCR) for Pb and Cd., Results: Snuff consumers had significantly higher blood-Pb and blood-Cd levels compared to non-consumers. Approximately 38.5 % of snuff consumers were found to exceed blood-Pb levels from the safe limits (10 µg dL⁻¹) of the World Health Organization (WHO), while 48.9 % of snuff consumers were found to exceed blood-Cd levels from the WHO safe range of 0.03-0.12 µg dL⁻¹. Snuff samples contained Pb and Cd concentrations ranging from 5.3 to 22.4 µg g⁻¹ and 0.5-2.2 µg g⁻¹, respectively. Drinking water and wheat flour samples were found within the safe limits for Pb and Cd concentration, suggesting snuff as the primary source of exposure. Health risk assessments indicated elevated HQ values, for Pb (38.9 %) and Cd (14.3 %) detected in snuff, surpassing the safe HQ threshold of 1. Concerning ILCR from snuff, values for Pb were within the acceptable range, but alarmingly 100 % of samples for Cd exceeded the safe limits of USEPA (1.0×10⁻⁴), indicating a significant cancer risk in snuff users., Conclusion: This study highlights the exceeded Pb and Cd concentrations in snuff as well as also reporting significant carcinogenic and non-carcinogenic health risks to consumers. These findings emphasized the urgent need for regulatory bodies to ensure the safety of snuff before selling in the market. Public Health Professionals should also initiate educational campaigns to raise awareness among consumers and manufacturers about various health risks associated with the snuff., Competing Interests: Declaration of Competing Interest I, being the Corresponding author, declared that there is no conflict of interest among the authors. I clearly disclosed no relationship with such a people and organization that could inappropriately influence our submitted work., (Copyright © 2024. Published by Elsevier GmbH.)

The present study revealed that various non-dietary items like pan masala, chewing tobacco, betel nuts, and toothpaste may contribute to the fluoride burden of the human body. In this line, an attempt was made to analyze the fluoride content of non-dietary items collected from Panipat City of Haryana. The study depicts that intake of pan masala, chewing tobacco, betel nuts, and toothpaste expose the consumers to 21.0 to 56.80, 18.00 to 36.20, 7.45 to 77.40, and 21.10 to 1118.20 µg fluoride/g respectively. The demand for these non-essential edible items is increasing daily, and people are unaware of the detrimental health effects of these products. The people of the study area are advised to prohibit these products as they contain an appreciable amount of fluoride., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Oral cancer is the most common malignancy in many developing countries, such as India, due to increased consumption of smokeless tobacco. The trace elemental components in commercially packaged forms of tobacco can play a significant role in the pathogenesis of oral cancer. To qualitatively assess the trace elements in various types of commercially packaged forms of tobacco using laser-induced breakdown spectroscopy (LIBS). Two popular varieties of 'Paan masala' that contained a mixture of slaked lime with areca nut, catechu, and other flavouring agents (tobacco was absent) and four types of packaged tobacco were obtained from 'Paan' shops. The contents in the packets were made into pellets using a hydraulic press and subjected to elemental analysis using LIBS. A ten-trial experiment was carried out on all six pellets. The National Institute of Standards and Technology (NIST) database was used to assess the emission lines. The elements obtained from commercially packaged tobacco and Paan masala were similar: calcium (Ca), iron (Fe), aluminium (Al), nickel (Ni), and chromium (Cr). Substances that cause DNA damage and carcinogenesis are inorganic elements such as nickel. Our study revealed that carcinogens such as nickel are present in the commercially packaged forms of tobacco and 'Paan masala' samples., (© 2024. The Author(s).)

Chewing tobacco may play a contributing role in complications during pregnancy as it contains various kinds of toxic metals such as lead (Pb), cadmium (Cd), cobalt (Co), manganese (Mn), chromium (Cr), and can cause alteration in serum metal concentration. Hence, the present study aimed to explore the effects of chewing tobacco consumption on serum metal contents in pregnant women. A total number of 200 chewing tobacco consumer pregnant women and 200 age-matched non-consumer pregnant women were selected for the study from the outpatient department of gynaecology at Rural Health Centre Tandojam, Pakistan. After obtaining the sociodemographic characteristics of all participants, 10 ml of venous blood was also drawn for serum metal analysis by atomic absorption spectroscopy. Different chewing tobacco samples consumed by consumer pregnant women were collected from local shops of Tandojam, Pakistan. Drinking water samples from the residential areas of consumer and non-consumer pregnant women were prepared and analysed for the same metal contents. In present study, serum Pb, Cd, K and Co were found significantly increased in CPW as compared to NCPW. Serum Pb was found significantly increased in gutkha consumers in comparison to mainpuri consumers. Serum Pb, Cd, and Co were present with significantly increased concentration in serum of CPW who were taking canal and well water for drinking purpose when compared with NCPW. Significant negative strong correlation of serum Pb with K and Cr of drinking water and Na of chewing tobacco samples were observed. Strong positive correlation of serum Cd and Cr with Co of drinking water had been observed. Whereas, serum maternal Co was strongly negatively correlated with Mn of chewing tobacco samples, and serum Cu of CPW had a strong positive correlation with K and Cr of drinking water and Na of chewing tobacco samples. In conclusion, consumption of chewing tobacco alters the serum metal contents in pregnant women at Tandojam and adjoining areas, Pakistan., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Nicotine pouches without tobacco are new products that deliver nicotine into the body via the oral mucosa. There is a lack of independent research on the chemical composition and product characteristics of these products, contributing to uncertainties regarding product regulation. This study sought to address knowledge gaps by assessing levels of nicotine and screening for tobacco-specific nitrosamines (TSNAs) in a sample of these products., Methods: Nicotine pouches (n=44) and nicotine-free pouches (n=2) from 20 different manufacturers were analysed regarding their contents of nicotine and TSNAs by gas chromatography with flame ionisation and liquid chromatography-tandem mass spectrometry, respectively. Product labelling and pH values of aqueous extracts were determined., Results: Nicotine contents of products ranged from 1.79 to 47.5 mg/pouch; median product weight, pH, and proportion of free-base nicotine were 0.643 g, 8.8, and 86%, respectively. A clear labelling of the nicotine content was missing on 29 products and nicotine strength descriptions were ambiguous. TSNAs were detected in 26 products, with a maximum of 13 ng N-nitrosonornicotine/pouch., Conclusion: Although nicotine pouches may potentially be a reduced risk alternative for cigarette smokers or users of some other oral tobacco products, nicotine contents of some pouches were alarmingly high. Presence of carcinogenic TSNAs in the nicotine pouches is of serious concern. Better manufacturing processes and quality control standards should be implemented. Labels of nicotine strength on most products are misleading. A strict regulation regarding nicotine contents and its labelling would be advisable., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The rapid proliferation of smokeless tobacco (SLT) in India has occurred without adequate information on the possible dangers and toxicity of these products. Tobacco flavors as well as nicotine (both protonated and un-protonated) are responsible for health dangers and addiction. The study aimed to offer information on the physical characteristics of commonly used smokeless tobacco products (including microscopic analysis), along with nicotine content (both total and un-protonated), pH, moisture, and flavors. The Standard Operating Procedures (SOPs) validated by the World Health Organization (WHO) recognized Tobacco Testing Laboratory TobLabNet) were applied for the analysis of various constituents of the SLTs. The microscopic analysis indicated that some of the SLT products like khaini were finely processed and available in filter pouches for users' convenience and prolonged use leading to prolonged retention and addiction potential. Nicotine absorption and availability (both protonated and un-protonated) are affected by moisture and pH. Essences provide a pleasant aroma and flavor, with an increased risk of misuse and other health problems. Few chewing tobacco and Zarda had the lowest levels of un-protonated nicotine (0.10-0.52% and 0.15-0.21%, respectively), whereas Gul, Gudhaku, and Khaini had the highest levels, ranging from 95.33 to 99.12%. Moisture and pH ranged from 4.54 to 50.19% and 5.25-10.07 respectively. Menthol (630.74-9681.42 µg/g) was the most popular flavour, followed by Eucalyptol (118.16-247.77 µg/g) and camphor (148.67 and 219.317 µg/g). SLT's health concerns and addiction dangers are exacerbated by the high proportion of bioavailable nicotine coupled with flavors. The findings of this study have important implications for the regulation and use of SLT in countries where use of SLT is prevalent., (© 2023. The Author(s).)

Smokeless tobacco products (STPs) contain several microbial communities which are responsible for the formation of carcinogens, like tobacco-specific nitrosamine (TSNAs). A majority of STPs are sold in loose/unpackaged form which can be loaded with a diverse microbial population. Here, the fungal population and mycotoxins level of three popular Indian loose STPs, Dohra, Mainpuri Kapoori (MK), and loose leaf-chewing tobacco (LCT) was examined using metagenomic sequencing of ITS1 DNA segment of the fungal genome and LC-MS/MS, respectively. We observed that Ascomycota was the most abundant phylum and Sterigmatomyces and Pichia were the predominant fungal genera in loose STPs. MK displayed the highest α-diversity being enriched with pathogenic fungi Apiotrichum, Aspergillus, Candida, Fusarium, Trichosporon, and Wallemia. Further, FUNGuild analysis revealed an abundance of saprotrophs in MK, while pathogen-saprotroph-symbiotroph were abundant in Dohra and LCT. The level of a fungal toxin (ochratoxins A) was high in the MK product. This study caution that loose STPs harbor various harmful fungi that can infect their users and deliver fungal toxins or disrupt the oral microbiome of SLT users which can contribute to several oral pathologies., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Smokeless tobacco (SLT) use is a significant cause of lip and oral cavity cancers. Globally, oral cancer prevalence is strongly linked to the types of tobacco products used, their chemical composition, and their pattern of use. Except snus, all SLT products sold in different World Health Organization regions are strongly associated with oral cancer incidence. Shammah showed the highest association OR with 95% confidence intervals (CI; OR, 38.74; 95% CI, 19.50-76.96), followed by oral snuff (OR, 11.80; 95% CI, 8.45-16.49), gutkha (OR, 8.67; 95% CI, 3.59-20.93), tobacco with betel quid (OR, 7.74; 95% CI, 5.38-11.13), toombak (OR, 4.72; 95% CI, 2.88-7.73), and unspecified chewing tobacco (OR, 4.72; 95% CI, 3.13-7.11). Most SLT products containing high levels of carcinogenic tobacco-specific nitrosamines (TSNA) exhibit a high risk of oral cancer. There is an urgent need to frame and implement international policies for oral cancer prevention through legal control of the TSNA levels in all SLT product types., Prevention Relevance: Most smokeless tobacco products sold worldwide, mainly shammah, toombak, gutkha, betel quid with tobacco, and dry snuff, are associated with a high risk of oral cancer. A high concentration of tobacco-specific nitrosamines in smokeless tobacco products is the major causative factor for oral cancer development., (©2022 American Association for Cancer Research.)

The microbial communities present in smokeless tobacco products (STPs) perform critical steps in the synthesis of carcinogens, mainly tobacco-specific nitrosamines (TSNAs). Most studies emphasize the bacterial component, and the mycobiome of STPs has not been well characterized. In this study, we investigated the fungal communities in the different categories of STPs by sequencing the internal transcribed spacer (ITS) rRNA region of the fungal genome. The ecological character of the fungal community associated with STPs was determined by using FUNGuild. Our results indicated that Ascomycota and Basidiomycota were the most abundant fungal phyla across all STPs. The predominant fungal genera in STPs were Pichia, Sterigmatomyces, and Mortierella. The α-diversity varied significantly across the STPs based on observed, Fisher, and Shannon indices. Using SparCC cooccurrence network analysis, significant positive correlations of 58.5% and negative connections of 41.5% were obtained among fungal genera identified in STPs. Furthermore, the functional predictions by FUNGuild determined that STPs possessed high abundances of saprotroph and pathotroph-saprotroph-symbiotroph fungal trophic groups. At the functional guild level, the qiwam samples contained high abundances of soil saprotrophs, while plant pathogens were prevalent in pan-masala samples. These results suggest that various fungal populations reside in STPs and interrelate with each other and can contribute to the synthesis of TSNAs. This study has established the basis for future large-scale investigations of STP-associated mycobiota and the impact of such mycobiota in oral carcinogenesis in STP users via inflammation and carcinogens (TSNAs and mycotoxins). IMPORTANCE Smokeless tobacco products (STPs) contain complex microbial communities that influence the synthesis of carcinogens, such as tobacco-specific nitrosamines (TSNAs). Research on STP-associated bacterial populations revealed connections between bacterial metabolism and TSNA synthesis. The abundance of the fungal population may also have an impact on the production of TSNAs. This study examined STPs popularly used in India, and diverse fungal communities were identified in these STPs. Pichia, Sterigmatomyces, and Mortierella were the predominant fungal genera in the STPs. High abundances of saprotroph and pathotroph-saprotroph-symbiotroph trophic groups in STPs could affect the degradation of tobacco products and the synthesis of TSNAs.

Smokeless tobacco (ST) products are used worldwide, and consumption is increasing in the USA. Although ST products are considered to occupy a different position on the tobacco product continuum of risk compared to combusted tobacco products, they can still lead to health problems, including cancer, dental problems and changes in heart rate and blood pressure. Therefore, the determination of harmful and potentially harmful constituents released from ST products into human saliva is important. Four certified reference ST products were tested in this study: loose leaf chewing tobacco (3S1), Swedish-style snus (1S4), snus (1S5) and moist snuff (3S3). These certified reference ST products are manufactured for research purposes, not for human consumption. The reference ST products were used in this study because they have been well characterized and are intended and designed to represent commercial ST products. The reference ST products were incubated in human saliva at 37°C with a range of incubation times for the evaluation of constituents released from these products into human saliva. In this study, alkaloids (nicotine and cotinine), tobacco-specific N-nitrosamines (TSNAs) (N'-nitrosornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) and benzo[α]pyrene (B[α]P) in the reference ST products and saliva samples were determined by gas chromatography--mass spectrometry (GC--MS), gas chromatography--flame ionization detection (GC--FID), or ultra-performance liquid chromatography--tandem mass spectrometry (UPLC--MS-MS). Our results indicate that the amounts of each constituent released from the reference ST products were altered by the tobacco cut size and product format (pouched or unpouched). The constituents (TSNAs and alkaloids) in moist snuff and loose leaf chewing tobacco were released faster compared to those in Swedish-style snus and snus. B[α]P was only detected in reference moist snuff samples, and only 3.4% of the total B[α]P was released into human saliva after incubation for 60 min, whereas higher percentages of total TSNAs and alkaloids were released at different rates from the four reference ST products., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Smokeless tobacco (ST) products are widely used throughout the world and contribute to morbidity and mortality in users through an increased risk of cancers and oral diseases. Bacterial populations in ST contribute to taste, but their presence can also create carcinogenic, Tobacco-Specific N-nitrosamines (TSNAs). Previous studies of microbial communities in tobacco products lacked chemistry data (e.g. nicotine, TSNAs) to characterize the products and identify associations between carcinogen levels and taxonomic groups. This study uses statistical analysis to identify potential associations between microbial and chemical constituents in moist snuff products., Methods: We quantitatively analyzed 38 smokeless tobacco products for TSNAs using liquid chromatography with tandem mass spectrometry (LC-MS/MS), and nicotine using gas chromatography with mass spectrometry (GC-MS). Moisture content determinations (by weight loss on drying), and pH measurements were also performed. We used 16S rRNA gene sequencing to characterize the microbial composition, and additionally measured total 16S bacterial counts using a quantitative PCR assay., Results: Our findings link chemical constituents to their associated bacterial populations. We found core taxonomic groups often varied between manufacturers. When manufacturer and flavor were controlled for as confounding variables, the genus Lactobacillus was found to be positively associated with TSNAs. while the genera Enteractinococcus and Brevibacterium were negatively associated. Three genera (Corynebacterium, Brachybacterium, and Xanthomonas) were found to be negatively associated with nicotine concentrations. Associations were also investigated separately for products from each manufacturer. Products from one manufacturer had a positive association between TSNAs and bacteria in the genus Marinilactibacillus. Additionally, we found that TSNA levels in many products were lower compared with previously published chemical surveys. Finally, we observed consistent results when either relative or absolute abundance data were analyzed, while results from analyses of log-ratio-transformed abundances were divergent., Competing Interests: The commercial affiliation of authors (LMK) does not alter their adherence to PLOS ONE policies on sharing data and materials.

N -Nitrosonornicotine (NNN) is a human carcinogen present in cigarette smoke and smokeless tobacco. Urinary NNN is usually measured in order to assess the exposure to this toxicant for tobacco users. NNN excretion in urine can be highly biased due to the formation of NNN by nitrosation of nornicotine under acidic conditions, both endogenously and exogenously. Hence, urinary NNN levels may not necessarily correctly reflect the product-specific exposure. Measurement of plasma NNN may be less prone to endogenous formation due to the stable pH (7.4) of blood. We developed an LC-MS/MS method for the quantification of NNN using 1 mL of human plasma. Validation according to FDA guidelines proved that the method is selective and highly sensitive with an LLOQ of 0.3 pg/mL. Accuracy and precision averaged to 98.7 and 7.5% (CV), respectively. The assay was applied to plasma samples collected from 10 experienced moist smokeless tobacco users during and after a single use of 2 g of the product for 40 min under controlled use conditions. Blood was drawn at 15 time points over a 6 h time course. The maximum NNN concentration ( C max ) ranged from 3.5 to 10 pg/mL (mean: 7.1 pg/mL) at a t max of 32 min. Plasma NNN and nicotine were found to have similar time courses. In conclusion, the determination of NNN in plasma may be fit-for-purpose to evaluate the product-use-specific exposure to this carcinogen.

Introduction: neffa, a form of air-dried smokeless tobacco used in North Africa, is spuriously perceived as a lower risk alternative to smoking. The objective of this study was to provide information on some harmful constituents of neffa and to use them for cancer risk assessment., Methods: a high-performance liquid chromatography method coupled with fluorescence detector was used to determine polycyclic aromatic hydrocarbons (PAHs) in one sample of neffa. An atomic absorption spectrometry was performed to determine the concentrations of lead and cadmium in three samples of neffa. The levels of toxicants found in neffa were used to assess for lifetime cancer risk as advocated by the US Environment Protection Agency., Results: the determination of PAHs in neffa allowed the identification of phenanthrene and anthracene. However, the higher molecular weight PAHs such as Benzo(a)Pyrene B(a)P were not detected. The concentrations of cadmium and lead varied between 1.3 to 2.8µg/g and 1.7 to 4.6µg/g respectively. Cancer risk for cadmium and lead varied between 4.2E-03 to 9.3E-03 and 2.5E-06 to 6.4E-06 respectively. Cancer risk for Cd exceeded the range of 10E-04 to 10E-06 of an acceptable risk., Conclusion: neffa is not a healthy alternative for overcoming smoking addiction. It contains mineral and organic pulmonary toxicants. This study could serve as a scientific basis to inform consumers about the products´ toxicity and help them to quit smokeless tobacco (SLT) use., Competing Interests: The authors declare no competing interests., (Copyright: Chukwukasi Wilson Kassy et al.)

Smokeless tobacco products possess a complex community of microorganisms. The microbial community ferment compounds present in the smokeless tobacco products and convert them into carcinogens like tobacco-associated nitrosamines. However, the potential of smokeless tobacco products associated bacteriome to manipulate systemic inflammation and other signaling pathways involved in the etiology of oral cancer will be a risk factor for oral cancer. Further, damage to oral epithelial cells causes a leaky oral layer that leads to increased infiltration of bacterial components like lipopolysaccharide, flagellin, and toxins, etc. The consumption of smokeless tobacco products can cause damage to the oral layer and dysbiosis of oral microbiota. Hence, the enrichment of harmful microbes due to dysbiosis in the oral cavity can produce high levels of bacterial metabolites and provoke inflammation as well as carcinogenesis. Understanding the complex and dynamic interrelation between the smokeless tobacco-linked bacteriome and host oral microbiome may help to unravel the mechanism of oral carcinogenesis stimulated by smokeless tobacco products. This review provides an insight into smokeless tobacco product-associated bacteriome and their potential in the progression of oral cancer. In the future, this will guide in the evolution of prevention and treatment strategies against smokeless tobacco products-induced oral cancer. Besides, it will assist the government organizations for better management and cessation policy building for the worldwide problem of smokeless tobacco addiction., Competing Interests: Declaration of competing interest The authors have confirmed that there is no conflict of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Several smokeless tobacco products are available in the market and comprise complex chemical matrices. Sample preparation for analysis of the multiple classes of harmful compounds in smokeless tobacco products is highly cumbersome. In this study, a simultaneous extraction scheme was developed for three toxic analyte classes in smokeless tobacco products using a two-phase solution consisting of 5% aqueous NaOH and dichloromethane in a 1:4 ratio. The dichloromethane extract was used to analyze four alkaloids directly at levels greater than parts per million; however, passing the layer through a silica cartridge for further purification and concentration was necessary for determining 18 polycyclic aromatic hydrocarbons and four volatile N-nitrosoamines at the ppt level. The multitargets were determined by using gas chromatography with tandem mass spectrometry. The limits of detection for the 18 polycyclic aromatic hydrocarbons, four volatile N-nitrosoamines, three minor alkaloids, and nicotine were 0.2-1.2, 0.2-0.4, 0.6-1.0, and 10.2 μg/g, respectively. Four different smokeless tobacco substrates were fortified with three levels of mixed standards, and the recoveries ranged between 83 and 110%. The method was highly efficient, reduced the sample amounts, solvents, and the time required by approximately 60%. The method was used to assay 18 smokeless tobacco products, and showed potentials in assaying drugs and other plant-based substrates., (© 2021 Wiley-VCH GmbH.)

Introduction: Based on arguments for harm reduction and health benefits, tobacco companies in the United States can apply for regulatory authorization to make "modified risk tobacco product" (MRTP) marketing claims. The impact of future MRTP claims may depend on whether they are noticed, believed, and lead to smokers switching products. This study provides baseline data about smokers' exposure to perceived MRTP claims ahead of any MRTP authorizations., Aims and Methods: We analyzed measures from Wave 3 of the US-based Population Assessment of Tobacco and Health (PATH) study which asked smokers to indicate if they had seen any e-cigarettes, snus, or other smokeless tobacco (SLT) products that claim to be "less harmful" in the past 12 months, and their likelihood of using products with these claims in the next 30 days., Results: Significantly fewer smokers noted having seen snus (5.1%) or other SLT (5.6%) with "less harmful" claims compared with e-cigarettes (29.1%). For each product, the prevalence of MRTP claim exposure was higher among smokers who perceived the product to be less harmful than smoking, who currently used the product, and who had higher rates of tobacco advertising exposure at the point of sale. Among smokers who noticed products with "less harmful" claims, about one-quarter said they would use them in the future (24%-27%)., Conclusions: Ahead of any Food & Drug Administration (FDA) authorization for MRTP claims, some smokers already perceive exposure to "less harmful" claims for e-cigarettes, but few do for SLT. MRTP claims may motivate some smokers to use these products., Implications: This study provides new baseline data about smokers' perceived exposure to MRTP claims in the United States ahead of any regulatory claim authorization. Using data from Wave 3 of the US PATH study, we found that some smokers already perceive exposure to "less harmful" claims for e-cigarettes (29%), but few do for SLT (5%-6%). Among smokers who noticed products with "less harmful" claims, about one-quarter said they would use them in the future (24%-27%), suggesting MRTP claims may motivate some smokers to use products described as "less harmful.", (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Tobacco-free 'modern' oral nicotine pouches (MOPs), are similar in appearance and use to Swedish-style snus, but without tobacco. There are few identified methods to create test samples for toxicologically assessment of MOPs in vitro. In this study we present a simple method for the extraction of pouch material in cell culture media, providing consistent nicotine concentration and easy in vitro assessment. A series of contemporary in vitro screening assays (viability, cell health markers, oxidative stress and genotoxicity) using human oral fibroblasts (HGF) and human lung epithelial cells (H292) were employed. Extracts were generated from LYFT and compared to snus (CRP1.1) and cigarette (1R6F) reference products. MOP and CRP1.1 extracts were generated by incubating one pouch in 20 ml of cell culture media, while 1R6F AqE was prepared by smoking 1 cigarette into 20 ml of cell culture media. 1R6F demonstrated toxicological responses in most assays; CRP1.1 had minimal to moderate effects while MOP demonstrated little or no response in all assays. This study demonstrated the generation of MOPs extracts and their toxicological evaluation using in vitro screening approaches. Future product usage, pharmacokinetics and clinical studies will further substantiate the reduced risk potential of MOPs., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bangladesh exhibits the second highest rate of smokeless tobacco (SLT) product usage in the world, and this has been associated with the high upper aerodigestive tract cancer incidence in this country. The goal of the present study was to examine the levels of the highly carcinogenic tobacco-specific nitrosamines (TSNAs) in Bangladeshi SLT products and compare these levels to that observed in SLT brands from southeast Asia and the USA. The levels of TSNAs and nicotine were determined by LC-MS/MS in twenty-eight SLT brands and several tobacco additives from Bangladesh, as well as several SLT brands from India, Pakistan and the USA. The levels of N-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoanatabine (NAT) and N-nitrosoanabasine (NAB) in Bangladeshi SLT brands ranged from 1.1-59, 0.15-34, 0.79-45, and 0.037-13 μg/g SLT powder, respectively. The mean levels of the highly carcinogenic TSNAs (NNN+NNK) were 7.4-, 2.4-, and 63-fold higher in Bangladeshi SLT products as compared to SLT brands from the USA, India and Pakistan, respectively; these trends were also observed for NAT and NAB. Similar mean levels of nicotine were observed in the Bangladeshi brands (31 mg/g powder) versus brands from the USA (25 mg/g powder) and India (20 mg/g powder); they were 3-fold higher than brands from Pakistan (10 mg/g powder). Gul SLT brands exhibited the highest pH and the highest levels of unprotonated nicotine. The high levels of TSNAs in Bangladeshi SLT brands may be an important factor contributing to the high rates of upper aerodigestive tract cancer in Bangladesh., Competing Interests: The authors have declared that no competing interests exist.

Developing dissolution testing methods to measure the nicotine release profiles from smokeless tobacco products is valuable for product assessment and product-to-product comparisons. In this work, we developed a robust dissolution method to study the in vitro release of nicotine from smokeless tobacco products using the U.S. Pharmacopeia flow-through cell dissolution apparatus 4 (USP-4). We further developed and validated a sensitive Ultra Performance Liquid Chromatography coupled to Photodiode Array detector (UPLC-PDA) method for the accurate quantitation of the released nicotine into artificial saliva, which is our selected dissolution medium. We have successfully shown the applicability of the validated method by investigating the release profiles of nicotine from various commercial and CORESTA reference smokeless tobacco products [CRP 1.1 (Swedish-style snus pouch), CRP 2.1 (American-style loose moist snuff), CRP 4 (loose-leaf chewing tobacco) and CRP 4.1 (chopped loose-leaf chewing tobacco)]. Nicotine release profiles were analyzed by calculating the difference factor (f 1 ) and similarity factor (f 2 ) by adopting a methodology referenced in the Guidance for Industry from FDA's Center for Drug Evaluation and Research (CDER) and by fitting the release profile curves using a first order kinetic model. Nicotine release was found to be dependent on the form and cut of the smokeless tobacco products, with a slower release observed for snus and loose-leaf, compared to chopped and loose moist snuff smokeless tobacco. This dissolution methodology can be extended to measure and compare release of other constituents from smokeless tobacco products and has the potential for method standardization., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Snus is an oral tobacco product that originated in Sweden. Snus products are available as fine-cut loose tobacco or in pre-portioned porous "pouches." Some snus products undergo tobacco pasteurization during manufacturing, a process that removes or reduces nitrite-forming microbes, resulting in less tobacco-specific nitrosamine content in the product. Some tobacco companies and researchers have suggested that snus is potentially less harmful than traditional tobacco and thus a potential smoking cessation aid or an alternative to continued cigarette consumption. Although snus is available in various countries, limited information exists on snus variants from different manufacturers., Methods: Moisture, pH, nicotine, and tobacco-specific N'-nitrosamines (TSNAs) were quantified in 64 snus products made by 10 manufacturers in the United States and Northern Europe (NE). Reported means, standard errors, and differences are least-square (LS) estimates from bootstrapped mixed effects models, which accounted for correlation among repeated measurements. Minor alkaloids and select flavors were also measured., Results: Among all product types, moisture (27.4%-59.5%), pH (pH 5.87-9.10), total nicotine (6.81-20.6 mg/g, wet), unprotonated nicotine (0.083-15.7 mg/g), and total TSNAs (390-4,910 ng/g) varied widely. The LS-mean unprotonated nicotine concentration of NE portion (7.72 mg/g, SE = 0.963) and NE loose (5.06 mg/g, SE = 1.26) snus were each significantly higher than US portion snus (1.00 mg/g, SE = 1.56). Concentrations of minor alkaloids varied most among products with the highest total nicotine levels. The LS-mean NNN+NNK were higher in snus sold in the US (1360 ng/g, SE = 207) than in NE (836 ng/g, SE = 132) countries. The most abundant flavor compounds detected were pulegone, eucalyptol, and menthol., Conclusion: Physical and chemical characteristics of US and NE products labeled as snus can vary considerably and should not be considered "equivalent". Our findings could inform public health and policy decisions pertaining to snus exposure and potential adverse health effects associated with snus., Competing Interests: The authors have declared no competing interests exist.

Background: Nicotine acts as major alkaloid of all tobacco products including smokeless tobacco (SLT) forms. The mode of SLT consumption is in the form of chewing under the cheek or lip and induced biochemical alterations in the plasma, saliva, and urine., Materials and Methods: The smokeless tobacco products like Raja or blue bull tobacco brands are widely consumed by human male volunteers under the age of 18-30 years for the period of 3 years consisting of 30g per day. The concentrations of nicotine and cotinine in samples of plasma, saliva, and urine are quantified by the method of HPLC. The remaining variables of plasma are evaluated by auto analyzer and spectrophotometric methods., Results: The analysis of results presented that significant increase in the levels of nicotine and cotinine in plasma, saliva, and urine of chewing tobacco users. The lipid profile (Cholesterol, triglycerides, HDL-C, and LDL-C), liver marker enzymes (SGOT, SGPT, and ALP), kidney markers (Creatinine, urea, and uric acid), glucose, and the remaining variables are present within normal range observed in SLT users. The lipid peroxidation (LPO), nitric oxide (NO) (NO2 and NO3), protein carbonyls (PCO), and peroxynitrites (ONOO-) are reported to be higher levels in the plasma of experimental subjects in comparison with normal controls. The various brands of tobacco varieties (Raja, madhu chhap, hans chhap, miraj, badshah, blue bull, and swagat gold tobacco) are presented., Conclusion: The chewing tobacco users exhibited greater amounts of nicotine and cotinine are at risk of cardiovascular due to nicotine has cardiovascular effects, and oral cancer disease complications in the future for chronic consumption of smokeless tobacco products  due to the presence of carcinogens of tobacco-specific N-nitrosamines.

Background: Despite the high prevalence of smokeless tobacco (ST) use in South Asia, ST dependence in this population has not been studied. Therefore, it is of interest to explore if ST dependence measures developed and validated in the western countries are of similar relevance to ST users who are culturally different and use distinct ST products. The aim of this study is to assess ST dependence among its users in Bangladesh by employing ST dependence scales based on three different approaches to measure tobacco dependence., Methods: Data collected from a community-based sample of exclusive ST users living in Dhaka, Bangladesh (n = 200) were used for this study. Three ST dependence measures, Tobacco Dependence Screener (TDS), Fagerström Test for Nicotine Dependence for ST users (FTND-ST), and Oklahoma Scale for Smokeless Tobacco Dependence (OSSTD), were translated in Bangla and administered to the study participants. Saliva samples were collected for cotinine measurement. Reliability and concurrent and construct validity of FTND-ST and OSSTD were examined., Results: Median cotinine concentration of the study participants was 440.2 (min = 2.9, max = 1874) ng/ml and 47% of them had TDS-based dependence diagnosis. Women had higher mean OSSTD, FTND-ST, and TDS scores as compared to men (p < 0.001). OSSTD demonstrated better reliability (α = 0.93) than FTND-ST, and TDS (α = 0.64 and 0.86, respectively). Concurrent validity of FTND-ST as evaluated by TDS, OSSTD, and cotinine concentration was affirmative., Conclusion: The FTND-ST is a valid and reliable tool to measure physical dependence among ST users in Bangladesh. The study was unable to replicate the structure of OSSTD., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Cigarette smoking is associated with an increase in cardiovascular disease risk, attributable in part to reactive volatile organic chemicals (VOCs). However, little is known about the extent of VOC exposure due to the use of other tobacco products., Methods: We recruited 48 healthy, tobacco users in four groups: cigarette, smokeless tobacco, occasional users of first generation e-cigarette and e-cigarette menthol and 12 healthy nontobacco users. After abstaining for 48 h, tobacco users used an assigned product. Urine was collected at baseline followed by five collections over a 3-h period to measure urinary metabolites of VOCs, nicotine, and tobacco alkaloids., Results: Urinary levels of nicotine were ≃2-fold lower in occasional e-cigarette and smokeless tobacco users than in the cigarette smokers; cotinine and 3-hydroxycotinine levels were similar in all groups. Compared with nontobacco users, e-cigarette users had higher levels of urinary metabolites of xylene, cyanide, styrene, ethylbenzene, and benzene at baseline and elevated urinary levels of metabolites of xylene, N,N-dimethylformamide, and acrylonitrile after e-cigarette use. Metabolites of acrolein, crotonaldehyde, and 1,3-butadiene were significantly higher in smokers than in users of other products or nontobacco users. VOC metabolite levels in smokeless tobacco group were comparable to those found in nonusers with the exception of xylene metabolite-2-methylhippuric acid (2MHA), which was almost three fold higher than in nontobacco users., Conclusions: Smoking results in exposure to a range of VOCs at concentrations higher than those observed with other products, and first generation e-cigarette use is associated with elevated levels of N,N-dimethylformamide and xylene metabolites., Implications: This study shows that occasional users of first generation e-cigarettes have lower levels of nicotine exposure than the users of combustible cigarettes. Compared with combustible cigarettes, e-cigarettes, and smokeless tobacco products deliver lower levels of most VOCs, with the exception of xylene, N,N-dimethylformamide, and acrylonitrile, whose metabolite levels were higher in the urine of e-cigarette users than nontobacco users. Absence of anatabine in the urine of e-cigarette users suggests that measuring urinary levels of this alkaloid may be useful in distinguishing between users of e-cigarettes and combustible cigarettes. However, these results have to be validated in a larger cohortcomprised of users of e-cigarettes of multiple brands., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Smokeless tobacco (SLT), a cause of potentially preventable diseases, has a diverse chemical composition encompassing toxicants as well as potent carcinogens. Though the chemical profile of SLT products has been analyzed earlier, this information is not available in a comprehensive and easily accessible format. Hence, there is an imperative felt need to develop a one-stop information source providing inclusive information on SLT products. SLTChemDB is the first such database that makes available detailed information on various properties of chemical compounds identified across different brands of SLT products. The primary information for the database was extracted through extensive literature search, which was further curated from popular chemical web servers and databases. At present, SLTChemDB contains comprehensive information on 233 unique chemical compounds and 82 SLT products. The database has been made user-friendly with facility for systematic search and filters. SLTChemDB would provide the initial data on chemical compounds in SLT products to various tobacco testing laboratories. The database also highlights research gaps and thus, would be a guide for researchers interested in chemistry and toxicology of SLT products. With regular update of information in the database, it shall be a valuable evidence base for policymakers to formulate stringent policies for SLT control.

The present study aims to quantify trace metals in different dokha (medwakh) and shisha tobacco products available in local markets. Recent research has shown that these products have higher amounts of nicotine and tar compared to various other tobacco products. No specific data are available on the concentration of trace elements in dokha (medwakh) and shisha tobacco products in the Middle East and North Africa (MENA) region. Harmful health effects due to the toxicity of these elements in tobacco and its smoke have not been adequately emphasized. Concentrations of trace elements were extensively studied using HORIBA XGT-7200 EDXRF fluorescence absorption spectroscopy. The mean concentrations of aluminum, calcium, chromium, copper, iron, magnesium, manganese, nickel, potassium, strontium and zinc in 13 dokha products in μg/g were 406.92 ± 41.72, 14703.27 ± 271.73, 11.73 ± 2.12, 25.58 ± 2.63, 753.85 ± 14.87, 5306.54 ± 134.94, 82.31 ± 4.55, 25.58 ± 2.50, 2212.12 ± 39.04, 816.92 ± 15.26 and 35.96 ± 2.63, respectively; and those in the three shisha products in μg/g were 244.83 ± 25.11, 8235.77 ± 144.51, 3.40 ± 0.38, 22.77 ± 4.50, 569.13 ± 10.22, 2096.20 ± 130.69, 72.13 ± 7.13, 27.67 ± 5.31, 4467.50 ± 168.06, 320.20 ± 6.03 and 36.40 ± 3.57, respectively. In our study, the quantified trace metal concentrations in dokha and shisha tobacco using the EDXRF method were ten times higher than the LODs. The percentage of RSD was <10%, validating the precision of the method. Tobacco smoking is a major source of consumption of toxic elements, not only in the smoker but also in non-smokers through passive smoking. In dokha (medwakh) and shisha tobacco products, compared with cigarettes, Ni levels were significantly higher, Cr, Cu and Zn levels were higher, and Fe levels were similar, while Al and Mn levels were lower. The dokha and shisha tobacco products have no filters; many toxic metals can quickly enter the lungs and cause different pulmonary diseases and oral infections. Tobacco smoking causes lung and oral cancers, chronic obstructive pulmonary disease and cardiovascular diseases., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: To evaluate microbiological contamination of areca nut-containing, ready-to-eat chewing substances easily accessible to vulnerable paediatric population., Methods: A pilot study was conducted at the Aga Khan University Medical College from June to October 2016 on twelve samples of areca nut-containing chewing substances (four supari, paan masala and gutka each) collected from various localities of Karachi. These were evaluated individually for total colony counts, hygiene indicator organisms, pathogenic organisms, and levels of aflatoxin. Microbial contamination was analysed using pour-plate method. Fungal aflatoxin levels were measured by enzyme-linked immunosorbent assay (ELISA).., Results: Wet gutka preparations were contaminated by Escherichia coli and Enterobacteriacaea. High levels of fungal aflatoxin (range: 0.43-1.84 mg/kg), a proven carcinogen, were identified in all the 12(100%) products. No sample contained pathogenic bacteria. However, 1(8.33%) sample did not meet hygiene criteria cut-off., Conclusions: Habitual use of unhygienic chewing substances containing fungal toxins is a public health concern that needs to be addressed through a preventative, behaviour-changing strategy..

Swedish snus is a smokeless tobacco product that contains reduced levels of harmful compounds compared with cigarette smoke. In Sweden, where snus use exceeds smoking among men, relatively low rates of major smoking-related diseases have been recorded. To better understand how snus use could align with current tobacco harm reduction strategies, its potential mechanisms of toxicity must be investigated. This study aimed to determine, via a systems toxicology approach, the biological impact of repeated 72-hour exposure of human gingival epithelial organotypic cultures to extracts from both a commercial and a reference snus and the total particulate matter (TPM) from cigarette smoke. At concentrations relevant for human use, cultures treated with snus extracts induced mild, generally reversible biological changes, while TPM treatment induced substantial morphological and inflammatory alterations. Network enrichment analysis and integrative analysis of the global mRNA and miRNA expression profiles indicated a limited and mostly transient impact of the snus extracts, in particular on xenobiotic metabolism, while the effects of TPM were marked and sustained over time. High-confidence miRNAs that might be related to pathological conditions in vivo were identified. This study highlights the limited biological impact of Swedish snus extract on human organotypic gingival cultures., (Copyright © 2018. Published by Elsevier Ltd.)

Objective: Foetuses and neonates of women who use tobacco are exposed to nicotine and tobacco-derived carcinogens. We determined the relationship between urine biomarkers of tobacco toxicant exposure postpartum and in the neonates of Alaska Native (AN) women, comparing smokers and smokeless tobacco (ST) users, including iqmik, a homemade ST product., Methods: AN women, including 36 smokers, 9 commercial ST and 16 iqmik users their neonates participated. Urine from the woman at the time of delivery and her neonate's first urine were analysed for cotinine, the major metabolite of nicotine, and 4-(methylnitrosamino)-1-(3) pyridyl-1-butanol (NNAL), a tobacco-specific carcinogen biomarker., Results: Maternal urine cotinine and neonatal urine cotinine were strongly correlated in all tobacco use groups (r from 0.83 to 0.9, p < 0.002). Correlations between maternal cotinine and neonatal NNAL were moderately strong for cigarettes and commercial smokeless but weaker for iqmik users (r 0.73, 0.6 and 0.36, respectively)., Conclusion: Correlations between maternal and neonatal biomarkers of tobacco toxicant exposure vary, dependent on tobacco product use., Significance: This study provides novel data on biomarkers of tobacco exposure among postpartum AN women and their neonates. The results could be useful to guide future epidemiological studies of health risks associated with use of various tobacco products during pregnancy.

A quantitative method for the analysis of aluminum in tobacco products was developed, validated and applied to select samples. Samples were prepared using standard microwave digestion of tobacco from various products. Detection and quantification utilized sector field inductively coupled plasma-mass spectrometry. Method applicability to analyze aluminum in a range of tobacco products was demonstrated with quantitative analyses of smokeless tobacco products, cigarette tobacco, little cigar tobacco and roll-your-own/pipe tobacco. Though these products represent a convenience sampling, we observed that smokeless tobacco products, as a category, had the lowest average aluminum concentrations. Roll-your-own or pipe tobacco and little cigar tobacco had higher median and ranges of aluminum concentrations than cigarette and smokeless tobacco samples.

Evaluating the source of nicotine in e-liquid is a problem. Tobacco-derived nicotine contains predominantly (S)-(-)-nicotine, whereas tobacco-free nicotine products may not. Thus, we developed a new normal phase high-performance liquid chromatography method to determinate the enantiomeric composition of nicotine in 10 kinds of flue-cured tobacco, 3 kinds of burley, 1 kind of cigar tobacco, 2 kinds of oriental tobacco, 5 kinds of Virginia cigarette, 5 kinds of blend cigarette, 10 kinds of e-liquid, and 4 kinds of smokeless tobacco. The amount of (R)-(+)-nicotine ranged from ~0.02% to ~0.76% of total nicotine. An e-liquid sample had the highest level of (R)-(+)-nicotine. The extraction and purification processes used to obtain commercial (S)-(-)-nicotine from the tobacco do not decrease the amount of (R)-(+)-nicotine in tobacco. So the amount of (R)-(+)-nicotine in samples in our work were the same as tobacco samples., (© 2018 Wiley Periodicals, Inc.)

The occurrence and mobility of different elements in oral smokeless tobacco products (STPs) were determined because the effects on human health must take into account their availability. In this research, the elemental analysis of 15 oral STPs of different brands purchased in local specialty stores in Europe, and the determination of % extraction of the different elements into an artificial salivary juice during the sucking or chewing operations were performed. In all samples analyzed, cobalt (Co) and chromium (Cr) (total) levels were <0.326 mg/kg. As far as non-essential or toxic elements, U was always <1.0 mg/kg, Th and Ti <0.1 mg/kg, Cd was <0.5. Pb was detectable in 60% of the samples, As in 33.3% and Ce in 20% of the samples; La was <1 mg/kg in 13 samples; Sb was <5 mg/kg in all sample with exception of sample 13; Al, Ni, Sr, Rb, Ba, Sn, Te, Ti and Hg were detectable in all samples. Using artificial saliva, the data of extractable levels show that the toxic elements, although poorly extracted, are not totally retained within the STPs, with a consequent potential health hazard associated with oral use of these products.

Smokeless tobacco (SLT) products are consumed by millions of people in over 130 countries around the world. Consumption of SLT has been estimated to cause a number of diseases accounting to more than 0.65 million deaths per year. There is sufficient epidemiological evidence on the association of SLT products with nicotine addiction, cancers of oral cavity and digestive systems but there is a lack of understanding of the role of toxic chemicals in these diseases. We provide the first comprehensive in-silico analysis of chemical compounds present in different SLT products used worldwide. Many of these compounds are found to have good absorption, solubility and permeability along with mutagenic and toxic properties. They are also found to target more than 350 human proteins involved in a plethora of human biological processes and pathways. Along with all the previously known diseases, the present study has identified the association of compounds of SLT products with a number of unknown diseases like neurodegenerative, immune and cardiac diseases (Left ventricular non compaction, dilated cardiomyopathy etc). These findings indicate far-reaching impact of SLT products on human health than already known which needs further validations using epidemiological, in-vitro and in-vivo methodologies. Thus, this study will provide one stop information for the policy makers in development of regulatory policies on toxic contents of SLT products., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: Smoking cigarettes and smokeless tobacco are one of the causes of oral cancer. This study compared the salivary level of cotinine in male smokeless tobacco users and smokers. Methods: In this cross-sectional (descriptive-analytical) study, stimulated saliva samples from 30 male smokers and 30 male smokeless tobacco consumers were collected and their cotinine contents were measured using the competitive ELISA method according the standard curve. The data was analyzed with independent t-test and linear regression using SPSS-19, and P<0.05 was considered significant. Result: Among the 60 subjects with the mean age of 21.27±2.6 years, the average level of cotinine in smokers (12.32±7.5 ng/ml) had no significant difference with that of smokeless tobacco consumers (11.23±4.4 ng/ml) (p=0.49). Conclusion: Salivary levels of cotinine were not significantly different in smokeless tobacco users and cigarette smokers. In addition, increases in the number of cigarettes smoked and in pack of smokeless tobacco used, were associated with increased salivary levels of cotinine. The increase was higher in smokeless tobacco consumers., (Creative Commons Attribution License)

The combination of multiple heart-cutting two-dimensional liquid chromatography (MHC-LC/LC) and quadrupole-orbitrap high-resolution mass spectrometry (HRMS) for simultaneous determination of the aflatoxins and ochratoxin A in snus is presented in this work. A C18 capillary column was used as the first dimension (1D) to isolate the aflatoxins and ochratoxin A from the complex matrices; then, a 2-position/10-port high-pressure valve equipped with two 60 μL loops was employed to transfer the heart-cuts of 1D-LC into a pentafluorophenyl (PFP) column for the second dimension (2D) separation. With the better separation of the MHC-LC/LC system, the sensitivity of the method was improved, which is essential for the trace mycotoxins analysis. Furthermore, HRMS performed in parallel reaction monitoring mode was employed to eliminate the interferences, and the sample pretreatment procedure was simplified. A new approach utilizing ethyl acetate with 1% formic acid/water solution was adopted to extract aflatoxins and ochratoxin A in snus, which provided parallel recoveries for aflatoxins and ochratoxin A with higher responses in comparison with the QuEChERS method. A dynamic range between 0.2 and 20 μg/kg was achieved with LOQs of 0.05 μg/kg for aflatoxin B 1 , 0.1 μg/kg for aflatoxin B 2 , G 1 , G 2 , and 1.0 μg/kg for ochratoxin A in dry mass of product. The results revealed that the established method exhibited good repeatability and recovery and could be used as a rapid and reliable approach for routine analysis of aflatoxins and ochratoxin A in snus.

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., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The pervasive smokeless tobacco (SLT) consumption and diseases related to its use is a hot topic for the public discussion. In this study, concentrations of chromium (Cr) were measured in different SLT products [snuff (dry and moist), mainpuri, and gutkha] offered and used in Pakistan. The current study was also designed to assess the Cr levels in the biological (scalp hair and blood) samples of male and female subjects, age ranged from 25 to 60 years, chewing different SLT products. For comparative purpose, the healthy persons of the same age group, who did not consume any SLT products, were selected as referents. The concentrations of Cr in SLT products and biological samples were measured by electrothermal atomic absorption spectrophotometer after microwave-assisted acid digestion. The validity and accuracy of the methodology were checked by certified reference materials (CRMs). The resulted data indicated that the adult persons, who consumed different SLT products, have 2-3 fold higher levels of Cr in biological samples as compared to referent subjects (p < 0.01). The persons, who chew/sniff different SLT products, have 50-80 and 42-82 % higher levels of Cr in their scalp hair and blood samples as related to referents. The daily intake of Cr is lower as compared to the recommended value of 50-200 μg/day. It was expected that 10 g consumption of various kinds of SLT products (snuff, mainpuri, and gutkha) may subsidize 21.2-220, 17.7-122, and 18.4-273 % of the recommended daily intake of Cr, respectively.

We identified and quantified a variety of mineral elements in 18 tobacco samples purchased from a Tunisian market. In total, 25 mineral elements have been measured in cigarettes, water pipe tobacco, and smokeless tobacco using inductively coupled plasma-optical emission spectroscopy following microwave-assisted digestion. Statistical analyses were performed using SPSSTM, version 18.0. The lowest concentrations of all studied elements were observed in water pipe tobacco. Significantly higher concentrations of Al, Fe, Mg, Na, Ca, Cr, and Co were found in smokeless tobacco, while cigarettes brands contained the highest concentrations of K, Mn, Ni, Ba, and Sr. There was no significant difference between the mineral contents of local and foreign cigarettes and conventional and light cigarettes. Our findings demonstrated that local smokeless tobacco appears to be the most hazardous tobacco type. The concentration of minerals in light cigarettes was not significantly different from the concentration in conventional cigarettes., (Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

The occurrence and mobility of Po in oral smokeless tobacco products (STPs) were determined because its effects on human health must be taken into account. This research was subdivided into two parts: determination by alpha spectrometry of the Po activity concentration in 16 oral smokeless tobacco products of different brands purchased in local specialty stores in Europe and evaluation of its percent extraction into an artificial salivary gland during sucking or chewing operations. Polonium-210 was detected in all samples, and its concentrations ranged from 3.46 to 14.8 Bq kg (mean value of 7.45 ± 3.82 Bq kg). The highest concentration was found in chewing tobacco. The samples showed no significant difference in the content of Po level. The data obtained in this study show that the polonium, although poorly extracted (12.8 ± 8.96%) by artificial saliva, is not totally retained within the smokeless tobacco products, with a consequent potential health hazard associated with oral use of these products.

A novel dispersive liquid-liquid microextraction (ILDLLμE) method using an extracting solvent (ionic liquid) and dispersant (Triton X-114) was developed for the separation and preconcentration of hexavalent chromium (Cr 6+ ) in artificial saliva extract (ASE) of chewing tobacco products, gutkha, and mainpuri (n = 23). In the proposed method, the extraction of Cr 6+ was accomplished by using ammonium pyrrolidinedithiocarbamate (APDC) as complexing agent and 1-butyl-3-methylimidazolium hexafluorophosphate [C 4 MIM] [PF 6 ] as extracting solvent. The tiny droplet of metal chelate was then dispersed into TX-114 emulsion, using vortex mixer. Various parameters such as concentration of APDC, pH of the solution, volume of [C 4 MIM] [PF 6 ], and TX-114 as well as extraction time were studied. Under the most favorable conditions, the limit of detection was found to be 0.068 μg/L with the relative standard deviation <5 %. The enrichment factor of developed method was found as 62.5, and method has been effectively applied for the analysis of Cr species in artificial saliva extract of gutkha and mainpuri products. The Cr 6+ was quantitatively recovered (<97 %) under optimal conditions, while the recovery of trivalent specie (Cr 3+ ), at the same experimental conditions, was observed to be <5 %. The Cr 3+ was determined by the difference of total Cr and Cr 6+ in artificial saliva extract of selected STP. Health risks associated with the intake of total Cr in gutkha and mainpuri were assessed in terms of estimated daily intake, such as carcinogenic and noncarcinogenic risks. Estimated daily intake of Cr via chewing 10 g/day of gutkha and mainpuri was found to be below the maximum tolerable daily intake, whereas the calculated risk of cancer for Cr was observed in the acceptable range of 10E -6 -10E -4 , except some brands of gutkha. Graphical Abstract ᅟ.

To evaluate the toxicity of smokeless tobacco products (STPs) on oral bacteria, seven smokeless tobacco aqueous extracts (STAEs) from major brands of STPs and three tobacco-specific N-nitrosamines (TSNAs) were used in a growth and viability test against 38 oral bacterial species or subspecies. All seven STAEs showed concentration-dependent effects on the growth and viability of tested oral bacteria under anaerobic culture conditions, although there were strain-to-strain variations. In the presence of 1 mg/ml STAEs, the growth of 4 strains decreased over 0.32-2.14 log 10 fold, while 14 strains demonstrated enhanced growth of 0.3-1.76 log 10 fold, and the growth of 21 strains was not significantly affected. In the presence of 10 mg/ml STAEs, the growth of 17 strains was inhibited 0.3-2.11 log 10 fold, 18 strains showed enhanced growth of 0.3-0.97 log 10 fold, and 4 strains were not significantly affected. In the presence of 50 mg/ml STAEs, the growth of 32 strains was inhibited 0.3-2.96 log 10 fold, 8 strains showed enhanced growth of 0.3-1.0 log 10 fold, and 2 strains were not significantly affected. All seven STAEs could promote the growth of 4 bacterial strains, including Eubacterium nodatum, Peptostreptococcus micros, Streptococcus anginosus, and Streptococcus constellatus. Exposure to STAEs modulated the viability of some bacterial strains, with 21.1-66.5% decrease for 4 strains at 1 mg/ml, 20.3-85.7% decrease for 10 strains at 10 mg/ml, 20.0-93.3% decrease for 27 strains at 50 mg/ml, and no significant effect for 11 strains at up to 50 mg/ml. STAEs from snuffs inhibited more tested bacterial strains than those from snus indicating that the snuffs may be more toxic to the oral bacteria than snus. For TSNAs, cell growth and viability of 34 tested strains were not significantly affected at up to 100 μg/ml; while the growth of P. micros was enhanced 0.31-0.54 log 10 fold; the growth of Veillonella parvula was repressed 0.33-0.36 log 10 fold; and the cell viabilities of 2 strains decreased 56.6-69.9%. The results demonstrate that STAEs affected the growth of some types of oral bacteria, which may affect the healthy ecological balance of oral bacteria in humans. On the other hand, TSNAs did not significantly affect the growth of the oral bacteria., (Published by Elsevier Ltd.)

Introduction: Sweeteners in tobacco products may influence use initiation and reinforcement, with special appeal to adolescents. Recent analytical studies of smokeless tobacco products (snuff, snus, dissolvables) detected flavorants identical to those added to confectionary products such as hard candy and chewing gum. However, these studies did not determine the levels of sweeteners. The objective of the present study was to quantify added sweeteners in smokeless tobacco products, a dissolvable product, electronic cigarette liquids and to compare with sweetener levels in confectionary products., Methods: Sweetener content of US-sourced smokeless tobacco, electronic cigarette liquid, and confectionary product samples was analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS)., Results: All smokeless products contained synthetic high intensity sweeteners, with snus and dissolvables exceeding levels in confectionary products (as much as 25-fold). All snus samples contained sucralose and most also aspartame, but no saccharin. In contrast, all moist snuff samples contained saccharin. The dissolvable sample contained sucralose and sorbitol. Ethyl maltol was the most common sweet-associated component in electronic cigarette liquids., Discussion: Sweetener content was dependent on product category, with saccharin in moist snuff, an older category, sucralose added at high levels to more recently introduced products (snus, dissolvable) and ethyl maltol in electronic cigarette liquid. The very high sweetener concentrations may be necessary for the consumer to tolerate the otherwise aversive flavors of tobacco ingredients. Regulation of sweetener levels in smokeless tobacco products may be an effective measure to modify product attractiveness, initiation and use patterns., Implications: Dissolvables, snus and electronic cigarettes have been promoted as risk-mitigation products due to their relatively low content of nitrosamines and other tobacco toxicants. This study is the first to quantify high intensity sweeteners in snus and dissolvable products. Snus and dissolvables contain the high intensity sweetener, sucralose, at levels higher than in confectionary products. The high sweetness of alternative tobacco products makes these products attractive to adolescents. Regulation of sweetener content in non-cigarette products is suggested as an efficient means to control product palatability and to reduce initiation in adolescents., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco.)

This investigation provides an updated survey measuring the levels of N'-nitrosonornicotine (NNN) and water content of a select number of smokeless tobacco products sold in the United States in 2015. A total of 34 smokeless tobacco products were collected and analyzed for NNN and water content using LC-MS/MS and GC-TCD, respectively. Smokeless tobacco products were chosen to obtain a representative sample of the different types of products on the U.S. market. These smokeless products represent 12 of the 25 top-selling smokeless tobacco products according to 2013 Nielsen net sales data while five of the smokeless tobacco products are of lower selling smokeless tobacco products. The NNN levels and the water content of the smokeless tobacco products were determined and compared to previous studies. Although the range of NNN levels found was broad for the examined smokeless tobacco products (0.64-12.0 μg/g dry weight), dry snuff had the highest levels of NNN observed (>5 μg/g dry weight). We observed a general decrease in NNN levels for the same six moist snuff products that were analyzed in 2004 compared to our current 2015 study. The water content of the smokeless tobacco products surveyed ranged from 3.92 to 54.8%.

A method was developed for the determination of nine volatile N-nitrosamines in tobacco and smokeless tobacco products. The targets are N-nitrosodimethylamine, N-nitrosopyrrolidine, N-nitrosopiperidine, N-nitrosomorpholine, N-nitrosoethylmethylamine, N-nitrosodiethylamine, N-nitrosodipropylamine, N-nitrosobuylmethylmine, and N-nitrosodibutylamine. The samples were treated by dispersive solid-phase extraction using 1 g of primary secondary amine and 0.5 g of carbon and then analyzed by gas chromatography with tandem mass spectrometry with an electron impact ion source. The recoveries for the targets ranged from 84 to 118%, with <16% relative standard deviations at three spiking levels of 0.5, 1.25, and 2.5 ng/g. The limits of detection ranged from 0.03 to 0.15 ng/g. With the use of the proposed method, we detected the presence of six nitrosamines in the range of 0.4-30.7 ng/g. The study demonstrated that the method could be used as a rapid, convenient, and high-throughput method for N-nitrosamines analysis in tobacco matrix., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Cigarette smoking is a major risk factor for several chronic diseases. Epidemiological data indicate the use of smokeless tobacco (ST) is associated with significantly lower risk for smoking-related diseases compared to cigarettes. Several biomarkers of exposure (BioExp) and effect (BioEff) associated with smoking and use of moist snuff (ST) were evaluated., Methods: A single site, cross-sectional clinical study enrolled three groups of generally healthy male smokers (SMK), moist snuff consumers (MSC), and non-tobacco consumers (NTC), and several BioExp and BioEff were evaluated., Results: Blood and urinary BioExp, including total nicotine equivalents and tobacco-specific nitrosamines, were higher in MSC compared to SMK. Biomarkers of combustion-related toxicants and cadmium were elevated in SMK. Elevated levels of some BioEff associated with oxidative stress (urinary isoprostanes and leukotriene E4), inflammation (white blood cell count), platelet activation (thromboxane metabolites), and lipid metabolism (apolipoprotein B100 and oxidized low-density lipoprotein) were observed in SMK relative to NTC and MSC (all p<0.05). The non-smoking groups (MSC and NTC) showed similar levels of combustion-related BioExp and BioEff., Conclusions: Higher levels of exposure to nicotine and some N'-nitrosamines may be observed in MSC, and SMK are exposed to higher levels of combustion-related toxicants. Changes in BioEff consistent with some aspects of inflammation, oxidative stress, and altered lipid metabolism were detected in SMK compared to the non-smoking groups. The biomarker data further improve our understanding of pathophysiological changes and the risk continuum associated with various tobacco products, and could be useful components of future assessments of tobacco products.

Background: India is the world's third largest consumer of tobacco. There are twice as many users of smokeless tobacco products (STPs) as cigarette smokers. The Government of Tamil Nadu has banned the sale of gutkha and pan masala in 2013. Our aim was to identify the varieties of illegal STP available in Chennai, India., Methods: In systematically chosen zones and regions of Chennai city, we randomly identified three kinds of kiosks (n = 18) and asked for "gutkha" and "pan masala," one of each product available. Details of each product were reviewed based on the information printed on the sachets., Results: Totally 65 STPs were collected; 26 distinct products and 23 brands. All products were claimed to be "tobacco" by the shop keepers. Sixty-five percent of the products informed to contain tobacco and 15.4% to contain pan masala. Five sachets did not inform about the content; 30.8% did not have a pictorial warning; a text warning was printed on 80.8%, but only two products had the messages in Tamil; 70% had promotional messages printed, and 57% had their registration numbers printed., Conclusion: The ban on STP is being systematically violated in Chennai. STP are cheap and easily available and due to promotional laudatory messages and lacking information about the content and warning of health damage, the consumers are left with the perception that they buy more or less harmless product. The Indian Government must introduce policies to control production, import, and sale of illicit STP but we also call for a coordinated international solution.

Use of smokeless tobacco products (STPs) is associated with oral cavity cancer and other health risks. Comprehensive analysis for chemical composition and toxicity is needed to compare conventional and newer STPs with lower tobacco-specific nitrosamines (TSNAs) yields. Seven conventional and 12 low-TSNA moist snuff products purchased in the U.S., Sweden, and South Africa were analyzed for 18 chemical constituents (International Agency for Research on Cancer classified carcinogens), pH, nicotine, and free nicotine. Chemicals were compared in each product using Wilcoxon rank-sum test and principle component analysis (PCA). Conventional compared to low-TSNA moist snuff products had higher ammonia, benzo[a]pyrene, cadmium, nickel, nicotine, nitrate, and TSNAs and had lower arsenic in dry weight content and per mg nicotine. Lead and chromium were significantly higher in low-TSNA moist snuff products. PCA showed a clear difference for constituents between conventional and low-TSNA moist snuff products. Differences among products were reduced when considered on a per mg nicotine basis. As one way to contextualize differences in constituent levels, probabilistic lifetime cancer risk was estimated for chemicals included in The University of California's carcinogenic potency database (CPDB). Estimated probabilistic cancer risks were 3.77-fold or 3-fold higher in conventional compared to low-TSNA moist snuff products under dry weight or under per mg nicotine content, respectively. In vitro testing for the STPs indicated low level toxicity and no substantial differences. The comprehensive chemical characterization of both conventional and low-TSNA moist snuff products from this study provides a broader assessment of understanding differences in carcinogenic potential of the products. In addition, the high levels and probabilistic cancer risk estimates for certain chemical constituents of smokeless tobacco products will further inform regulatory decision makers and aid them in their efforts to reduce carcinogen exposure in smokeless tobacco products., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Introduction: Recently, a tobacco product, Chaini Khaini, identified as snus appeared in India. The product marketing emphasises its discreet nature and explicitly claims safety by referring to the existing evidence on Swedish snus. We analysed tobacco-specific nitrosamines and nicotine in 12 samples of Chaini Khaini purchased in 2013 at open markets in India., Methods: Samples were purchased twice: in March 2013 from Mumbai and in November 2013 from Mumbai and Ahmedabad. Chemical constituents were measured by our routine validated methods., Results: Levels of carcinogenic nitrosamines NNN, NNK and NNAL averaged 22.9 (±4.9), 2.6 (±1.0) and 3.1 (±1.5) µg/g tobacco (wet weight), respectively. The levels of NAB, which is normally present in trace levels in tobacco products, ranged from 3.9 to 12.9 µg/g tobacco. Total nicotine levels in all samples averaged 10.0 mg/g tobacco and unprotonated nicotine accounted for an average 95.4% of the total nicotine content., Conclusions: Chaini Khaini, which is labelled as snus and is marketed as a safe alternative to other tobacco products contains very high levels of carcinogenic nitrosamines and biologically available nicotine. Interventions are urgently needed to educate current and potential consumers of this product., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)