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The use of reconstituted human airway (RHuA) epithelial tissues to assess functional endpoints is highly relevant in respiratory toxicology, but standardised methods are lacking. In June 2015, the Institute for In Vitro Sciences (IIVS) held a technical workshop to evaluate the potential for standardisation of methods, including ciliary beat frequency (CBF). The applicability of a protocol suggested in the workshop was assessed in a multi-laboratory ring study. This report summarises the findings, and uses the similarities and differences identified between the laboratories to make recommendations for researchers in the absence of a validated method. Two software platforms for the assessment of CBF were used — Sisson-Ammons Video Analysis (SAVA; Ammons Engineering, Clio, MI, USA) and ciliaFA (National Institutes of Health, Bethesda, MD, USA). Both were utilised for multiple read temperatures, one objective strength (10×) and up to four video captures per tissue, to assess their utility. Two commercial RHuA tissue cultures were used: MucilAir™ (Epithelix, Geneva, Switzerland) and EpiAirway™ (MatTek, Ashland, MA, USA). IL-13 and procaterol were used to induce CBF-specific responses as positive controls. Further testing addressed the impact of tissue acclimation duration, the number of capture fields and objective strengths on baseline CBF readings. Both SAVA and ciliaFA reliably collected CBF data. However, ciliaFA failed to generate accurate CBF measurements above ∼10 Hz. The positive controls were effective, but were subject to inter-laboratory variability. CBF endpoints were generally uniform across replicate tissues, objective strengths and laboratories. Longer tissue acclimation increased the percentage active area, but had minimal impact on CBF. Taken together, these findings support the development and validation of a standardised CBF measurement protocol.

Consumer demands and innovation have led to an increasingly diverse range of nicotine delivery systems, driven by a desire to reduce risk associated with traditional combustible cigarettes. This speed of change provides a mandate for rapid new product assessment. We have used the validated technology ToxTracker®, to assess biomarkers of DNA damage, protein misfolding, oxidative and cellular stress, across the categories of cigarette (1R6F), tobacco heating product (THP 1.4) and electronic cigarette (ePen 3). In addition, we compared the commonly used test matrices for tobacco and nicotine products; whole aerosol aqueous extracts (AqE) and gas vapour phase (GVP), determining their suitability across the product categories. We demonstrated a significant reduction in oxidative stress and cytotoxicity for THP 1.4 over cigarette, further reduced for ePen 3, when assessed by both dilution and nicotine dosimetry. We also identified that while the extraction matrices AqE and GVP from combustible products were equivalent in the induced responses, this was not true of the other category examples, moreover THP 1.4 GVP demonstrates a50 % reduction in both toxicity and cytotoxicity endpoints over AqE. This indicates that unlike cigarette, the active components or toxicants for THP and electronic cigarette are associated with the aerosol fraction of these categories.

Monocytes exhibiting a pro-inflammatory phenotype play a key role in adhesion and development of atherosclerotic plaques. As an alternative to smoking, next-generation tobacco and nicotine products (NGP) are now widely used. However, little is known about their pro-inflammatory effects on monocytes. We investigated cell viability, anti-oxidant and pro-inflammatory gene and protein expression in THP-1 monocytes after exposure to aqueous smoke extracts (AqE) of a heated tobacco product (HTP), an electronic cigarette (e-cig), a conventional cigarette (3R4F) and pure nicotine (nic). Treatment with 3R4F reduced cell viability in a dose-dependent manner, whereas exposure to alternative smoking products showed no difference to control. At the highest non-lethal dose of 3R4F (20%), the following notable mRNA expression changes were observed for 3R4F, HTP, and e-cig respectively, relative to control; HMOX1 (6-fold, < 2-fold, < 2-fold), NQO1 (3.5-fold, < 2-fold, < 2-fold), CCL2 (4-fold, 3.5-fold, 2.5-fold), IL1B (4-fold, 3-fold, < 2-fold), IL8 (5-fold, 2-fold, 2-fold), TNF (2-fold, 2-fold, < 2-fold) and ICAM1 was below the 2-fold threshold for all products. With respect to protein expression, IL1B (3-fold, < 2-fold, < 2-fold) and IL8 (3.5-fold, 2-fold, 2-fold) were elevated over the 2-fold threshold, whereas CCL2, TNF, and ICAM1 were below 2-fold expression for all products. At higher doses, greater inductions were observed with all extracts; however, NGP responses were typically lower than 3R4F. In conclusion, anti-oxidative and pro-inflammatory processes were activated by all products. NGPs overall showed lower responses relative to controls than THP-1 cells exposed to 3R4F AqE.

Summary Tobacco heating products (THPs) have reduced toxicant emissions relative to cigarettes. THPs are continually evolving, but safety and efficacy studies on each new variant involve considerable resources. As employed by the pharmaceutical industry, a “bridging” process could be used to demonstrate product equivalence. Therefore, we investigated the feasibility of a bridging approach by evaluating aerosol emissions and in vitro cytotoxicity of five variant THPs in relation to a base product. All products were compared to a reference cigarette and a commercial benchmark. Relative to smoke, chemical reductions in THP aerosols were comparable among the THPs at 94–97%. The aerosols showed similar cytotoxicity in human lung tissues exposed at the air-liquid interface (p = 0.8378) but were significantly less toxic than smoke (p = 0.04). Relative to the THP benchmark, variant THPs showed lower cytotoxicity (p = 0.0141). Emissions and cytotoxicity data demonstrated that the variant THPs were comparable to the base THP, irrespective of consumable format or flavour. This dataset demonstrates the feasibility of a bridging approach and can inform an evidence-based strategy in developing sufficient data to predict similarity against an already established dataset. Therefore, avoiding repetition of vast data generation could ease authorisation requirements of newer products. Finally, we propose that more work is required to understand chemical, biological (in vitro), human consumption, and clinical data before the equivalence of these products (and others) can be definitively demonstrated. Future studies maybe needed to assess additional chemical and biological outputs and all data will need to be contextualised against human consumption data in terms of a bridging framework.

Goblet cell hyperplasia and overproduction of airway mucin are characteristic features of the lung epithelium of smokers and COPD patients. Tobacco heating products (THPs) are a potentially less risky alternative to combustible cigarettes, and through continued use solus THPs may reduce smoking-related disease risk. Using the MucilAir™ in vitro lung model, a 6-week feasibility study was conducted investigating the effect of repeated cigarette smoke (1R6F), THP aerosol and air exposure. Tissues were exposed to nicotine-matched whole aerosol doses 3 times/week. Endpoints assessed were dosimetry, tight-junction integrity, cilia beat frequency (CBF) and active area (AA), cytokine secretion and airway mucin MUC5AC expression. Comparison of incubator and air exposed controls indicated exposures did not have a significant effect on the transepithelial electrical resistance (TEER), CBF and AA of the tissues. Cytokine secretion indicated clear differences in secretion patterns in response to 1R6F and THP exposure. 1R6F exposure resulted in a significant decrease in the TEER and AA (p=0.000 and p=0.000, respectively), and an increase in MUC5AC positive cells (p=0.002). Repeated THP exposure did not result in a significant change in MUC5AC positive cells. This study demonstrates repeated cigarette smoke whole aerosol exposure can induce these morphological changes in vitro.

Graphical abstract, Highlights • Tobacco-free nicotine pouches are an emerging nicotine product category. • RTCA provides a real-time assessment of the potential cytotoxicity of these products. • Gingival fibroblasts and epithelial cells showed comparable responses. • The BAT products assessed showed little to no cytotoxicity, with increasing flavours and nicotine strengths. • RTCA could differentiate between a commercial product and a reference snus product., In many regulated industries there is an increasing pressure to provide timely and robust risk assessment data to support product launches. Real-time cell analysis (RTCA) is a tool that allows for the fast and relatively labour-free cytotoxic assessment of test compounds, compared to traditional methods. Here, we propose an application for the RTCA platform to provide a screening approach, to evaluate the cytotoxic potential of tobacco-free nicotine pouches, also termed modern oral product (MOP), to determine the contribution of differing nicotine strengths (4−11 mg) and a range of available flavour types from multiple markets, on overall product toxicity. Aqueous extracts were prepared for all products using 1 pouch in 20 mL cell culture media and applied to the cell system for 24 h. Test extract nicotine concentrations reflected the increases in product nicotine strength; however, these changes were not present in the same magnitude in the cytotoxicity data obtained from both primary human gingival fibroblasts (HGF) and an NCI-H292 human bronchial epithelial continuous cell line. Furthermore, across the range of flavours and product nicotine strengths tested, H292 cells whilst not the target organ for oral product use, accurately predicted the results seen in HGFs and could be considered a useful surrogate for fast screening studies. H292 cells are more easily cultured and for longer periods, offering a more compatible test system. In conclusion, the data demonstrate the utility of the RTCA platform for the quick assessment of a large range of product variants. Furthermore, for a cytotoxicity measure with this test product, the simple H292 cell line can predict outcomes in the more complex HGF and provide useful pre-clinical cytotoxicity screening data to inform the risk assessment of MOPs and the relative contribution of flavourings, nicotine and other components.

Graphical abstract, We have developed a novel vaping product (NVP) IS1.0(TT), which utilises a stainless-steel mesh to transfer and vaporise the e-liquid, mitigating some of the potential sources of toxicants that can be generated using the more traditional ‘wick and coil’ approach. The emissions from IS1.0(TT) have previously been found to have lower levels of toxicants overall when directly compared with a commercial wick and coil e-cig. This current study assessed the toxicological responses to aerosols from this NVP. Responses induced by IS1.0(TT)were compared to those from a 3R4F reference cigarette, using in vitro test methods which included regulatory genetic toxicological assays as well as some more contemporary screening approaches. The experimental conditions were designed to facilitate the testing of aerosol from this vaping product at doses that in most cases greatly exceeded those of the 3R4F comparator showed little to no toxicological responses and demonstrated significantly reduced effects in these in vitro assays when compared to 3R4F. Furthermore, the extreme doses tested in the present study indicate that the toxicant profile of this NVP translates to lower biological activity in vitro, and suggests that the absolute risk hazard level associated with electronic cigarettes can be reduced through continuous improvement as the technology evolves.

In vitro testing can facilitate the rapid assessment of next generation nicotine delivery products (NGPs) with comparisons to combustible tobacco products. In vitro assays for cytotoxicity and oxidative stress were employed at BAT (UK) and JT (Japan) to test total particulate matter (TPM) of a scientific reference cigarette and aerosol collected mass (ACM) of a commercially available E-cigarette and two tobacco heating products (THP). 3R4F TPMs were generated using the Health Canada intense (HCI) regimen, a modified regime (mHCI) for the THP ACMs and the CORESTA recommended method no. 81 for the E-cigarette ACM. Human lung cells were exposed to the test product TPM/ACMs at concentrations between 0–200 μg/ml followed by the employment of commercially available assays for endpoint analysis that included reactive oxygen species (ROS) generation, the glutathione ratio (GSH:GSSG), activation of the antioxidant response elements (ARE) and cellular viability. TPM/ACM nicotine concentrations were quantified using a UPLC-PDA technique. At both laboratories the 3R4F TPM induced significant and dose-dependent responses in all in vitro assays, whereas no significant responses could be measured for the NGP ACMs. In conclusion, both laboratories obtained comparable results across all endpoints therefore demonstrating the utility of the in vitro techniques combined with standardised test products to support the assessment of NGPs.

Tobacco smoking and hemodynamic forces are key stimuli for the development of endothelial dysfunction. As an alternative to smoking, next generation tobacco and nicotine products (NGP) are now widely used. However, little is known about their potential pro-inflammatory and atherogenic effects on the endothelium. In this study, we analyzed key parameters of endothelial function after exposure to aqueous smoke extracts (AqE) of a heated tobacco product (HTP), an electronic cigarette (e-cig), a conventional cigarette (3R4F) and pure nicotine. All experiments were performed under atheroprotective high laminar or atherogenic low flow with primary human endothelial cells. Treatment with 3R4F, but not alternative smoking products, reduced endothelial cell viability and wound healing capability via the PI3K/AKT/eNOS(NOS3) pathway. Laminar flow delayed detrimental effects on cell viability by 3R4F treatment. 3R4F stimulation led to activation of NRF2 antioxidant defense system at nicotine concentrations ≥0.56 μg/ml and increased expression of its target genes HMOX1 and NQO1. Treatment with HTP revealed an induction of HMOX1 and NQO1 at dosages with ≥1.68 μg/ml nicotine, whereas e-cig and nicotine exposure had no impact. Analyses of pro-inflammatory genes revealed an increased ICAM1 expression under 3R4F treatment. 3R4F reduced VCAM1 expression in a dose-dependent manner; HTP treatment had similar but milder effects; e-cig and nicotine treatment had no impact. SELE expression was induced by 3R4F under static conditions. High laminar flow prevented this upregulation. Stimulation with laminar flow led to downregulation of CCL2 (MCP-1). From this downregulated level, only 3R4F increased CCL2 expression at higher concentrations. Finally, under static conditions, all components increased adhesion of monocytes to endothelial cells. Interestingly, only stimulation with 3R4F revealed increased monocyte adhesion under atherosclerosis-prone low flow. In conclusion, all product categories activated anti-oxidative or pro-inflammatory patterns. NGP responses were typically lower than in 3R4F exposed cells. Also, 3R4F stimulation led to an impaired endothelial wound healing and induced a pro-inflammatory phenotype compared to NGP treatment., Graphical abstract Image 1, Highlights • 3R4F, but not NGP and nicotine, impair endothelial cell viability and wound healing. • All tested product categories induce anti-oxidative enzymes and adhesion molecules in HUVEC. • Only 3R4F increases monocyte adhesion to endothelial cells under low laminar flow. • Protective effects of high laminar flow counteract with deleterious effects of smoking.

No cigarette smoke test matrix is without limitation, due to the complexity of the starting aerosol and phase to phase dynamics. It is impossible to capture all chemicals at the same level of efficiency, therefore, any test matrix will inadvertently or by design fractionate the test aerosol. This case study examines how four different test matrices derived from cigarette smoke can be directly compared. The test matrices assessed were as follows, total particulate matter (TPM), gas vapour phase (GVP), a combination of TPM + GVP and whole aerosol (WA). Here we use an example assay, the mouse lymphoma assay (MLA) to demonstrate that data generated across four cigarette smoke test matrices can be compared. The results show that all test matrices were able to induce positive mutational events, but with clear differences in the biological activity (both potency and toxicity) between them. TPM was deemed the most potent test article and by extension, the particulate phase is interpreted as the main driver of genotoxic induced responses in the MLA. However, the results highlight that the vapour phase is also active. MLA appeared responsive to WA, with potentially lower potency, compared to TPM approaches. However, this observation is caveated in that the WA approaches used for comparison were made on a newly developed experimental method using dose calculations. The TPM + GVP matrix had comparable activity to TPM alone, but interestingly induced a greater number of mutational events at comparable relative total growth (RTG) and TPM-equivalent doses when compared to other test matrices. In conclusion, this case study highlights the importance of understanding test matrices in response to the biological assay being assessed and we note that not all test matrices are equal.

Atherosclerosis is a complex process involving progressive pathological events, including monocyte adhesion to the luminal endothelial surface. We have developed a functional in vitro adhesion assay using BioFlux microfluidic technology to investigate THP-1 (human acute monocytic leukaemia cell) monocyte adhesion to human aortic endothelial cells (HAECs). The effect of whole smoke conditioned media (WSCM) generated from University of Kentucky reference cigarette 3R4F, electronic cigarette vapour conditioned media (eVCM) from an electronic nicotine delivery system (ENDS) product (Vype ePen) and nicotine on monocyte adhesion to HAECs was evaluated. Endothelial monolayers were grown in microfluidic channels and exposed to 0–1500 ng/mL nicotine or nicotine equivalence of WSCM or eVCM for 24 h. Activated THP-1 cells were perfused through the channels and a perfusion, adhesion period and wash cycle performed four times with increasing adhesion period lengths (10, 20, 30 and 40 min). THP-1 cell adhesion was quantified by counting adherent cells. WSCM induced dose-dependent increases in monocyte adhesion compared to vehicle control. No such increases were observed for eVCM or nicotine. Adhesion regulation was linked to increased ICAM-1 protein expression. Staining of ICAM-1 in HAECs and CD11b (MAC-1) in THP-1 cells demonstrated adhesion molecule co-localisation in BioFlux plates. The ICAM-1 adhesion response to WSCM was downregulated by transfecting HAECs with ICAM-1 siRNA. We conclude that the BioFlux system is able to model human monocyte adhesion to primary human endothelial cells in vitro and WSCM drives the greatest increase in monocyte adhesion via a mechanism involving endothelial ICAM-1 expression.

Electronic cigarettes (e-cigarettes) and tobacco heating products (THPs) have reduced yields of toxicants and have recently emerged as a potentially safer alternative to combustible cigarettes. To understand if reduced toxicant exposure is associated with reductions in biological responses, there is a need for high-quality pre-clinical in vitro studies. Here, we investigated the cytotoxic response of human umbilical vein endothelial cells to conventional cigarette aqueous aerosol extracts (AqE) and highly concentrated AqEs from e-cigarettes (two generations of atomisers) and THPs (two variants). All AqE samples were generated by a standardized methodology and characterized for nicotine, propylene glycol and vegetable glycerol. The cigarette AqE caused a maximum 100 ± 0.00 % reduction in cell viability at 35 % dose (2.80 puffs) as opposed to 96.63 ± 2.73 % at 50 % (20 puffs) and 99.85 ± 0.23 % at 75 % (30 puffs) for the two THP variants (glo Bright Tobacco, glo Rich Tobacco), and 99.07 ± 1.61 % at the neat ePen2.0 e-cigarette (200 puffs). The AqE of the remaining e-cigarettes either resulted in an incomplete dose-response or did not elicit any response. The methods utilized were suitably sensitive to not only differentiate between cigarette, THP and e-cigarette aerosols but also to distinguish between products within each product category.

Endothelial cell migration is a critical process in the maintenance of healthy blood vessels. Impaired endothelial migration is reportedly associated with the development of cardiovascular diseases. Here, we report on the development of a 96-well in vitro endothelial migration assay for the purpose of comparative toxicological assessment of a novel THP relative to cigarette smoke, to be able to rapidly inform regulatory decision making. Uniform scratches were induced in confluent human umbilical vein endothelial cells using the 96-pin wound maker and exposed to 3R4F cigarette or THP aqueous extracts (AqE). Endothelial migration was recorded over 24 h, and the rate of wound closure calculated using mean relative wound density rather than migration rate as previously reported. This self-normalising parameter accounts for starting wound size, by comparing the density of the scratch to the outer region at each time-point. Furthermore, wound width acceptance criteria was defined to further increase the sensitivity of the assay. 3R4F and THP AqE samples were tested at comparable nicotine concentrations. 3R4F showed significant cytotoxicity and inhibition of wound healing whereas THP AqE did not show any response in either endpoint. This 96-well endothelial migration assay was suitably sensitive to distinguish combustible cigarette and THP test articles.

Tobacco-free ‘modern’ oral nicotine pouches (MOPs), are similar in appearance and use to Swedish-style snus, but without tobacco. There are few identified methods to create test samples for toxicologically assessment of MOPs in vitro. In this study we present a simple method for the extraction of pouch material in cell culture media, providing consistent nicotine concentration and easy in vitro assessment. A series of contemporary in vitro screening assays (viability, cell health markers, oxidative stress and genotoxicity) using human oral fibroblasts (HGF) and human lung epithelial cells (H292) were employed. Extracts were generated from LYFT and compared to snus (CRP1.1) and cigarette (1R6F) reference products. MOP and CRP1.1 extracts were generated by incubating one pouch in 20 ml of cell culture media, while 1R6F AqE was prepared by smoking 1 cigarette into 20 ml of cell culture media. 1R6F demonstrated toxicological responses in most assays; CRP1.1 had minimal to moderate effects while MOP demonstrated little or no response in all assays. This study demonstrated the generation of MOPs extracts and their toxicological evaluation using in vitro screening approaches. Future product usage, pharmacokinetics and clinical studies will further substantiate the reduced risk potential of MOPs.

In vitro studies have been widely used to support the toxicological evaluation of chemicals and complex mixtures including cigarette smoke. In this study, the total particulate matter and whole aerosol from a Kentucky reference 3R4F cigarette and two commercially available tobacco heating products (THPs) were assessed using in vitro mutagenicity, cytotoxicity and tumour-promoting activity assays. The Ames assay assessed mutagenicity using Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and TA102 ± metabolic activation (S9). The mouse lymphoma assay was used with short 3 h and longer 24 h exposures. The Bhas 42 cell transformation assay was incorporated as an in vitro alternative for detecting tumour promoters, and the neutral red uptake cell viability assay provided an acute measure of cytotoxicity. To complement the approach, the Ames assay was also employed with S. typhimurium tester strains TA98, TA100, TA1535, TA97 and TA102 using a scaled down methodology for the assessment of aerosols. All the in vitro techniques employed produced a clear positive response with cigarette smoke and in contrast, a negative response to THPs at doses equivalent to or higher than a cigarette smoke test matrix. The data show little difference between the THPs assessed suggesting parity between products.

Tobacco heating products (THPs) represent a subset of the next-generation nicotine and tobacco product category, in which tobacco is typically heated at temperatures of 250-350 °C, thereby avoiding many of the harmful combustion-related toxicant emissions of conventional cigarettes. In this study, we have assessed aerosol generation and cytotoxicity from two commercially available THPs, THP1.0 and THS, relative to tobacco smoke from 3R4F reference cigarettes, using an adapted Borgwaldt RM20S Smoking Machine. Quantification of nicotine in the exposed cell-culture media showed greater delivery of nicotine from both THPs than from the cigarette. Using Neutral Red Uptake assay, THPs demonstrated reduced in vitro cytotoxicity in H292 human bronchial epithelial cells as compared with 3R4F cigarette exposure at the air-liquid interface (p 0.0001). Both THPs demonstrated a statistically similar reduction in biological response, with87% viability relative to 3R4F at a common aerosol dilution (1:40, aerosol:air). A similar response was observed when plotted against nicotine; a statistical difference between 3R4F and THPs (p 0.0001) and no difference between the THPs (p = 0.0186). This pre-clinical in vitro biological testing forms part of a larger package of data to help assess the safety and risk reduction potential of next-generation tobacco products relative to cigarettes, using a weight of evidence approach.

In vitro cell transformation assays (CTA) are used to assess the carcinogenic potential of chemicals and complex mixtures and can detect nongenotoxic as well as genotoxic carcinogens. The Bhas 42 CTA has been developed with both initiation and promotion protocols to distinguish between these two carcinogen classes. Cigarette smoke is known to be carcinogenic and is positive in in vitro genotoxicity assays. Cigarette smoke also contains nongenotoxic carcinogens and is a tumour promoter and cocarcinogen in vivo. We have combined a suite of in vitro assays to compare the relative biological effects of new categories of tobacco and nicotine products with traditional cigarettes. The Bhas promotion assay has been included in this test battery to provide an in vitro surrogate for detecting tumor promoters. The activity of an electronic cigarette (e-cigarette; Vype ePen) was compared to that of a reference cigarette (3R4F) in the promotion assay, using total particulate matter (TPM)/aerosol collected matter (ACM) and aqueous extracts (AqE) of product aerosol emissions. 3R4F TPM was positive in this assay at concentrations ≥6 µg/mL, while e-cigarette ACM did not have any promoter activity. AqE was found to be a lesssuitable test matrix in this assay due to high cytotoxicity. This is the first study to use the Bhas assay to compare tobacco and nicotine products and demonstrates the potential for its future application as part of a product assessment framework. These data add to growing evidence suggesting that e-cigarettes may provide a safer alternative to traditional cigarettes. Environ. Mol. Mutagen. 58:190-198, 2017. © 2017 Wiley Periodicals, Inc.

Tobacco smoking is a risk factor for various diseases. The underlying cellular mechanisms are not fully characterized, but include oxidative stress, apoptosis, and necrosis. Electronic-cigarettes (e-cigarettes) have emerged as an alternative to and a possible means to reduce harm from tobacco smoking. E-cigarette vapor contains significantly lower levels of toxicants than cigarette smoke, but standardized methods to assess cellular responses to exposure are not well established. We investigated whether an in vitro model of the airway epithelium (human bronchial epithelial cells) and commercially available assays could differentiate cellular stress responses to aqueous aerosol extracts (AqE) generated from cigarette smoke and e-cigarette aerosols. After exposure to AqE concentrations of 0.063-0.500 puffs/mL, we measured the intracellular glutathione ratio (GSH:GSSG), intracellular generation of oxidant species, and activation of the nuclear factor erythroid-related factor 2 (Nrf2)-controlled antioxidant response elements (ARE) to characterize oxidative stress. Apoptotic and necrotic responses were characterized by increases in caspase 3/7 activity and reductions in viable cell protease activities. Concentration-dependent responses indicative of oxidative stress were obtained for all endpoints following exposure to cigarette smoke AqE: intracellular generation of oxidant species increased by up to 83%, GSH:GSSG reduced by 98.6% and transcriptional activation of ARE increased by up to 335%. Caspase 3/7 activity was increased by up to 37% and the viable cell population declined by up to 76%. No cellular stress responses were detected following exposure to e-cigarette AqE. The methods used were suitably sensitive to be employed for comparative studies of tobacco and nicotine products.

Conduct of the mouse lymphoma assay (MLA) is underpinned by Organisation for Economic Co-operation and Development (OECD) Test Guideline 490 and International Conference on Harmonisation S2(R1) guidance and is a recognised in vitro genotoxicity test battery assay. It has been used on a limited number of occasions for the assessment of some tobacco and nicotine products, such as e-cigarettes and tobacco heating products (THP). The aim of this study was to assess the suitability of the MLA for genotoxicity testing with a variety of tobacco and nicotine products. Total particulate matter (TPM) from a 3R4F cigarette was compared against a commercial electronic cigarette liquid (e-liquid), electronic cigarette (e-cigarette) aerosol matter captured from the same e-liquid, and TPM from a commercial THP. Treatment conditions included 3 h exposures with and without metabolic activation and a longer 24 h exposure without metabolic activation (-S9) at concentrations up to 500 μg/mL. Under all treatment conditions, 3R4F produced a clear positive response with regard to induction of mutation. In contrast, no marked induction of mutation was observed for the e-liquid, e-cigarette aerosol or THP. Additionally, data are presented as a function of nicotine equivalents for comparisons between these different tobacco products and test matrices.

In this study, a variety of test matrices from tobacco and nicotine delivery products were assessed against a 3R4F Kentucky reference cigarette using the in vitro micronucleus assay. Testing was conducted using two Chinese hamster cell lines (CHO and V79), and a human lymphoblastoid cell line (TK6), in accordance with established guidelines. Total particulate matter (TPM) from a 3R4F Reference cigarette was compared to an electronic cigarette e-liquid, electronic cigarette TPM and TPM from a commercial tobacco heating product using a standard and an extended treatment condition with recovery period. Cells were assessed with 3R4F TPM prior to assessment of the other tobacco and nicotine product test matrices. These cell lines gave varied responses to 3R4F TPM with the most robust response using V79 cells. The use of an extended exposure/recovery period was seen to increase assay sensitivity for CHO and V79 cell lines but was less clear for TK6 cells. Negative responses were observed for all products except 3R4F across all treatment conditions in V79 cells. The most potent response to cigarette smoke was following extended treatment with recovery, suggesting this may be a more appropriate treatment for the future assessment of tobacco and nicotine product test matrices.

Cigarette smoking is a major risk factor for many adverse health conditions. Novel tobacco heating products (THPs) heat tobacco, reducing exposure to many of the harmful combustion toxicants in conventional cigarette emissions. In vitro studies have been employed to support the toxicological evaluation of chemicals and complex mixtures, including cigarette smoke. The use of automated robotics platforms for in vitro toxicological screening complements traditional testing approaches. Multiparametric toxicity and oxidative stress endpoints were used to assess in vitro biological responses elicited after exposure to total particulate matter (TPM) from two commercially available THPs, and the reference tobacco product 3R4F, in human bronchial epithelial cells. A luciferase-based reporter gene assay was used to assess antioxidant response element (ARE) transcriptional activation in stably transfected H292 cells after 6 and 24 h exposures. High-content screening was used to assess 10 endpoints normal human bronchial epithelial cells after 4 or 24 h exposures. 3R4F TPM stimulated significant increases in ARE activation (p 0.005) and moderate activity in HCS cell-based assays compared to THP at comparable doses. THPs showed little or no activity in all assays. HCS techniques can extend safety assessments providing information quickly in the early stages of product innovation and development.

Tobacco smoke is a complex mixture of over 6,000 individual chemical constituents. Approximately 150 of these have been identified as ‘tobacco smoke toxicants’ due to their known toxicological effects. A number of these toxicants are present in the gaseous phase of tobacco smoke. This presents a technical challenge when assessing the toxicological effects of these chemicals in vitro. We have adapted a commercially available tobacco smoke exposure system to enable the assessment of the contribution of individual smoke toxicants to the overall toxicological effects of whole mainstream cigarette smoke (WS). Here we present a description of the exposure system and the methodology used. We use the example of a gaseous tobacco smoke toxicant, ethylene oxide (EtO), a Group 1 IARC carcinogen and known mutagen, to illustrate how this methodology can be applied to the assessment of genotoxicity of gaseous chemicals in the context of WS. In the present study we found that EtO was positive in Salmonella typhimurium strain YG1042, a strain that is sensitive to tobacco smoke. However, EtO did not increase the mutagenicity of the WS mixture when it was added at greatly higher concentrations than those found typically in WS. The findings presented here demonstrate the suitability of this exposure system for the assessment of the mutagenic potential of gases in vitro. Whilst we have focused on tobacco smoke toxicants, this system has broad application potential in studying the biological effects of exposure to a wide range of gaseous compounds that are present within complex aerosol mixtures. Environ. Mol. Mutagen. 55:662–672, 2014. © 2014 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc., on behalf of Environmental Mutagen Society

Cigarette smoking causes many human diseases including cardiovascular disease, lung disease and cancer. Novel tobacco products with reduced yields of toxicants compared to cigarettes, such as tobacco-heating products, snus and electronic cigarettes, hold great potential for reducing the harms associated with tobacco use. In the UK several public health agencies have advocated a potential role for novel products in tobacco harm reduction. Public Health England has stated that "The current best estimate is that e-cigarettes are around 95% less harmful than smoking" and the Royal College of Physicians has urged public health to "Promote e-cigarettes widely as substitute for smoking". Health related claims on novel products such as 'reduced exposure' and 'reduced risk' should be substantiated using a weight of evidence approach based on a comprehensive scientific assessment. The US FDA, has provided draft guidance outlining a framework to assess novel products as Modified Risk Tobacco Products (MRTP). Based on this, we now propose a framework comprising pre-clinical, clinical, and population studies to assess the risk profile of novel tobacco products. Additionally, the utility of this framework is assessed through the pre-clinical and part of the clinical comparison of a commercial e-cigarette (Vype ePen) with a scientific reference cigarette (3R4F) and the results of these studies suggest that ePen has the potential to be a reduced risk product.

Cigarette smoking is a risk factor for several diseases. There has been a steep increase in the use of e-cigarettes that may offer a safer alternative to cigarette smoking. In vitro models of smoking-related diseases may provide valuable insights into disease mechanisms associated with tobacco use and could be used to assess e-cigarettes. We previously reported the application of a 'scratch wound' assay, measuring endothelial cell migration rate following artificial wounding, in the presence or absence of cigarette smoke extracts. This study reports the comparative effects of two commercial e-cigarette products (Vype ePen and Vype eStick) and a scientific reference cigarette (3R4F) on endothelial migration in vitro. Puff-matched extracts were generated using the Health Canada Intense (HCI) regime for cigarettes and a modified HCI for e-cigarettes. Exposure to 3R4F extract (20h) induced concentration-dependent inhibition of endothelial cell migration, with complete inhibition at concentrations >20%. E-cigarette extracts did not inhibit migration, even at double the 3R4F extract nicotine concentration, allowing cells to migrate into the wounded area. Our data demonstrate that e-cigarettes do not induce the inhibition of endothelial cell migration in vitro when compared to 3R4F. The scratch wound assay enables the comparative assessment between tobacco and nicotine products in vitro.

In vitro cell transformation assays (CTA) are used to assess the carcinogenic potential of chemicals and complex mixtures and can detect nongenotoxic as well as genotoxic carcinogens. The Bhas 42 CTA has been developed with both initiation and promotion protocols to distinguish between these two carcinogen classes. Cigarette smoke is known to be carcinogenic and is positive in in vitro genotoxicity assays. Cigarette smoke also contains nongenotoxic carcinogens and is a tumour promoter and cocarcinogen in vivo. We have combined a suite of in vitro assays to compare the relative biological effects of new categories of tobacco and nicotine products with traditional cigarettes. The Bhas promotion assay has been included in this test battery to provide an in vitro surrogate for detecting tumor promoters. The activity of an electronic cigarette (e‐cigarette; Vype ePen) was compared to that of a reference cigarette (3R4F) in the promotion assay, using total particulate matter (TPM)/aerosol collected matter (ACM) and aqueous extracts (AqE) of product aerosol emissions. 3R4F TPM was positive in this assay at concentrations ≥6 µg/mL, while e‐cigarette ACM did not have any promoter activity. AqE was found to be a lesssuitable test matrix in this assay due to high cytotoxicity. This is the first study to use the Bhas assay to compare tobacco and nicotine products and demonstrates the potential for its future application as part of a product assessment framework. These data add to growing evidence suggesting that e‐cigarettes may provide a safer alternative to traditional cigarettes. Environ. Mol. Mutagen. 58:190–198, 2017. © 2017 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.

This study assessed the toxicological and biological responses of aerosols from a novel hybrid tobacco product. Toxicological responses from the hybrid tobacco product were compared to those from a commercially available Tobacco Heating Product (c-THP), a prototype THP (p-THP) and a 3R4F reference cigarette, using in vitro test methods which were outlined as part of a framework to substantiate the risk reduction potential of novel tobacco and nicotine products. Exposure matrices used included total particulate matter (TPM), whole aerosol (WA), and aqueous aerosol extracts (AqE) obtained after machine-puffing the test products under the Health Canada Intense smoking regime. Levels of carbonyls and nicotine in these matrices were measured to understand the aerosol dosimetry of the products. The hybrid tobacco product tested negative across the in vitro assays including mutagenicity, genotoxicity, cytotoxicity, tumour promotion, oxidative stress and endothelial dysfunction. All the THPs tested demonstrated significantly reduced responses in these in vitro assays when compared to 3R4F. The findings suggest these products have the potential for reduced health risks. Further pre-clinical and clinical assessments are required to substantiate the risk reduction of these novel products at individual and population levels.

The mechanism(s) by which cigarette smoke contributes to lung diseases, such as cancer, remains unclear. Recent developments in our knowledge of cell signalling events suggest that cigarette smoke causes oxidative stress and proinflammatory responses in cells of the lung. Cigarette smoke is a complex mixture of over 4000 compounds and high levels of oxidants and reactive oxygen species (ROS) have been detected in both mainstream and sidestream smoke. Oxidative stress that ensues, when the antioxidant defences are depleted, is accompanied by increases in ROS production in lung epithelial cells. Cigarette smoke-mediated oxidative stress produces DNA damage and activates survival signalling cascades resulting in uncontrolled cell proliferation and transformation. Intervention studies using antioxidants have provided compelling evidence that oxidative stress plays a critical role in the aetiology of smoking-related disorders.

Carcinogenesis is a highly complex, multi-stage process that can occur over a relatively long period before its clinical manifestation. While the sequence in which a cancer cell acquires the necessary traits for tumour formation can vary, there are a number of mechanisms that are common to most, if not all, cancers across the spectrum of possible causes. Many aspects of carcinogenesis can be modelled in vitro. This has led to the development of a number of mechanistically driven, cell-based assays to assess the pro-carcinogenic and anti-carcinogenic potential of chemicals. A review is presented of the current in vitro models that can be used to study carcinogenesis, with examples of cigarette smoke testing in some of these models, in order to illustrate their potential applications. We present an overview of the assays used in regulatory genotoxicity testing, as well as those designed to model other aspects that are considered to be hallmarks of cancer. The latter assays are described with a view to demonstrating the recent advances in these areas, to a point where they should now be considered for inclusion in an overall testing strategy for chemical carcinogens.

The aim of research in the FRAME Alternatives Laboratory at the University of Nottingham Medical School is summarised, i.e. to use human cell culture-based projects and in vivo studies in human volunteers as alternatives to the use of rodent models in the study of human disease. This is especially important when the available animal models do not adequately represent the pathophysiological situation in humans. The approach is exemplified by summaries of studies on the effects of starvation on skeletal muscle in human volunteers, and on lipid metabolism in obese female volunteers.

The induction of transformation in Syrian hamster embryo (SHE) cells is a multifactorial process, in comparison to endpoints induced in in vitro genotoxicity assays such as Ames, mouse lymphoma and cytogenetics [Y. Berwald, L. Sachs, In vitro cell transformation with chemical carcinogens, Nature (London) 200 (1963) 1182-1184]. Furthermore, a number of non-genotoxic carcinogens and promoters such as clofibrate and diethylhexylphthalate, have been positively identified in this assay, while giving false negative results in traditional genotoxicity assays [H. Yamasaki, J. Ashby, M. Bignami, W. Jongen, K. Linnainmaa, R.F. Newbold, G. Nguyen-Ba, S. Parodi, E. Rivedal, D. Schiffmann, J.W.I.M. Simons, P. Vasseur, Nongenotoxic carcinogens: development of detection methods based on mechanisms: a European project, Mutat. Res. 353 (1996) 47-63]. A high concordance between results obtained in this assay when compared with rodent carcinogenesis bioassays has also been noted [R.J. Isfort, G.A. Kerckaert, R.A. LeBoeuf, Comparison of the standard and reduced pH Syrian hamster embryo (SHE) in vitro cell transformation assays to predict the carcinogenic potential of chemicals, Mutat. Res. 356 (1996) 11-63]. Carcinogenesis is known to be a multistage process, with agents potentially acting at each stage. Specifically, mouse skin painting experiments established that tumour induction could be mechanistically divided into two distinct phases, termed initiation and promotion. Initiation, is defined as the stage at which a normal cell is converted to a latent tumour cell, followed by promotion where the latent tumour cell progresses to a tumour [W.F. Friedwald, P. Rous, The initiating and promoting elements in tumour production: analysis of the effects of tar, benzpyrene and methylcholanthrene on rabbit skin, J. Exp. Med. 80 (1944) 101-125]. A protocol for the pH 6.7 SHE transformation assay has been developed which allows separation of cell transformation process into two phases, potentially analogous to initiation and promotion in vivo. This allows chemicals found to be positive in the traditional SHE cell transformation assay to be further classified as initiators or promoters. Following validation with known initiators, benzo(a)pyrene and N-methyl-N'-nitro-N-nitrosoguanidine and promoters, 12-O-tetradecanoyl-phorbol-13-acetate and phenobarbitone, the two-stage model was applied to cigarette smoke particulates which was found to act both at the initiation and promotion stage of cell transformation.

The conventional method for assessing the safety of products, ranging from pharmaceuticals to agrochemicals, biocides and industrial and household chemicals — including cosmetics — involves determining their toxicological properties by using experimental animals. The aim is to identify any possible adverse effects in humans by using these animal models. Providing safe products is undoubtedly of the utmost importance but, over the last decade or so, this aim has come into conflict with strong public opinion, especially in Europe, against animal testing. Industry, academia and the regulators have worked in partnership to find other ways of evaluating the safety of products, by non-animal testing, or at least by reducing the numbers of animals required and the severity of the tests in which they are used. There is a long way to go before products can be evaluated without any animal studies, and it may be that this laudable aim is an impossible dream. Nevertheless, considerable progress has been made by using a combination of in vitro tests and the prediction of properties based on chemical structure. The aim of this review is to describe these important and worthwhile developments in various areas of toxicological testing, with a focus on the European regulatory framework for general industrial and household chemicals.

The epithelial-mesenchymal transition (EMT) program was first identified from studies on embryogenesis and organ development, but has since been found to play vital roles in tissue remodelling and tumorigenesis. EMT is a biological process during which epithelial cells change their morphology to adopt fibroblast-like characteristics. During EMT, cell adhesion is lost while cells become more motile. A number of distinct molecular events are required for a cell to undergo EMT including transcription factor activation, cytoskeletal rearrangement, changes in cell adhesion molecule expression and increased production of extracellular matrix-degrading enzymes. EMT has recently been linked to chronic inflammation and cigarette smoke exposure in the lung and is believed to be involved in lung carcinogenesis. As the specific genes and proteins involved in EMT have been well defined, they can serve as markers to demonstrate the passage of cells through EMT in the context of cigarette smoking. The purpose of this study was to develop an in vitro model of EMT using immortalised human bronchial epithelial (BEAS-2B) cells. EMT was induced in BEAS-2B cells using a 72 hour treatment with 5 ng/mL TGF-β1. Expression of the following EMT markers were assessed at the transcript level using reverse transcription real time-polymerase chain reaction (RT-PCR): alpha smooth muscle actin (α-SMA), collagen 1, fibronectin, vimentin, e-cadherin, slug and snail. Protein levels of α-SMA, fibronectin, β-catenin, e-cadherin and zonular occludin-1 were evaluated using immunocytochemistry techniques. This model was then used to determine the effects of acute (72 hour) cigarette smoke particulate matter treatment on EMT in BEAS-2B cells. Cells were treated with particulate matter alone, and in combination with TGF-β1. Particulate matter did not induce EMT directly, nor did it influence EMT induced by TGF-β1 under the treatment conditions used in this study, based on marker expression. It is possible that a more prolonged exposure to particulate matter is required for EMT induction in BEAS-2B cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2423. doi:1538-7445.AM2012-2423