BRITISH AMERICAN TOBACCO Science & Technology

2015 report Foreword 1 Dr David O’Reilly, Group Scientific and R&D Director

RESEARCH PRIORITIES Stewardship and next generation products 2 Dr Christopher Proctor

PLANT BIOTECHNOLOGY From seed to smoke 4 Dr Kieron Edwards

GOOD RESEARCH PRACTICE Good research practice — securing innovation 7 John McBrearty

ANALYTICAL METHOD DEVELOPMENT Chemical characterisation of next generation Science & products 8 Dr Christopher Wright Technology POPULATION STUDIES Assessing the impact of new products 11 2015 Oscar Camacho STEWARDSHIP Leading the way in product risk assessment 14 About this Report This is a British American Tobacco p.l.c. report describing Dr Audrey Richter some of the activity of British American Tobacco Group Research & Development in the UK. References to ‘British TOBACCO HEATING PRODUCTS American Tobacco’, ‘we’, ‘us’ and ‘our’ when denoting Eliminating combustion to potentially opinion or activity refer to British American Tobacco Group Research & Development. reduce risk 16 Whilst reasonable steps have been taken to ensure that Dr Chuan Liu the information contained within this publication is correct, the authors, British American Tobacco, its agents, ELECTRONIC CIGARETTES contractors and sub-contractors give no warranty and make no representation as to its accuracy and aaccept no liability Rapid evolution presents new challenges 20 for any errors or omissions. Dr Kevin McAdam Any trademarks, service marks or logos used in this publication, and copyright in it, are the property of British PRODUCT TESTING American Tobacco. Building the evidence base 26 Also available on bat-science.com Dr Marianna Gaça

QUANTIFYING RISK @BAT_Sci Improved understanding of toxicants and disease 29 Fiona Cunningham

AEROSOL SCIENCE Understanding consumer product interaction 32 John McAughey Design and production JamesMcQuat.com SCIENCE ENGAGEMENT Our open approach 36 Main image photography JohnnyJGallagher.com Sarah Cooney

Printing INSIDE BAT — PEOPLE hobbs.uk.com Bring your difference to BAT 40

INSIDE BAT — ENGINEERING ® MIX Focusing on practical solutions 42 Paper from responsible sources FSC® C020438 FOREWORD

e have spoken for some years about the understood, and are as safe as possible. importance of tobacco harm reduc- New nicotine products should con- W tion for British American Tobacco, for tinue to increase in relevance, which our consumers and for regulators. We is why we have expanded our research believe that developing less risky tobacco efforts to focus on these emerging product and nicotine products is transforma- categories. The swift evolution of these tional in many dimensions, and know we categories and the diversity of available must take a strong lead in doing this. products means that we are constantly In 2013, we were the first international pushing the boundaries to develop more tobacco company to launch an e-cigarette, advanced mechanics and better perform- Vype, in the UK. Since then we have ing products to give our consumers what continued to innovate in the category, and they want. Our research efforts continue have since launched three new prod- to focus on understanding how the dif- ucts, Vype ePen, Vype eStick and Vype ferent product categories perform under eTank and a wide range of liquids, and various conditions and how consumers are expanding our geographic footprint. use them; the constituents of the aerosol, We are also the first tobacco company as well as the possible creation of ther- to receive, in 2014, a medicines licence mal breakdown products; and the effects for a nicotine inhalator called Voke, and on the body of inhaling the aerosol. DR DAVID O’REILLY in 2015 we launched a tobacco heating By working together with our consum- Group Scientific and product. To develop less risky choices ers, we better understand their needs and R&D Director for smokers that give them a true alter- can offer greater expertise and insight David first joined us in 1991 native choice to cigarettes will require into areas such as materials science, with a PhD in molecular strong consumer insights, tremendous formulation chemistry, and engineer- virology and he was appointed innovation, and comprehensive science ing. We must also play our part in the to the Management Board of to ensure these next generation products ongoing regulatory debate, providing BAT in 2012. He directs the company’s scientific research, do offer reduced-risk potential. We think the data to enable regulators around the which seeks to develop it is important to offer a range of prod- world to make evidence-based decisions. innovative reduced-risk products ucts, including tobacco heating prod- We have embraced and will continue along with the tools to test ucts, e-cigarettes and licenced nicotine to embrace new scientific disciplines to and substantiate their risk products because consumer needs vary. create the knowledge needed to ensure potential. He has also been the driving force behind the The next few years are incredibly the potential of this category is realised investment and growth of BAT’s important and will determine whether the as well as our intention to be a leader R&D into a forward-looking, opportunity of tobacco harm reduction in these new product categories. consumer-led, multidisciplinary becomes widely adopted. Just as we saw Through this our second Science and environment. Previously, he led with snus, some regulators have prohib- Technology Report we continue to build on our programmes in corporate social responsibility and public ited e-cigarettes, and the World Health our heritage of scientific engagement and health policy and engagement. Organisation has not been very support- transparency, and maintain our desire to ive. We are working hard to ensure that work with others to deliver the opportuni- our products are fully characterised and ties that tobacco harm reduction offer. ☐

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 1 RESEARCH PRIORITIES

STEWARDSHIP AND NEXT GENERATION PRODUCTS

he risks of cigarette smoking to human chemical characterisation in a manner health are very well understood. Given that consistent with consumer use. These data T we have had a R&D unit at BAT since 1956, were all used to build our knowledge on we have decades of expertise, as well as how to apply our own stewardship stand- published research, on tobacco and tobacco ards to snus, ensuring that we minimised smoke toxicants. We have completed a risks associated with our products to every multi-year research programme, aimed at extent possible. However, as a product trying to reduce the risks associated with with potential for reducing risk, snus conventional cigarettes by using a number received little regulatory support, being of toxicant-reducing technologies. Although banned in the European Union outside this research gave some encouraging results, Sweden, and little consumer interest in the we found few differences in clinical studies markets where we tried to introduce it. between biomarkers of biological effect in More recently, we have focused on two volunteers who were switched to reduced- other potentially reduced-risk product cat- toxicant prototype cigarettes and those egories, tobacco heating products (THPs) who continued to smoke regular cigarettes. and electronic cigarettes (e-cigarettes). Through well-established principles of toxi- We also hope to add licensed medicinal cology and safety risk assessments, our prod- nicotine products, but will not discuss this uct stewardship approach, therefore, focuses category further in this report as these on ensuring that any ingredient, material, or types of products are subject to quite dif- design feature used in our products do not ferent regulatory oversight and procedures add to the inherent risk of cigarette smoking. from the THP or e-cigarette categories. The research taught us that if we hoped We were the first international tobacco to find tobacco or nicotine alternatives for company to launch an e-cigarette when cigarette smokers that might be deter- Vype was marketed in the UK in July, mined to be less risky than cigarettes, we 2013. Just as with snus, entering into a probably had to substantially reduce the new product category required an ac- toxicant emissions over and above what celerated research programme to develop DR CHRISTOPHER PROCTOR Chief Scientific Officer is possible, even with multiple toxicant- understanding of how the products work, reducing technologies, in cigarettes. what emissions they produce, and how Chris is our Chief Scientific We began with Swedish snus, given the consumers were likely to use them, so Officer and also Group Head of well-documented epidemiological data that our testing regimes could be set Scientific Product Stewardship based at R&D Southampton. accumulated in Sweden showing sole use up to be relevant to consumer use. His teams look at product to be far less risky than cigarette smok- To add to the complexity of test- safety issues and are working ing. Snus is not risk free, and, as we began ing, the e-cigarette category is much on research methodologies to developing our own products, even though broader than that of, for example, snus. substantiate the reduced-risk they complied with industry standards for We began with disposable ‘cig-a-like’ potential of next generation products. Chris regularly manufacturing and low levels of toxi- devices with simple formulations, but engages on various topics cants, such as nitrosamines, we needed to rapidly developed a range of modular related to tobacco regulatory develop our own understanding of how devices with a wider range of flavours science and has presented the products worked and were used by and battery sizes, and are currently at multiple workshops of consumers. We completed and published introducing new innovations at a rapid the US Food and Drug Administration in recent years. surveys of behavioural use, developed and rate. Emissions from e-cigarettes are used new toxicological tests, undertook a very different from those of cigarettes; pharmacokinetic study, and did extensive they are much simpler and substantially

2 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 RESEARCH PRIORITIES RESEARCH PRIORITIES

lower in certain toxicants that are as- regulators with confidence in the poten- sociated with conventional cigarettes. tial for next generation products, through There is the potential for the presence robust science. A key challenge is agree- of toxicants not seen in cigarettes, given ing methodologies and implementing possible interactions between formula- standardisation across the various tests. tions and device materials, and, therefore, As such, we are working with standards we have had to take a fresh approach to organisations, such as the British Stand- e-cigarette stewardship, with a strong focus ards Institute and the French organisa- on materials and formulations use. We tion, AFNOR, to contribute to what may have taken the approach of non-targeted constitute sensible industry-wide standards. chemical characterisation of the aerosols to It would be beneficial if a single scien- ensure we could develop a full understand- tific framework could be agreed upon by ing of what our e-cigarettes are emitting. all stakeholders (ranging from regulators, In parallel, we have had to develop new to public health bodies, to industry). This techniques to measure the ways in which should in turn allow an evidence-based vapers use these products and to estab- regulatory approach for next genera- lish toxicological tests sensitive enough tion tobacco and nicotine products that to deal with the significantly reduced would include an agreed mechanism for levels of toxicants produced by them. substantiating exposure- or health-related In stewarding these new product cat- claims. Transparency is critical and can be egories, it is critical for us to demonstrate facilitated through the publication of data that innovations don’t give rise to unin- and working in an open and collaborative tended consequences, including increased manner. The recent advent of workshops exposure and risk. We readily share our moderated by independent organisations, approach to stewardship of next generation such as the Institute for In Vitro Sciences, products with the regulatory and scientific is an example of where stakeholders can community. We have already published come together with a view to debating and several papers on this topic and expect to then agreeing a science framework that publish many more in the coming years. could be used for evidence-based regula- In addition, we expect to undertake a tion of products across the risk spectrum. broader set of research studies, including Today we stand at a defining moment in chemical toxicological, clinical, and popula- the development and future regulation of tion studies, to assess next generation prod- tobacco and nicotine products. We believe ucts as potentially reduced-risk products. As it is critical for everyone with an interest strong supporters of the move to replace ani- in this emerging field to remain open to mal testing we will continue to invest in and dialogue and collaborative working where collaborate on in vitro models, and are excit- possible, to ensure the advancement of an ed about the emergence of systems science. evidence-based approach to substantiating We believe that it is essential to provide risk for next generation products. ☐

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 3 PLANT BIOTECHNOLOGY

4 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 PLANT BIOTECHNOLOGY PLANT BIOTECHNOLOGY

TOXICANT REDUCTION – FROM SEED TO SMOKE

obacco (Nicotiana tabacum), a member a suite of biotechnology tools (including of the nightshade family (Solanaceae), is molecular biology, genomics, biochemis- T an important plant biology model. It was try, and systems biology) to understand the first plant adapted for tissue culture and modify these traits. The use of trans- and to be genetically transformed, which genic and marker-assisted breeding tech- were crucial advances in the early de- niques means we use genetic modification velopment of molecular plant biology. (GM) and non-GM (molecular breeding) Our tobacco biotechnology research approaches in our research. We under- programme focuses on basic research on stand that the use of GM plants remains the tobacco genome and practical plant controversial in some countries, and it re- modification research. R&D Cambridge mains BAT’s policy to not use GM tobacco seeks to develop biotechnology solutions in any of its commercial products world- that will help to push characteristics of wide. However, GM is an important tech- tobacco leaf in ways that are consumer nology within our research programme, as acceptable. Our research programme has it provides opportunities for toxicant re- several key areas: toxicant reduction strat- duction to be explored and enables investi- egies, sustainability of tobacco agriculture, gation into solutions to address challenges and genomics and molecular breeding that other technologies may not be able to tools for faster delivery of solutions. tackle so effectively. GM technologies al- The levels of toxicants in tobacco low the rapid investigation of target genes aerosol come not only from the physi- or pathways as a research tool to help nar- cal design parameters of cigarettes or row down the areas of interest, before they tobacco-heating products (THPs), but also are translated into non-GM solutions that from the physical and chemical proper- BAT can then look to apply commercially. ties of the tobacco and other ingredients As well as changing the quality of to- that are burned or heated. The diverse bacco leaf, BAT is developing solutions to DR KIERON EDWARDS Molecular Breeding Manager range of effects that can impact the levels help its farmers grow tobacco sustainably, of toxicants make modifying elements of by breeding plants that will give increased Kieron’s background includes tobacco leaf to reduce levels of harmful yields and reducing the required resources biological sciences; ranging toxicants while preserving the integrity (chemicals, water etc) for growth. from genetics to bioinformatics and systems biology. After of the plant itself a major challenge. The his PhD in plant genetics, properties can vary due to multiple fac- Toxicant reduction strategies — Kieron’s post-doctoral work tors, including the environment in which tobacco-specific nitrosamines (TSNAs). focused on circadian plant the tobacco is grown (climate, soil, etc.), Nitrogen is a nutrient essential for growth. biology. He joined us in 2008 position of the tobacco leaf on the plant, Tobacco, like other plants, is very efficient and is now responsible for developing and implementing the genetic characteristics of the tobacco at taking up and using nitrogen or related molecular breeding capabilities plant, and the way that the tobacco leaf compounds. Excess nitrogen is stored in in tobacco. He also manages has been harvested, cured, and processed. the leaves and what the plant does not use several critical projects, A key part of our work lies in under- during its life cycle remains in the leaf including identification of standing the genetics of the tobacco plant through to harvest, largely as nitrate and genetic markers and predictive biomarkers for complex traits. and which metabolic or biochemical nitrite oxides. The remaining nitrogen pathways and genes drive the characteris- compounds contribute to the formation tic traits of interest, so that we can apply of TSNAs, which in turn contribute to

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 5 PLANT BIOTECHNOLOGY

the toxicity of tobacco smoke; exposure protection chemicals (so-called sucker- to these compounds has been linked to cides) and improving the sustainability several cancers. TSNAs are formed when of tobacco agriculture. The promoter nicotine and related nitrogen-containing is linked to genes that either interrupt compounds undergo a chemical reaction the cell cycle, which impairs growth, or during harvesting and curing of the leaves. produces termination signals that can Using the genomics programme as lead to cell death and no sucker growth. well as available scientific literature, we identified sets of genes that influence the Recent successes uptake, transportation, and metabolism We have sequenced the large and com- of nitrogen in plants. We then identi- plex tobacco genome (around 50% larger fied a suitable target gene (for an enzyme than the human genome), which has in the nitrogen pathway) and devised a enabled the mapping of 69,500 genes and strategy for developing a new tobacco advanced our understanding of how the variety that would produce reduced tobacco plant functions. Genome-scale levels of TSNAs through incorporation sequencing, assembly, and characterisa- of the additional gene in the tobacco tion of the tobacco genome is a major plant’s DNA. Biochemical analysis of accomplishment that took nearly 3 years. these new plants confirmed a decrease As well as providing insights into in the levels of some TSNAs (specifically, gene-directed growth, regulation, and 4-(methylnitrosamino)-1-(3-pyridyl)-1- biochemistry, the sequence can be used butanone or NNK, and N’-nitrosonor- as a reference to identify genetic dif- nicotine or NNN) in the cured tobacco ferences between different varieties of when compared with control plants). tobacco, providing the potential to breed new cultivars with improved character- Toxicant reduction strategies – heavy istics for industrial applications. These metal reduction. Heavy metals are a could include cultivars that are less major concern in tobacco smoke because likely to produce harmful compounds they are linked to cancer and cardiovas- upon burning or, through understand- cular disease. Many plants absorb heavy ing the genetic regulation of metabolism, metals (for example, cadmium, mercury, for example, with improved yields. lead, arsenic, and chromium) through We aim to utilise the genome data in their roots. In most plant species, the combination with other ‘-omics’ data heavy metals are stored within the roots, types, such as transcriptomics, which but tobacco is unusual because the heavy measures gene expression, to better un- metals are translocated to and stored derstand the metabolic and biochemical in the leaves. We have been working to pathways driving the qualities of tobacco identify the genes responsible for heavy leaf. Improving the quality of the genome metal translocation to identify whether sequence and extent of annotation and tobacco could be modified so that the information related to the genome will heavy metals remain in the root. Research aid this process greatly. Such improve- has been on target genes linked to a ment has many facets and will require a promoter (gene switch) that is specific for significant amount of work, so there is the roots; engineering of this gene might a great deal of potential for collabora- ultimately result in any metals transported tion in this area; it is too big a task for into the plant root remaining there. any one party to carry out in isolation.

Agricultural sustainability — Aims for future reducing use of chemicals. Side shoots »» Develop greater understanding of the or ‘suckers’ have to be manually or genetic drivers for toxicant precursors chemically removed by the farmer, in tobacco, sustainability traits, and leaf which is a significant outlay for them. quality If suckers are allowed to grow, however, »» Improve the quality of the tobacco ge- yield, productivity, and quality of the nome sequence to a gold standard level leaf will decrease. Our aim is to prevent to support continued greater under- or decrease this growth, using a spe- standing of the tobacco plant cific promoter that is active in the leaf »» Grow our molecular breeding pro- axil (the point in the plant of side shoot gramme, developing genetic markers growth), thereby reducing use of crop for key traits of interest ☐

6 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 GOOD RESEARCH PRACTICE

GOOD RESEARCH PRACTICE — SECURING INNOVATION

Good Research Practice (GRP) describes data-capture technologies. In the the culture and processes that R&D ever-changing R&D environment, this have adopted to ensure that our scien- software platform has been aligned to tific research is conducted in a manner support product development design that aligns with industry, business, and control documentation needs. consumer, and regulatory expectations. This allows our researchers across R&D Originally introduced by the Medical to access the necessary procedures Research Council in the UK to estab- and information for conducting their lish common standards among its work and publishing their results. many research partners across the As well as building common un- JOHN MCBREARTY globe, GRP is now accepted in most derstanding across R&D to improve Quality Assurance Manager well-known research institutes and the integrity of our data and results is becoming a minimum standard and how they are used, GRP also Before joining us, John worked for 20 years in the benchmark for conducting any type provides a common research platform pharmaceutical industry of scientific research. Although not already used in other organisations. at global companies such a regulatory requirement itself, GRP Thus, GRP ensures transparency in the as Cyanamid, Wyeth, and is a standard that enables R&D to be research standards applied in R&D. Pfizer, where he gained a responsive in meeting current and Ensuring that GRP underpins all our wide range of qualifications, skills, and experience to future regulatory requirements, such as scientific research not only provides ensure processes and good laboratory, manufacturing, and confidence when publishing in peer- systems adhere to regulatory clinical practices, and ISO standards. reviewed scientific journals, but also standards within the R&D We build the principles of GRP into enables us to attain external licences environment. As R&D Quality our standard operating procedures and or accreditation for performing our Manager, John is responsible for ensuring our systems test methods. We have incorporated research work. Most recently our and processes meet existing GRP into the R&D electronic quality analytical laboratories achieved accredi- regulatory requirements, management system that manages tation from United Kingdom Accredita- are well prepared for future and governs our study plans, scien- tion Service in maintaining, a global requirements, and adhere to tific reports, procedures and training standard for competence in testing and Good Research Practices. records, and is integrated into our calibration laboratories ☐

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 35 ANALYTICAL METHOD DEVELOPMENT

8 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 ANALYTICAL METHOD DEVELOPMENT ANALYTICAL METHOD DEVELOPMENT

CHEMICAL CHARACTERISATION OF NEXT-GENERATION PRODUCTS

n accordance with our commitment established targeted methods are used to to the stewardship of new technolo- look for and measure very accurately a I gies and products, we have invested in small number of substances, non-targeted leading-edge capabilities for the evalu- methods are designed to detect the wid- ation of product chemistries. Assessing est possible range of substances and to the chemical and toxicological profiles of provide semi-quantitative data for initial e-cigarettes and tobacco-heating products toxicological assessment. For example, (THPs) presents technical challenges, the aerosol emitted by a THP might yield especially when a comparison with to- over 700 chromatographic peaks, for all bacco cigarettes is required for context. of which the chemical composition and Historically, the chemical evaluation approximate concentrations are estimated. of cigarettes and cigarette smoke has focused on a relatively small number (44) Recent successes of substances, the so called Hoffmann An area in which we have made significant analytes. These substances largely com- advances is the application of adsorption prise members of several chemical classes sampling of aerosols, gas chromatographic (including tobacco-specific nitrosamines separation, and mass-selective detection. or TSNAs, carbonyl compounds, aromatic Briefly, our approach comprises drawing compounds, and trace elements) that have the aerosol through a small tube contain- been frequently identified by numerous ing chemical adsorbents, which retain DR CHRISTOPHER WRIGHT researchers in tobacco smoke, are known most of its constituents by a combination Head of Analytical Science human carcinogens or other human of impaction and chemisorption. When toxicants, and are present at abundances As the Head of Analytical the tube is heated, the aerosol constitu- Science, Chris is the chemistry expected to cause adverse health effects ents are released, trapped, and passed lead for analytical strategy in humans. In addition to the Hoffmann into a gas chromatograph (GC). After and scientific practice. He list, however, over 6,000 other chemicals separation into their individual compo- is an experienced analytical have been detected in cigarette smoke. nents in the GC, the aerosol constituents chemist with nearly 30 years In contrast, our knowledge of the trace of research experience in the are eluted and passed into a time-of- areas of food, environment, chemistry of e-cigarettes and THPs is less flight mass spectrometer (TOF-MS). consumer products, and complete, largely because these products The comprehensive information tobacco. He joined us in 2008, are based on relatively new technolo- produced using this thermal desorp- with a focus on improving gies that are still undergoing significant tion GC-TOF-MS method has enabled the robustness of toxicant analysis and the development evolution. For example, e-cigarettes are us to detect substances in aerosols from of exploratory chemical available in several formats, including e-cigarettes and THPs at very low concen- screening programmes. Chris disposable ‘cig-a-like’ products, replace- trations – a few nanograms per puff, or has worked on committees for able cartomiser systems, and refillable less. The sensitivity of the method allows the International Organization designs, each with many variations in the structured evaluation of potential con- for Standardization, Royal Society of Chemistry, and materials used and power. We evaluate sumer exposure (for example, by applying Ministry of Agriculture, Fisheries the chemistry of new products as compre- thresholds of toxicological concern) and and Food/Department of hensively as possible in order to capture/ the review of chemistry data to priori- Health scientific committees. analyse as many substances as possible, tise substances for further investigation. and to do this we have developed non- Although not intended to be accurately targeted analytical techniques. Although quantitative, the method is internally

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 9 ANALYTICAL METHOD DEVELOPMENT

standardised and gives us orders of mag- optimised an approach that automatically nitude of the concentrations of the aerosol diverts large chromatographic peaks away constituents. Results to date are consistent from the TOF-MS to a different (flame with other published research demon- ionisation) detector. This approach, which strating that e-cigarette aerosols contain is highly reproducible, avoids overload- much lower concentrations of certain ing the TOF analyser and provides toxic substances than cigarette smoke. data that can be processed further. A major technical challenge has been We have also collaborated with the very wide range of amounts of aerosol researchers at the University of Liège in constituents. For instance, glycerol can Belgium to develop two-dimensional comprise more than 50% of the mass GC (GCxGC) methods with which to of an e-cigarette aerosol, whereas sub- evaluate our technologies. The advan- stances of potential concern may require tage of GCxGC is significantly increased monitoring at levels below 0.0000001%. chromatographic capacity to separate Measurement at both ends of this range complex mixtures into their individual simultaneously is physically impossi- components, which in turn improves our ble. Therefore, we have developed and confidence in the chemical identity of the detected substances. These techniques allow us to demonstrate bulk and specific Cigarette chemical differences between our technologies, visually and statistically (Figure 1). The processing and interpretation of such complex data sets also presents technical challenges, which we have ad- dressed by developing, in conjunction with a commercial partner, Genedata, a suite of chemical informatics tools to visualise and statistically analyse our data. The Genedata Expressionist software platform allows us to take data from many replicate analyses of a sample, build composite data files that capture all the relevant THP features and sort, cluster, and compare the information with a wide range of peer- validated statistical algorithms. These analyses enable us to make incremental chemical profile comparisons between established or well-characterised products and novel products or new variants to identify key chemical differences or correlations between different suites of chemicals. This new knowledge supports product assessment, product development, and the extension of technical standards.

E-cigarette Aims for the future »» Apply next generation multidimen- sional chromatography and accurate mass technologies to increase the confidence of identification of substances in e-cigarette and THP aerosols to 90% or greater; this approach has already enabled us to identify substances in e-cigarette aerosols quickly that would otherwise be extremely difficult, and in some cases almost impossible, to identify Figure 1: GCxGC-TOFMS chromatograms of cigarette smoke (solid phase »» Develop high-mass-accuracy spectral micro-extraction sampling) and aerosols from next generation products libraries for high performance liquid (thermal desorption sampling). The dynamic range of the e-cigarette chromatography/TOF mass spectrom- chromatogram is compressed by the high levels of glycerol in the aerosol. etry applications ☐

10 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 POPULATION STUDIES POPULATION STUDIES

ASSESSING THE IMPACT OF NEW PRODUCTS

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 11 POPULATION STUDIES

hen the Family Smoking Prevention and on a compartmental model using system Tobacco Control Act was introduced in dynamics. We think this is the best way of W the United States in 2009, the US Food and realistically representing complex behav- Drug Administration (FDA) described iours in an intuitive and transparent way. it as a population approach to minimis- The model takes into account all types of ing the harm caused by tobacco use. The smokers, including dual users of cigarettes agency was explicit in its wish to ensure and other tobacco products; factors such as that any regulations introduced should age, gender, and consumption; and types of have positive public health outcomes, not behaviour, including experimentation, initi- only for smokers but also for ex-smokers, ation, regular use, and cessation (Figure 1). non-smokers, adults, and vulnerable We try to reflect patterns of usage, utilising populations. The FDA’s industry guidance what’s happened in the past to predict what for introducing new products – whether might happen in the future. The model through substantial equivalents, pre-market is updated every year with birth, death, tobacco applications, or modified-risk and migration rates to provide long-term tobacco product (MRTP) applications projections of the impact of a new product. – expects tobacco companies to provide By using a status quo scenario (in which data on the likely impact a new product nothing changes) as a benchmark, such will have on the population as a whole. models can answer ‘what if?’ questions and This guidance seems consistent with predict how outcomes will change over time. approaches taken by regulators outside the The uptake of a new product is likely to USA. It could be argued, for example, that be influenced by a wide range of factors, the EU’s ban on snus outside Sweden is un- including fundamental characteristics, related to the product’s effect on individual actual performance, availability, price, risk, but the fear that its introduction will smoking history of the consumer, and, OSCAR CAMACHO increase initiation into tobacco, and eventu- potentially, for MRTPs, perception of its Computational Tools and ally lead consumers to cigarette smoking. relative health risks. Thus, any studies Statistics Manager This fear has proven to be unfounded in performed before the product is launched Oscar holds a BSc in the case of snus consumers in Sweden1. can only broadly suggest what might Mathematics with Statistics, as The potential impact of a new product happen after the product is introduced. well as an MSc in Biometrics, is highly complex, as it is contingent on In the case of MRTPs, FDA guidance and Applied Bioinformatics, before joining us in 2010 as a not only who consumes the product and in suggests post-market surveillance as a technical specialist in statistics. what amounts, but also how and why it is way of determining the actual popula- He now leads a group of data used (eg, in combination with other nico- tion impact a product has after launch. scientists providing support in tine products or as an aid to quit smoking). As ‘real’ post-market data are acquired statistics, bioinformatics and Moreover, behaviours are dynamic and are on usage patterns and fed into our mathematical modelling to R&D; guiding our scientists likely to change over time, for instance as model, we should be able to estimate to ensure sound data analysis consumers switch products or quit. To as- the potential long-term morbidity and techniques, data dissemination sess fully the potential impact of a product mortality effects on the population. and design of efficient on a population, a model must encompass To enable information to be collected experiments are applied many variables and be sensitive to changes. soon after a new product is introduced, throughout studies’ life cycle. Our approach, which aligns with previous an active sampling approach could be models recognised by regulators, is based used to obtain information directly from

12 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 POPULATION STUDIES POPULATION STUDIES

Birth rate Relapse to NGP

Quit NGP NGP user Former NGP user Relapse to dual use Never smoker Switch to smoking (never smoker) (never smoker)

Relapse to smoking NGP to dual use

Initiate smoking

Initiate dual use

Initiate smoking

Current smoker Relapse to smoking

Relapse to smoking

Revert to smoking Former NGP NGP dual user dual user Smoker to dual use Quit

Relapse to smoking Switch to NGP NGP to dual use NGP user (smoking history) Dual use to NGP Quit smoking Initiate NGP

Revert to NGP Quit NGP

Relapse to NGP Former smoker

Former NGP user Relapse to dual use (smoking history)

Relapse to dual use

consumers on current and historical made good progress in developing ques- Figure 1. System dynamics model use of tobacco and nicotine products. tionnaire-based tools to assess the impact illustrating the introduction of Parallel to this, the introduction of a of novel products on current smokers2. a novel reduced-risk tobacco or system to gather consumer comments, Additionally, we are analysing the nicotine product in a market. including any adverse health events, integrated output from population stud- could also provide valuable insights. ies and other data (eg, relative risk with The ability to collect meaningful post- respect to specific endpoints) to develop market information will depend on how a dynamic population model for pre- popular a new product becomes. As popu- dicting the impact of novel products. larity grows, cross-sectional studies could Our population model has been built and observe the use of the product in the context is currently undergoing validation testing of other products. Prospective observational with data sets on e-cigarette use in the UK studies could also be used to look at differ- and with historic data on snus use in Sweden. ent usage patterns, and if the numbers of study participants are sufficient, changes in Aims for the future health-related measures could be assessed. »» In collaboration with other external The appropriateness of tobacco com- groups, develop suitable protocols for; panies undertaking surveys with non- • Abuse liability assessment smokers, and particularly those who are • Consumer (smokers and non-smokers) underage, presents a challenge. Our mar- risk perception and comprehension keting principles do not allow such research »» Build a framework for potential product and, therefore, if necessary, we would uptake by various groups need to develop, with regulators, third- »» Develop a tiered approach for post- party approaches to collecting such data. market surveillance »» Publish the first analysis using the Recent successes dynamic population model ☐ We are working with external experts to develop and validate questionnaires and 1. ESTOC. Factsheet on Gateway to Smoking. http://estoc.org/library/fact-sheets (accessed Nov other research tools that can provide insights 17, 2015). into the likely uptake of novel products 2. Ashley, M et al., Pilot study to evaluate selected before they are actually launched. These questions of subjective measures of individuals’ motivations for, and use of, tobacco and nicotine address factors such as intent to use, risk products. Presented at CORESTA SSPT 2015; Jeju, perception, and abuse potential. We have South Korea: 4–8 October 2015.

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 13 STEWARDSHIP

LEADING THE WAY IN PRODUCT RISK ASSESSMENT

&D’s Product Stewardship function consists techniques to assess emissions from of scientists and regulatory professionals next-generation products to identify R who have a global remit to ensure that our and assess risks related to any com- products are developed and manufactured pounds above thresholds of toxicological to meet our duty-of-care standards, and concern are compliant with all relevant regulations »» A three-dimensional in vitro model of in the country or jurisdiction of sale. lung epithelium to assess the irritation We aim to be a recognised leader in the potential of product ingredients responsible stewardship of all tobacco and nicotine products, including e-cigarettes, We presented our contact sensitisation tobacco heating products and snus. We risk assessment approaches for snus and actively guide research and development e-liquid ingredients at the Congress of activities enabling product risk to be care- the European Societies of Toxicology in fully managed and overseeing the delivery 2014. Additionally, we have developed of quality products to the consumer. and published our risk assessment ap- Our approaches are based on well- proach to the screening of flavours for established principles of toxicological and e-liquids. This information formed part of safety risk assessment. They enable us a presentation to the FDA in early 2015. to define acceptability criteria for many We have used self-reported vaping aspects of product specification and behaviour and consumption data from performance, in the interests of consumer recent surveys of e-cigarettes users to safety. These in turn form the basis of the increase the robustness of the evidence- company’s stewardship guidelines and based exposure paradigms that inform our DR AUDREY RICHTER standards across our product portfolio. quantitative toxicological risk assessments. Head of Product Stewardship Our stewardship remit incorpo- Product Stewardship partnered with Audrey started her career in rates the entire product lifecycle and, Nicoventures to propose product standards Medical Laboratory Sciences as such, we provide expert advice and for electronic cigarettes; these have been before moving to academia. She support to the development, manufac- shared with some external scientific and spent 15 years at the University ture and distribution of products. regulatory parties, including the Brit- of Southampton both in Medical Oncology, where she gained The product stewardship team oversees ish Standards Institute (BSI). Following her PhD, and in Respiratory Cell the compilation of key product data and consultation with a number of interested and Molecular Biology. Audrey scientific support packages required for parties, the BSI has now published a vol- joined us in 2002 and set up an regulatory reporting. We provide expert untary standard for the product category. in vitro models work programme technical guidance to all our group com- before taking on a series of leadership roles in Biosciences, panies, for example, on chemicals legisla- Challenges requiring collaboration Smokeless Tobacco Science tion, ingredients restrictions and bans, and The field of toxicology is evolving rapidly as and Clinical Studies. In her legislated disclosure processes to enable research into in silico and in vitro methods current role, as Head of Product regulatory engagement and compliance. and systems biology, for example, reveal Stewardship, she now ensures that we perform rigorous valuable approaches to risk assessment. We stewardship assessments Recent successes continue to keep abreast of the latest devel- on product innovations. We have incorporated new methods into opments and apply new techniques when our risk assessment approach, including: they add value to our current approaches. »» Advanced non-targeted analysis One particular challenge is translating in

14 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 STEWARDSHIP STEWARDSHIP

vitro data into quantitative risk assessment on a regular basis. External experts in cesses and supporting data sets for next- to support specific ingredient levels in new consumer research with a strong interest generation products as they become products for all product categories. As in tobacco and nicotine products provide mainstream commercial products this issue impacts the field of toxicology invaluable guidance on methodologies »» Contribute to the development of global generally, we will engage in the develop- and approaches to gathering relevant data voluntary and regulatory product stand- ment of new approaches through scientific as well as alerting us to trends and behav- ards for e-cigarettes and other emerging forums and follow qualified recommenda- iours in particular consumer groups. product categories tions and best practices as they emerge. »» Quantify the risks and potential toxicant Consumer behaviour changes con- Aims for the future reduction benefits of next-generation stantly in relation to many factors, includ- »» Continue to work in partnership with products with use of the latest scientific ing lifestyle choices, environmental and our Analytical Development Centre to data, in the context of tobacco harm financial pressures, and becoming accus- develop and deliver new methodologies reduction tomed to new products. Exposure estimates for the characterisation and quantifica- »» Continue to publish and communicate are a key factor in our risk assessment tion of emissions from next-generation externally on our approaches to the risk paradigms, so we need to be mindful of products to address new challenges and assessment of next-generation products trends and collect data that will enable us knowledge gaps to enable dialogue and engagement with to review our guidelines and standards »» Standardise our risk assessment pro- scientists and regulators ☐

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 15 TOBACCO HEATING PRODUCTS

Tobacco heating devices Combustion of cigarettes

Distillation Pyrolysis Combustion

O N N H H N N 2 O H H N H H C C O N OH O H C C H N H HH HO OH H N H 2 N OH H H

HO H Tobacco thermal decomposition Tobacco

0 100 200 300 400 500 600 700 800 900 1000

Temperature (°C)

Figure 1: Generation of toxicants at different temperatures. Heating at lower temperatures rather than burning generates fewer and lower levels of toxicants.

16 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 TOBACCO HEATING PRODUCTS TOBACCO HEATING PRODUCTS

ELIMINATING COMBUSTION TO POTENTIALLY REDUCE RISK

he scientific community widely agrees that reductions was unclear. To reduce toxicant the majority of smoking-related diseases formation but retain a taste resembling T are caused not by nicotine but by toxi- cigarette smoke, therefore, we have been cants formed during the combustion and developing tobacco heating products pyrolysis of tobacco. Research in tobacco (THPs) that heat tobacco to between thermochemistry has established that 200°C and 300°C, a temperature range different classes of tobacco constituents sufficient to vaporise the nicotine (and decompose at different temperatures, some other volatile compounds) into an releasing chemical compounds into the inhalable aerosol stream, but not enough aerosol as they do so. Because the tip of a to burn the tobacco and cause pyrolysis burning cigarette undergoes a very wide or combustion. As a result, the aerosol range of temperatures (from ambient to produced by these products contains about 1000°C), the smoke it produces con- nicotine and compounds that provide tains a vast array of chemicals (Figure 1). the consumer with taste and flavour but Studies have shown, for example, that with far fewer or lower levels of toxicants at lower temperatures (between 100°C than are present in cigarette smoke. and 200°C), water and volatile substances, There are two main types of THPs. including flavour molecules, loosely bound The first is based on a battery-powered to the surface of the tobacco leaf are the heating source and often resembles an first to be released through distillation and e-cigarette (Figure 3). A user inserts the evaporation. Although the boiling point tobacco rod into the heating chamber, of nicotine is around 250°C, it starts to va- which contains the control circuitry porise and enter the aerosol between 170°C necessary for regulating temperature and and 200°C. At 300–400°C, some decompo- is turned on by pressing a button. The sition components start to form from the second, a carbon-tip-fuelled product, nor- breakdown of cellulose and other struc- mally looks like a conventional cigarette tural components of the tobacco plant, but and is lit in the conventional manner. DR CHUAN LIU it is not until temperatures of more than During use, air is heated as it is drawn Head of Tobacco Heating 400°C are reached that the main pyrolysis through the lit carbon source. The hot air Product Science occurs. Leaf components, such as amino passes through the tobacco, extracting acids and esters, decompose at 400–600°C, flavour and releasing nicotine (and other Chuan holds a PhD in materials and carboxymethylcelluloses and carbon- aerosol constituents) to form a vapour. This science from Cambridge University, and since joining us ates at 600–900°C, producing the bulk of vapour passes through a filter or mouth- in 2000, has become one of the carbon monoxide, polycyclic aromatic piece and is delivered as an aerosol stream. our key experts on pyrolysis, hydrocarbons, and other combustion prod- These products do not burn down or emit smoke formation, and toxicant- ucts that form the tar in cigarette smoke. a strong smell like conventional cigarettes, reduction technologies for and the tobacco within, which may also cigarettes. Since 2014, Chuan has been the lead scientist Recent successes be blended and treated to ensure nicotine for our tobacco-heating Attempts to selectively modify the complex release, maintains its shape after use. products, ensuring high-quality combustion process in conventional ciga- Although THPs generate aerosols standards of research for this rettes have not been successful. Although containing significantly fewer toxicants emerging product category. a reduction in toxicant exposure was than cigarette smoke, the exact fractions achieved, the biological impact of these depend on many variables, including

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 17 TOBACCO HEATING PRODUCTS

91% 90% 89% reduction reduction reduction

WHO FDA HEALTH CANADA 9 chemicals 18 chemicals 44 chemicals

TOBACCO HEATING PRODUCT

REFERENCE CIGARETTE

no no 87% mutagenicity* mutagenicity* reduction

MUTAGENICITY MUTAGENICITY CYTOTOXICITY (particles) (whole aerosol)

TOBACCO HEATING PRODUCT

REFERENCE CIGARETTE * No observed mutagenicity under these test conditions

99% no 94% reduction genotoxicity* reduction

OXIDATIVE γ-H2AX BHAS STRESS (DNA damage) (Tumour promotion)

TOBACCO HEATING PRODUCT

REFERENCE CIGARETTE * No genotoxicity, oxidative stress or tumour promotion was found under these test conditions

Figure 2. Testing of aerosols from prototype THPs shows fewer toxicant emissions and substantially reduced mutagenicity, genotoxicity and oxidative stress compared with smoke from conventional cigarettes.

operating temperature and the tobacco It must be remembered that the stew- blend used. To guide the responsible ardship of cigarettes is not suitable for development and appropriate oversight of THPs. The lower temperatures involved these next-generation products, we need means that some of the processes seen in to develop a full understanding of how cigarettes do not occur in heated tobacco THPs operate, how consumers use them, and vice versa. Testing regimes might and a suitable approach to stewardship. need to be adapted and standardised for

18 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 TOBACCO HEATING PRODUCTS TOBACCO HEATING PRODUCTS

Battery/electronics Heat source Tobacco rod Mouthpieces Tobacco vapour

Figure 3. An illustration of how a rechargeable battery-powered tobacco heating product could function, where a tobacco rod is heated to a controlled temperature for aerosol generation.

THP aerosols, which are considerably less assays to measure oxidative stress, inflam- requiring additional research into prod- complex than cigarette smoke. In addi- mation, and cytotoxicity in lung epithelial uct science, stewardship, and substantiation, research must focus on potential new cells, and have shown that these models tion of their potential for reduced risk. toxicants as well as those already associated are suitably sensitive to compare different We are keen to engage with academic, with smoking cigarettes. New sampling nicotine-delivery products. Studies using industrial and entrepreneurial partners techniques and methodologies for chemical these models show that cellular responses who can help us achieve the best possible analyses are needed to thoroughly charac- indicative of oxidative stress are lower in THPs in terms of risk and satisfaction. terise these products and their potential to cells exposed to THP aerosol than in cells reduce the toxic effects of tobacco smoking. exposed to cigarette smoke (Figure 2c). Aims for the future At our Analytical Development Centre, However, there is still more to be done »» Continue to evaluate how types of we have already developed a range of sensi- to fully characterise THP emissions, in tobacco and processing and blending tive methods for characterising emissions particular to enable the use of these data influence the chemical and sensory from THPs and used them to demonstrate in risk assessments. Further evaluation profiles of THP aerosols that levels of known smoke toxicants are will require a series of multi-disciplinary »» Continue to develop our understanding of significantly lower than in cigarette smoke studies that must also consider patterns of the THP category to ensure that our THP (Figure 2a). Using a smoking machine consumer use. Successful development will products are stewarded to meet state-of- adapted to generate, dilute, and deliver require tobacco science, portable electron- the-art product safety assessments THP aerosols to a bronchial epithelial- ics, and consumer insights to be combined »» Engage with regulators, policy makers cell cytotoxicity model, we have shown in such a way that meets modern societal and public-health advocates, and publish significantly less cytotoxicity than with requirements for nicotine consumption. relevant, robust scientific data to ensure conventional cigarette smoke (Figure 2b). This novel category of tobacco product that THP regulation is appropriate and We have also developed a panel of in vitro continues to present new challenges based on sound scientific evidence ☐

innovation in harm reduction discover bat-science.com

@BAT_Sci

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 19 ELECTRONIC CIGARETTES

20 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 ELECTRONIC CIGARETTES ELECTRONIC CIGARETTES

RAPID EVOLUTION PRESENTS NEW CHALLENGES

he types of e-cigarettes sold, the science findings. For example, one study has shown base examining their impact on users’ that mice exposed to e-cigarette vapour T health, the emergence of product stand- display significantly impaired pulmonary ards and regulations have seen rapid bacterial clearance compared with mice developments over the last 12 months. exposed to air4, whereas a study conducted Continuing innovation and commit- by our own scientists has found little cyto- ment to product stewardship are needed toxic response from e-cigarette aerosols to deliver on the public-health promise in human reconstituted airway tissues5. of e-cigarettes and provide consum- Essentially, all e-cigarettes are ers with the safest possible products. battery-powered devices that heat a We have previously described the liquid (e-liquid) to produce an inhal- emerging popularity of e-cigarettes able aerosol. E-liquid usually contains as a potential tool for reducing the excipient (glycerol, propylene glycol and/ harm associated with cigarette smok- or water), flavourings, and, in many cases, ing. Evidence to date suggests that, nicotine, although nicotine-free prepara- although their use may not be entirely tions are available. The amount, content, safe, e-cigarettes have the potential to be and characteristics of the aerosol depend substantially less risky than cigarettes. mainly on the power applied to the heating Nevertheless, debate about e-cigarette coil and the physical characteristics of the safety continues, and we have seen a sharp e-liquid formulation (such as composition, rise in numbers of associated scientific viscosity, and its specific heat capacity). studies published. Many have focused on A huge variety of e-cigarettes is now DR KEVIN MCADAM contents of the aerosol, possible health available. These include disposable and Head of Nicoventures effects associated with e-cigarette use, rechargeable devices, many of which Research the potential of e-cigarettes to act as a resemble conventional cigarettes in

Kevin is an experienced chemist, gateway to cigarette smoking, and their size and shape; larger, more-complex who has spent the past effectiveness as smoking-cessation aids. closed modular systems with replace- 30 years researching tobacco As researchers develop a deeper able flavour cartridges; open modular harm reduction. Kevin understanding of these issues, discrepan- devices to which users add their own specialised in combustion cies between study findings are emerg- e-liquids; and dripping devices. A vast chemistry during his PhD and atmospheric pollution ing. A recent example of this was the assortment of flavours (estimated at more during his postdoctoral widely disseminated research purporting than 7,000) is also sold worldwide. training. Kevin joined BAT to show higher levels of formaldehyde With the range of devices and ways of in 1987, and has worked on and acetaldehyde in e-cigarette aerosol customising them evolving rapidly, there the science underpinning than in cigarette smoke1.Those studies, is a real concern about the lack of product tobacco harm reduction, including smokeless tobacco however, have been strongly criticised quality standards across the e-cigarette in- products, reduced-toxicant by some e-cigarette and public health dustry. Manufacturing methods, materials cigarettes, tobacco-heating experts for their unrealistic heating and device performance can differ greatly, products, and e-cigarettes. He conditions2 and their failure to take into and few devices are properly tested before now heads our Nicoventures account likely consumer behaviour3. sale. To safeguard consumers, we continue research programme. Research into the health effects of e-cig- to advocate standardisation and ensure arette use is also providing contradictory that our devices, e-liquids, and the vapour

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 21 ELECTRONIC CIGARETTES

22 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 ELECTRONIC CIGARETTES ELECTRONIC CIGARETTES

Laser testing of e-cigarette aerosol Droplet size and distribution are important characteristics of an aerosol. Malvern Instrument’s Spraytec laser diffraction system measures the intensity of light scattered as a laser beam passes through an aerosol. Because droplets of a particular size will diffract light in a certain way, these diffraction measurements can be used to calculate droplet size distributions in real-time, enabling efficient characterization of aerosols to generate regulatory-compliant data and support product development.

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 23 ELECTRONIC CIGARETTES

products, taking into account not only the a device materials and e-liquid ingredients, but also compounds in the vapour that result from heating the e-liquid. We use a wide range of analytical techniques, spe- 99.5% 99% 95% reduction reduction reduction cialised laboratory technology, and expertise to first develop and then conduct detailed tests of the vapour from the flavours and e-liquids we formulate, the products we manufacture, and the combinations WHO FDA HEALTH CANADA 9 chemicals 18 chemicals 44 chemicals in which they are sold. We also study the way consumers actually use e-cigarettes to ELECTRONIC CIGARETTE make sure our risk assessments are based on and reflect consumers’ behaviours. REFERENCE CIGARETTE Only when we are fully satisfied that a product meets our high quality standards do we allow a product on the market. Concerns over e-cigarettes has led to b the development of a global regulatory divide, with some countries (eg Singapore) instigating bans on all e-cigarettes, others no no 91% (eg Canada) restricting only those con- mutagenicity* mutagenicity* reduction taining nicotine, and still others allowing free use of e-cigarettes. Recently, Wales an- nounced a ban on e-cigarette use in public.

MUTAGENICITY MUTAGENICITY CYTOTOXICITY Recent successes (particles) (whole aerosol) To promote good practice across the industry, we believe it is our responsibil- ELECTRONIC CIGARETTE ity to share our knowledge and contribute REFERENCE CIGARETTE to the science of vaping. Our scientists * No observed mutagenicity under these test conditions have presented our scientific insights and approach to stewardship of e-cigarette flavours at major science conferences c hosted by the US Food and Drug Admin- istration (FDA), the Society for Research on Nicotine and Tobacco, and the Global no no tumour Forum on Nicotine workshops. We also oxidative no genotoxicity* promotion recently published the first practical guide stress activity on how to develop flavours responsibly, based on sound toxicological principles6. We have conducted a wide ranging comparison of the relative toxicology of e-ciga- OXIDATIVE γ-H2AX BHAS STRESS (DNA damage) (Tumour promotion) rettes and a conventional cigarette (Figure 1). Measurement of a number of toxicants ELECTRONIC CIGARETTE corresponding to toxicant lists identified REFERENCE CIGARETTE by the WHO, FDA and Health Canada * No observed mutagenicity under these test conditions showed 95-99% lower levels of these toxicants in e-cigarette emissions than in cigarette smoke. Similarly, a series of in vitro Figure 1. Testing of Vype ePen aerosols shows fewer toxicant emissions toxicology tesets examing a number of bi- and substantially reduced mutagenicity, genotoxicity and oxidative stress oassays covering mutagenicity, cytotoxicy, compared with smoke from conventional cigarettes. oxidtative stress, genotoxicity or tumour promition activity, in which cigarette produced meet our high quality standards. smoke gives clear responses, showed that BAT’s and Nicoventures’ entire ap- e-cigarette aerosols were either inactive proach to e-cigarettes is underpinned by or 90% less activity than cigarette smoke. responsible product stewardship. We be- We continue to participate fully in the lieve that it is vital to know and rigorously development of national and international test exactly what each e-cigarette contains standards aimed at phasing out poorly and produces. We carefully assess our manufactured products and safeguarding

24 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 ELECTRONIC CIGARETTES ELECTRONIC CIGARETTES

consumers. In April, 2015, for example, Looking forward constituents of e-liquids and e-cigarette France’s association for standardisa- We anticipate that the year ahead will see aerosols, as part of our commitment to tion, AFNOR, published the world’s first significant developments in the world of effective product stewardship national standards for e-cigarettes and e-cigarettes. For example the national e- »» Continue to publish our scientific e-liquids 7. We were active contributors cigarette standards will serve as a roadmap research on e-cigarettes and participants in the development of for draft European product standards, »» As ever, we welcome the opportunity these rigorous product standards, which on which the European Committee for for collaborative research with external set out a series of voluntary safety and Standardisation has begun work. In scientists in the field ☐ quality criteria for designing and testing addition, voluntary global standards products before they reach the mar- aimed at analytical laboratories are cur- 1. Jensen, RP et al., Hidden formaldehyde in e-cigarette aerosols. N. Engl. J. Med. 2015; 372: ket. The standards aim to improve the rently being developed by CORESTA 392–394. practices of all manufacturers, suppliers, and the International Organization for 2. Bates, C. Spreading fear and confusion with misleading formaldehyde studies. http://www. test laboratories, and distributors with Standardization and are expected to be clivebates.com/?p=2706 (accessed Aug 11, 2015). a view to minimising the risks of use to published over the next few years. 3. Farsalinos, KE et al. Nicotine absorption from consumers by defining construction and The FDA is working to finalise e- electronic cigarette use: comparison between experienced consumers (vapers) and naïve users performance criteria for e-cigarettes, cigarette regulations and the European (smokers). Sci. Rep. 2015; 5: 11269. identifying purity standards for author- Tobacco Product Directive will be imple- 4. Sussan, TE et al., Exposure to electronic cigarettes ised ingredients, and defining a list of mented fully in national laws in 2016. The impairs pulmonary anti-bacterial and anti-viral defenses in a mouse model. Plos One 2015; 10: banned substances in e-liquids. These first legally binding regulatory frameworks e0116861. standards were the result of work by close in Europe and the USA, therefore, will 5. Neilson, L et al., Development of an in vitro cytotoxicity model for aerosol exposure using 3D to 60 organisations, including consumer be clarified throughout 2016 and 2017. reconstructed human airway tissue; application for associations, test laboratories, health assessment of e-cigarette aerosol. Toxicol. In Vitro organisations, and manufacturers. A vol- Aims for the future 2015; 29: 1952–1962. 6. Costigan, S & Meredith, C, An approach to untary standard focused on characterising »» Continue to understand what our con- ingredient screening and toxicological risk e-cigarette emissions is expected shortly. sumers are seeking from our products assessment of flavours in e-liquids. Regul. Toxicol. The UK’s British Standards Institution and how they use them Pharmacol. 2015; 72: 361–369. 7. AFNOR. AFNOR publishes the world’s first published similar e-cigarette guidelines »» Continue to research flavour and sen- voluntary standards for electronic cigarettes in June 2015, and, once again, BAT and sory science and e-liquids. http://www.afnor.org/en/news/ » news/2015/avril-2015/afnor-publishes-the- Nicoventures were strong contributors » Continue to refine and develop analyti- world-s-first-voluntary-standards-for-electronic- to the development of these standards. cal approaches to characterise low-level cigarettes-and-e-liquids (accessed Aug 11, 2015).

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 25 PRODUCT TESTING

26 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 PRODUCT TESTING PRODUCT TESTING

BUILDING THE EVIDENCE BASE

e are using cutting-edge science and order: conventional cigarettes > THPs technology to develop both a range of > smokeless tobacco > e-cigarettes > W innovative tobacco heating products licensed medicinal products (Figure 1). (THPs) and nicotine products, such as The aim of our research is to expand electronic cigarettes (e-cigarettes) and understanding of the science supporting the science to evaluate them. We are the evaluation of next-generation prod- driving the science in characterisation of ucts and a determination of whether their toxicants in smoke and aerosols, learning placement on a risk spectrum is justified. how exposure to these toxicants trans- To this end, and in line with likely lates to risk of developing disease, and regulatory requirements, BAT has working on substantiating the reduced- established a framework for assess- risk potential of THPs and e-cigarettes ing new products for their potential for compared with conventional cigarettes. risk reduction by integrating chemi- The risks of smoking conventional cal, toxicological, and human studies at combustible cigarettes are well known. It is the individual and population levels. firmly established that the combustion of We have established a range of tools to tobacco results in a smoke containing more characterise rigorously toxicant yields in than 6,000 different constituents, of which next generation product emissions and approximately 150 are known human toxi- to evaluate responses to these products cants. Most of the diseases associated with in in vitro toxicity tests compared with DR MARIANNA GAÇA smoking, such as cardiovascular disease, results for conventional cigarettes. Our Pre-Clinical Assessment chronic obstructive pulmonary disease, and toxicity testing involves a battery of in Manager cancer, are caused by inhaling this smoke. vitro assays, including those already

Marianna holds a PhD in cell In an effort to reduce the risks associ- recommended by the Organisation for and molecular medicine and ated with tobacco use, we are focusing Economic Cooperation and Develop- did her postdoctoral work at our research and development on new ment (OECD), such as the Ames, in vitro Yale School of Medicine and products such as THPs, e-cigarettes, and micronucleus, and mouse lymphoma University of Pennsylvania before low-toxicant oral tobacco products (eg assays for genotoxicity the Neutral Red joining us in 2004. She has held various roles investigating snus). These products deliver nicotine Uptake assay for cytotoxicity. We are also the biological effects of without the smoke that is generated by developing other in vitro assays to assess cigarette smoke, including the combustible cigarettes and, as such, are key tobacco-related disease endpoints development of in vitro models considered to hold potential for reducing in commercially available human tissue of tobacco related-diseases the risks associated with tobacco use. If systems and models, including the use of and whole-smoke exposure systems. Marianna has also the reduced risk potential is substantiated, primary cell types and three-dimensional represented BAT (and the wider it will be important that it is supported reconstituted human tissue models, tobacco industry) at board by a robust body of evidence, both for such as MucilAirTM and EpiAirwayTM. level on many in vitro societies, consumer confidence in our products including IVTIP and ASCCT. and for evidence-based regulation. Recent successes She is passionate about the development and application of In 2014, we adopted McNeill and Although some of the toxicity assays 1 in vitro models as alternatives to Munafo’s tobacco and nicotine risk we employ are commonly used by the product assessment in animals. continuum and adapted it for our use, tobacco industry and the methodologies ranking products by exposure of con- are consolidated through CORESTA (an sumers to toxicants in the following association founded in 1956 to promote

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 27 PRODUCT TESTING

CONVENTIONAL TOBACCO-HEATING LOW-TOXICANT ELECTRONIC LICENSED CIGARATTES PRODUCTS SMOKELESS CIGARETTES MEDICINAL TOBACCO PRODUCTS

HIGH EXPOSURE TO TOXICANTS LOW

Figure 1: Adapted tobacco and nicotine risk continuum.

international cooperation in scientific re- research at many scientific conferences search relative to tobacco), our research- and welcome dialogue with other scien- ers are adapting them where necessary tists. All material we have presented at and applicable to assess next generation scientific conferences can be accessed on products more accurately. For example, our science website, bat-science.com. we recently modified the Ames test to enable the assessment of aerosols and Aims for the future whole smoke2,3 and have applied existing »» Publish the latest datasets on next irritancy testing standards to the testing generation products, including the of e-cigarette vapour in the EpiAirwayTM science underpinning the chemical and model4,showing e-cigarette vapour pro- biological tests that we are using ducing results similar to those for air. »» Do more to integrate in silico and in We have also started to participate in vitro toxicology assessments a new series of workshops initiated and »» Look at the scientific ways to bridge moderated by independent organisa- between datasets, facilitating the more tions, such as the Institute for In Vitro efficient development of products by Sciences (IIVS). This represents a good avoiding duplication of tests ☐ example of how public health, academic and industry scientists, and regulators, 1. McNeill, A & Munafò, MR, Reducing harm from tobacco use. J. Psychopharmacol. 2013; 27: 13–18. can come together with a view to debating 2. Kilford, J et al., A method for assessment of the potential science frameworks that could genotoxicity of mainstream cigarette-smoke by be used for evidence-based regulation use of the bacterial reverse-mutation assay and an aerosol-based exposure system. Mutat. Res. 2014; of products across the risk continuum. 769: 20–28. We have carried out a variety of tests 3. Azzopardi, D et al., Evaluation of an air–liquid on Vype ePen, one of our commercially interface cell culture model for studies on the inflammatory and cytotoxic responses to tobacco available e-cigarettes, and a prototype smoke aerosols. Toxicol. in Vitro 2015; 29: THP, which is under development in 1720–1728. 4. Neilson, L et al., Development of an in vitro R&D, and the results are encouraging. cytotoxicity model for aerosol exposure using 3D Initial studies published by other groups5,6 reconstructed human airway tissue; application are contributing to the establishment of an for assessment of e-cigarette aerosol. Toxicol. in Vitro 2015; 29: 1952–1962. evidence base with the potential to sub- 5. Zenzen, V et al., Reduced exposure evaluation of stantiate that THPs and e-cigarettes gen- an electrically heated cigarette smoking system. Part 2: smoke chemistry and in vitro toxicological erate substantially lower levels of toxicant evaluation using smoking regimens reflecting emissions than conventional cigarettes. human puffing behaviour. Regul. Toxicol. We have a strong heritage in scien- Pharmacol. 2012; 64 (suppl. 1): S11–S34. 6. Cheng, T, Chemical evaluation of electronic tific engagement, and to promote best cigarettes. Tob. Control 2014; 23 (suppl. 2): ii11– practice we openly share and present our ii17.

28 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 QUANTIFYING RISK QUANTIFYING RISK

IMPROVED UNDERSTANDING OF TOXICANTS AND DISEASE

e have set out a framework of studies that calculated MOE and ascribing categories we think need to be considered when try- for toxicants with limited or insufficient W ing to determine whether tobacco heating data to generate a MOE2.We also described products or e-cigarettes could be described the concept of calculating cumulative as reduced risk products in comparison MOEs for groups of toxicants with similar with cigarettes (Figure 1). One part of this toxicological properties, illustrated with framework is computational toxicology. aldehydes present in tobacco smoke3. We have for several years been us- The World Health Organization (WHO) ing computational toxicology to try and Study Group on Tobacco Product Regula- prioritise toxicants found in tobacco smoke. tion has proposed a list of 18 tobacco smoke Although aerosols from next generations toxicants for reduction and monitoring. products have fewer chemicals, there Some of these toxicants are found in the are still some toxicants present. As this vapour phase of tobacco smoke, others in is the case, improved understanding of the particulate phase, and the remainder in the role and dose–response relationships both phases. We have recently completed between toxicants in aerosols and smoke an assessment of all the particulate-phase from our products and tobacco-related toxicants with our suite of genotoxicity diseases will help us focus the develop- tests, which includes in vitro micronu- ment of technologies for reducing the cleus, Ames, and mouse lymphoma assays. levels of those toxicants. It will also help We have also evaluated a number of the us quantify residual risks as we continue particulate-phase toxicants with our in vitro to develop a range of new products aimed models of disease. We have adapted the at reducing exposure to certain toxicants. Ames mutagenicity and neutral red uptake Our quantitative risk assessment ap- cytotoxicity assays for analysing gases4, proach (Figure 2) is based on integrat- and we are currently using this methodol- ing predictive and experimental models. ogy to assess the remainder of the WHO Computer modelling approaches, includ- toxicant list with use of the appropriate FIONA CUNNINGHAM ing margin of exposure (MOE) calcula- genotoxicity tests and disease models. Combustible Toxicology tions, mode of action (MOA) reviews, and Scientist physiologically based pharmacokinetic Aims for the future Fiona joined our in vitro (PBPK) modelling, are supplemented where »» Identify tobacco smoke toxicants that are models team in 2005, with necessary with data from in vitro models of known to be present in the aerosol from a BSc in chemistry, and has disease and conventional in vitro toxicol- next generation products and apply our since completed an MSc in ogy assays, as directed by the MOA. computational tools to predict their likely environmental toxicology and pollution monitoring. impact on biological activity Recently Fiona’s main focus has Recent successes »» Continue to generate and refine MOEs been prioritisation of tobacco In 2011, we published a paper outlining our where possible for all toxicants of interest smoke toxicants with use of use of MOEs to segregate tobacco smoke »» Use the gaseous exposure system within several in silico techniques, toxicants and prioritise our research1. R&D to test gaseous tobacco smoke toxi- and she has become one of the leading experts in this field This was followed by a presentation at the cants in our in vitro models of disease within the tobacco industry. Tobacco Science Research Conference, »» Use the MOA reviews to direct the as- in which we proposed the categorising of sessment of individual tobacco smoke tobacco smoke toxicants according to the toxicants within relevant in vitro models

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In vitro regulatory In vitro models toxicology of disease

Chemical and Exposure and physical pharmacokinetic Post market characterisation study Systems Science surveillance

TYPE OF Product design Computational Biomarker of Consumer STUDY stability Toxicology effect study perception study

Stewardship Toxicant exposure Individual risk Population risk PURPOSE science reduction reduction reduction

Figure 1: Studies required to substantiate reduced-risk potential of next generation tobacco and nioctine products.

01110001 01110101 01100001 01101110MOE 01110100 01101001 01100110 01111001 01101001 01101110 01100111 00100000analysis 01110010 01101001 01110011 01101011 01110001 01110101 01100001 01101110 01110100 01101001 01100110 01111001 01101001 01101110 01100111 00100000Range of 01110010 01101001 01110011 01101011 High priority <10,000 >10,000 High priority 01110001 01110101 01100001 01101110 MOEs 01110100 01101001 01100110 01111001 01101001 01101110 01100111 00100000 01110010 01101001 01110011 01101011 01110001 01110101 01100001 01101110 01110100 01101001 01100110 01111001 MOE 01101001 01101110 01100111 00100000analysis 01110010 01101001 01110011 01101011 01110001 01110101 01100001 01101110 01110100 01101001 01100110 01111001 01101001 01101110 01100111 00100000 01110010 01101001 01110011 01101011 01110001 01110101Key event 01100001Key event01101110 Key01110100 event 01101001Key event 01100110 01111001 01101001 01101110I 01100111 II00100000 01110010III 01101001IV 01110011 01101011 01110001 01110101 01100001 01101110 01110100 01101001 01100110 01111001 01101001 01101110 01100111 00100000 01110010 01101001 01110011 01101011 01110001 01110101 01100001 01101110In vitro 01110100 01101001 01100110 01111001 01101001 01101110 01100111 00100000testing 01110010 01101001 01110011 01101011 01110001 01110101 01100001 01101110 01110100 01101001 01100110 01111001 01101001 01101110 01100111 00100000 01110010 01101001High 01110011 priority 01101011 01110001 01110101 01100001 01101110PoD 01110100 01101001 01100110 01111001 PBPK 01101001 01101110 01100111 00100000analysis 01110010 01101001 01110011 01101011 01110001 01110101 01100001 01101110 01110100 01101001Low 01100110 priority 01111001

Figure 2: Our quantitative risk assessment approach.

of disease and/or traditional genetic tobacco smoke toxicants. Food Chem Toxicol 2011; 49: 2921–2933. toxicology assays 2. Cunningham, F et al., Tools for the prioritisation of »» Develop toxicokinetic and PBPK models, tobacco smoke toxicants: an overview. Recent Adv. Tob. Sci. 2012; 39: 21–31. where possible, for all of the 18 WHO 3. Cunningham, F et al., Cumulative risk assessment Study Group on Tobacco Product Regu- of three aldehydes present in tobacco smoke: using lation chemicals and adapt an established margin of exposure (MOE) and mode of action 5 (MOA) evaluations. Toxicologist 2012; 126: 25. model for nicotine for use with nicotine 4. Breheny, D et al., Application of a modified gaseous delivery devices, such as e-cigarettes exposure system to the in vitro toxicological » assessment of tobacco smoke toxicants. Environ. » Develop an approach to the risk assess- Mol. Mutagen. 2014; 55: 662–672. ment of mixtures of toxicants present in 5. Teeguarden, JG et al., A multi-route model smoke/aerosol ☐ of nicotine-cotinine pharmacokinetics, pharmacodynamics and brain nicotinic 1. Cunningham, FH et al., A novel application of acetylcholine receptor binding in humans. Regul. the margin of exposure approach: Segregation of Toxicol. Pharmacol. 2013; 65: 12–28.

30 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 QUANTIFYING RISK QUANTIFYING RISK

In vitro exposure machine syringes In the smoking machine, these syringes generate and dilute cigarette, tobacco heating product or e-cigarette aerosols and deliver this to cell cultures located at the air-liquid interface. To avoid any cross-contamination of different aerosol types, each syringe is designated to one particular product.

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 31 AEROSOL SCIENCE

32 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 AEROSOL SCIENCE AEROSOL SCIENCE

UNDERSTANDING CONSUMER PRODUCT INTERACTION

etermining an appropriate and reproduc- to deposit. A broader understanding of ible measurement of dose is an important nicotine delivery is also needed in order to D step in gauging uptake of whole aerosol and support the production of novel alterna- individual components of aerosol by the tives to combustible cigarettes that are consumer. Measurement helps us to under- acceptable to and suitable for consumers. stand nicotine delivery and enables analysis of the toxicological action of aerosol at Recent successes cellular, organ, and whole body levels. We We have undertaken collaborative work have focused on the development of real- with external researchers to improve time measurement tools to better define understanding of the physical processes and replicate human puffing and inhala- in smoke, and have shown the overriding tion behaviours, which allows equivalent importance of coagulation and condensa- doses to be assessed in laboratory-based tion (phase change) during the early puffing biological assays. Through reassessing phase of smoking when the mouth fills with previously published data and the use of smoke1,2. We have also shown that physical computational modelling, we are help- changes of the aerosol during subsequent ing to improve sensitivity analyses, align inhalation into the lungs are dominated by laboratory findings with dose effects in hygroscopic growth (accretion of water on humans, and characterise next-generation to the smoke droplet)3 in parallel with evap- tobacco-heating products and e-cigarettes. oration of soluble volatile and semi-volatile The size of particles in tobacco smoke chemicals, including nicotine. Rapid drop- and the partition of individual chemical let growth, effective dilution with water, and components between particles and the diffusion within the droplet all combine vapour change constantly. Several chemi- to slow the rate of nicotine release1,4. JOHN MCAUGHEY cals have been identified by regulators Additionally, we have measured the Principal (Aerosol) Scientist and scientists as likely key toxicants that density of smoke droplets under steady- John is a world-leading aerosol potentially influence disease risk owing state and transient (puff-by-puff) sam- scientist with more than 30 to their observed biological actions in pling5,6. Data from our experimental design years of experience. Early humans. Thus, it is important to identify confirms that smoke aerosol comprises in his career, he moved to the doses of smoke components, individu- spherical droplets with density that is unaf- AEA Technology, where he researched and published ally and in whole smoke, that are delivered fected by cigarette design parameters, such in multiple fields of aerosol to specific tissues and organs. Focusing as format, filtration, yield, and ventilation. science, including tobacco mainly on the lungs, we assess the loca- These findings are consistent with the smoke science, particulate tion of deposition, concentration, duration dominance of early coagulation processes air pollution, automotive of exposure and mechanisms of removal. giving chemically homogenous droplets. emissions, pharmaceutical inhalers, radiation dosimetry, This characterisation is even more im- Through other collaborations we have and occupational medicine. portant with novel products, the aerosols investigated the relative deposition, uptake, John advocates effective of which might be quite different from and reactions of formaldehyde, acetalde- science communication and cigarette smoke. To assess new products, hyde, and acrolein in the upper airways has previously served on a we may need to develop new techniques through hybrid computational fluid dy- number of UK Government scientific committees. and models that provide information on namic and physiologically based pharma- how the products are used and where in the cokinetic models7. We have compared the respiratory system the aerosols are likely data with previously published non-adverse

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sion for size and concentration measurements on an individual-puff basis12. Use of laser diffraction also allows alignment of our measurements with existing published standards, which enables us to validate our procedures for assessment of inhaled aerosol.

Aims for the future »» Extend the measurement and modelling of specific processes that determine initial particle size, concentration, and chemical composition to tobacco heating products and e-cigarettes »» Further characterise coagulation, condensation, evaporation, and hygroscopic growth processes to improve understanding of particle dynamics and partition in regional deposition »» Extend regional deposition estimates to key toxicants in the vapour phase of Image coming effect levels and confirmed a risk hierarchy inhaled smoke and exhaled breath to im- 27 Nov of acrolein > formaldehyde > acetaldehyde. prove computational toxicology models This sophisticated combination approach »» Support specific dosimetry assess- offers scope for aligning real-world expo- ments for key toxicants at the air–liquid sures and doses of key chemicals at the interface for tobacco and novel nicotine cellular level when replicating these with products tested within in vitro models of doses delivered to in vitro disease models. disease ☐ Recognising that detailed dose mod- 1. Pichelstorfer, L et al., Lagrangian/Eulerian model of els covering all harmful and potentially coagulation and deposition of inhaled particles in the human lung. J. Aerosol Sci. 2013; 64: 125–142. harmful constituents of tobacco smoke 2. Asgharian, B et al., Component-specific, cigarette requires significant resources, we have particle deposition modelling in the human developed an initial empirical approach respiratory tract. Inhal. Toxicol. 2014; 26: 36–47. 3. Johnson, TJ et al., Hygroscopic effects on the to separating particle deposition and mobility and mass of cigarette smoke particles. J. vapour deposition. Particle deposition Aerosol Sci. 2015; 86: 69–78. is significantly influenced by inhalation 4. Pichelstorfer, L & Hofmann, W Modelling aerosol dynamics of cigarette smoke in a denuder tube. J. behaviour, such as depth and duration. Aerosol Sci. 2015; 88: 72–89. Vapour deposition is generally independ- 5. Johnson, TJ et al., Steady-state measurement of the effective particle density of cigarette smoke. J. ent of inhalation behaviour and increases Aerosol Sci. 2014; 75: 9–16. with increasing vapour pressure of the 6. Johnson, TJ et al., Transient measurement of the compound. Deposition of semi-volatiles, effective particle density of cigarette smoke. J. Aerosol Sci. 2015; 87: 63–74. such as polyaromatic hydrocarbons, lies 7. Corley, RA et al., Modelling of aldehydes from between these extremes. Recognition of cigarette smoke: comparative risks of aldehyde constituents in cigarette smoke using transient this range offers an effective screening computational fluid dynamics/physiologically and prioritisation approach to dosing that based pharmacokinetic models of the rat and complements the margins of exposure human respiratory tracts. Toxicol. Sci. 2015; 146: 65–88. 8,9 approach we use in risk assessment . 8. Charles, FK et al., Methodologies for the Our real-time measurements of tobacco quantitative estimation of toxicant dose to cigarette smokers using physical, chemical and bioanalytical smoke aerosol have been cited on a number data. Inhal. Toxicol. 2013; 25: 383–397. of occasions10,11 and adopted more broadly 9. Cunningham, FH et al., A novel application of within the tobacco research community. We the margin of exposure approach: Segregation of tobacco smoke toxicants. Food Chem. Toxicol. 2011; have adapted these systems for measuring 49: 2921–2933. e-cigarette aerosol, the droplets of which 10. Adam, T et al., Simultaneous on-line size and chemical analysis of gas phase and particulate phase are larger than those in fresh cigarette of cigarette mainstream smoke. Anal. Bioanal. smoke (150–250 nm), having a median Chem. 2009; 394: 1193–1203. diameter of approximately 500 nm. One 11. Adam, T et al., Influence of filter ventilation on the chemical composition of cigarette mainstream consequence of this larger size is that the smoke. Anal. Chim. Acta 2010; 657: 36–44. droplets become amenable to measurement 12. Cabot, R et al., (2014). E-cigarettes: aerosol sampling and droplet size measurement. American by optical techniques, such as laser diffrac- Association for Aerosol Research (AAAR) 33rd tion, and electrical mobility techniques, Annual Conference, October 20–24, 2014; Orlando, both of which demonstrate excellent preci- FL, USA; poster No. 2PH.5.

34 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 GOOD RESEARCH PRACTICE

A SCIENTIFIC FRAMEWORK TO ASSESS NEXT GENERATION PRODUCTS

A framework for the assessment of A range of in vitro tests are required to novel tobacco and nicotine products assess the mutagenicity, genotoxicity with the potential to reduce health risks and cytotoxicity of the emissions. compared to cigarettes has been under The clinical phase is comprised of discussion for over a decade, after the studies that determine the potential US Institute Of Medicine issued its of the product to reduce risk at the report Clearing the smoke – the scientific individual level and require data basis for tobacco harm reduction. The from human volunteers to show US Food and Drug Administration nicotine uptake and assess changes in (FDA), is the only national regulator biomarkers of exposure and biomarkers to have provided a draft framework of biological effect. For the prediction with which to assess novel tobacco of health-related outcomes an and nicotine products for their approach is proposed that uses systems harm reduction potential through science to integrate disease relevant in their Modified Risk Tobacco Product vitro biological and clinical responses. application process. In addition to the It is likely that potentially reduced FDA’s proposed regulatory assessment risk tobacco and nicotine products framework, a recent publication from will continue to be upgraded as new the Tobacco Product Assessment technologies become available, as Consortium (TobPRAC) presented has been the case with electronic a four-stage model inclusive of: cigarettes. Scientific studies that bridge pre-market evaluation; pre-claims from a data set of pre-clinical and evaluation; post-market activities; and clinical studies for similar potentially monitoring and re-evaluation. Their reduced risk products should be framework highlighted key tests and sufficient to allow products with reference products that would be new technologies to be determined required to demonstrate reduction in as potentially reduced risk. DR JAMES MURPHY risk and product stability by chemical, A weight-of-evidence approach, Head of Reduced Risk toxicological and human studies at based on the output of the pre-clinical Substantiation the individual and population levels. and clinical studies would be used James holds a PhD in polymer Our approach builds on those to determine whether the product chemistry and joined us proposals and sets out an integrated had the potential to reduce risk. For in 2005 after postdoctoral framework using pre-clinical, clinical a new-to-world product, reduced risk research at the Nara Institute and population studies to assess determination would require longer- of Science & Technology in Japan developing novel the risk reduction potential of novel term population-based studies. polymer-based drug delivery tobacco and nicotine products, To assess the impact of the product systems. He has held a variety including electronic cigarettes, at the on the population as a whole, a Post of roles in R&D, including individual and population level. Market Surveillance (PMS) plan would leading the development The key elements of the pre-clinical be agreed pre-marketing. A PMS and assessment of a Reduced Toxicant Prototype cigarette, phase include the demonstration programme could include information as well as development and of the stability of the product and about product usage patterns, commercialisation of Vype the toxicology of the emissions of consumer perception; provide data e-cigarettes. He now heads up the product. Analytical chemistry with respect to the health risks, and our Reduced Risk Substantiation studies of the emissions of the the effect on morbidity and mortality unit, overseeing all of our pre- clinical and clinical research product comprising both targeted as compared with using other products programs. His group measurement of the US FDA listed or quitting use of tobacco products. is working on research harmful and potentially harmful Specific information could also be methodologies to substantiate constituents, and untargeted analytical collected such as health care visits, the reduced-risk potential of chemistry studies to ensure no physiological measurements and next-generation products. unexpected compounds are formed. adverse events. ☐

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 35 SCIENCE ENGAGEMENT

our

approach

36 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 SCIENCE ENGAGEMENT SCIENCE ENGAGEMENT

eveloping a scientific underpinning to materials science and electronics to ad- tobacco harm reduction is too urgent and vanced analytical chemistry, through to tox- D too important to be left to any one sector icology and risk assessment, clinical studies, of the scientific community. We think that and modelling of population effects, as well regulators, academia and industry all have as genomics, metabolomics, and agricul- roles to play. That is why, particularly over ture. Although the tobacco industry was the past decade, we have tried to be open not strong historically in many of these and transparent about the science we do, disciplines, the fact that several companies and have sought to develop scientific col- are now developing novel products that aim laborations with a wide range of groups. to be less risky than conventional cigarettes Tobacco and nicotine science is emerg- has required a multi-disciplinary approach ing as an exciting interdisciplinary field to innovating and assessing such products. that encompasses subjects ranging from As you can see in this report, we are

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 37 SCIENCE ENGAGEMENT

650 600 Welcomed over 1500 visitors to our R&D Live Centre at Global R&D since 2011

300 Over 150 peer-reviewed articles published since 2008

Seven scientiﬁc conferences hosted at our world-class facilities on site

VISITORS 2012 2013 2014

researching products across the risk (AAAS)). All our conference posters and spectrum, from electronic cigarettes (e- presentations are available to download cigarettes) and medically licensed nicotine from the library section of bat-science.com. products, to oral products and tobacco- We are publishing much of our work heating products. We also made a public in peer-reviewed journals. More than commitment in 2007, as part of our duties 150 BAT-authored papers have been as a responsible tobacco company, to a published in at least 50 different journals much more open and transparent approach since 2008 (again, all the manuscripts can that involves sharing information about be found in the library on bat-science. our research into tobacco harm reduction. com). It is heartening that the vast major- In 2008, we launched our dedicated ity of journal editors and editorial boards science website, www.bat.science.com, continue to believe that good science through which we share a great deal of speaks for itself and should be judged information about the non-competitive objectively by the peer-review process. aspects of our research programmes. In Most journals today have straightfor- an effort to encourage genuine two-way ward processes that ensure that potential engagement, we launched, in summer conflicts of interest are clearly disclosed. 2015, a Twitter handle (@BAT_Sci), which In addition, our senior scientists SARAH COONEY Head of Scientific Collaboration is allowing us to directly engage with have acted as peer reviewers for more & Communication scientists, policy-makers, and our consum- than 40 different journals; this reflects ers. By removing barriers to engagement, an external recognition of the wealth Sarah oversees BAT R&D’s we have seen an overwhelmingly positive of knowledge and expertise on tobacco external scientific engagement strategy. She joined us in 2010 response; we have also been invited to and nicotine science that sits within the with over 10 years’ experience participate in robust scientific discussion tobacco industry (and BAT in particu- in science publishing, ranging via Twitter. Both these tools have been lar). We expect these requests to increase from editorial and production welcomed by the scientific community further over the next few years as the through to editorial director as valuable sources of information. output from the growing tobacco regula- and new journal and web development. She holds an MSc Our scientists attend international tory science community also increases. in agricultural biotechnology, scientific conferences to present our work We were the first tobacco company to and her research has been and engage in dialogue with other sci- allow external visitors to tour our R&D published in Science and entists in the field, including academics, facilities in the UK (in Southampton and Genetics. Sarah has held regulators, and public-health stakehold- Cambridge), developing a science exhibi- positions at most of the world’s leading publishers, including ers. These conferences range from niche tion centre in 2011 that we updated in late Nature Publishing Group, tobacco events (including the Society for 2015; here visitors spend time with our Elsevier, BioMed Central and Research in Nicotine and Tobacco and the practicing scientists to learn about their Springer. She also managed Global Forum on Nicotine) to subject- areas of expertise. We have hosted visit- the publishing programme specific meetings (Society of Toxicology, ing academics, journalists, scientists from of the Society of Chemical Industry in collaboration the Association of Inhalation Toxicologists, standards bodies, scientists from consumer with Wiley-Blackwell. and the European Aerosol Conference), goods companies, regulatory scientists, to mainstream fora (eg American Asso- learned societies, politicians, business ciation for the Advancement of Science people and many others. We have seen

38 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 SCIENCE ENGAGEMENT SCIENCE ENGAGEMENT

steady growth in the number of visitors are also increasing their levels of open- to the centre over the past few years. ness and transparency by launching We have also begun to host local busi- dedicated science websites and open- ness, national and international scientific ing up their R&D facilities to visitors. conferences in a modern 300-seat audito- rium at our Southampton site, including Recent successes meetings of the Chromatography Society, BAT senior scientists are regularly invited the British Mass Spectrometry Society, to contribute to the US Food and Drug the British Carbon Group, the Institute Administration’s Center for Tobacco for Food Science & Technology, and the Products workshops on tobacco regulatory In Vitro Testing Industrial Platform. science. We are also actively participating Our first Science & Technol- in the work of standards bodies, includ- ogy Report, published in early 2014, also ing the International Organization for marked an industry first. Available in Standardization, and, in the context of both print and app versions, we gave an e-cigarettes, the British Standards Insti- overview of BAT’s progress and aspira- tute and the French organisation, AFNOR tions in 10 different areas of science. (see pages 24–25). Our senior staff have We follow best practice regarding clini- presented on tobacco harm reduction and cal studies, as established by the pharma- e-cigarettes at mainstream scientific confer- ceutical industry, for instance obtaining ences including the AAAS and the EuroSci- ethics approval from the relevant geo- ence Open Forum (ESOF). We continue graphical independent ethics committees, to regularly publish good manuscripts in and registering our clinical studies on the high-quality peer-reviewed journals. International Standard Randomised Con- Our Chief Scientific Officer, Chris trolled Trial Number (ISRTCN; isrctn.com) Proctor, was part of the American or ClinicalTrials.gov databases before study Chemical Society 2015 writing com- initiation. It is well accepted (by the scien- mittee to revise the society’s scientific tific community) that registration of a clini- integrity policy through his work with cal trial in this way creates a commitment their Corporation Associates division. to publish the results of the study. Also, every academic who receives funding for Aims for the future fundamental research from BAT is encour- »» Refresh the bat-science.com website aged to publish the results that arise from »» Continue to explore new social media ini- the project, irrespective of the findings, tiatives and further develop our presence and to acknowledge the funding source. on Twitter These activities have given BAT a very »» Continue to present at mainstream and strong heritage of scientific engagement niche scientific conferences in the tobacco industry. We are pleased »» Continue to host visitors and appropriate to see that other tobacco companies scientific conferences at our R&D site ☐

m .co bat-science

DAY 1: August 25, 2011 Afternoon session Panel 2: Comparison of MRTPs to other Products

WORKSHOP David Burns, M.D. Timothy McAfee, M.D., M.P.H. Christopher Proctor, PhD Lars F Rutqvist, M.D., PhD SCIENCE & TECHNOLOGY Karla Sneegas, M.P.H.

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 39 INSIDE BAT R&D

40 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 INSIDE BAT R&D INSIDE BAT — PEOPLE

Bring your difference to BAT

i Chloe, first of all, could you tell us a level at which it can be used. Although H bit about what you do at BAT? this might sound simple, a huge amount I work in Product Stewardship as an evalu- of work actually goes into making sure an ator. Our department conducts toxico- ingredient doesn’t add to the toxicological logical risk assessments of materials and risk of any of our products. A typical risk additives that go into all our products, assessment takes about 6 weeks, although from commercially available combustibles we can do them sooner if necessary. to those in development, like e-cigarettes and tobacco-heating products (THPs). What does a typical day look like? Although I am a toxicologist, my job How did you come to work for BAT? is largely desk-based. A typical day After growing up in Qatar, I did a BSc depends on deadlines but usually in- in biological sciences at Birmingham volves a combination of commercial University, followed by a lab-based MRes evaluations, work on novel projects (like in toxicology at the University of Not- e-cigarettes), some countersigning (when tingham. Initially I had wanted to be we double check each other’s work), an NHS pathologist but found it very and providing some ad-hoc technical difficult to gain the necessary experience. guidance to other team members on a Through a recruitment agency looking particular assessment. I enjoy the vari- for toxicology graduates, I discovered ety of different challenges every day. the toxicology job at BAT and a similar one at the Food Standards Agency. My How has your role changed? granddad had worked for Ceylon Tobacco Technically I’ve been an evaluator in the Company [a BAT subsidiary] a long time same department for the 7 years, but my ago and said how great it was, and I also role has developed as I’ve become more Are you interested in thought my quality of life would be better experienced and I now also manage a working at BAT R&D? We in Southampton than in central London, small team. I started out on our more have interviewed a member so I chose the position at BAT. That was straightforward commercial cigarette of R&D staff so that you in 2008 and I am still here 7 years later! portfolio; whether we can or can’t sup- can hear first-hand what port ingredients for these products is a motivates our world-class Overview of what you do lot more black and white. I then moved team, their backgrounds and A project team will come to us with a new onto more novel products and now I lead how they feel about working ingredient or material they’d like to use. It on e-cigarettes. When a project comes here. might be a new type of paper in a cigarette in, I’m expected to know where to look Chloe Winnington is a or a flavour for an e-liquid, for example. for relevant data and evidence and draw scientist in the Product We then evaluate the ingredient based on from previous experience to find ways Stewardship team. Chloe how it is going to be used, what sort of of contextualising exposure and sup- joined BAT fresh out product it is used in and the potential ex- porting that ingredient by myself. of university and has posure of that ingredient is to the consum- progressed up the ranks over er. We use toxicology data and scientific What experience have you gained in the past 6 years, developing evidence currently available to help us the job? into a highly valued and determine whether the ingredient is suit- I’ve grown in confidence as my role has talented BAT scientist. able for such use and, if so, the maximum progressed, and am constantly trying to

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 41 INSIDE BAT — PEOPLE

find creative solutions to problems and more efficient ways of working. In addition to the toxicology side of things, I’ve also learnt a lot about the products, including technical features that might change smoke chemistry and functionality.

Where do you see yourself in 5 years? I’d like to continue developing into a more managerial and advisory role, but at the same time I’d never want to give up the science side of things as I enjoy the continuous learning I get from my from my job – I am never bored!

What is the biggest challenge you face in your job? Not many people know that we’re a global department, supporting products not just from R&D but from all over the company. Often we’re seen as the people who slow progress down and put the brakes on ideas, but it’s crucial that we take the time to rigorously risk assess everything that goes into our products to ensure that the ingredients we use are of the highest quality and do not add to the toxicological risk of the products, whether they’re already commercially available or in development. Our department is the only one that knows every single detail about every product (tobacco, flavours, paper ingredients, filters etc) — that’s why we’re the only department that can deal with legal disclo- sures as well. Although we may sometimes seem to slow things down, we are actually working hard to protect consumers, product developers, and the company.

Is there anything that surprises you about working for BAT? I was initially surprised by just how much care goes into assessment of the ingredients we use and making sure that they do not make our products more harmful, even when used Focusing in only tiny amounts. There is a lot of negativity around this industry and we’re under a lot of scrutiny, but I can on honestly say I’m proud of what I do. practical Have you got any advice for someone following in your footsteps? Certainly in my department, if you are solutions interested and motivated there’s a lot to understand and learn from experienced toxicologists as well as opportunities for career progression. As always, working here is what you make it. ☐

42 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 INSIDE BAT — PEOPLE INSIDE BAT R&D

he unseen heroes of R&D must be those products in various formats, or develop- T in the Innovation Development Centre ing methods to optimise a process, the (IDC). This team has a dynamic, agile, team applies its engineering aptitude and and flexible manufacturing capability that technical expertise to drive innovation. can support R&D in launching new-to- They have done a lot of work, for exam- world products, innovations, and tech- ple, finding ways to manufacture reconsti- nologies. These troubleshooters are the tuted tobacco-sheet materials to improve people who not only keep things running the performance of tobacco heating prod- smoothly but can also turn ideas into ucts (THPs). They’ve supported the work to reality, as such, they are vital to R&D. manage moisture levels, and have adapted IDC takes responsibility for the plan- standard processing for manufacturing ning and deployment of all technical THPs, shaping the future manufacturing resources, aiming to increase capabilities demands of this emerging product category. and flexibility but without affecting the “Good process control with flexibility level of support and productivity it offers, helps support developing new products to reduce its physical space footprint, and – it is critical to everything,” says Richard to increase space for new equipment. Hepworth, Manufacturing Engineer. Mark Goodeve, Senior Engineer, Manufacturing innovation explained that often project managers With an increasing focus on innovative come into IDC for an initial informal technologies in recent years, the manu- chat about an idea’s potential. There will facturing facility has become ever more then be a more technical discussion and, integral to R&D, helping project teams by once the engineer has given the project setting up or evolving the processes that manager a steer on what can be manu- lead to new or improved products. Whether factured, the department either adapts it is modifying equipment to manufacture current equipment or procures what is a new product, adjusting machines to make necessary, dependent on the format and

BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 43 INSIDE BAT — ENGINEERING

How the Innovation Development Centre helped to design the cover of this report

1 2

3 4

1. Planned and drawn using computer-aided design 2. High-pressure water jets cut shape into aluminium sheet 3. Shape is cut 4. Shape is extracted from sheet 5. Final cover image created by shining light 5 through metal shape and photographing

specification. “We like a challenge with manufacturing and processing Their expertise has been vital in and everyone here has the same of equipment. The work conducted enabling the business to develop attitude – whatever we can do to by the Engineering Workshop prototypes for exploratory work help, we will,” he says. “We are very ranges from conducting preventive into e-cigarettes and THP devices, prototype-led here and can produce maintenance, acceptance testing, and continues to provide unique a machine-made sample that has the fault finding, interfacing new pieces solutions for unique requirements. potential to be developed further of kit with current equipment, and “Anyone can come to us and talk or scaled up and commercialised.” calibrating and testing devices, to through ideas,” says Gary. “We en- manufacturing bespoke solutions. joy new challenges and try to bring Engineering workshop These engineers really are even whimsical ideas to life and Gary Baker and Richard Vezey, both the background problem solvers support them whenever we can.” Senior Engineers, are in charge of who support the entire business. By actively seeking new solutions work on the electrical, control, and As well as sorting machinery, and embracing change, the IDC mechanical sides of things. They are the engineering workshop is the keeps the core business running and responsible for all machinery (me- go-to place for a quick turna- is able to provide innovative solu- chanical, electrical, control systems, round of custom-made parts for tions that support the development hardware, and software) associated new products in development. of next-generation products. ☐

44 BRITISH AMERICAN TOBACCO | SCIENCE AND TECHNOLOGY REPORT 2015 Selected publications from 2014-2015 smoke using the Ames assay: A multi-strain approach. Mutation Research/Genetic Toxicology and Environmental 1. Neilson L, Mankus C, Thorne D, Jackson G, Debay J Mutagenesis 782, 9 -17. and Meredith C. (2015). Development of an in vitro 5. Baxter A, Thain S, Banerjee A, Haswell L, Parmar cytotoxicity model for aerosol exposure using 3D A, Phillips G. and Minet E. (2015). Targeted omics reconstructed human airway tissue; application for analyses, and metabolic enzyme activity assays assessment of e-cigarette aerosol. Toxicology in Vitro 29, demonstrate maintenance of key mucociliary 1952-1962. characteristics in long term cultures of reconstituted 2. Costigan S and Meredith C. (2015). “An approach to human airway epithelia. Toxicology in Vitro 29, 864-875. ingredient screening and toxicological risk assessment 6. Murphy J, Lowe F, Fearon I, Camacho O, Minet E. of flavours in e-liquids. Regulatory Toxicology and (2015). A framework for the assessment of reduced Pharmacology 72, 361-369. risk tobacco and nicotine products. Recent Advances in 3. Forster M, Liu C, Duke M, McAdam K and Proctor C. Tobacco Science 41, 51–81. (2015). An experimental method to study emissions 7. Proctor C, Shepperd C, McAdam K, Eldridge A, from heated tobacco between 100-200°C. Chemistry Meredith C. (2014). Integrating chemical, toxicological Central Journal 9, 20. and clinical research to assess the potential of reducing 4. Thorne D, Kilford J, Hollings M, Dalrymple A, health risks associated with cigarette smoking through Ballantyne M, Meredith C and Dillon D. (2015). toxicant regulation. Recent Advances in Tobacco Science The mutagenic assessment of mainstream cigarette 40, 1-34. Group Research & Development Southampton and Cambridge