Protocol for the "magnitude of cigarette substitution after initiation of e-cigarettes and its impact on biomarkers of exposure and potential harm in dual users" (MAGNIFICAT) study.

Introduction: Many 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. Objective: Herein 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 analyses: For 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 dissemination: The 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.

A sensitive GC-MS/MS method for the quantification of benzo[a]pyrene tetrol in urine.

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.

Human metabolism and excretion kinetics of the surfactant 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) after oral and dermal administration.

2,4,7,9-Tetramethyl-5-decyne-4,7-diol (TMDD) is a non-ionic surfactant with a wide range of applications. TMDD is considered a high-production chemical and, due to its low biodegradation rate, possesses a potentially high prevalence in the environment. However, despite its widespread use, toxicokinetic data and data on internal exposure to TMDD in the general population are completely lacking. Hence, we developed a human biomonitoring (HBM) method for TMDD. Our approach included a metabolism study with four subjects, who were administered an oral dose of 75 µg TMDD/kg body weight and a dermal dose of 750 µg/kg body weight. Terminal methyl-hydroxylated TMDD (1-OH-TMDD) was previously identified as the main urinary metabolite in our lab. The results of the oral and dermal applications were used to determine the toxicokinetic parameters of 1-OH-TMDD as a biomarker of exposure. Finally, the method was applied to 50 urine samples from non-occupationally exposed volunteers. Results show that TMDD was rapidly metabolized, with an average tmax of 1.7 h and a rapid and almost complete (96%) excretion of 1-OH-TMDD until 12 h after oral dosage. Elimination was bi-phasic, with half-lives of 0.75-1.6 h and 3.4-3.6 h for phases 1 and 2, respectively. The dermal application resulted in a delayed urinary excretion of this metabolite with a tmax of 12 h and complete excretion after about 48 h. The excreted amounts of 1-OH-TMDD represented 18% of the orally administered TMDD dose. The data of the metabolism study demonstrated a fast oral as well as substantial dermal resorption of TMDD. Moreover, the results indicated an effective metabolism of 1-OH-TMDD, which is excreted rapidly and completely via urine. Application of the method to 50 urine samples revealed a quantification rate of 90%, with an average concentration of 0.19 ng/mL (0.97 nmol/g creatinine). With the urinary excretion factor (Fue) derived from the metabolism study, we estimated an average daily intake of 1.65 µg TMDD from environmental and dietary sources. In conclusion, 1-OH-TMDD in urine is a suitable biomarker of exposure to TMDD and can be applied for biomonitoring of the general population.

Monitoring the exposure to ethoxyquin between 2000 and 2021 in urine samples from the German Environmental Specimen Bank.

Human Biomonitoring (HBM) of emerging chemicals gained increasing attention within the EU in recent years. After evaluating the metabolism, we established a new HBM method for ethoxyquin (EQ), a feed additive, which was banned in 2017 due to concerns regarding the possible exposure of the general population to it and its highly toxic precursor p-phenetidine. The method was applied to 250 urine samples from the Environmental Specimen Bank collected between 2000 and 2021. The major metabolite EQI was quantified in the majority of the study samples illustrating the ubiquitous exposure of the non-occupationally exposed population. A rather constant exposure was observed until 2016 with a significant decline from 2016 to 2021. This drop falls within the EU wide ban of the chemical as a feed additive from June 2017 which led to a gradual removal until its complete suspension in June 2020. The daily intake (DI) was evaluated with respect to the reported derived no-effect level (DNEL) to estimate the potential health risks from EQ exposure. The median DI of 0.0181 µg/kg bw/d corresponds to only 0.01 % of the DNEL. Even the observed maxima up to 13.1 µg/kg bw/d only accounted for 10 % of the DNEL. Nevertheless, the values suggest a general exposure with the risk of higher burden in a low fraction of the population. In regard to the EQ associated intake of the carcinogen and suspected mutagen p-phenetidine, this level of exposure cannot be evaluated as safe. The recent decrease and the broad exposure substantiate the need for future HBM campaigns in population representative studies to further investigate the observed reductions, potentially find highly exposed subgroups and clarify the impact of the ban as feed additive on EQ exposure.

Determination of a specific metabolite for the non-ionic surfactant 2,4,7,9-Tetramethyl-5-decyne-4,7-diol (TMDD) by UPLC-MS/MS.

2,4,7,9-Tetramethyldec-5-yne-4,7-diol (TMDD) is a non-ionic surfactant commonly used as defoaming agent and numerous other applications. Effluents of wastewater treatment plants have been identified as one of the main sources of TMDD emissions into the environment. Due to its broad application in various fields, TMDD was selected for the development of a biomonitoring method for assessing human exposure within the frame of the cooperation project of the German Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety (BMUB) and the German Chemical Industry Association (VCI) in 2020. This study aimed to identify a urinary metabolite for TMDD by UPLC-Q-Orbitrap-MS which can be used as a biomarker of TMDD exposure. Monohydroxylated TMDD (1-OH-TMDD) was deciphered as the most prominent metabolite of TMDD in humans in a series of in vitro and in vivo experiments. In a next step, a quantitative method for the determination of 1-OH-TMDD was developed and validated. Quantification was achieved by isotope dilution using D3-1-OH-TMDD as internal standard. The method is characterized by a simple sample clean-up procedure and an enzymatic hydrolysis of possible metabolite conjugates with ß-glucuronidase. Method validation was performed according to international guidelines for bioanalytical method validation. The method proved its robustness, precision, accuracy and sensitivity for the intended purpose, i.e. the assessment of TMDD exposure in the general population by means of human biomonitoring.

Time trend of exposure to secondhand tobacco smoke and polycyclic aromatic hydrocarbons between 1995 and 2019 in Germany - Showcases for successful European legislation.

Starting in 2002, regulations and legislative amendments in Germany focused on the non-smoker protection with several measures to reduce exposure to secondhand tobacco smoke (SHS). The present work aimed to evaluate the relationship between polycyclic aromatic hydrocarbons (PAHs) and SHS exposure and to determine to which extent enforced non-smoking regulations and smoking bans affected the exposure of the non-smoking population in Germany since their implementation in the early 2000s until today. For this purpose, cotinine and selected monohydroxylated PAHs (OH-PAHs) were analyzed by means of (UP)LC-MS/MS in 510 24-h-urine samples of the Environmental Specimen Bank collected over a time span of 24 years from 1995 to 2019. Median urinary cotinine levels were found to steadily and significantly decline by 82% from 1995 to 2019. A significant decrease of urinary 3-hydroxybenzo[a]pyrene (19%), 1-OH-pyrene (39%), 1-naphthol (66%), 1- (17%), 2- (25%), and 3-OH-phenanthrene (22%) was also observed throughout the same time span. The decline in urinary levels of cotinine and several OH-PAHs can most likely be attributed to smoking bans and regulations limiting SHS and PAH exposure. This study therefore emphasizes the relevance of human biomonitoring to investigate the exposure of humans to chemicals of concern, assess the effectiveness of regulatory measures, and help policies to enforce provisions to protect public health.

Comparison of urinary mercapturic acid excretions in users of various tobacco/nicotine products.

Urinary mercapturic acids (MAs) are detoxification products for electrophiles occurring in the human body. They are suitable biomarkers of exposure to directly acting electrophilic chemicals or to chemicals which generate the electrophile during its metabolism. We determined the urinary excretion of 19 MAs in habitual users of combustible cigarettes (CCs), electronic cigarettes (ECs), heated tobacco products (HTPs), oral tobacco (OT), and nicotine replacement therapy (NRT) products, and nonusers (NUs) of any tobacco/nicotine products. The 19 MAs are assumed to be physiologically formed primarily from 15 toxicants with three of them belonging to IARC Group 1 (human carcinogen), seven to Group 2A (probable human carcinogen), four to Group 2B (possible human carcinogen), and one to Group 3 (not classifiable as carcinogen). Smoking (CC) was found to be associated with significantly elevated exposure to ethylene oxide (or ethylene), 1,3-butadiene, benzene, dimethylformamide, acrolein, acrylamide, styrene, propylene oxide, acrylonitrile, crotonaldehyde, and isoprene compared with the other user groups and NU. Users of HTPs revealed slight elevation in the MAs related to acrolein, acrylamide, and crotonaldehyde compared with the other non-CC groups. Vaping (EC) was not found to be associated with any of the MAs studied. In conclusion, the determination of urinary MAs is a useful tool for assessing the exposure to toxicants (mainly potential carcinogens) in users of various tobacco/nicotine products. Our data also give cause to clarify the role of vaping (EC) in urinary excretion of DHPMA (precursor: glycidol).

Urinary cotinine and exposure to passive smoke in children and adolescents in Germany - Human biomonitoring results of the German Environmental Survey 2014-2017 (GerES V).

Passive smoking is a preventable and significant cause of many serious health problems, with children being particularly at risk. In the fifth German Environmental Survey (GerES V), conducted from 2014 to 2017, information reflecting the extent of passive smoke exposure in children and adolescents was collected by interview-based questionnaires and human biomonitoring (HBM) analyses of cotinine in urine from 2260 participants, aged 3-17 years. Based on these population-representative data, we describe current passive smoke exposure stratified by different subgroups and identify specific exposure determinants using multivariate logistic regression. The questionnaire data revealed that 42% of children and adolescents lived with at least one smoker in the household. Quantifiable concentrations of cotinine could be detected in 56% of the participants. The overall median concentration of cotinine was 0.2 µg/L, with children and adolescents of low socioeconomic status found to be a group particularly affected by passive smoke with higher cotinine concentrations (median = 1.2 µg/L). In the multiple analysis, the most significant predictor of cotinine levels derived from the questionnaire was passive smoking at home (odds ratio (OR) 13.07 [95CI: 4.65, 36.70]). However, parental smoking and passive smoking among friends and relatives could also be identified as independent factors influencing elevated cotinine levels. The comparison between the previous cycle GerES IV (2003-2006) on 3-14-year-olds and GerES V shows that tobacco smoke exposure of children decreased significantly. This decrease is likely an effect of extensive non-smoker protection laws being enforced 2007-2008 on federal and state level. This is reflected by a halving of urinary cotinine concentrations. Nevertheless, our results indicate that passive smoke is still a relevant source of harmful pollutants for many children and adolescents in Germany, and thus support the need for further efforts to reduce passive smoke exposure, especially in the private environment.

Assessment of the Exposure to Aromatic Amines in Users of Various Tobacco/Nicotine Products.

Aromatic amines such as ortho-toluidine (o-Tol), 2-aminonaphthalene (2-AN), and 4-aminobiphenyl (4-ABP) are human bladder carcinogens and occur at various workplaces, in ambient air, in food products, as well as in tobacco smoke. In a clinical study comprising a period of 74 h under confinement, we investigated the exposure to these three aromatic amines as well as to 3-aminobiphenyl (3-ABP) by measuring them in urine of habitual users of combustible cigarettes (CCs), electronic cigarettes (ECs), heated tobacco products (HTPs), oral tobacco (OT), and nicotine replacement therapy products (NRTs). Non-users (NU) of any tobacco/nicotine products served as (negative) control group. Smokers (CC) exhibited the highest levels for all four aromatic amines measured, significantly elevated compared to NU and non-CC users. Urinary levels in users of EC, HTP, NRT (mostly nicotine gum), and OT (mostly snus) were not significantly different from those in NU. Users of HTP showed slightly elevated urinary excretion levels of o-Tol, 3-ABP, and 4-ABP compared to some other non-CC groups. Dose markers such as daily consumption, urinary nicotine equivalents (Nequ), and plasma cotinine (CotP) were found to be consistently and significantly correlated with the excretion of aromatic amines for smokers (CC) only. Excretion levels of 3- and 4-ABP in smokers were significantly lower in the urine collected overnight compared to that collected during the day, which is just the opposite of what we observed for other biomarkers in this study. The possible reason for this observation is discussed. In conclusion, in contrast to smoking of CCs, the use of ECs, HTPs, nicotine gum, and oral tobacco was not observed to be associated with significant exposure to the aromatic amines o-Tol, 2-AN, 3-ABP, and 4-ABP. The observed slight increase in o-Tol, 3-ABP, and 4-ABP excretions in HTP users has to be verified in larger studies.

Assessment of the exposure to polycyclic aromatic hydrocarbons in users of various tobacco/nicotine products by suitable urinary biomarkers.

Polycyclic aromatic hydrocarbons (PAHs) occur naturally (bitumen and oils) and are formed during all incomplete combustions of organic materials. PAH exposure sources are manifold and include specific workplaces, ambient air, various foodstuffs, tobacco smoke and some medications. At least four members of this class of chemicals have been classified as proven or probable human carcinogens. Assessment of the exposure to PAHs with suitable methods is of importance, particularly in users of new-generation tobacco/nicotine products, which are intended to replace combustible cigarettes (CCs), a major source of non-occupational exposure to PAHs. In a clinical study comprising a period of 74 h under confinement, we investigated the exposure to naphthalene (Nap), fluorene (Flu), phenanthrene (Phe), pyrene (Pyr) and benzo[a]pyrene (BaP) by measuring urinary monohydroxy-PAH (OH-PAH) derived from these parent compounds in habitual users of CCs, electronic cigarettes (ECs), heated tobacco products (HTPs), oral tobacco (OT), and nicotine replacement therapy products (NRTs). Non-users (NU) of any tobacco/nicotine products served as (negative) control group. Smokers exhibited the highest levels for all PAH biomarkers measured, almost all of which were significantly different from the NU and user groups of all other products investigated. CC smokers were the only group which showed a significant relationship between almost all PAH biomarkers and dose markers such as daily consumption, urinary nicotine equivalents (Nequ) and plasma cotinine (CotP). The ratios in urinary OH-PAH between CC and all other groups were dependent on the biomarker and range from < 2 to > 10. These ratios could at least partly be explained by the enzymes involved, their region-selectivity and inducibility by smoking. In conclusion, cigarette smokers (CC) were the only group, which showed product use dependent exposure to PAHs, whereas users of EC, HTP, NRT and OT were not distinguishable from NU of any tobacco/nicotine products.

Assessment of nicotine delivery and uptake in users of various tobacco/nicotine products.

Today various tobacco and nicotine products are available, many of them can be regarded as potentially risk-reduced products when compared to the most frequently used product, combustible cigarettes (CCs). A commonality of these products is that they deliver nicotine, although in quite different amounts and uptake routes, the most common of which are inhalation through the lung and absorption through the oral mucosa. Product-specific nicotine delivery as well as the subject-related use patterns are important factors which determine the pharmacokinetics and achieved internal dose levels of the alkaloid. The latter two parameters are highly relevant for the long-term product loyalty and, consequently, for the implicated health risks, since the risk-reduced products will replace CCs in the long-term only when users will experience a similar level of satisfaction. We measured nicotine and its major metabolites in plasma, saliva and urine samples collected in a controlled clinical study with habitual users (10 per group) of CCs, electronic cigarettes (ECs), heated tobacco products (HTP), oral tobacco (OT), and nicotine replacement therapy (NRT). Non-users (NU) of any tobacco/nicotine products served as (negative) control group. Moderate to strong correlations were observed between the daily consumption and the urinary nicotine equivalents (comprising nicotine and its 10 major metabolites, Nic + 10) or plasma and saliva cotinine concentrations. The average daily nicotine dose as measured by the urinary excretion of Nic + 10 (reflecting approximately 95 % of the absorbed nicotine) amounted to 17 and 22 mg/24 h for smokers (CC) and OT users, respectively, while it was in the range of 6-12 mg/24 h for users of ECs, HTP and NRT products, with high inter-individual variations in each user group. The individual daily nicotine intake, which was calculated by applying product-specific models, showed none to good agreement with the corresponding internal nicotine dose measured by Nic + 10 excretion. Possible reasons for the observed deviations between calculated and objectively measured nicotine doses are discussed.

Assessment of the Exposure to Tobacco-Specific Nitrosamines and Minor Tobacco Alkaloids in Users of Various Tobacco/Nicotine Products.

Tobacco-specific nitrosamines (TSNAs), in particular, the human carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), are important toxicants in tobacco and also (as contaminants) in nicotine products. In a clinical study comprising a period of 74 h under confinement, we investigated the exposure to NNK, NNN, N'-nitrosoanabasine (NAB), and N'-nitrosoanatabine (NAT) as well as to the minor tobacco alkaloids anabasine (AB) and anatabine (AT) by measuring suitable biomarkers in habitual users of combustible cigarettes (CCs), electronic cigarettes (ECs), heated tobacco products (HTPs), oral tobacco (OT), and nicotine replacement therapy products (NRTs). Non-users (NU) of any tobacco/nicotine products served as the (negative) control group. Smokers exhibited the highest levels for all biomarkers measured, except for AB in urine, which was found to be highest in OT users. Somewhat elevated levels compared to NU, EC, and NRT groups were also observed in the users of HTPs. In the users of tobacco-containing products (CC, HTP, and OT), most frequently the biomarkers significantly correlated with the dose markers such as daily consumption, urinary nicotine equivalents (Nequ), and plasma cotinine (CotP). In conclusion, except for smokers (CC) and OT users, exposure of users of ECs, HTPs, and NRTs to TSNAs as well as the minor tobacco alkaloids AB and AT is marginal and statistically not distinguishable from that of NU. Finally, our results for NNN in the saliva provide preliminary evidence for the formation of NNN from the precursor nornicotine in the presence of thiocyanate as a catalyst. The latter hypothesis requires experimental verification.

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.

Time trend of the exposure to geraniol in 24-h urine samples derived from the German Environmental Specimen Bank from 2004 to 2018.

Geraniol (trans-3,7-dimethyl-2,6-octadiene-1-ol) is an acyclic isoprenoid monoterpene with a widespread use as fragrance in consumer products, agrochemicals and pharmaceuticals. The class of terpene chemicals has been associated with varying sensitizing potencies. A recently developed sensitive LC- MS/MS method for the analysis of geraniol metabolites was further improved and validated for the two metabolites, 8-carboxygeraniol and Hildebrandt acid. The successfully validated method was applied to 250 urine samples derived from the Environmental Specimen Bank (ESB) collected between 2004 and 2018. Both metabolites of this allergen of special concern were quantified in all urine samples of this study. Correlation analysis revealed that 8-carboxygeraniol appears to be the sole specific biomarker in urine for geraniol exposure. Overall, the excreted amounts of 8-carboxygeraniol remained unchanged in urine samples collected from 2004 to 2018. However, a significantly higher 8-carboxygeraniol excretion per 24 h was observed in females compared to males across the sampling years from 2004 to 2012. This trend equalized in the years 2015 and 2018. We could demonstrate that 8-carboxygeraniol may be a suited biomarker for assessing the geraniol exposure in the general population. Regardless of the fact that additional, preferably population representative studies combining HBM and health examination were helpful to further elucidate the risks of a geraniol exposure, the current study adds important data for identifying time trends and body burden of geraniol in the environment and shows the ubiquitous exposure towards mixtures of sensitizing chemicals.

Identification of biomarkers specific to five different nicotine product user groups: Study protocol of a controlled clinical trial.

BACKGROUND: Assessing biomarker profiles in various body fluids is of large value to discern between the sole use of nicotine products. In particular, the assessment of the product compliance is required for long-term clinical studies. The objective of this study was the identification of biomarkers and biomarker patterns in body fluids, to distinguish between combustibles, heated tobacco products, electronic cigarettes, oral tobacco and oral/dermal nicotine products used for nicotine replacement therapy (NRT), as well as a control group of non-users. METHODS: A controlled, single-center study was conducted with 60 healthy subjects, divided into 6 groups (5 nicotine product user groups and one non-user group) based on their sole use of the products of choice. The subjects were confined for 76 h, during which, free and uncontrolled use of the products was provided. Sample collections were performed according to the study time schedule provided in Table 2. The primary outcome will be validated through analysis of the collected biospecimens (urine, blood, saliva, exhaled breath and exhaled breath condensate) by means of untargeted omics approaches (i.e. exposomics, breathomics and adductomics). Secondary outcome will include established biomarker quantification methods to allow for the identification of typical biomarker patterns. Statistical analysis tools will be used to specifically discriminate different product use categories. RESULTS/CONCLUSIONS: The clinical trial was successfully completed in May 2020, resulting in sample management and preparations for the quantitative and qualitative analyses. This work will serve as a solid basis to discern between biomarker profiles of different nicotine product user groups. The knowledge collected during this research will be required to develop prototype diagnostic tools that can reliably assess the differences and evaluate possible health risks of various nicotine products.

Assessment of the potential vaping-related exposure to carbonyls and epoxides using stable isotope-labeled precursors in the e-liquid.

The formation of carbonyls and epoxides in e-cigarette (EC) aerosol is possible due to heating of the liquid constituents. However, high background levels of these compounds have inhibited a clear assessment of exposure during use of ECs. An EC containing an e-liquid replaced with 10% of 13C-labeled propylene glycol and glycerol was used in a controlled use clinical study with 20 EC users. In addition, five smokers smoked cigarettes spiked with the described e-liquid. Seven carbonyls (formaldehyde, acetaldehyde, acrolein, acetone, crotonaldehyde, methacrolein, propionaldehyde) were measured in the aerosol and the mainstream smoke. Corresponding biomarkers of exposure were determined in the user's urine samples. 13C-labeled formaldehyde, acetaldehyde and acrolein were found in EC aerosol, while all seven labeled carbonyls were detected in smoke. The labeled biomarkers of exposure to formaldehyde (13C-thiazolidine carboxylic acid and 13C-N-(1,3-thiazolidine-4-carbonyl)glycine), acrolein (13C3-3-hydroxypropylmercapturic acid) and glycidol (13C3-dihydroxypropylmercapturic acid) were present in the urine of vapers indicating an EC use-specific exposure to these toxicants. However, other sources than vaping contribute to a much higher extent by several orders of magnitude to the overall exposure of these toxicants. Comparing data for the native (unlabeled) and the labeled (exposure-specific) biomarkers revealed vaping as a minor source of user's exposure to these toxicants while other carbonyls and epoxides were not detectable in the EC aerosol.

Benzene metabolite SPMA and acrylamide metabolites AAMA and GAMA in urine of children and adolescents in Germany - human biomonitoring results of the German Environmental Survey 2014-2017 (GerES V).

Benzene and acrylamide are carcinogenic substances contained inter alia in tobacco smoke. The mercapturic acid metabolites of benzene, N-acetyl-S-phenyl-L-cysteine (SPMA), and of acrylamide, N-acetyl-S-(3-amino-3-oxopropyl)-cysteine (AAMA) and N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)-cysteine (GAMA), were analysed in 2260 first-morning void urine samples from children and adolescents aged 3-17 years, participating in the population-representative German Environmental Survey on Children and Adolescents, GerES V 2014-2017. SPMA was detected in 98% of the participants with a geometric mean (GM) of 0.097 µg/L urine. Smokers had about 10-fold higher levels of the benzene metabolite SPMA than non-smokers. The sample comprises of 48 self-reported smokers, mainly in the oldest age group (14-17-year-olds). Second-hand smoke exposure, living near busy or very busy roads, and using domestic fuels for heating were additionally associated with higher benzene metabolite levels. SPMA levels in GerES V were lower compared to levels found in other countries, which in part however may reflect different proportions of smokers. The acrylamide metabolites AAMA and GAMA were detected in 100% of the participants with a GM of 72.6 µg/L urine for AAMA and 15.0 µg/L urine for GAMA. Smoking children and adolescents had about 2.5-fold higher AAMA levels than non-smoking ones. The frequency of consumption of french-fried potatoes and potato crisps consumption was also positively associated with urinary AAMA and GAMA levels. Compared to the urinary AAMA and GAMA levels in Germany and other countries, levels in GerES V tended to be higher than in the few studies reported. The urinary levels of the benzene biomarker SPMA, and the acrylamide biomarkers AAMA and GAMA build the basis to derive reference values for the exposure of children and adolescents in Germany. The results reveal options for exposure reduction mainly in personal choices regarding smoking and diet, but also requiring policy to maintain efforts in non-smoking regulations and improving ambient air quality. Providing these results also to the European HBM Initiative HBM4EU will contribute to gain knowledge on the exposure of the European population, the health impact of carcinogens and thus providing support for substantiated exposure assessment.

Human biomonitoring in urine samples from the Environmental Specimen Bank reveals a decreasing trend over time in the exposure to the fragrance chemical lysmeral from 2000 to 2018.

2-(4-tert-butylbenzyl)propionaldehyde (trade names, e.g. lysmeral or lilial) is a fragrance chemical frequently used in cosmetic products where it is labelled as Butylphenyl methylpropional. A recently developed LC-MS/MS method for the analysis of four lysmeral metabolites (tert-butylbenzoic acid (TBBA), lysmerol, lysmerylic acid, and hydroxy-lysmerylic acid) was applied to 329 urine samples from the Environmental Specimen Bank collected between 2000 and 2018. The two major metabolites TBBA and lysmerol were found in quantifiable concentrations in almost all samples in this study and correlated significantly. Hence, both analytes proved to be specific biomarkers indicating the broad exposure to lysmeral. A significant decline was found for TBBA and lysmerol for the monitored years with the most pronounced decrease from 2012 to 2015. The daily intake (DI) was used to evaluate potential health risks with respect to the derived no-effect level (DNEL) as a threshold for exposure of the general population. The median DI (1.63 µg/kg bw/d) and the 95th percentile (4.69 µg/kg bw/d) corresponded to 2.6% and 7.5% of the lowest DNEL (62.5 µg/kg bw/d for oral administration), respectively. Even though a decreasing trend in exposure was observed the data still calls for efforts to reduce the exposure towards lysmeral since metabolites of lysmeral were detected in nearly all samples and adverse effects cannot be excluded. Clearly, these results need to be substantiated by HBM campaigns in population representative samples like the German Environmental Survey in adults (GerES VI) to provide more robust data for the adult population.

Development of a human biomonitoring method for assessing the exposure to ethoxyquin in the general population.

Ethoxyquin (EQ) is commonly used as an antioxidant in animal feeds. Although EQ is not permitted for usage in food products for humans within the EU, residues of EQ and its transformation products could be determined in food of animal origin. Despite its widespread use and concerns on its toxicological profile, no information about the systemic exposure to EQ in the general population is available. Hence, we developed a human biomonitoring (HBM) method for EQ. Our approach included a metabolism study with five subjects, who were administered an oral dose of 0.005 mg EQ/kg body weight. Unchanged EQ and the major metabolite 2,2,4-trimethyl-6(2H)-quinolinone (EQI) were identified as urinary excretion products of EQ. While small amounts of EQ could be determined in high concentrated samples from the metabolism study only, 28.5% of the orally applied EQ dose could be recovered as EQI. Toxicokinetic parameters were determined for EQI, the potential biomarker of exposure. In addition, an analytical method for EQI (LOQ = 0.03 µg/L) in urine based on UHPLC-MS/MS comprising enzymatic glucuronide hydrolysis and salt-assisted liquid-liquid extraction was developed, validated and applied to 53 urine samples from the general population. EQI could be quantified in 11 (21%) of the samples in levels up to 1.7 µg/L urine, proving the suitability of the developed method for the intended purpose.

A novel quantification method for sulfur-containing biomarkers of formaldehyde and acetaldehyde exposure in human urine and plasma samples.

A novel method for the quantification of the sulfur-containing metabolites of formaldehyde (thiazolidine carboxylic acid (TCA) and thiazolidine carbonyl glycine (TCG)) and acetaldehyde (methyl thiazolidine carboxylic acid (MTCA) and methyl thiazolidine carbonyl glycine (MTCG)) was developed and validated for human urine and plasma samples. Targeting the sulfur-containing metabolites of formaldehyde and acetaldehyde in contrast to the commonly used biomarkers formate and acetate overcomes the high intra- and inter-individual variance. Due to their involvement in various endogenous processes, formate and acetate lack the required specificity for assessing the exposure to formaldehyde and acetaldehyde, respectively. Validation was successfully performed according to FDA's Guideline for Bioanalytical Method Validation (2018), showing excellent performance with regard to accuracy, precision, and limits of quantification (LLOQ). TCA, TCG, and MTCG proved to be stable under all investigated conditions, whereas MTCA showed a depletion after 21 months. The method was applied to a set of pilot samples derived from smokers who consumed unfiltered cigarettes spiked with 13C-labeled propylene glycol and 13C-labeled glycerol. These compounds were used as potential precursors for the formation of 13C-formaldehyde and 13C-acetaldehyde during combustion. Plasma concentrations were significantly lower as compared to urine, suggesting urine as suitable matrix for a biomonitoring. A smoking-related increase of unlabeled biomarker concentrations could not be shown due to the ubiquitous distribution in the environment. While the metabolites of 13C-acetaldehyde were not detected, the described method allowed for the quantification of 13C-formaldehyde uptake from cigarette smoking by targeting the biomarkers 13C-TCA and 13C-TCG in urine.Graphical abstract.