Key Challenges and Recommendations for In Vitro Testing of Tobacco Products for Regulatory Applications: Consideration of Test Materials and Exposure Parameters.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and 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 nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24-26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol. These three types of samples can be tested individually, or the PCM and GVP can be combined. Whole smoke/aerosol can be bubbled through media or applied directly to cells at the air-liquid interface. Summaries of the speaker presentations and the recommendations developed by the workgroup are presented. Following discussion, the workshop concluded the following: that there needs to be greater standardisation in aerosol generation and collection processes; that methods for testing the NGPs need to be developed and/or optimised, since simply mirroring cigarette smoke testing approaches may be insufficient; that understanding and quantitating the applied dose is fundamental to the interpretation of data and conclusions from each study; and that whole smoke/aerosol approaches must be contextualised with regard to key information, including appropriate experimental controls, environmental conditioning, analytical monitoring, verification and performance criteria.

Bridging: Accelerating Regulatory Acceptance of Reduced-Risk Tobacco and Nicotine Products.

INTRODUCTION: The number and variety of alternative tobacco and nicotine products that can potentially provide reduced-risk choices for cigarette smokers who switch completely to such products instead of continued smoking have grown substantially in the past decade. Innovation and choice are likely to improve the prospects of smokers making the switch, but this provides challenges to regulators and manufacturers to ensure that changes to regulations and products promote and do not hinder contributions to tobacco harm reduction. AIMS AND METHODS: This paper looks at where bridging data sets for tobacco heating products, closed system vaping products, and oral nicotine products might enable innovation while protecting the interests of consumers. RESULTS: We review product data from chemical studies and a toxicological study showing how bridging can be applied and consider what product development changes might allow bridging from existing datasets or trigger the need for new ones. CONCLUSIONS: Bridging across specific product ranges can increase the speed of innovation, foster competition, and limit the burden of assessment for regulators while maintaining product safety and quality. IMPLICATIONS: Bridging partial data sets is an established practice within other industries, that aims to improve efficiency with regulatory approvals, accepts natural product variation, and supports product innovation. We review product data from chemical studies and a toxicological study showing how bridging can be applied and consider what product development changes might allow bridging from existing datasets or trigger the need for new ones. This in turn can increase the speed of innovation, foster competition, and limit the burden of assessment for regulators while maintaining product safety and quality.

Assessment of novel tobacco heating product THP1.0. Part 7: Comparative in vitro toxicological evaluation.

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.

Assessment of novel tobacco heating product THP1.0. Part 6: A comparative in vitro study using contemporary screening approaches.

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.

Assessment of tobacco heating product THP1.0. Part 5: In vitro dosimetric and cytotoxic assessment.

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, with >87% 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.

Assessment of enamel discoloration in vitro following exposure to cigarette smoke and emissions from novel vapor and tobacco heating products.

PURPOSE: To evaluate in vitro enamel sample discoloration following exposure to a scientific reference cigarette (3R4F) or emissions from next generation tobacco and nicotine products (NGPs) such as electronic cigarettes (EC) and tobacco heating products (THP). METHODS: Bovine enamel blocks (6.5 × 6.5 mm) were prepared and pre-incubated with human or artificial saliva, to form a pellicle layer before exposure to either particulate matter (PM) or whole aerosols. PM was prepared by capturing 3R4F cigarette smoke (CS), a commercial THP (THP1.0) or a novel vapor product (NVP)/next generation e-cigarette aerosols on Cambridge filter pads followed by elution with dimethyl sulfoxide (DMSO). Ten enamel samples were exposed to each PM for 14 days. For aerosol exposure, 12 enamel samples were exposed (200 puffs per day, for 5 consecutive days) to 3R4F CS or THP1.0 and NVP aerosols. Control samples were incubated with DMSO (PM study) or phosphate buffered saline (PBS, aerosol study). Individual enamel sample color readings (L*, a*, b*) were measured at baseline and on each exposure day. Mean ΔL*, Δa*, Δb* and ΔE values were calculated for each product or control. A one-way ANOVA was used to assess the differences between the products and controls. The Tukey procedure for pairwise comparisons was also used. RESULTS: At all timepoints, 3R4F PM and CS induced enamel discoloration that was statistically significant (< 0.0001) when compared to THP1.0 or NVP. After 14-day PM exposure, mean ΔE values were 29.4± 3.6, 10.5 ± 2.3, 10.7 ± 2.6 and 12.6 ± 2.0 for 3R4F, THP1.0, NVP and DMSO control respectively. After 5-day CS or aerosol exposure, mean ΔE values were 26.2 ± 3.2, 3.6 ± 1.9, 3.4 ± 1.3, 5.3 ± 0.8 for 3R4F CS, THP1.0, NVP or PBS control, respectively. Both exposure methods demonstrated that THP1.0 and NVP induced minimal staining, mean ΔL* , Δa* , Δb* and ΔE values were comparable to DMSO or PBS controls. CLINICAL SIGNIFICANCE: For the first time, diverse NGPs across the risk continuum were assessed in vitro for their impact on enamel staining. CS exposure significantly increased the level of bovine enamel sample discoloration, whereas THP1.0 or NVP exposure resulted in values comparable to the controls.

Assessing modified risk tobacco and nicotine products: Description of the scientific framework and assessment of a closed modular electronic cigarette.

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.

Assessment of in vitro COPD models for tobacco regulatory science: Workshop proceedings, conclusions and paths forward for in vitro model use.

The Family Smoking Prevention and Tobacco Control Act of 2009 established the Food and Drug Administration Center for Tobacco Products (FDA-CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 8-10 December 2014, IIVS organised a workshop conference, entitled Assessment of In Vitro COPD Models for Tobacco Regulatory Science, to bring together stakeholders representing regulatory agencies, academia, industry and animal protection, to address the research priorities articulated by the FDA-CTP. Specific topics were covered to assess the status of current in vitro technologies as they are applied to understanding the adverse pulmonary events resulting from tobacco product exposure, and in particular, the progression of chronic obstructive pulmonary disease (COPD). The four topics covered were: a) Inflammation and Oxidative Stress; b) Ciliary Dysfunction and Ion Transport; c) Goblet Cell Hyperplasia and Mucus Production; and d) Parenchymal/Bronchial Tissue Destruction and Remodelling. The 2.5 day workshop included 18 expert speakers, plus poster sessions, networking and breakout sessions, which identified key findings and provided recommendations to advance the in vitro technologies and assays used to evaluate tobacco-induced disease etiologies. The workshop summary was reported at the 2015 Society of Toxicology Annual Meeting, and the recommendations led to an IIVS-organised technical workshop in June 2015, entitled Goblet Cell Hyperplasia, Mucus Production, and Ciliary Beating Assays, to assess these assays and to conduct a proof-of-principle multi-laboratory exercise to determine their suitability for standardisation. Here, we report on the proceedings, recommendations and outcomes of the December 2014 workshop, including paths forward to continue the development of non-animal methods to evaluate tissue responses that model the disease processes that may lead to COPD, a major cause of mortality worldwide.

E-cigarette aerosols induce lower oxidative stress in vitro when compared to tobacco smoke.

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.

Electronic cigarette aerosol induces significantly less cytotoxicity than tobacco smoke.

Electronic cigarettes (E-cigarettes) are a potential means of addressing the harm to public health caused by tobacco smoking by offering smokers a less harmful means of receiving nicotine. As e-cigarettes are a relatively new phenomenon, there are limited scientific data on the longer-term health effects of their use. This study describes a robust in vitro method for assessing the cytotoxic response of e-cigarette aerosols that can be effectively compared with conventional cigarette smoke. This was measured using the regulatory accepted Neutral Red Uptake assay modified for air-liquid interface (ALI) exposures. An exposure system, comprising a smoking machine, traditionally used for in vitro tobacco smoke exposure assessments, was adapted for use with e-cigarettes to expose human lung epithelial cells at the ALI. Dosimetric analysis methods using real-time quartz crystal microbalances for mass, and post-exposure chemical analysis for nicotine, were employed to detect/distinguish aerosol dilutions from a reference Kentucky 3R4F cigarette and two commercially available e-cigarettes (Vype eStick and ePen). ePen aerosol induced 97%, 94% and 70% less cytotoxicity than 3R4F cigarette smoke based on matched EC50 values at different dilutions (1:5 vs. 1:153 vol:vol), mass (52.1 vs. 3.1 µg/cm2) and nicotine (0.89 vs. 0.27 µg/cm2), respectively. Test doses where cigarette smoke and e-cigarette aerosol cytotoxicity were observed are comparable with calculated daily doses in consumers. Such experiments could form the basis of a larger package of work including chemical analyses, in vitro toxicology tests and clinical studies, to help assess the safety of current and next generation nicotine and tobacco products.

Applying new approach methodologies to assess next-generation tobacco and nicotine products.

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.

Comparative toxicological assessment of cigarettes and new category products via an in vitro multiplex proteomics platform.

Cigarette smoking is a risk factor for several diseases such as cancer, cardiovascular disease (CVD), and chronic obstructive pulmonary diseases (COPD), however, the underlying mechanisms are not fully understood. Alternative nicotine products with reduced risk potential (RRPs) including tobacco heating products (THPs), and e-cigarettes have recently emerged as viable alternatives to cigarettes that may contribute to the overall strategy of tobacco harm reduction due to the significantly lower levels of toxicants in these products' emissions as compared to cigarette smoke. Assessing the effects of RRPs on biological responses is important to demonstrate the potential value of RRPs towards tobacco harm reduction. Here, we evaluated the inflammatory and signaling responses of human lung epithelial cells to aqueous aerosol extracts (AqE) generated from the 1R6F reference cigarette, the glo™ THP, and the Vype ePen 3.0 e-cigarette using multiplex analysis of 37 inflammatory and phosphoprotein markers. Cellular exposure to the different RRPs and 1R6F AqEs resulted in distinct response profiles with 1R6F being the most biologically active followed by glo™ and ePen 3.0. 1R6F activated stress-related and pro-survival markers c-JUN, CREB1, p38 MAPK and MEK1 and led to the release of IL-1α. glo™ activated MEK1 and decreased IL-1ß levels, whilst ePen 3.0 affected IL-1ß levels but had no effect on the signaling activity compared to untreated cells. Our results demonstrated the reduced biological effect of RRPs and suggest that targeted analysis of inflammatory and cell signaling mediators is a valuable tool for the routine assessment of RRPs.

Advances in whole aerosol approaches for in vitro e-cigarette testing.

Electronic-cigarette regulation and risk assessment is a prominent and developing field, as the popularity and prevalence of this product category increases. Over the last 10 years since their emergence, there have been many advances and adaptations to current in vitro testing techniques to better assess and predict absolute consumer risk. However, there are still requirements to create a cross-field harmonised approach to appropriate exposure and experimental design. With many assessments still being carried out using methods developed and optimised for cigarette smoke, there must first be an acknowledgement regarding the differences between cigarette smoke and tobacco-free e-cigarette aerosols before we can accurately assess these distinct products. Here, we discuss five published studies from within our own research to demonstrate how in vitro testing techniques have evolved to improve determination of risk by considering appropriate dosimetry and exposure for both e-cigarette and cigarette aerosols and how we can contextualise the data through human consumption and dose extrapolation, ultimately giving more relevance to in vitro data. Furthermore, we have demonstrated the evolution of techniques, which has allowed us to bridge between platforms, simplify exposure set-up, experimental design and demonstrate technology evolution within our products, thus fulfilling a responsible duty of care to consumers via an appropriate and robust in vitro product assessment.

Application of ToxTracker for the toxicological assessment of tobacco and nicotine delivery products.

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 a >50 % 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.

Hepatic stellate cells require a stiff environment for myofibroblastic differentiation.

The myofibroblastic differentiation of hepatic stellate cells (HSC) is a critical event in liver fibrosis and is part of the final common pathway to cirrhosis in chronic liver disease from all causes. The molecular mechanisms driving HSC differentiation are not fully understood. Because macroscopic tissue stiffening is a feature of fibrotic disease, we hypothesized that mechanical properties of the underlying matrix are a principal determinant of HSC activation. Primary rat HSC were cultured on inert polyacrylamide supports of variable but precisely defined shear modulus (stiffness) coated with different extracellular matrix proteins or poly-L-lysine. HSC differentiation was determined by cell morphology, immunofluorescence staining, and gene expression. HSC became progressively myofibroblastic as substrate stiffness increased on all coating matrices, including Matrigel. The degree rather than speed of HSC activation correlated with substrate stiffness, with cells cultured on supports of intermediate stiffness adopting stable intermediate phenotypes. Quiescent cells on soft supports were able to undergo myofibroblastic differentiation with exposure to stiff supports. Stiffness-dependent differentiation required adhesion to matrix proteins and the generation of mechanical tension. Transforming growth factor-ß treatment enhanced differentiation on stiff supports, but was not required. HSC differentiate to myofibroblasts in vitro primarily as a function of the physical rather than the chemical properties of the substrate. HSC require a mechanically stiff substrate, with adhesion to matrix proteins and the generation of mechanical tension, to differentiate. These findings suggest that alterations in liver stiffness are a key factor driving the progression of fibrosis.

The development of an in vitro 3D model of goblet cell hyperplasia using MUC5AC expression and repeated whole aerosol exposures.

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.

Application of text mining to develop AOP-based mucus hypersecretion genesets and confirmation with in vitro and clinical samples.

Mucus hypersecretion contributes to lung function impairment observed in COPD (chronic obstructive pulmonary disease), a tobacco smoking-related disease. A detailed mucus hypersecretion adverse outcome pathway (AOP) has been constructed from literature reviews, experimental and clinical data, mapping key events (KEs) across biological organisational hierarchy leading to an adverse outcome. AOPs can guide the development of biomarkers that are potentially predictive of diseases and support the assessment frameworks of nicotine products including electronic cigarettes. Here, we describe a method employing manual literature curation supported by a focused automated text mining approach to identify genes involved in 5 KEs contributing to decreased lung function observed in tobacco-related COPD. KE genesets were subsequently confirmed by unsupervised clustering against 3 different transcriptomic datasets including (1) in vitro acute cigarette smoke and e-cigarette aerosol exposure, (2) in vitro repeated incubation with IL-13, and (3) lung biopsies from COPD and healthy patients. The 5 KE genesets were demonstrated to be predictive of cigarette smoke exposure and mucus hypersecretion in vitro, and less conclusively predict the COPD status of lung biopsies. In conclusion, using a focused automated text mining and curation approach with experimental and clinical data supports the development of risk assessment strategies utilising AOPs.

Evaluation of precision and accuracy of the Borgwaldt RM20S(®) smoking machine designed for in vitro exposure.

The Borgwaldt RM20S(®) smoking machine enables the generation, dilution, and transfer of fresh cigarette smoke to cell exposure chambers, for in vitro analyses. We present a study confirming the precision (repeatability r, reproducibility R) and accuracy of smoke dose generated by the Borgwaldt RM20S(®) system and delivery to exposure chambers. Due to the aerosol nature of cigarette smoke, the repeatability of the dilution of the vapor phase in air was assessed by quantifying two reference standard gases: methane (CH(4), r between 29.0 and 37.0 and RSD between 2.2% and 4.5%) and carbon monoxide (CO, r between 166.8 and 235.8 and RSD between 0.7% and 3.7%). The accuracy of dilution (percent error) for CH(4) and CO was between 6.4% and 19.5% and between 5.8% and 6.4%, respectively, over a 10-1000-fold dilution range. To corroborate our findings, a small inter-laboratory study was carried out for CH(4) measurements. The combined dilution repeatability had an r between 21.3 and 46.4, R between 52.9 and 88.4, RSD between 6.3% and 17.3%, and error between 4.3% and 13.1%. Based on the particulate component of cigarette smoke (3R4F), the repeatability (RSD = 12%) of the undiluted smoke generated by the Borgwaldt RM20S(®) was assessed by quantifying solanesol using high-performance liquid chromatography with ultraviolet detection (HPLC/UV). Finally, the repeatability (r between 0.98 and 4.53 and RSD between 8.8% and 12%) of the dilution of generated smoke particulate phase was assessed by quantifying solanesol following various dilutions of cigarette smoke. The findings in this study suggest the Borgwaldt RM20S(®) smoking machine is a reliable tool to generate and deliver repeatable and reproducible doses of whole smoke to in vitro cultures.

The genotoxicological assessment of a tobacco heating product relative to cigarette smoke using the in vitro micronucleus assay.

In vitro studies have supported the toxicological evaluation of chemicals and complex mixtures including cigarette smoke and novel tobacco and nicotine products which include tobacco heating products (THP). This new environment requires faster testing, higher throughput and appropriate in vitro studies, to support product innovation and development. In this study, total particulate matter (TPM) from a commercially available THP and a reference cigarette (3R4F) were assessed up to 500 µg/mL using two in vitro micronucleus techniques. V79 and TK6 cells were assessed using conventional OECD 487 manual scoring techniques, whereas, CHO cells were assessed using contemporary, automated high content screening approaches (Cellomics ArrayScan® VTI). V79 cells gave the most consistent response with all three treatment conditions producing a clear positive genotoxic response. Human TK6 cells only produced dose-dependent response, indicative of a weak-positive response. CHO cells demonstrated a positive response with TPM using long (24 h) -S9 conditions. All three cell lines equally demonstrated a negative response with THP TPM up to 500 µg/mL. In conclusion, THP TPM did not increase micronuclei formation above control levels even at doses far exceeding that tested with reference cigarette smoke, in most cases up to 10x the dose delivered compared to that of cigarette smoke. This study supports the growing belief that THPs are less risky than conventional cigarettes and that 21st century screening techniques can be employed to support product design and decision making, as a potential 1st screen prior to more traditional assessments.

Optimization of aqueous aerosol extract (AqE) generation from e-cigarettes and tobacco heating products for in vitro cytotoxicity testing.

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.