Reference Cigarette Research Articles

The in vitro assessment of a novel vaping technology

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.

Particulate matter emissions of less harmful-looking super-slim size cigarettes appealing to women: a laser spectrometric analysis of second-hand smoke.

Slim tobacco products shall appear by their look less harmful. In 2013, the European Union ministers discussed to ban them. However, only a ban on small package sizes was realized. To add more data for exposure risk assessment of slim tobacco products, the particulate matter (PM) amount in second-hand smoke (SHS) of super-slim size cigarettes compared with a king size brand was investigated. PM amount of four super-slim size cigarette types of the brand Couture was analysed in comparison with the king-size reference cigarette. Therefore, SHS was produced in an enclosed space with a volume of 2.88m3 by an automatic environmental tobacco smoke emitter. PM size fractions PM10, PM2.5 and PM1 were measured in real time using a laser aerosol spectrometer. SHS of Couture Gold contained about 36% and Couture Purple about 28% more PM than the reference cigarette. In contrast, Couture Green emitted about 37% and Couture Silver about 53% less PM than the reference cigarette. Depending on the brand, the PM2.5 mean concentrations increased up to 1538μg/m3. This exceeds the 24-h mean concentration of at most 25μg/m3 according to the WHO Air quality guidelines about 62-fold. Smoking in enclosed rooms leads to a massive increase of PM. The PM pollution by slim-size tobacco products are substantial and sometimes higher than by king size tobacco products. Therefore, SHS exposure from slim-size tobacco products is not less harmful to health. Decision makers should take this aspect in consideration.

A Screening Method by Gas Chromatography-Mass Spectrometry for the Quantification of 24 Aerosol Constituents from Heat-Not-Burn Tobacco Products

Summary A screening method allowing the quantification of 24 aerosol constituents using gas chromatography-mass spectrometry has been developed to assess the aerosol chemistry of heat-not-burn tobacco products. The aim of this method was to quantify phenol, o-cresol, m-cresol, p-cresol, catechol, resorcinol, hydroquinone, 1,3-butadiene, isoprene, benzene, acrylonitrile, toluene, pyridine, styrene, 1,2-propylene glycol, menthol, 2-furanmethanol, acrylamide, naphthalene, nicotine, acetamide, quinoline, triacetin, and glycerine in the aerosol emitted by heated tobacco products. The aerosol was generated by an electrically heated tobacco system (PMI’s Heated Tobacco System (THS 2.4)) with one single aerosol collection method, using the Health Canada smoking regimen and analyzed with two analytical methods. The method was validated according to the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use and the Association of Official Analytical Chemists guidelines. A regression model based on a linear relationship between concentration and response ratio with a 1/x weighting factor was selected for phenol, o-cresol, m-cresol, p-cresol, 1,3-butadiene, isoprene, benzene, acrylonitrile, toluene, pyridine, styrene, 2-furanmethanol, acrylamide, naphthalene and acetamide. A quadratic regression model with a 1/x weighting factor was chosen for catechol, resorcinol, hydroquinone, 1,2-propylene glycol, menthol, nicotine, quinoline, triacetin and glycerine. Coefficients of variation for repeatability were determined between 7.9% and 17.8% and for intermediate precision between 8.1% and 19.9%. The matrix effect of the heated tobacco aerosol extract was assessed by performing a recovery study, where the aerosol extracts were spiked at different concentrations for the compounds to be analyzed. In addition, the mainstream smoke from 3R4F reference cigarettes was analyzed, and results were compared with previously published studies. The method was successfully validated, providing data consistent with published data and it was shown to be selective, precise and accurate.

Structural, functional, and molecular impact on the cardiovascular system in ApoE-/- mice exposed to aerosol from candidate modified risk tobacco products, Carbon Heated Tobacco Product 1.2 and Tobacco Heating System 2.2, compared with cigarette smoke

AimTo investigate the molecular, structural, and functional impact of aerosols from candidate modified risk tobacco products (cMRTP), the Carbon Heated Tobacco Product (CHTP) 1.2 and Tobacco Heating System (THS) 2.2, compared with that of mainstream cigarette smoke (CS) on the cardiovascular system of ApoE-/- mice. MethodsFemale ApoE-/- mice were exposed to aerosols from THS 2.2 and CHTP 1.2 or to CS from the 3R4F reference cigarette for up to 6 months at matching nicotine concentrations. A Cessation and a Switching group (3 months exposure to 3R4F CS followed by filtered air or CHTP 1.2 for 3 months) were included. Cardiovascular effects were investigated by echocardiographic, histopathological, immunohistochemical, and transcriptomics analyses. ResultsContinuous exposure to cMRTP aerosols did not affect atherosclerosis progression, heart function, left ventricular (LV) structure, or the cardiovascular transcriptome. Exposure to 3R4F CS triggered atherosclerosis progression, reduced systolic ejection fraction and fractional shortening, caused heart LV hypertrophy, and initiated significant dysregulation in the transcriptomes of the heart ventricle and thoracic aorta. Importantly, the structural, functional, and molecular changes caused by 3R4F CS were improved in the smoking cessation and switching groups. ConclusionExposure to cMRTP aerosols lacked most of the CS exposure-related functional, structural, and molecular effects. Smoking cessation or switching to CHTP 1.2 aerosol caused similar recovery from the 3R4F CS effects in the ApoE-/- model, with no further acceleration of plaque progression beyond the aging-related rate.

CARDIORESPIRATORY IMPACT OF E-VAPOR AEROSOLS AND CIGARETTE SMOKE EXPOSURE IN THE APOE KNOCKOUT MOUSE MODEL

Chronic exposure to cigarette smoke (CS) causes cardiorespiratory diseases in smokers. Considerable attention has been given to the reduced harm potential of alternative e-vapor products. ApoE-/- mice were used to evaluate lung inflammation, emphysematous changes, atherosclerosis development, and cardiovascular function upon chronic (6-month) exposure to fresh air (Sham), CS, or e-vapor aerosols. Mice were exposed to CS from 3R4F reference cigarettes or e-vapor aerosols generated using capillary aerosol generators from various e-liquids (“CARRIER” containing humectants [propylene glycol, glycerin], “BASE” containing humectants and nicotine, and “TEST” containing humectants, nicotine, and flavors) via a whole-body inhalation system. Exposure to CS at a nicotine concentration of 35 μg/L causes adverse effects on the lungs (including increased lung volume and inflammation), accelerates atherosclerotic plaque formation, and alters the cardiorespiratory transcriptome. CS exposure causes an impairment of both systolic and diastolic cardiac function, as assessed by ejection fraction, fractional shortening, isovolumic relaxation time, and E/A ratio. In comparison with CS, exposure to e-vapor aerosols lower atherosclerotic plaque formation, was associated with limited to absent inflammatory cells in the lung tissue, and induced substantially less molecular changes in cardiorespiratory transcriptomes. Ultrasound analysis highlighted that cardiac dysfunction and aortic stiffness were less prominent in nicotine-containing e-vapor aerosol-exposed groups than in the CS-exposed group. This study suggests that in comparison with CS, e-vapor aerosols induce substantially lower biological responses associated with smoking-related cardiovascular and pulmonary diseases.

Comparison of cytotoxicity of IQOS aerosols to smoke from Marlboro Red and 3R4F reference cigarettes

This study compared the cytotoxicity of IQOS aerosols to smoke from Marlboro Red (MR) and 3R4F reference cigarettes. Aerosol/smoke solutions were tested as the gas vapor phase (GVP), particulate phase (total particulate matter or TPM), or whole aerosol/smoke (WA), the latter being what smokers actually inhale. Cytotoxicities were evaluated using the LDH, MTT and neutral red uptake (NRU) assays in conjunction with eight different cell types, mainly from the respiratory system. Most test solutions did not compromise the plasma membranes of cells (LDH). However, mitochondrial activity (MTT) and dye uptake/lysosomal activity (NRU) were equally depressed by IOQS aerosols and cigarette smoke solutions at the high concentrations. Our NRU data with mouse NIH/3T3 transformed fibroblasts were similar to those previously reported by the IQOS manufacturer and showed little cytotoxicity in the NRU assay. In both studies with NIH/3T3 cells, the results were significantly different from 3RF4 cigarette smoke, suggesting reduced toxicity with IQOS. However, by expanding evaluations to a broader spectrum of cells that included respiratory system cells and by including higher concentrations of GVP, as well as WA, cytotoxicity equivalent to that of Marlboro Red and 3R4F cigarettes was frequently observed with IQOS aerosols in the MTT and NRU assays.

Application of a multi-layer systems toxicology framework for in vitro assessment of the biological effects of Classic Tobacco e-liquid and its corresponding aerosol using an e-cigarette device with MESH\u2122 technology

We previously proposed a systems toxicology framework for in vitro assessment of e-liquids. The framework starts with the first layer aimed at screening the potential toxicity of e-liquids, followed by the second layer aimed at investigating the toxicity-related mechanism of e-liquids, and finally, the third layer aimed at evaluating the toxicity-related mechanism of the corresponding aerosols. In this work, we applied this framework to assess the impact of the e-liquid MESH Classic Tobacco and its aerosol compared with that of cigarette smoke (CS) from the 3R4F reference cigarette. In the first layer, we evaluated the cytotoxicity profile of the MESH Classic Tobacco e-liquid (containing humectants, nicotine, and flavors) and its Base e-liquid (containing humectant and nicotine only) in comparison with total particulate matter (TPM) of 3R4F CS using primary bronchial epithelial cell cultures. In the second layer, the same culture model was used to explore changes in specific markers using high-content screening assays to identify potential toxicity-related mechanisms induced by the MESH Classic Tobacco and Base e-liquids beyond cell viability in comparison with the 3R4F CS TPM-induced effects. Finally, in the third layer, we compared the impact of exposure to the MESH Classic Tobacco or Base aerosols with 3R4F CS using human organotypic air–liquid interface buccal and small airway epithelial cultures. The results showed that the cytotoxicity of the MESH Classic Tobacco liquid was similar to the Base liquid but lower than 3R4F CS TPM at comparable nicotine concentrations. Relative to 3R4F CS exposure, MESH Classic Tobacco aerosol exposure did not cause tissue damage and elicited lower changes in the mRNA, microRNA, and protein markers. In the context of tobacco harm reduction strategy, the framework is suitable to assess the potential-reduced impact of electronic cigarette aerosol relative to CS.

Establishment of rapid risk assessment model for cigarette smoke extract exposure in chronic obstructive pulmonary disease

Rapid risk assessment models for different types of cigarette smoke extract (CSE) exposure are critical to understanding the etiology of chronic obstructive pulmonary disease. The present study investigated inflammation of cultured tracheal tissues with CSE exposure. Rat trachea rings were isolated, cultured, then exposed to various concentrations of CSE from 3R4 F reference cigarettes for 4 h. Tissue/cellular morphology, ultrastructure, viability and damage, inflammatory cell infiltration, and inflammatory protein levels were measured and compared to untreated controls. Human bronchial epithelial cells (BEAS-2B) exposed to 0 or 300 μg/mL CSE were cocultured with macrophages to assess extent of mobilization and phagocytosis. Endotracheal epithelium cilia densities were significantly reduced with increasing CSE concentrations, while mucous membranes became increasingly disordered; both eventually disappeared. Macrophages became larger as the CSE concentration increased, with microvilli and extended pseudopodium covering their surface, and many primary and secondary lysosomes present in the cytoplasm. Inflammatory cell infiltration also increased with increasing CSE dose, as did intracellular adhesion molecule-1(ICAM-1), interleukin-6(IL-6). The method described here may be useful to qualitatively characterized the effects of the compound under study. Then, we use BEAS-2B cell line system to strength the observation made in the cultured tissues. Probably, an approach to integrate results from both experiments will facilitate its application. These results demonstrate that cultured rat tracheal rings have a whole-tissue structure that undergoes inflammatory processes similar to in vivo tissues upon CSE exposure.

An inter-laboratory in vitro assessment of cigarettes and next generation nicotine delivery products

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.

Chemical characterization of nanoparticles and volatiles present in mainstream hookah smoke

Waterpipe smoking is becoming more popular worldwide and there is a pressing need to better characterize the exposure of smokers to chemical compounds present in the mainstream smoke. We report real-time measurements of mainstream smoke for carbon monoxide, volatile organic compounds and nanoparticle size distribution and chemical composition using a custom dilution flow tube. A conventional tobacco mixture, a dark leaf unwashed tobacco and a nicotine-free herbal tobacco were studied. Results show that carbon monoxide is present in the mainstream smoke and originates primarily from the charcoal used to heat the tobacco. Online measurements of volatile organic compounds in mainstream smoke showed an overwhelming contribution from glycerol. Gas phase analysis also showed that very little filtration of the gas phase products is provided by the percolation of mainstream smoke through water. Waterpipe smoking generated high concentrations of 4-100 nm nanoparticles, which were mainly composed of sugar derivatives and especially abundant in the first 10 min of the smoking session. These measured emissions of volatiles and particles are compared with those from a reference cigarette (3R4F) and represent the equivalent of the emission of one or more entire cigarettes for a single puff of hookah smoke. Considerations related to the health impacts of waterpipe smoking are discussed.

Combustible Cigarette and Smokeless Tobacco Product Preparations Differentially Regulate Intracellular Calcium Mobilization in HL60 Cells

Changes in the level of intracellular calcium ([Ca2+]i) are central to leukocyte signaling and immune response. Although evidence suggests that cigarette smoking affects inflammatory response via an increase in intracellular calcium, it remains unclear if the use of smokeless tobacco (e.g., moist snuff) elicits a similar response. In this study, we evaluated the effects of tobacco product preparations (TPPs), including total particulate matter (TPM) from 3R4F reference cigarettes, smokeless tobacco extract (STE) from 2S3 reference moist snuff, and nicotine alone on Ca2+ mobilization in HL60 cells. Treatment with TPM, but not STE or nicotine alone, significantly increased [Ca2+]i in a concentration-dependent manner in HL60 cells. Moreover, TPM-induced [Ca2+]i increase was not related to extracellular Ca2+ and did not require the activation of the IP3 pathway nor involved the transient receptor potential (TRP) channels. Our findings indicate that, in cells having either intact or depleted endoplasmic reticulum (ER) Ca2+ stores, TPM-mediated [Ca2+]i increase involves cytosolic Ca2+ pools other than thapsigargin-sensitive ER Ca2+ stores. These results, for the first time, demonstrate that TPM triggers [Ca2+]i increases, while significantly higher nicotine equivalent doses of STE or nicotine alone, did not affect [Ca2+]i under the experimental conditions. In summary, our study suggests that in contrast with STE or nicotine preparations, TPM activates Ca2+ signaling pathways in HL60 cells. The differential effect of combustible and non-combustible TPPs on Ca2+ mobilization could be a useful in vitro endpoint for tobacco product evaluation.

Cigarette and ENDS preparations differentially regulate ion channels and mucociliary clearance in primary normal human bronchial 3D cultures.

Cigarette smoking is known to disrupt the normal mucociliary function of the lungs, whereas the effect of electronic nicotine delivery systems (ENDS) is not completely understood. This study aimed to compare the effects of acute exposure of primary normal human bronchial epithelial (NHBE) 3D cultures at air-liquid interface to combustible cigarette and ENDS preparations on mucociliary function, including ion channel function, ciliary beat frequency (CBF), and airway surface liquid (ASL) height. Differentiated NHBE cultures were exposed to whole smoke-conditioned media (WS-CM) or total particulate matter (TPM) prepared from 3R4F reference cigarettes, whole aerosol-conditioned media (ACM) or e-TPM generated from a marketed ENDS product, or nicotine alone. We found that a dose of 7 μg/mL equi-nicotine units of cigarette TPM and WS-CM significantly decreased cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) function, which regulates fluid homeostasis in the lung. Conversely, higher (56 µg/mL) equi-nicotine units of ENDS preparations or nicotine alone had no effect on CFTR and ENaC function. Despite a significant decrease in ion channel function, cigarette smoke preparations did not alter CBF and ASL. Similarly, ENDS preparations and nicotine alone had no effect on ASL and CBF. This study demonstrates that acute exposures of cigarette smoke preparations exert a notable inhibitory effect on CFTR and ENaC function compared with ENDS preparations. In summary, the functional assays described herein are potentially useful for tobacco product evaluations.

Genotoxicity evaluation of tobacco and nicotine delivery products: Part Two. In vitro micronucleus assay

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 Smoke Disrupts Calcium Homeostasis in Airway Smooth Muscle Cells

Tobacco smoking is the largest risk factor for developing chronic obstructive pulmonary disease (COPD), and is associated with airway hyperresponsiveness. Hyperresponsiveness is linked to abnormal calcium metabolism, which regulates the contractility and hence the tone of airway smooth muscle (ASM). However, whether cigarette smoke (CS) directly alters or regulates calcium signaling in ASM cells (ASMC) is not fully known. In airway epithelial cells, acute CS exposure increases intracellular Ca2+ concentrations ([Ca2+]i). We hypothesise that CS impedes ASM function by disrupting calcium homeostasis in ASMC, and the present study aims to unravel the effects of CS on calcium handling in ASMC. Primary rat and human ASMC were isolated via enzymatic digestion, and maintained in culture up to 8 passages. ASMC were seeded onto glass coverslips, and deprived of serum for 24–48 hours prior to calcium imaging studies using fura‐2‐AM. ASMC were exposed to either whole CS or CS extract (CSE) from 3R4F reference cigarettes (University of Kentucky) with the particulate phase filtered out. We found that both CS and CSE elevated [Ca2+]i in rat and human ASMC, and this increase was not abolished following depletion of sarcoplasmic‐reticulum calcium stores with thapsigargin or cyclopiazonic acid. The increase in [Ca2+]i in response to CSE was concentration‐dependent. Removal of extracellular calcium attenuated the CSE‐induced elevation of [Ca2+]i in hASMC, suggesting that CSE may activate calcium influx pathways in cultured hASMC. We speculate that this elevation of [Ca2+]i would lead to exacerbated ASM contraction, especially under inflamed conditions in COPD, and hence narrowing of the airway lumen. Further investigations into the source of [Ca2+]i increase, identity of potential calcium influx channels activated, and the direct effects of CS/CSE on ASM contraction are on‐going.Support or Funding InformationThis work is supported by Newcastle University's JJ Hunter Bequest scholarship.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High Content Screening in NHBE cells shows significantly reduced biological activity of flavoured e-liquids, when compared to cigarette smoke condensate

There is scientific agreement that the detrimental effects of cigarettes are produced by the formation of Harmful and Potentially Harmful Constituents from tobacco combustion and not by nicotine. For this reason numerous public health bodies and governments worldwide have indicated that e-cigarettes have a central role to play in tobacco harm reduction.In this study, high content screening (HCS) was used to compare the effects of neat e-liquids and 3R4F reference cigarette smoke condensate (CSC), which served as a positive control, in Normal Human Bronchial Epithelial (NHBE) cells. The endpoints measured covered cellular health, energy production and oxidative stress. Base liquids, with or without nicotine, and commercial, flavoured, nicotine-containing e-liquids (CFs), had little or no effect on cell viability and most HCS endpoints even at significantly higher concentrations (typically 100 times or higher) than 3R4F CSC. CSC induced a dose-dependent decrease of cell viability and triggered the response in all HCS endpoints. Effects of CFs were typically observed at or above 1%. CF Menthol was the most active flavour, with minimum effective concentrations 43 to 659 times higher than corresponding 3R4F CSC concentrations. Our results show a lower biological activity of e-liquids compared to cigarette smoke condensate in this experimental setting, across wide range of cellular endpoints.

A lower impact of an acute exposure to electronic cigarette aerosols than to cigarette smoke in human organotypic buccal and small airway cultures was demonstrated using systems toxicology assessment

In the context of tobacco harm-reduction strategy, the potential reduced impact of electronic cigarette (EC) exposure should be evaluated relative to the impact of cigarette smoke exposure. We conducted a series of in vitro studies to compare the biological impact of an acute exposure to aerosols of “test mix” (flavors, nicotine, and humectants), “base” (nicotine and humectants), and “carrier” (humectants) formulations using MarkTen® EC devices with the impact of exposure to smoke of 3R4F reference cigarettes, at a matching puff number, using human organotypic air–liquid interface buccal and small airway cultures. We measured the concentrations of nicotine and carbonyls deposited in the exposure chamber after each exposure experiment. The deposited carbonyl concentrations were used as representative measures to assess the reduced exposure to potentially toxic volatile substances. We followed a systems toxicology approach whereby functional biological endpoints, such as histopathology and ciliary beating frequency, were complemented by multiplex and omics assays to measure secreted inflammatory proteins and whole-genome transcriptomes, respectively. Among the endpoints analyzed, the only parameters that showed a significant response to EC exposure were secretion of proteins and whole-genome transcriptomes. Based on the multiplex and omics analyzes, the cellular responses to EC aerosol exposure were tissue type-specific; however, those alterations were much smaller than those following cigarette smoke exposure, even when the EC aerosol exposure under the testing conditions resulted in a deposited nicotine concentration approximately 200 times that in saliva of EC users.

Assessment of priority tobacco additives per the requirements of the EU Tobacco Products Directive (2014/40/EU): Part 3, Smoking behavior and plasma nicotine pharmacokinetics

This publication is part of a series of 3 publications and describes the clinical assessment performed to fulfill the regulatory requirement per Art. 6 (2) of the EU Tobacco Products Directive 2014/40/EU under which Member States require manufacturers and importers of cigarettes and Roll Your Own tobacco containing an additive that is included in the priority list established by Commission Implementing Decision (EU) 2016/787 to carry out comprehensive studies (European Union, 2016). In our clinical study, two distinct end points were investigated, namely measuring plasma nicotine pharmacokinetics as a measure of nicotine uptake, and analyses of changes in smoker puffing behavior as a measure of cigarette smoke inhalation. This clinical study indicated that the inclusion of none of the priority additives either as single additive or as part of a chemical mixture, facilitated nicotine uptake. Furthermore, the data did not suggest that differences in the inhalation pattern of cigarette smoke of any of the Priority Additives tested occurred when compared to the additive-free reference cigarette. Finally, it is concluded that neither the scientific literature nor our study gave circumstantial indications of increased addictiveness for cigarettes containing these priority additives.

Assessment of priority tobacco additives per the requirements of the EU Tobacco Products Directive (2014/40/EU): Part 1: Background, approach, and summary of findings

This paper is part of a series of 3 publications and describes the non-clinical and clinical assessment performed to fulfill the regulatory requirement per Art. 6 (2) of the EU Tobacco Products Directive 2014/40/EU; under which Member States shall require manufacturers and importers of cigarettes and roll-your-own tobacco containing an additive that is included in the priority list established by Commission Implementing Decision (EU) 2016/787 to carry out comprehensive studies. The Directive requires manufacturers and importers of cigarettes and Roll Your Own tobacco to examine for each additive whether it; contributes to and increases the toxicity or addictiveness of tobacco products to a significant or measurable degree; if it leads to a characterizing flavor of the product; if it facilitates inhalation or nicotine uptake, and if it results in the formation of CMR (carcinogenic, mutagenic and reprotoxic) constituents and if these substances increase the CMR properties of the respective tobacco product to a significant or measurable degree. This publication gives an overview on comprehensive smoke chemistry, in vitro toxicity, and human clinical studies commissioned by the members of the Priority Additives Tobacco Consortium to independent Contract Research Organizations (CROs) where the emissions of test cigarettes containing priority additives were compared to emissions emerging from an additive-free reference cigarette. Whilst minor changes in smoke chemistry parameters were observed when comparing emissions from test cigarettes with emissions from additive-free reference cigarettes, only two of the additives (sorbitol and guar gum) tested led to significant increases in a limited number of smoke constituents. These changes were not observed when sorbitol or guar gum were tested in a mixture with other priority additives. None of the priority additives resulted in increases in in vitro toxicity (Ames, Micronucleus, Neutral Red Uptake) or led to changes in smoking behavior or absorption (rate or amount) of nicotine measured during the human clinical study as compared to the additive-free reference cigarette.

A six-month systems toxicology inhalation/cessation study in ApoE−/− mice to investigate cardiovascular and respiratory exposure effects of modified risk tobacco products, CHTP 1.2 and THS 2.2, compared with conventional cigarettes

Smoking is one of the major modifiable risk factors in the development and progression of chronic obstructive pulmonary disease (COPD) and cardiovascular disease (CVD). Modified-risk tobacco products (MRTP) are being developed to provide substitute products for smokers who are unable or unwilling to quit, to lessen the smoking-related health risks. In this study, the ApoE-/- mouse model was used to investigate the impact of cigarette smoke (CS) from the reference cigarette 3R4F, or aerosol from two potential MRTPs based on the heat-not-burn principle, carbon heated tobacco product 1.2 (CHTP1.2) and tobacco heating system 2.2 (THS 2.2), on the cardiorespiratory system over a 6-month period. In addition, cessation or switching to CHTP1.2 after 3 months of CS exposure was assessed. A systems toxicology approach combining physiology, histology and molecular measurements was used to evaluate the impact of MRTP aerosols in comparison to CS. CHTP1.2 and THS2.2 aerosols, compared with CS, demonstrated lower impact on the cardiorespiratory system, including low to absent lung inflammation and emphysematous changes, and reduced atherosclerotic plaque formation. Molecular analyses confirmed the lower engagement of pathological mechanisms by MRTP aerosols than CS. Both cessation and switching to CHTP1.2 reduced the observed CS effects to almost sham exposure levels.

Mainstream smoke constituents and in vitro toxicity comparative analysis of 3R4F and 1R6F reference cigarettes.

A new Kentucky reference cigarette, 1R6F, has been manufactured to replace the depleting 3R4F reference cigarette. The 3R4F Kentucky reference cigarettes have been widely used as monitor or comparator cigarettes for mainstream smoke analysis and in vitro and in vivo toxicological data of cigarettes and novel tobacco products. Both reference cigarettes were analyzed in the same laboratory during the same period of time with the goal of performing a comparison of 3R4F and 1R6F. On the basis of the results obtained from aerosol chemistry and in vitro assays, we consider that the 1R6F reference cigarette is a suitable replacement for the 3R4F reference cigarette as a comparator/monitor cigarette. Its specific use as a comparator for novel tobacco products was checked on the basis of a comparative test with the Tobacco Heating System 2.2 as an example.