Levels of nicotine and tobacco-specific nitrosamines in oral nicotine pouches.

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.

Consumption of chewing tobacco alters the serum metal contents in pregnant women at Tandojam and adjoining areas.

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.

Physical and chemical characterization of smokeless tobacco products in India.

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.

Fungal Community Composition and Function Associated with Loose Smokeless Tobacco Products.

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.

Oral Cancer Risk Assessment for Different Types of Smokeless Tobacco Products Sold Worldwide: A Review of Reviews and Meta-analyses.

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.

Composition and Ecological Functionality of Fungal Communities Associated with Smokeless Tobacco Products Mainly Consumed in India.

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.

Associations between microbial communities and key chemical constituents in U.S. domestic moist snuff.

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.

Assessment of the Exposure to NNN in the Plasma of Smokeless Tobacco Users.

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 (Cmax) ranged from 3.5 to 10 pg/mL (mean: 7.1 pg/mL) at a tmax 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.

An in Vitro Study of Constituents Released from Smokeless Tobacco Products into Human Saliva.

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.

Chemical characteristics and cancer risk assessment of smokeless tobacco used in Tunisia (neffa).

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.

Impact of smokeless tobacco-associated bacteriome in oral carcinogenesis.

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.

Smokeless tobacco analysis: Simultaneous extraction and purification of alkaloids, volatile N-nitrosamines, and polycyclic hydrocarbons for GC-MS/MS.

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.

Smokers' Exposure to Perceived Modified Risk Claims for E-Cigarettes, Snus, and Smokeless Tobacco in the United States.

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."

An approach for the extract generation and toxicological assessment of tobacco-free 'modern' oral nicotine pouches.

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.

Comparison of tobacco-specific nitrosamine levels in smokeless tobacco products: High levels in products from Bangladesh.

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.

Method development and validation of dissolution testing for nicotine release from smokeless tobacco products using flow-through cell apparatus and UPLC-PDA.

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 (f1) and similarity factor (f2) 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.

Chemical analysis of snus products from the United States and northern Europe.

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.

Quantification of Nicotine and Cotinine in Plasma, Saliva, and Urine by HPLC Method in Chewing Tobacco Users.

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.

Evaluation of tobacco dependence measures in South Asian smokeless tobacco users.

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.

SLTChemDB: A database of chemical compounds present in Smokeless tobacco products.

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.