It has previously been found that, compared with cigarette smoke, the aerosols generated by heated tobacco products contain fewer and lower harmful and potentially harmful constituents (HPHCs) and elicit lower biological activity in in vitro models and lower smoking-related exposure biomarker levels in clinical studies. It is important to accumulate such scientific evidences for heated tobacco products with a novel heating system, because different heating system may affect the quantitative aspect of the amount of HPHCs and the qualitative aspect of the biological activity of the aerosol generated. Here, the chemical properties of, and toxicological responses to aerosols emitted by DT3.0a, a new heated tobacco product with a novel heating system, and cigarette smoke (CS) were compared, using chemical analyses, in vitro battery (standardized genotoxicity and cytotoxicity) assays, and mechanistic (ToxTracker and two-dimensional cell culture) assays. Regular- and menthol-flavored DT3.0a and standard 1R6F reference cigarettes were tested. Selected HPHC yields were lower in DT3.0a aerosol than 1R6F CS. The genotoxicity-related assays indicated that DT3.0a aerosol was not genotoxic, regardless of metabolic activation. The other biological assays indicated that less cytotoxicity induction and oxidative stress response were elicited by DT3.0a aerosol compared with 1R6F CS. Similar results were found for both regular and menthol DT3.0a. Like previous reports for heated tobacco products with other heating systems, the results of this study indicated that DT3.0a aerosols have chemical and biological properties less likely to be harmful than 1R6F CS.

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.

A growing number of public health bodies, regulators and governments around the world consider electronic vapor products a lower risk alternative to conventional cigarettes. Of critical importance are rapid new approach methodologies to enable the screening of next generation products (NGPs) also known as next generation tobacco and nicotine products. In this study, the activity of conventional cigarette (3R4F) smoke and a range of NGP aerosols (heated tobacco product, hybrid product and electronic vapor product) captured in phosphate buffered saline, were screened by exposing a panel of human cell-based model systems using Biologically Multiplexed Activity Profiling (BioMAP® Diversity PLUS® Panel, Eurofins Discovery). Following exposure, the biological activity for a wide range of biomarkers in the BioMAP panel were compared to determine the presence of toxicity signatures that are associated with specific clinical findings. NGP aerosols were found to be weakly active in the BioMAP Diversity PLUS Panel (≤3/148 biomarkers) whereas significant activity was observed for 3R4F (22/148 biomarkers). Toxicity associated biomarker signatures for 3R4F included immunosuppression, skin irritation and thrombosis, with no toxicity signatures seen for the NGPs. BioMAP profiling could effectively be used to differentiate between complex mixtures of cigarette smoke or NGP aerosol extracts in a panel of human primary cell-based assays. Clinical validation of these results will be critical for confirming the utility of BioMAP for screening NGPs for potential adverse human effects.

devTOX quickPredict (devTOX qP ) is a metabolomics biomarker-based assay that utilises human induced pluripotent stem (iPS) cells to screen for potential early stage embryonic developmental toxicity in vitro. Developmental toxicity potential is assessed based on the assay endpoint of the alteration in the ratio of key unrelated biomarkers, ornithine and cystine (o/c). This work aimed to compare the developmental toxicity potential of tobacco-containing and tobacco-free non-combustible nicotine products to cigarette smoke. Smoke and aerosol from test articles were produced using a Vitrocell VC10 smoke/aerosol exposure system and bubbled into phosphate buffered saline (bPBS). iPS cells were exposed to concentrations of up to 10% bPBS. Assay sensitivity was assessed through a spiking study with a known developmental toxicant, all-trans-retinoic acid (ATRA), in combination with cigarette smoke extract. The bPBS extracts of reference cigarettes (1R6F and 3R4F) and a heated tobacco product (HTP) were predicted to have the potential to induce developmental toxicity, in this screening assay. The bPBS concentration at which these extracts exceeded the developmental toxicity threshold was 0.6% (1R6F), 1.3% (3R4F), and 4.3% (HTP) added to the cell media. Effects from cigarette smoke and HTP aerosol were driven largely by cytotoxicity, with the cell viability and o/c ratio dose-response curves crossing the developmental toxicity thresholds at very similar concentrations of added bPBS. The hybrid product and all the electronic cigarette (e-cigarette) aerosols were not predicted to be potential early developmental toxicants, under the conditions of this screening assay.

The expression of some microRNAs (miRNA) is modulated in response to cigarette smoke (CS), which is a leading cause of major preventable diseases. However, whether miRNA expression is also modulated by the aerosol/extract from potentially reduced-risk products is not well studied. The present work is a meta-analysis of 12 in vitro studies in human organotypic epithelial cultures of the aerodigestive tract (buccal, gingival, bronchial, nasal, and small airway epithelia). These studies compared the effects of exposure to aerosols from electronic vapor (e-vapor) products and heated tobacco products, and to extracts from Swedish snus products (in the present work, will be referred to as reduced-risk products [RRPs]) on miRNA expression with the effects of exposure to CS or its total particulate matter fraction. This meta-analysis evaluated 12 datasets of a total of 736 detected miRNAs and 2775 exposed culture inserts. The t-distributed stochastic neighbor embedding method was used to find similarities across the diversity of miRNA responses characterized by tissue type, exposure type, and product concentration. The CS-induced changes in miRNA expression in gingival cultures were close to those in buccal cultures; similarly, the alterations in miRNA expression in small airway, bronchial, and nasal tissues resembled each other. A supervised clustering was performed to identify miRNAs exhibiting particular response patterns. The analysis identified a set of miRNAs whose expression was altered in specific tissues upon exposure to CS (e.g., miR-125b-5p, miR-132-3p, miR-99a-5p, and 146a-5p). Finally, we investigated the impact of RRPs on miRNA expression in relation to that of CS by calculating the response ratio r between the RRP- and CS-induced alterations at an individual miRNA level, showing reduced alterations in miRNA expression following RRP exposure relative to CS exposure (94 % relative reduction). No specific miRNA response pattern indicating exposure to aerosols from heated tobacco products and e-vapor products, or extracts from Swedish snus was identifiable.