Human biomonitoring (HBM) plays a pivotal role in assessing exposure to toxicologically relevant chemicals, with urinary metabolites serving as key indicators. Despite the widespread implementation of HBM programs globally, certain metabolites, such as mercapturic acids (MAs) derived from volatile organic compounds (VOCs), remain understudied in non-occupationally exposed populations. To bridge this data gap, we analyzed 18 MAs in 360 24-h urine samples collected over a time span of 21 years from 2000 to 2021 in Germany. Two LC-MS/MS methods were utilized to quantify MAs in urine samples obtained from the Environmental Specimen Bank. Statistical analyses, including Kruskal-Wallis tests and Spearman correlations, were employed to evaluate temporal trends, sex-specific differences, and correlations between MAs. Quantification rates between 95 and 100% were obtained for 14 of the 18 MAs, with notable variations in concentrations among different metabolites. The most pronounced decrease in MA levels was observed from 2010/2015 to 2019 with a significant trend for 8 MAs, potentially reflecting changes in environmental exposures and regulations. Moreover, significant differences in urinary excretion per 24 h between males and females were observed for several MAs, highlighting the importance of considering sex in exposure assessments. Comprehensive human biomonitoring (HBM) data with regard to the exposure to volatile organic compounds (VOCs) in Europe is lacking. This prompted us to quantify 18 mercapturic acids as urinary VOC metabolites in 360 samples from the Environmental Specimen Bank collected between 2000 and 2021 in Germany. Our study demonstrates the ubiquitous exposure to numerous VOCs of high toxicological relevance, albeit with a decreasing trend over time for most of the metabolites. This emphasizes the need for a broader HBM to better understand the risk of VOC exposure in Germany and more general in Europe.

The working group "Analyses in Biological Materials" of the German Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (MAK Commission) developed and verified this biomonitoring method for the determination of the most important urinary metabolite of ethoxyquin - 2,2,4‑trimethyl‑6(2H)-quinolinone (EQI). Ethoxyquin is a quinoline-based synthetic antioxidant. Its use as active substance in pesticides was prohibited in 2011. Nevertheless, it continues to be used as a feed additive and is mainly introduced into the environment by feeding treated fishmeal to fish in aquaculture. A human metabolism study showed that ethoxyquin is first metabolised to 1,2‑dihydro-2,2,4‑trimethyl-6‑quinolinol (hydroxyquin) and is then further oxidised to the more stable EQI. In the biomonitoring method presented here, the glucuronide of hydroxyquin in the urine sample is enzymatically hydrolysed using β‑glucuronidase from E. coli. Thereafter, hydroxyquin oxidises spontaneously to EQI. The hydrolysate is subject to salt-assisted liquid-liquid extraction (SALLE) with ethyl acetate. Analysis is performed by UPLC‑MS/MS after positive electrospray ionisation (ESI+). Calibration is performed using standards prepared in pooled urine and processed analogously to the samples. EQI‑D10 is applied as an internal standard. The method provides reliable and accurate analytical results, as shown by the good precision data with standard deviations no greater than 6%. Good accuracy data were obtained with mean relative recoveries in the range of 103-110%. The method is both selective and sensitive, whereby a quantitation limit of 0.03 μg EQI/l was achieved.

Respiratory symptoms are common among smokers without COPD, yet symptom instruments are often optimized for established disease and may under-capture early changes relevant to cessation and tobacco harm reduction. We conducted a repeatability and replicability study of the Respiratory Symptom Experience Scale (RSES) in a rigorously phenotyped cohort to confirm both measurement stability and reproducible discrimination by smoking status. Healthy adults were recruited at a UK hospital and classified as current, former, or never smokers using self-report, exhaled CO, and a comprehensive panel of biomarkers of exposure. RSES was administered at Visit 1 and repeated after 7 ± 3 days at Visit 2 to assess test–retest repeatability. Known-group validity (discriminability by smoking status) was evaluated by comparing RSES scores across biochemically verified groups using non-parametric tests, with ANCOVA for exploratory covariate adjustment. True replicability across independent datasets was not formally tested in the present study. Of 180 enrolled participants, 170 were included (55 current smokers, 60 former smokers, 55 never smokers) after excluding clinical ineligibility and biomarker-inconsistent smoking status. RSES demonstrated high repeatability (Pearson r = 0.87; Spearman ρ = 0.85) with minimal bias (− 0.07) and good agreement. RSES also showed consistent known-group discrimination by smoking status, differing between current and never smokers (p < 0.0001) and across all three groups (p < 0.0001). Current smokers reported higher scores than never (median 2.0 vs. 1.0) and former smokers (2.0 vs. 1.2), whereas former and never smokers did not differ (p = 0.686). Covariate adjustment did not materially alter results. RSES shows strong repeatability and consistent known-group discrimination by smoking status, supporting its use as a patient-relevant endpoint for cessation, and harm-reduction trials, as well as for clinical respiratory and regulatory research.

Cigarette smokers are exposed to over 7,000 chemicals, including many toxic and carcinogenic agents. Alternative non-combustible nicotine products considerably reduce exposure to harmful constituents. Extensive research on biomarkers of potential harm is crucial for assessing the early health impacts of smoking and the reduction of harm after quitting or switching to alternative products. However, such clinical trials are constrained by poor verification of self-reported product use, as common procedures using exhaled carbon monoxide (eCO) and cotinine strip tests are limited by short detection periods and non-specificity, respectively. Therefore, product-use-specific biomarkers of exposure (BoEs) are needed for accurate biochemical verification in studies evaluating the health impact of new products, such as electronic cigarettes. We conducted a cross-sectional study in 180 participants, including current, former, and never-smokers, and applied several BoEs to verify their use behavior. Our multi-biomarker approach monitored exposure to nicotine, tobacco-specific nitrosamines, acrylonitrile and propylene glycol (PG), which were significantly elevated in the current smoker group except PG, identifying six non-compliant subjects (4 current, 1 former and 1 never-smoker). The proposed biomarker panel outperformed eCO and was able to distinguish distinct use patterns, such as vaping vs. smoking, thereby enhancing data accuracy. Moreover, biochemically determined exposure variables such as carboxyhemoglobin and cotinine showed stronger correlations with BoEs than self-reported cigarette consumption. Therefore, the suggested panel is particularly valuable for non-controlled studies, where reliance on self-report can bias outcomes. Implementing the proposed verification strategy can improve study validity and strengthen evidence on the health impacts of switching to alternative products.

Human biomonitoring (HBM) has become a crucial tool for assessing exposure to emerging chemicals. We analyzed 250 24-h urine samples from the German Environmental Specimen Bank (ESB), collected between 2000 and 2021, for exposure to diethylamino hydroxybenzoyl hexyl benzoate (DHHB), a UV filter increasingly used in sunscreens. Three major metabolites were examined: 2-(4-diethylamino)-, 2-(4-ethylamino)-, and 2-(4-amino)-2-hydroxybenzoyl)benzoic acid (DHB, EHB, AHB), with detection rates of 18°%, 13°%, and 87°%, respectively. While EHB and DHB were specific to DHHB, AHB suggested other exposure sources, making it unreliable for assessing DHHB exposure. DHB and EHB were first detected in 2012, with increased detection rates thereafter. The median daily intake of 37 ng/kg bw/d was much lower than the derived no-effect level of 2900 mg/kg bw/d, indicating low risk from DHHB exposure. However, since the analyzed ESB samples were collected in winter, they likely reflect exposure from other products and the environment rather than sunscreen-related exposure. Recently, concerns have emerged regarding the DHHB impurity di-n-hexylphthalate (DnHexP), a reproductive toxicant not authorized in the EU. Retrospective analysis of oral DHHB dosing experiments indeed revealed impurity related dose-dependent excretion of DnHexP metabolites (MnHexP, oxidized 5-OH-MnHexP, and 5-oxo-MnHexP). Due to uncertainties in dose allocation, only a rough excretion fraction of 45°% for MnHexP was derived. Our findings suggest that the DHHB impurity DnHexP may contribute to DnHexP exposure in sunscreen users applying products with contaminated DHHB. Given DnHexP's toxicity, this warrants re-assessment of DHHB's safety in cosmetics and enhanced surveillance of both DHHB and DnHexP in HBM studies.

Rose oxide (RO) is used in cosmetic, hygiene, laundry, cleaning, and household products as a synthetic fragrance. Synthetic rose oxide differs from naturally occurring rose oxide in its enantiomer composition. While natural RO consists mainly of the (-)-cis isomer, synthetic RO contains approximately equal parts of (-)-cis and (+)-cis isomers, as well as small amounts of (-)-trans and (+)-trans isomers. Since in this project, the exposure to industrially produced chemicals by human biomonitoring (HBM) was in the focus, the method development aimed at suitable urinary metabolites derived from the (+)-cis isomer of synthetic RO. For the identification of biomarkers of exposure to synthetic RO as well as for assessing its toxicokinetics, 5 subjects were administered a single oral (0.1 mg/kg body weight) and dermal (0.5 mg/kg) dose of synthetic RO. Analysis of urine fractions by means of high-resolution mass spectrometry (HRMS, conducted with an Orbitrap instrument) revealed that 9-hydroxy-RO (9-OH-RO), 2-hydroxy-4-carboxy-RO (HC-RO), and di-hydroxy-RO and a lactone derived from ring-hydroxylated RO were potential biomarkers of exposure to synthetic RO. Unfortunately, attempts to develop and validate a HBM method for one of these metabolites failed due to insufficient sensitivity (9-OH-RO) or unavailability of standard materials for the required enantiomers (HC-RO, di-OH-RO), lactone). Alternatively, a headspace-solid-phase-micro-extraction-GC-MS (HS-SPME-GC-MS) method for the parent compound (+)-cis-RO was developed and validated. DVB (divinyl benzene)/PDMS (polydimethylsiloxane) fibers suited best for the extraction. Enantiomeric separation was achieved on a Lipodex G column with a temperature gradient. (+)-cis-RO was detected in electron impact ionization (EI) mode. The method performed well with a lower limit of quantification (LLOQ) of 1 ng/L, precision of <10 % and accuracy of >90 %. After oral application, urinary excretion of (+)-cis-RO was fast (mean half-life: 4.1 h) and almost complete after 48 h. However, the mean urinary excretion rate in the 5 subjects amounted to only 7 ppm. (+)-cis-RO was found to be at or slightly above the LLOQ in about half of 14 urine samples from the general population. In conclusion, our investigations for the first time provide data for RO metabolism and toxicokinetics in humans. Despite good to excellent performance, the newly developed and validated HS-SPME-GC-MS method for (+)-cis-RO in urine is only of limited value for HBM of exposure to synthetic RO in the general population, due to the tiny urinary excretion rate of unmetabolized RO and the potential risk of contamination with environmental RO during urine collection and subsequent analytical steps. For a suitable HBM method, potential RO metabolites would need to be synthesized in enantiomerically pure form. Our study provides possible candidates for this approach.

Summary Wastewater-based epidemiology (WBE) is a recent methodology for assessing the exposure to and (intentional) use of all kinds of chemicals in defined populations (communities) by analysis of suitable biomarkers in wastewater (WW) samples, which are commonly collected at the inlet of wastewater treatment plants (WWTPs) serving this population. WBE has been also applied to determine the use of life-style products containing caffeine, alcohol and nicotine on a global basis. This paper outlines the methodological principles of WBE and presents the outcome of a systematic review on WBE studies that utilize cotinine as biomarker in WW samples to assess tobacco/nicotine product use prevalence. For the latter purpose, 60 studies conducted on four continents (no studies were performed in Africa) could be evaluated. Back-calculated WBE-derived mean nicotine consumptions were found to be in the range of 2000–3000 mg/d/1000 persons, with large variations. WBE-derived nicotine consumption data were mostly in fair agreement with the results of ‘classical’ methods such as surveys, (official) statistics, cigarette sales data and epidemiological studies. As reasons for deviations between WBE and classical methods, most frequently the use of illicit tobacco products, nicotine replacement therapy (NRT) products, electronic cigarettes (ECs), reporting bias or outdated statistics were discussed by the study authors. Hitherto available long-term WBE studies covered time periods of 1–8 years showing mostly constant or decreasing time trends for nicotine consumption, depending on the country and time period investigated. The decrease is mostly interpreted as a consequence of lower smoking rates due to tobacco control activities. In a few studies, the minor tobacco alkaloid anabasine (AB) was measured in WW samples as a specific indicator for tobacco-containing products. The currently available results imply that AB in WW is decreasing faster over time than cotinine in WW does, suggesting that the use of tobacco products (mainly combustible cigarettes, CCs) is gradually replaced by tobacco-free nicotine products such as NRT products, ECs and others. Taking up this approach, we conducted a Monte Carlo simulation (MCS) by which the time courses over 10 years of cotinine (indicator of the consumption of (any) nicotine product), AB (indicator of the consumption of (any) tobacco-product) and 2-cyanoethyl-mercapturic acid (2CyEMA, indicator of the exposure to the combustion product acrylonitrile, assumed to originate mainly from CC use) concentrations in WW samples collected at the inlet of a (hypothetical) WWTP serving a large city were modelled. The outcome of the MCSs for two simplified, but, in our view, not unrealistic scenarios revealed that time courses, in particular those of the WW concentration ratios AB/cotinine and 2CyEMA/cotinine, can provide useful information on the use habits for conventional (CCs and oral tobacco products) and smokeless nicotine products (ECs, HTPs, oral nicotine pouches, NRT products) in a population over time. These changes are the net result of transitions between user groups of the various nicotine/tobacco products and non-users and thus would allow to deduce the success (or failure) of tobacco harm reduction (THR) measures in a population. From the presented evidence we conclude that WBE is a potential tool to monitor the progress of THR in populations, provided that suitable biomarkers are measured in WW samples collected over a time period of several years.

Background:The substitution of combustible cigarettes (CC) with non-combustible nicotine alternatives, such as e-cigarettes (EC), significantly lowers exposure to harmful chemicals. However, many individuals who use ECs continue smoking CCs, becoming dual users and remaining at increased risk of toxin exposure. This study will examine how the reduction in cigarettes smoked per day (CPD) after switching to ECs correlates with changes in biomarkers of exposure (BoE) and potential harm (BoPH) to evaluate the extent of harm in dual users. Methods:The study is a prospective, non-randomised, observational, longitudinal cohort study in which 250 CC smokers will be invited to use EC as part of a smoking reduction/cessation strategy. Participants will be grouped in five dual use categories based on the change from baseline in CPD at 1, 3 and 6 months, based on self-reported use in the preceding 30 days. The primary outcome of interest is the mean absolute urinary 2-cyanoethyl mercapturic acid (2CyEMA), a well-validated BoE, also assessed at 1, 3 and 6 months. Other BoE will be assessed, together with exploration of the association between varying intensities of dual use and BoPH. The association between the dual use category and levels of BoE will be evaluated using analysis of variance (ANOVA), in order to understand which BoE are best suited to identifying different levels of dual use. Exploratory assessment of the value of BoPH will similarly be evaluated, to assess whether their predictive power extends from the binary smoker/non-smoker categorization to a more nuanced dual use categorization. The specific details of the own-brand CC will be available once smokers' enrollment in the study is complete. The EC used in the study will be the KIWI 2 Pen refillable pod system, with participants given the option to choose from three different flavored e-liquids: LEAF, MIDWAY, and GLACIAL, all containing 0.9 % nicotine. For specialized equipment used for biomarker analysis please refer our previous publication: https://pubmed.ncbi.nlm.nih.gov/38584934/. Conclusion:EC are increasingly adopted as a harm reduction strategy in CC smokers who wish to quit smoking, the outlined Statistical Analysis Plan (SAP) provides a robust approach to understand the impact of EC on CC use and to quantify the impact of dual use on clinical outcomes using BoPH. For statistical calculations we will use R software.

The global prevalence of electronic cigarettes, heated tobacco products, and other smokeless alternatives has grown significantly in the last ten years. These products have been suggested as combustion-free alternatives for conventional tobacco products like cigarettes, aiming to reduce the negative health impacts associated with smoking. However, the impact of those products on the health and safety of the general population are still unclear, as the absolute exposure from those products has not been thoroughly studied, yet. In this project, a non-targeted LC-HRMS method was developed comprising four different analytical modes for the investigation of the exposure profile in urine of the product users. The method is characterized by its high sensitivity and reproducibility, as shown during method validation. As a proof of concept, we first applied this method to detect significant differences in biomarkers of exposure (BoEs) between smokers and non-smokers. We observed a total of 171 BoEs significantly elevated in smokers, including several well-known biomarkers of smoke exposure like nicotine and its metabolites, mercapturic acid derivatives, and phenolic compounds. Some of the detected biomarkers are present at low ng/mL concentrations in urine, proving the high sensitivity needed for a holistic exploration of the exposome. Moreover, we were able to identify BoEs that have not been reported previously for smoking, such as 2,6-dimethoxyphenol and 7-methyl-1-naphthol glucuronide.

Abstract Background Use of combustible cigarettes (CCs) and smokeless oral tobacco products are well documented risk factors for a variety of oral diseases. However, the potential oral health risks of using recently introduced (since about 2000) non-combustible tobacco/nicotine products (NCPs: electronic cigarettes (ECs), heated tobacco products (HTPs) and oral nicotine pouches (ONPs), remain poorly established. Methods This review evaluates published human studies on detrimental oral health effects in people who use NCPs compared to those smoking cigarettes and those not using any tobacco/nicotine product (NU). We identified 52 studies, predominantly focusing on adults who used electronic cigarettes as an NCP. The studies exhibited significant heterogeneity regarding design, populations, endpoints and quality. Reported outcomes, based on both single and grouped endpoints were qualitatively evaluated by comparing people who use NCPs with NU and with people smoking CCs. Significant increases (indicating a worsening in oral health), significant decreases (indicating a lower level of detrimental effects) and no significant difference between groups were assigned scores of + 1, -1 and 0, respectively. Scores from studies belonging to the same single or grouped endpoints were averaged to a summary score ranging from − 1 to + 1. Results The qualitative meta-analysis revealed that comparisons of EC versus NU groups yielded mean scores of 0.29 for pre-cancerous lesions (N = 14 observations), 0.27 for inflammatory processes (N = 83), 0.43 for oral clinical parameters (N = 93) and 0.70 for shifts in the oral microbiome (N = 10). The corresponding values for the EC versus CC group comparisons amounted to -0.33 (N = 15), -0.14 (N = 76), -0.27 (N = 78) and 0.57 (N = 7). Most studies had significant limitations regarding group sizes, duration of NCP use (mostly only a few years) and validity of self-reported exclusive NCP use. Notably, the implications of dual use (EC + CC) and prior CC use were often not adequately considered. Conclusions The evaluated studies suggest that use of ECs is associated with relatively fewer detrimental oral health effects compared to smoking, yet oral health status remains poorer compared to not using any tobacco/nicotine products. These results have to be interpreted with caution due to a number of limitations and uncertainties in the underlying studies, particularly the potential biases and confounding factors inherent in cross-sectional study designs.

Exposure to aromatic amines may occur via tobacco smoke, hair dyes or tattoo inks, but also in the workplace during certain manufacturing processes. As some aromatic amines are known or suspected carcinogens, human biomonitoring (HBM) is essential to assess their exposure. Aromatic amines were among the selected chemicals in HBM4EU, a European-wide project to harmonise and advance HBM within 30 European countries. For this purpose, the analytical comparability and accuracy of participating laboratories were assessed by a QA/QC programme comprising interlaboratory comparison investigations (ICIs) and external quality assurance schemes (EQUASs). This paper presents the evaluation process and discusses the results of three ICI/EQUAS rounds for the determination of aromatic amines in urine conducted in 2019 and 2020. The final evaluation included ten participants which analysed the following six targeted aromatic amines over three rounds: aniline, ortho-toluidine (TOL), 4,4 '-methylenedianiline (MDA), 4,4 '-methylenebis(2-chloroaniline) (MOCA), 2,4-diaminotoluene (2,4-TDA), and 2,6-diaminotoluene (2,6-TDA). Most participants achieved satisfactory and highly comparable results, although low quantification limits were required to quantify the parameters at the level of exposure in the general population. Hydrolysis of the sample followed by liquid-liquid extraction and subsequent analysis of the derivatised analytes by means of GC-MS/MS were preferred for the sensitive and precise determination of aromatic amines in urine. This QA/QC programme succeeded in establishing a network of laboratories with high analytical comparability and accuracy for the analysis of aromatic amines in Europe.

New types of nicotine and tobacco products like electronic cigarettes (ECs), heated tobacco products or nicotine pouches have been discussed as less harmful alternatives to combustible cigarettes and other toxic forms of tobacco products. Their harm reduction potential lay in the efficient transition away from smoking to those new products. Numerous studies addressing the cessation efficacy of ECs have been published with contradictory outcomes. Yet, a comprehensive Cochrane review concluded with high certainty on the cessation efficacy of ECs. This prompted us to perform a review to identify weaknesses in common study designs and to summarize best practices for the study design on the potential of new nicotine products as cessation aids. 120 articles retrieved from Medline were found to be eligible. Most of the studies in the field were interventional trials while observational studies played a minor role in the evaluation of smoking cessation. Efficacy was predominantly assessed for ECs in 77% of the reports while heated tobacco (17%) and non-combustible products (11%) were less frequently investigated up to now. Measures to determine the efficacy were questionnaire-based assessments as well as use documentation/prevalence and abstinence rates. Studies varied largely in their duration and sample size with medians of 3 months and 156.5 participants, respectively. With the help of this review, we identified several weaknesses in the common study designs. One major limitation in longitudinal trials was the lack of compliance measures suited to verify the use status over longer time periods, relying solely on self-reports. Moreover, the motivation of the participants to quit was rarely defined and a profound familiarization period was not taken into account for the majority of the studies. To what extent such weaknesses influence the outcome of the studies was beyond the scope of this review. We encourage researchers to consider the recommendations which resulted from this review in order to determine the abuse liability and cessation efficacy of the products in a more robust manner. Finally, we like to call attention to the missing data for low- and middle-income countries which would require quitting strategies most urgently to combat the tobacco smoking epidemic.

Tobacco smoke contains several electrophilic constituents which are capable of forming adducts with nucleophilic sites in DNA and proteins like hemoglobin (Hb) and albumin. New nicotine and tobacco products are discussed as less harmful forms of tobacco use compared to smoking combustible cigarettes (CC) due to reduced exposure to harmful constituents. Hence, the adduct profile in users of various tobacco/nicotine products is expected to differ characteristically. In this article, we present a novel nontargeted screening strategy using GC-MS/MS for Hb adducts based on the analysis of the respective derivatized N-terminal valine adducts after modified Edman degradation. We analyzed blood samples from a clinical study with habitual users of CCs, electronic cigarettes, heated tobacco products (HTPs), oral tobacco, nicotine replacement therapy products and nonusers of any tobacco/nicotine products. Our nontargeted approach revealed significant differences in the Hb adduct profiles of the investigated tobacco/nicotine product user groups. Adduct identification was performed by means of an internal database, retention time estimations based on the theoretical boiling points, as well as in-house synthesized reference compounds. Several chemicals that form adducts with Hb could be identified: methylating and ethylating agents, ethylene oxide, acrylonitrile, acrylamide, glycidamide and 4-hydroxybenzaldehyde. Levels were elevated in smokers compared to all other groups for Hb adducts from methylating agents, ethylene oxide, acrylonitrile, acrylamide and glycidamide. Our approach revealed higher concentrations of Hb adducts formed by ethylation, acrylamide and glycidamide in users of HTPs compared to nonusers. However, concentrations for the latter two were still lower than in smokers. Due to their long half-lives, Hb adducts related to acrylonitrile, acrylamide (glycidamide), and ethylene oxide exposure may be useful for the biochemical verification of subjects̀ compliance in longitudinal and cross-sectional studies with respect to smoking and HTP use/abstinence.

Abstract Background Use of traditional tobacco products, including combustible cigarettes (CCs) and smokeless oral products, is an established risk factor for various oral diseases. A potential oral health risk of using new generation tobacco/nicotine products (NGPs) such as electronic cigarettes (ECs), heated tobacco products (HTPs) and oral nicotine pouches (ONPs) is not yet well established. Methods In this systematic review, we evaluated published human studies on detrimental oral health effects in NGP users compared to CC smokers and non-users (NU). We identified 52 studies, of which almost all investigations were on EC users. The studies were extremely heterogeneous in terms of design, subjects, endpoints and quality. Reported outcomes, based on both single and grouped endpoints were qualitatively evaluated by comparing NGP users with NU and CC users. Significant increases (indicating a worsening in oral health), significant decreases (indicating an improvement) and no significant difference between groups were assigned scores of + 1, -1 and 0, respectively. Results With this approach, comparisons of EC versus NU yielded mean scores of 0.29 (pre-cancerous lesions, N = 14 observations), 0.27 (inflammatory processes, N = 83), 0.43 (oral clinical parameters, N = 93) and 0.70 (shifts in the oral microbiome, N = 10). The corresponding values for the EC versus CC comparisons amounted to: -0.33 (N = 15), -0.14 (N = 76), -0.27 (N = 78) and 0.57 (N = 7). Most of the evaluated studies have severe limitations in terms of group sizes, duration of NGP use and validity of self-reported exclusive NGP use. In particular, any dual use (EC + CC) was mostly not adequately taken into account. Conclusions The evaluated studies suggest that use of ECs is associated with some improvement of oral health effects compared to cigarette smoking (CC), but oral health is still found to be worse compared to NU. These results have to be interpreted with caution due to a number of limitations and uncertainties in the underlying studies.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants formed during the incomplete combustion of organic matter such as tobacco. Among these, benzo[a]pyrene (BaP) has been classified as a known carcinogen to humans. It unfolds its effect through metabolic activation to BaP-(7R,8S)-diol-(9S,10R)-epoxide (BPDE), the ultimate carcinogen of BaP. In this article, we describe a simple and highly sensitive GC–NICI–MS/MS method for the quantification of urinary BaP-(7R,8S,9R,10S)-tetrol (( +)-BPT I-1), the hydrolysis product of BPDE. The method was validated and showed excellent results in terms of accuracy, precision, and sensitivity (lower limit of quantification (LLOQ): 50 pg/L). In urine samples derived from users of tobacco/nicotine products and non-users, only consumption of combustible cigarettes was associated with a significant increase in BPT I-1 concentrations (0.023 ± 0.016 nmol/mol creatinine, p < 0.001). Levels of users of potentially reduced-risk products as well as non-users were all below the LLOQ. In addition, the urine levels of six occupationally exposed workers were analyzed and showed the highest overall concentrations of BPT I-1 (844.2 ± 336.7 pg/L). Moreover, comparison with concentrations of 3-hydroxybenzo[a]pyrene (3-OH-BaP), the major detoxification product of BaP oxidation, revealed higher levels of 3-OH-BaP than BPT I-1 in almost all study subjects. Despite the lower levels, BPT I-1 can provide more relevant information on an individual’s cancers susceptibility since BPDE is generated by the metabolic activation of BaP. In conclusion, BPT I-1 is a suitable biomarker to distinguish not only cigarette smokers from non-smokers but also from users of potentially reduced-risk products.

Summary Background Next generation of nicotine/tobacco products (NGPs) include electronic cigarettes (ECs), heated tobacco products (HTPs), oral nicotine pouches (NPs) and smokeless tobacco (SLT) products (in particular snus). These products commonly contain nicotine and are intended to replace combustible cigarettes (CCs) and thus can be regarded as tobacco harm reduction products. To fulfill this role, it is essential that nicotine, which has well established addictive properties, is not causally related to health risks upon chronic use. Objectives The purpose of this review is to evaluate the scientific literature to answer the question, whether nicotine is involved in the development of any diseases or disorders associated with the acute, short, mid- and long-term use of NGPs. Appropriate results from studies with nicotine replacement therapy (NRT) products (gum, patches, inhalers, lozenges) are included as reference basis for inferring the health effects of NGPs. Furthermore, suggestions for filling identified gaps and for avoiding or minimizing limitations and weaknesses in study design are provided. Methods Literature databases such as MEDLINE, Google Scholar and an in-house ABF library (containing about 180,000 articles) were searched for relevant articles. Furthermore, pertinent monographs (such as the US Surgeon General Reports) and recent reviews were screened for further publications. Inclusion criteria were: all human studies investigating the association between use (preferably chronic use) of the nicotine/tobacco products mentioned above and health effects, including diseases, disorders, changes in biomarkers of biological effect (BOBEs). In vivo (animal) and in vitro studies were also considered, provided effects of NGPs in the presence and absence of nicotine or in relation to the nicotine exposure dose were reported. Also, reference lists of recent suitable articles were screened. In total, about 500 articles were retrieved by this approach. The role of nicotine was evaluated by considering the article authors’ statements and their cited references as well as by own judgement of reported results. Human studies are presented in a standardized table format. Results In total, 183 human studies were evaluated, with cardiovascular diseases (CVD) ranking highest (N = 75 studies), followed by respiratory diseases (43), oral health disorders (23), cancer (10), metabolic syndrome (7), reproduction disorders (5) and several other diseases (< 5). The majority of studies do not provide evidence for a participation of nicotine in the pathogenesis. Some (weak) evidence was found that nicotine might be involved in some CVD-related effects and metabolic syndrome. This would be also supported by results from animal and in vitro studies. Discussion Human studies showed some severe limitations and weaknesses with respect to the study design and time of availability of NGPs on the market. A severe flaw is the insufficient consideration of dual use (NGP + CC), particularly in studies on chronic use, which could have led to erroneously increased risks for NGPs with direct consequences also for the role of nicotine. Additionally, prior effects from using CC have an impact. Both circumstances could have led to inaccurate conclusions in terms of elevated risk levels, which require changes in method designs. Suggestions for methodological improvements are provided for future studies. Conclusions A final evaluation of the role of nicotine in disease development in NGP users is currently not possible because use durations are too short. Chronic studies often suffer from insufficient separation between NGP only and dual use together with CCs, which may falsely increase the observed health risk. There is some limited evidence that nicotine may be involved in CVD-related effects, which, however, has to be verified in well controlled long-term studies. The potential involvement of nicotine in other patho-mechanisms also requires further research.

IntroductionMany smokers who use e-cigarettes (ECs) to quit continue smoking alongside vaping. The impact on health among individuals who simultaneously smoke conventional cigarettes (CCs) and use ECs remains unclear. The varying patterns of dual use present differing levels of overall toxin exposure and relative risks concerning smoking-related diseases. Understanding these complexities is vital to assessing the implications for human health.ObjectiveHerein we describe a protocol designed to analyze the impact of different level of substituting CCs with ECs on exposure to toxicants. We’ll use biomarkers to measure this exposure and assess harm reduction in dual users through clinical endpoints, harm-related biomarkers, and behavioral correlations. We expect to observe progressive changes with varying patterns of dual use.Methods and analysesFor this purpose, we planned to recruit a group of 250 smokers who will be asked to reduce their CC consumption by adopting ECs (intervention group). A separate group of 50 smokers will continue to smoke CC (reference group). Study groups will be followed up for 6 months during which biospecimens will be collected for biomarker analyses, and clinical endpoints will be assessed. The trial is structured to characterize subjects’ usage patterns over time using robust biomarkers of exposure and a standardized mobile phone application to facilitate the precise categorization of dual users along the risk continuum based on their usage behaviors. Subject recruitment will start in February 2024 and enrolment is expected to be completed by August 2024. Results will be reported early in 2025. Study findings may provide valuable insights into health benefits or risks associated with varying patterns of dual use.Ethics and disseminationThe study protocol and informed consent forms will be approved by the local Ethical Review Boards. Study results will be disseminated through articles published in reputable, peer-reviewed, open access, scientific journals, presentations at conferences, and the University website.

In the context of the evolving landscape of nicotine consumption, the assessment of biomarkers plays a crucial role in understanding the health impact of different product categories. Exhaled breath (EB) emerges as a promising, non-invasive matrix for biomarker analysis, complementary to conventional urine and plasma data. This study explores distinctive EB biomarker profiles among users of combustible cigarettes (CC), heated tobacco products (HTP), electronic cigarettes (EC), smokeless/oral tobacco (OT), and oral/dermal nicotine products (NRT). We have successfully developed and validated a non-targeted GC-TOF-MS method for the analysis of EB samples across the aforementioned product categories. A total of 66 compounds were identified, with significantly elevated levels in at least one study group. The study found that CC users had higher levels of established VOCs associated with smoking, which supports the proof-of-concept of the method. Breathomic analysis identified increased levels of p-cymene and α-pinene in EC users, while HTP users showed potential biomarker candidates like γ-butyrolactone. This study underscores the utility of EB biomarkers for a comprehensive evaluation of diverse nicotine products. The unique advantages offered by EB analysis position it as a valuable tool for understanding the relationship between exposure and health outcomes.

Background: Despite decades of research on pharmacological and behavioural smoking cessation treatments, current quit aids are of limited success. The introduction of new, combustion-free nicotine and tobacco products extended the tool kit for people who smoke to switch away from their risky habit. We performed a systematic review including 120 studies resulting in several recommendations for a robust study design to determine the cessation efficacy of a new nicotine or tobacco product. Consequently, we prepared this study protocol to assess the cessation efficacy of heated tobacco products (HTPs) and nicotine pouches (NPs). Methods: 250 subjects (125 exclusive smokers and 125 exclusive smokeless tobacco (SLT) users) will be recruited and offered a choice of HTPs in case of smokers and a choice of NPs in case of SLT users in order to switch. Subjects will undergo four visits (baseline, 1, 3, and 6 months) to collect biospecimens and for physical examinations. Use behaviour and questionnaires will be monitored on a regular basis by means of a smartphone-app. We describe a sensitive and specific compliance monitoring using suitable biomarkers of exposure. The sample size of 250 subjects and duration of 6 months will allow the quit rates to be assessed with sufficient statistical power. Finally, the choice between different products shall reflect the individuals’ preferences. Conclusions: This protocol can be applied generically, providing a robust determination of a products’ cessation efficacy. Trial Registration: The trial will be registered in the International Clinical Trials Registry Platform.