Smoking is a cause of serious diseases in adult smokers. Next generation products (NGP) can provide potentially reduced harm alternatives of nicotine delivery, due to the reductions in numbers and levels of toxicants associated with their use. These include heated tobacco and herbal products (HTPs/ HHPs) and electronic vapour products (EVPs). This study assessed the effects of a range of NGP (an HTP, an HHP and two EVPs) and a reference cigarette (1R6F) in the ToxTracker Antioxidant (AO) and ToxProfiler Mitotox Assessment Xtension (MAX) assays, which provide mechanistic insights via a range of cell stress endpoints. Aqueous extracts of smoke/ aerosol were generated by bubbling through phosphate-buffered saline solution, which was analysed for nicotine and carbonyl content. In the ToxTracker AO assay, 1R6F induced oxidative stress, genotoxicity and protein damage responses. Addition of N-acetyl cysteine (NAC) and glutathione (GSH) partially reduced selected responses, indicating oxidative stress contributes but does not fully account for all observed effects. Oxidative stress responses followed a similar trend for HTP; but was not classed as genotoxic. The HHP induced one oxidative stress marker, which was reduced by the addition of NAC/GSH. In the ToxProfiler MAX assay, 1R6F induced oxidative, endoplasmic reticulum and cell cycle stress. HTP induced the same stress pathways but was less potent than 1R6F. The HHP only induced an oxidative stress response, and at higher concentrations than 1R6F. The EVPs did not induce any of the cell stress markers across both assays and none of the test products induced mitochondrial toxicity. Overall, biological activity followed a decreasing gradient from 1R6F to HTP, HHP and EVPs. The findings of the assays used in this study reflect the proposed placement of such products on the relative reduced risk scale based on their smoke/aerosol chemistry and thus highlights the tobacco harm reduction potential of NGP.

Data from pre-clinical and clinical studies form part of an integrated assessment of the tobacco harm reduction (THR) potential of novel products that may act as cigarette alternatives for adult smokers. We report data from pre-clinical (emissions chemistry and in vitro toxicology) and clinical (nicotine pharmacokinetics and subjective effects) studies conducted with the iSENZIA™ heated herbal system (HHS; PULZE™ 2.0 device with iSENZIA™ sticks), which utilizes electronic heating of a tea-based substrate to generate an inhalable nicotine-containing aerosol. The aerosols from the iSENZIA™ HHS contained significantly lower levels, by up to 99.8%, of the nine World Health Organization Study Group on Tobacco Product Regulation (WHO TobReg) analytes compared with 1R6F reference cigarette smoke and elicited significantly lower in vitro cytotoxicity, genotoxicity, and mutagenicity responses. The clinical study demonstrated that the iSENZIA™ HHS delivers satisfactory levels of nicotine to users and has lower abuse liability than cigarettes. Overall, our data suggest that iSENZIA™ has the potential to offer substantially reduced toxicant exposure, as well as a reduction in toxicity, compared to cigarettes, while delivering satisfactory levels of nicotine. These findings support the THR potential of the iSENZIA™ HHS as a reduced-risk, acceptable alternative product for adult smokers.

In vitro toxicology research has accelerated with the use of in silico, computational approaches and human in vitro tissue systems, facilitating major improvements evaluating the safety and health risks of novel consumer products. Innovation in molecular and cellular biology has shifted testing paradigms, with less reliance on low-throughput animal data and greater use of medium- and high-throughput in vitro cellular screening approaches. These new approach methodologies (NAMs) are being implemented in other industry sectors for chemical testing, screening candidate drugs and prototype consumer products, driven by the need for reliable, human-relevant approaches. Routine toxicological methods are largely unchanged since development over 50 years ago, using high-doses and often employing in vivo testing. Several disadvantages are encountered conducting or extrapolating data from animal studies due to differences in metabolism or exposure. The last decade saw considerable advancement in the development of in vitro tools and capabilities, and the challenges of the next decade will be integrating these platforms into applied product testing and acceptance by regulatory bodies. Governmental and validation agencies have launched and applied frameworks and “roadmaps” to support agile validation and acceptance of NAMs. Next-generation tobacco and nicotine products (NGPs) have the potential to offer reduced risks to smokers compared to cigarettes. These include heated tobacco products (HTPs) that heat but do not burn tobacco; vapor products also termed electronic nicotine delivery systems (ENDS), that heat an e-liquid to produce an inhalable aerosol; oral smokeless tobacco products (e.g., Swedish-style snus) and tobacco-free oral nicotine pouches. With the increased availability of NGPs and the requirement of scientific studies to support regulatory approval, NAMs approaches can supplement the assessment of NGPs. This review explores how NAMs can be applied to assess NGPs, highlighting key considerations, including the use of appropriate in vitro model systems, deploying screening approaches for hazard identification, and the importance of test article characterization. The importance and opportunity for fit-for-purpose testing and method standardization are discussed, highlighting the value of industry and cross-industry collaborations. Supporting the development of methods that are accepted by regulatory bodies could lead to the implementation of NAMs for tobacco and nicotine NGP testing.

With the use of in vitro new approach methodologies (NAMs) for the assessment of non-combustible next-generation nicotine delivery products, new extrapolation methods will also be required to interpret and contextualize the physiological relevance of these results. Quantitative in vitro to in vivo extrapolation (QIVIVE) can translate in vitro concentrations into in-life exposures with physiologically-based pharmacokinetic (PBPK) modelling and provide estimates of the likelihood of harmful effects from expected exposures. A major challenge for evaluating inhalation toxicology is an accurate assessment of the delivered dose to the surface of the cells and the internalized dose. To estimate this, we ran the multiple-path particle dosimetry (MPPD) model to characterize particle deposition in the respiratory tract and developed a PBPK model for nicotine that was validated with human clinical trial data for cigarettes. Finally, we estimated a Human Equivalent Concentration (HEC) and predicted plasma concentrations based on the minimum effective concentration (MEC) derived after acute exposure of BEAS-2B cells to cigarette smoke (1R6F), or heated tobacco product (HTP) aerosol at the air liquid interface (ALI). The MPPD-PBPK model predicted the in vivo data from clinical studies within a factor of two, indicating good agreement as noted by WHO International Programme on Chemical Safety (2010) guidance. We then used QIVIVE to derive the exposure concentration (HEC) that matched the estimated in vitro deposition point of departure (POD) (MEC cigarette = 0.38 puffs or 11.6 µg nicotine, HTP = 22.9 puffs or 125.6 µg nicotine) and subsequently derived the equivalent human plasma concentrations. Results indicate that for the 1R6F cigarette, inhaling 1/6th of a stick would be required to induce the same effects observed in vitro, in vivo. Whereas, for HTP it would be necessary to consume 3 sticks simultaneously to induce in vivo the effects observed in vitro. This data further demonstrates the reduced physiological potency potential of HTP aerosol compared to cigarette smoke. The QIVIVE approach demonstrates great promise in assisting human health risk assessments, however, further optimization and standardization are required for the substantiation of a meaningful contribution to tobacco harm reduction by alternative nicotine delivery products.

BackgroundSmoking cigarettes is a cause of serious diseases in smokers, including cardiovascular disease. Through a pathway of endothelial dysfunction, lipid infiltration, macrophage recruitment and vascular remodeling, atherosclerosis is fundamental in the development of most cardiovascular diseases. There is an increasing number of next-generation products (NGP) which provide potentially reduced harm forms of nicotine delivery to adult smokers. This study aimed to optimise an in vitro cardiovascular model to assess such products. Human Coronary Artery Endothelial Cells (HCAECs) were cultured on an OrganoPlate®2-lane chip (Mimetas BV) combined with THP-1 monocytes under flow conditions.MethodsAn aqueous aerosol extract from the 1R6F reference cigarette was compared with two categories of NGP, (a heated tobacco product (HTP) and an electronic nicotine delivery system (ENDS)), to assess relative effects on select atherogenic endpoints (oxidative stress, monocyte adhesion, ICAM-1 expression, and inflammatory markers). Following exposure of THP-1 monocytes with the aqueous extracts, the resulting conditioned medium was then added to the HCAEC vessels.Results1R6F was consistently the most potent test article, eliciting observed responses at 4x lower concentrations than applied for both the HTP and ENDS. The HTP was more potent than the ENDS product across all endpoints, however, all test articles increased monocyte adhesion. ICAM-1 did not appear to be a main driver for monocyte adhesion, however, this could be due to replicate variability. Upon comparison to an extract-only control exposure, THP-1-medium pre-conditioning was an important mediator of the responses observed.ConclusionIn conclusion, the data suggests that the NGP extracts, containing primary aerosol chemical constituents exhibit a marked reduction in biological activity in the early key events associated with atherogenesis when compared to a cigarette, adding to the weight of evidence for the tobacco harm reduction potential of such products.

Heated tobacco products represent a novel category of tobacco products in which a tobacco consumable is heated to a temperature that releases nicotine from the tobacco leaf but not to a temperature sufficient to cause combustion. Heated tobacco products may therefore have the potential to be a less harmful alternative for adult smokers who would otherwise continue to smoke cigarettes, as their use should result in exposure to substantially fewer and lower levels of toxicants. This update represents a two-year extension to our previous narrative review, which covered peer-reviewed journal articles published up to August 31, 2021. The scientific evidence published between 2021 and 2023 continues to indicate that aerosols produced from heated tobacco products contain fewer and substantially lower levels of harmful and potentially harmful constituents and that these observed reductions consistently translate to reduced biological effects in both in vitro and in vivo toxicological studies. Biomarker and clinical data from studies in which product use is controlled within a clinical setting continue to suggest changes in levels of biomarkers of exposure, biomarkers of potential harm, and clinical endpoints indicating the potential for reduced harm with switching to exclusive use of heated tobacco products in adult smokers. Overall, the available peer-reviewed scientific evidence continues to indicate that heated tobacco products offer promise as a potentially less harmful alternative to cigarettes, and as such, the conclusions of our original narrative review remain valid.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to develop recommendations for optimal scientific and technical approaches for conducting in vitro assays to assess potential toxicity within and across tobacco and various next-generation products (NGPs) including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDSs). This publication was developed by a working group of the workshop members in conjunction with the sixth workshop in that series entitled “Dosimetry for conducting in vitro evaluations” and focuses on aerosol dosimetry for aerosol exposure to combustible cigarettes, HTP, and ENDS aerosolized tobacco products and summarizes the key challenges as well as documenting areas for future research.

In vitro testing is important to characterise biological effects of consumer products, including nicotine delivery products such as cigarettes, e-cigarettes and heated tobacco products. Users' cells are exposed to these products' aerosols, of variant chemical compositions, as they move along the respiratory tract. In vitro exposure systems are available to model such exposures, including delivery of whole aerosols to cells, and at the air-liquid interface. Whilst there are clear advantages of such systems, factors including time to aerosol delivery, aerosol losses and number of cell cultures that can be exposed at one time could be improved. This study aimed to characterise a custom-built smoke/ aerosol exposure in vitro system (SAEIVS) using 1R6F reference cigarette smoke. This system contains five parallel smoking chambers and delivers different dilutions of smoke/ aerosol to two separate cell culture exposure chambers in <10 s. Using two dosimetry measures (optical density 400 nm [OD400 ]; mass spectrometric nicotine quantification), the SAEIVS demonstrated excellent linearity of smoke dilution prior to exposure (R2 = 0.9951 for mass spectrometric quantification; R2 = 0.9965 for OD400 ) and consistent puff-wise exposures across 24 and 96 well plates in cell culture relevant formats (e.g., within inserts). Smoke loss was lower than previously reported for other systems (OD400 : 16%; nicotine measurement: 20%). There was good correlation of OD400 and nicotine measurements, indicating that OD was a useful surrogate for exposure dosimetry for the product tested. The findings demonstrated that the SAEIVS is a fit-for-purpose exposure system for the reproducible dose-wise exposure assessment of nicotine delivery product aerosols.

Tobacco harm reduction (THR) involves providing adult smokers with potentially reduced harm modes of nicotine delivery as alternatives to smoking combustible cigarettes. Heated tobacco products (HTPs) form a category with THR potential due to their ability to deliver nicotine and flavours through heating, not burning, tobacco. By eliminating burning, heated tobacco does not produce smoke but an aerosol which contains fewer and lower levels of harmful chemicals compared to cigarette smoke. In this study we assessed the in vitro toxicological profiles of two prototype HTPs' aerosols compared to the 1R6F reference cigarette using the 3D human (bronchial) MucilAir™ model. To increase consumer relevance, whole aerosol/smoke exposures were delivered repeatedly across a 28 day period (16, 32, or 48 puffs per exposure). Cytotoxicity (LDH secretion), histology (Alcian Blue/H&E; Muc5AC; FoxJ1 staining), cilia active area and beat frequency and inflammatory marker (IL-6; IL-8; MMP-1; MMP-3; MMP-9; TNFα) levels were assessed. Diluted 1R6F smoke consistently induced greater and earlier effects compared to the prototype HTP aerosols across the endpoints, and in a puff dependent manner. Although some significant changes across the endpoints were induced by exposure to the HTPs, these were substantially less pronounced and less frequently observed, with apparent adaptive responses occurring over the experimental period. Furthermore, these differences between the two product categories were observed at a greater dilution (and generally lower nicotine delivery range) for 1R6F (1R6F smoke diluted 1/14, HTP aerosols diluted 1/2, with air). Overall, the findings demonstrate the THR potential of the prototype HTPs through demonstrated substantial reductions in toxicological outcomes in in vitro 3D human lung models.

This study aimed to compare the aerosol chemistry and in vitro toxicological profiles of two prototype Heated Tobacco Product (p-HTP) variants to the 1R6F Reference Cigarette. In the neutral red uptake screen the p-HTPs were 37-39-fold less potent than 1R6F, in the micronucleus assay, responses to the p-HTPs were 8-22-fold less, and in the Ames test mutagenicity was weak or removed compared to 1R6F. The cardiovascular scratch wound assay revealed 58-fold greater wound healing impairment following exposure to 1R6F smoke extracts than the p-HTPs. Furthermore, in seven cell stress-related high content screening endpoints (cell count, cytochrome c release, mitochondrial membrane potential, GSH depletion, NFkB translocation, phosphorylation of c-jun and phosphorylation of H2AX), at 4 and 24 h, responses were substantially greater to 1R6F smoke extracts at comparable nicotine levels. The reduced in vitro effects of the p-HTPs were attributed to substantial reductions (90-97%) in selected HPHCs measured compared to in 1R6F smoke. The multiple endpoint in vitro assessment approach provides greater mechanistic insight and the first reported toxicological characterisation of these p-HTPs in the literature. Overall, the findings contribute to the growing weight of evidence that HTPs may offer a reduced harm mode of nicotine delivery to adult smokers.

Food-producing animals are infected by a number of ectoparasites that can result in production losses. Control of these parasites is largely based on the use of chemicals that are either applied topically to the skin or sometimes given parenterally. Most ectoparasiticides are neurotoxins, exerting their effect on the nervous system of the target parasite. As a result of frequent and inappropriate use, resistance of arthropods to the lethal effects of parasiticides has been reported in many parts of the world. Ectoparasiticides can also be toxic to mammals and restrictions are therefore applied to many of the compounds indicated for use in food-producing animals to ensure that unacceptable residues are not present in products intended for human consumption. Consequently many international organizations and countries have issued their own pesticide maximum residual limits in international trade.

Introduction: Tobacco harm reduction (THR) represents one of the most promising public health policies with continued product innovations crucial to making THR a reality. Tobacco-free nicotine pouches (TFNPs) are a recent THR product innovation available in a growing number of countries as a potentially less harmful alternative to traditional tobacco products. Materials and Methods: In this study, we compared the in vitro biological activity of two commercially available TFNPs, one snus product, and combustible cigarette smoke in three in vitro toxicological assays (neutral red uptake [NRU], in vitro micronucleus [IVM], and Ames test). An extraction method for TFNPs and snus in phosphate-buffered saline based on the International Organization of Standardization 10993-12 standard to assess the in vitro biological activity is also presented. Results and Discussion: Combustible cigarette smoke total particle matter (TPM) induced a statistically significant positive response in all three in vitro assays. By contrast, the TFNPs and snus extracts were negative in both the Ames and IVM assays, and demonstrated weak cytotoxicity in the NRU assay compared with TPM, under the conditions of test. When using the HepG2 cell line in the NRU assay, it was possible to differentiate between TFNP and snus extracts, with only snus extracts resulting in a measurable EC50 response. Conclusion: This initial preclinical in vitro toxicity assessment of TFNPs compared with both tobacco snus and combustible cigarette smoke indicates that the tested TFNPs have a substantially reduced in vitro toxicity activity compared with traditional tobacco products. Given this initial data, both further scientific exploration and public health discussion on these products are warranted.

Combustible cigarette smoking is an established risk factor for cardiovascular disease. By contrast, the cardiotoxicity potential of non-combustible next generation nicotine products (NGPs), which includes heated tobacco products (HTPs) and electronic vaping products (EVPs), and how this compares relative to combustible cigarettes is currently an area of scientific exploration. As such, there is a need for a rapid screening assay to assess this endpoint. The Cardio quickPredict is a metabolomics biomarker-based assay that uses human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) to screen for potential structural and functional cardiac toxicants based on the changes of four metabolites, lactic acid, arachidonic acid, thymidine, and 2′-deoxycytidine. The study aims were to investigate the cardiotoxicity potential of NGPs compared to cigarettes, in addition to nicotine. To accomplish this, hiPSC-CM were exposed to smoke or aerosol bubbled PBS samples: reference cigarette (1R6F); three variants of HTP; and three EVP variants. The 1R6F bPBS was the most active, having cardiotoxic potential at 0.3–0.6% bPBS (0.4–0.9 μg/mL nicotine), followed by HTP, which displayed cardiotoxic potential at a 10 times higher concentration, 3.3% bPBS (4.1 μg/mL nicotine). Both 1R6F and HTP bPBS (at 10-fold higher concentration than 1R6F) affected all four predictive metabolites, whereas none of the EVP bPBS samples were active in the assay up to the maximal concentration tested (10% bPBS). Nicotine tested on its own was predicted to have cardiotoxic potential at concentrations greater than 80 μg/mL, which is higher than expected physiological levels associated with combustible cigarette smoking. The application of this rapid screening assay to NGP research and the associated findings adds to the weight-of-evidence indicating that NGPs have a tobacco harm reduction potential when compared to combustible cigarettes. Additionally, this technique was shown to be sensitive and robust for the assessment of different NGPs and may be considered as part of a larger overall scientific framework for NGP assessments.