WHO study group on tobacco product regulation: report on the scientific basis of tobacco product regulation: sixth report of a WHO study group

WHO study group on tobacco product regulation Report on the scientific basis of tobacco product regulation: Sixth report of a WHO study group W H O T e c h n i c a l R e p o r t S e r i e s 1001 The World Health Organization was established in 1948 as a specialized agency of the United Nations serving as the directing and coordinating authority for international health matters and public health. One of WHO’s constitutional functions is to provide objective and reliable information and advice in the field of human health, a responsibility that it fulfils in part through its extensive programme of publications. The Organization seeks through its publications to support national health strategies and address the most pressing public health concerns of populations around the world. 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The responsibility for the interpretation and use of the material lies with the reader. In no event shall WHO be liable for damages arising from its use. This publication contains the collective views of an international group of experts and does not necessarily represent the decisions or the policies of the World Health Organization. Printed in Switzerland WHO Library Cataloguing-in-Publication Data WHO study group on tobacco product regulation: report on the scientific basis of tobacco product regulation: sixth report of a WHO study group (WHO technical report series; nº. 1001) 1. Tobacco Use Disorder – prevention and control. 2. Tobacco Industry – legislation. 3. Tobacco Control Campaigns. 4. Tobacco – chemistry. I. World Health Organization. II. WHO Study Group on Tobacco Product Regulation. III. Series. ISBN 978-92-4-121001-0 ISBN 978-92-4-069660-0 (PDF) ISSN 0512-3054 iii Contents Participants in the eighth meeting of the WHO Study Group on Tobacco Product Regulation vii Acknowledgements ix Abbreviations and acronyms x 1. Introduction 1 2. Cigarette characteristics and design features 3 2.1 Introduction 4 2.2 Cigarette characteristics that influence perception and use 5 2.2.1 Overview 5 2.2.2 Cigarette characteristics that influence user perception 6 2.2.3 Cigarette characteristics that influence user behaviour 9 2.3 Cigarette characteristics that affect the content of smoke emissions 12 2.3.1 Tobacco 12 2.3.2 Paper 13 2.3.3 Filter 14 2.3.4 Physical dimensions 16 2.4 Design features and additives that modify smoke pH and addictiveness 19 2.4.1 Overview 19 2.4.2 Ammonia, sugars and reconstituted tobacco 20 2.4.3 Other ingredients 21 2.4.4 Tobacco blend and physical characteristics 21 2.4.5 Measuring “smoke pH” 22 2.5 Innovations that could influence either perception or delivery 22 2.5.1 Overview 22 2.5.2 Reduced-nicotine cigarettes 23 2.5.3 Coloured cigarette paper 24 2.5.4 Specialty filters 25 2.5.5 Tobacco industry research on delivery through special filters and with treated tobacco 26 2.6 Research that would inform scientific evaluation of the public health impact of design characteristics 28 2.7 Conclusions 29 2.8 Recommendations 31 2.8.1 Policy recommendations 32 2.8.2 Research recommendations 32 2.9 References 33 iv 3. Possible application of WHO Tobacco Laboratory Network standard operating procedures to evaluation of electronic nicotine delivery systems 41 3.1 Background 42 3.2 General methodological considerations in evaluating electronic nicotine delivery systems (ENDS) 44 3.3 Nicotine 45 3.3.1 Nicotine in ENDS liquid 45 3.3.2 Nicotine in ENDS aerosol 46 3.4 Tobacco-specific nitrosamines 47 3.4.1 Tobacco-specific nitrosamines in ENDS liquid 47 3.4.2 Tobacco-specific nitrosamines in ENDS aerosol 48 3.5 Benzo[a]pyrene 48 3.5.1 Benzo[a]pyrene in ENDS liquid 48 3.5.2 Benzo[a]pyrene in ENDS aerosol 49 3.6 Additional analytes 49 3.6.1 Carbonyls 49 3.6.2 Solvents 51 3.6.3 Volatile organic compounds 52 3.6.4 Phenolic compounds 53 3.6.5 Metals 53 3.6.6 Flavours 54 3.7 Recommendations for extension of methods 55 3.7.1 Nicotine 57 3.7.2 Tobacco-specific nitrosamines 58 3.7.3 Benzo[a]pyrene 58 3.7.4 Volatile organic compounds 59 3.7.5 Carbonyls 59 3.8 Research that will inform future regulatory use of data on ENDS 59 3.9 Conclusions 60 3.10 Recommendations 63 3.11 References 65 4. Waterpipe toxicant content and emissions 71 4.1 Introduction 71 4.2 Puff topography and emissions testing regimens 73 4.3 Toxicant content and emissions 75 4.4 Influence of testing protocols on measurements of toxicant emissions from waterpipes 79 4.4.1 Puffing regimen 81 4.4.2 Heat source 81 4.4.3 Temperature of tobacco 82 4.4.4 Effect of water 82 4.5 Influence of waterpipe design on levels of emissions of waterpipe tobacco products 82 4.5.1 Components and accessories 82 4.5.2 “Real-world” and research-grade waterpipes 83 v 4.5.3 Waterpipe hose 84 4.5.4 Waterpipe tray versus foil 84 4.6 Conclusions 86 4.7 Recommendations for regulators 87 4.8 References 87 5. Applicability and adaptability of the WHO Tobacco Laboratory Network standard operating procedures for cigarettes to waterpipe tobacco 91 5.1 Introduction 91 5.2 Smoking methods 92 5.2.1 Heat sources 92 5.2.2 Head 93 5.2.3 Head covering 93 5.2.4 Water 94 5.2.5 Hose 94 5.2.6 Filter 94 5.3 Smoking machines 95 5.4 Sampling of waterpipe tobacco 96 5.5 Sample preparation 97 5.6 Determination of contents and emissions 99 5.6.1 Contents of waterpipe tobacco 99 5.6.2 Emissions of tar, nicotine and carbon monoxide 100 5.7 Discussion 102 5.8 Conclusions and recommendations 105 5.8.1 Recommendations for regulators 105 5.8.2 Recommendation for researchers 106 5.9 References 106 6. Toxic contents and emissions of smokeless tobacco products 109 6.1 Introduction 109 6.1.1 Global prevalence 111 6.1.2 Diversity in the manufacture and physical properties of smokeless tobacco products 111 6.2 Product composition 112 6.2.1 Tobacco 112 6.2.2 Additives 112 6.3 Emissions from smokeless tobacco products 114 6.3.1 Nicotine 114 6.3.2 Toxic and carcinogenic agents 116 6.3.3 Microbes and their constituents 120 6.4 Reducing the concentrations of toxicants in smokeless tobacco products 121 6.5 Conclusions and recommendations 123 6.6 References 125 vi 7. Applicability or adaptability of standard operating procedures for nicotine, tobacco-specific N-nitrosamines and benzo[a]pyrene in cigarette contents and emissions to tobacco products other than cigarettes, particularly smokeless tobacco products 131 7.1 Introduction 131 7.2 Nicotine, tobacco-specific N-nitrosamines and benzo[a]pyrene in smokeless tobacco products 133 7.2.1 Nicotine 133 7.2.2 Tobacco-specific N-nitrosamines 133 7.2.3 Benzo[a]pyrene 133 7.3 Evaluation of applicability of WHO standard operating procedures for analysis of smokeless tobacco products 133 7.3.1 Analytical considerations 133 7.3.2 Determination of nicotine 134 7.3.3 Determination of tobacco-specific N-nitrosamines 134 7.3.4 Determination of benzo[a]pyrene 135 7.4 Discussion and recommendations 136 7.5 References 138 8. Overall recommendations 141 vii Participants in the eighth meeting of the WHO Study Group on Tobacco Product Regulation Rio de Janeiro, Brazil, 9–11 December 2015 Members Dr D.L. Ashley, Director, Office of Science, Center for Tobacco Products, Food and Drug Administration, Rockville, Maryland, United States of America Professor O.A. Ayo-Yusuf, Dean, School of Oral Health Sciences, Sefako Makgatho Health Sciences University, MEDUNSA, Pretoria, South Africa Professor A.R. Boobis, Professor of Biochemical Pharmacology, Centre for Pharmacology and Therapeutics, Department of Medicine, Imperial College, London; Director of Public Health England Toxicology Unit, Imperial College, London, United Kingdom Professor Mike Daube, Professor of Health Policy, Curtin University; Director, Public Health Advocacy Institute Western Australia, Perth, Western Australia, Australia Dr M.V. Djordjevic, Program Director/Project Officer, Tobacco Control Research Branch, Behavioral Research Program, Division of Cancer Control and Population Sciences, National Cancer Institute, Bethesda, Maryland, United States of America Dr P. Gupta, Director, Healis Sekhsaria Institute for Public Health, Mumbai, India Dr S.K. Hammond, Professor of Environmental Health Sciences, School of Public Health, University of California at Berkley, Berkley, California, United States of America Dr D. Hatsukami, Professor of Psychiatry, University of Minnesota, Minneapolis, Minnesota, United States of America Dr A. Opperhuizen, Director, Office for Risk Assessment and Research, Utrecht, The Netherlands Dr G. Zaatari (Chair), Professor and Chairman, Department of Pathology and Laboratory Medicine, American University of Beirut, Beirut, Lebanon Presenters Dr Nuan Ping Cheah, Director, Cosmetics and Cigarette Testing Laboratory, Pharmaceutical Division, Applied Sciences Group, Health Sciences Authority, Singapore Dr Gregory Connolly, Professor, Northeastern University, Boston, Massachusetts, United States of America Dr Thomas Eissenberg, Professor of Psychology and Co-Director, Center for the Study of Tobacco Products, Virginia Commonwealth University, Richmond, Virginia, United States of America Dr Esteve Fernández, Head, Tobacco Control Unit, Catalan Institute of Oncology, Bellvitge viii Institute of Biomedical Research; Associate Professor of Epidemiology and Public Health, University of Barcelona, Barcelona, Spain Dr Patricia Richter, Deputy Chief, Tobacco and Volatile Branch, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America Dr Alan Shihadeh, Professor of Mechanical Engineering, Faculty of Architecture and Engineering, American University of Beirut, Beirut, Lebanon Dr Reinskje Talhout, National Institute for Public Health and Environment, Centre for Health Protection, Bilthoven, The Netherlands Mr Geoffrey Ferris Wayne, Research Consultant, Sebastopol, California, United States of America Ms Ana Claudia Bastos de Andrade, Head, Tobacco Products Control Department, National Health Surveillance Agency, Rio de Janeiro, Brazil Dr Katja Bromen, Policy Officer, Tobacco Control Team, European Commission, Directorate General Health and Food Safety, Substances of Human Origin and Tobacco Control, Brussels, Belgium Mr Denis Choniere, Director, Tobacco Products Regulatory Office, Controlled Substances and Tobacco Directorate, Health Canada, Ottawa, Ontario, Canada Mrs Nalan Yazicioğlu, Engineer, Tobacco and Alcohol Market Regulatory Authority, Ankara, Turkey Convention Secretariat of the WHO FCTC Dr Carmen Audera-Lopez, Technical Officer, WHO, Geneva, Switzerland Secretariat (Prevention of Noncommunicable Diseases, WHO, Geneva, Switzerland) Ms M. Aryee-Quansah, Administrative Assistant, Tobacco Free Initiative Dr A. Peruga, Programme Manager, Tobacco Free Initiative Ms G. Vestal, Technical Officer (Legal), Tobacco Free Initiative ix Acknowledgements The WHO Study Group on Tobacco Product Regulation (TobReg) expresses its gratitude to the authors of the background papers used as the basis for this report. Production of the report was coordinated by Ms Sarah Emami, with the supervision and support of Dr Vinayak Prasad and Dr Douglas Bettcher. Dr Armando Peruga and Ms Gemma Vestal assisted in organizing the meeting. Administrative support was provided by the following WHO personnel: Ms Miriamjoy Aryee-Quansah, Mr Gareth Burns, Mr Luis Madge, Ms Rosane Serrao, Ms Moira Sy, Ms Elizabeth Tecson and Ms Angeli Vigo. TobReg acknowledges the facilitators of the Working Group on Articles 9 and 10 of the WHO Framework Convention on Tobacco Control (WHO FCTC), who helped ensure that WHO and TobReg adequately responded to the request of the Conference of the Parties: Ms Ana Claudia Bastos de Andrade (Brazil), Dr Katja Bromen, Mr Denis Chonière (Canada) and Mrs Nalan Yazicioğlu (Turkey). TobReg would like to express its gratitude to the Agência Nacional de Vigilância Sanitária (ANVISA) for hosting the meeting and to Ms Ana Claudia Bastos de Andrade (ANVISA) and Dr Adriana Blanco (Tobacco Control Regional Adviser, WHO Regional Office for the Americas) for ensuring a smooth, productive TobReg meeting in Brazil. TobReg thanks colleagues in the WHO FCTC Secretariat who assisted throughout production of this document, namely: Dr Carmen Audera-Lopez, Ms Guangyuan Liu and Dr Tibor Szilagyi (Technical Officers) and Dr Vera da Costa e Silva (current Head of the Convention Secretariat). xAbbreviations and acronyms CDC Centers for Disease Control and Prevention (USA) CFP Cambridge filter pad CI confidence interval CO carbon monoxide COP Conference of the Parties CORESTA Cooperation Centre for Scientific Research Relative to Tobacco ENDS electronic nicotine delivery systems FCTC Framework Convention on Tobacco Control FEMA Flavor and Extract Manufacturers Association (USA) FID flame ionization detection GC gas chromatography GRAS generally recognized as safe HPLC high-performance liquid chromatography IARC International Agency for Research on Cancer ISO International Standards Organization MS mass spectrometry NNAL 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone NNN N’-nitrosonornicotine PAH polycyclic aromatic hydrocarbon ppm parts per million RIVM National Institute for Public Health and the Environment (Netherlands) SOP standard operating procedure TobLabNet Tobacco Laboratory Network TobReg WHO Study Group on Tobacco Product Regulation TPM total particulate matter TSNA tobacco-specific nitrosamine VOC volatile organic compounds 11. Introduction Effective tobacco product regulation is an essential component of a comprehensive tobacco control programme. It includes regulation of contents and emissions by mandated testing, disclosure of test results, setting limits, as appropriate, and imposing restrictions on packaging and labelling. Tobacco product regulation is covered under Articles 9, 10 and 11 of the WHO Framework Convention on Tobacco Control (WHO FCTC) and in the partial guidelines on implementation of Articles 9 and 10. The WHO Study Group on Tobacco Product Regulation (TobReg) was formally constituted by the WHO Director-General in 2003 to address regulatory gaps. Its mandate is to provide evidence-based policy recommendations on tobacco product regulation to the Director-General. TobReg is composed of national and international scientific experts on product regulation, treatment of tobacco dependence and laboratory analysis of tobacco ingredients and emissions. The experts are from countries in all six WHO regions. As a formalized entity of WHO, TobReg submits technical reports to the WHO Executive Board through the Director-General to draw the attention of Member States to the Organization’s work in tobacco product regulation. The technical reports are based on unpublished background papers that have been discussed by TobReg. The eighth meeting of TobReg was held in Rio de Janeiro, Brazil on 9–11 December 2015. The discussions covered priorities for tobacco product regulation and addressed the request of the COP of the WHO FCTC at its sixth session to: ■ prepare a report based on scientific evidence on specific characteris- tics of cigarettes, including slim and “super-slim” designs, filter venti- lation and innovative filter design features such as flavour-delivering mechanisms in capsules, to the extent that those characteristics affect the public health objectives of the WHO FCTC, for consideration by TobReg at its first meeting after the sixth session of the COP; ■ assess options for regulating electronic nicotine and non-nicotine delivery systems in order to achieve the objectives outlined in reso- lution FCTC/COP6(9) and to consider methods for measuring the contents and emissions of these products; ■ assess, within two years, whether the standard operating procedures (SOPs) for determining nicotine, tobacco-specific N-nitrosamines (TSNAs) and benzo[a]pyrene in cigarette contents and emissions are applicable or adaptable, as appropriate, to tobacco products other than cigarettes, including waterpipe smoke and smokeless tobacco; and 2 ■ prepare reports on the toxic contents and emissions of waterpipe and smokeless tobacco products. At the meeting, a background paper on the aerosol of electronic nicotine delivery systems (ENDS) was also discussed, which is published separately.1 TobReg also discussed the prevalence and use of menthol in tobacco products and, after the meeting, published an advisory note2 containing evidence-based conclusions and recommendations for policy-makers and regulators, including for a ban on menthol (and its analogues, derivatives and precursors) in cigarettes. TobReg hopes that the conclusions and recommendations in this report and the advisory note will be helpful to countries in implementing the product regulation provisions of the WHO FCTC. 1 http://www.who.int/tobacco/industry/product_regulation/eletronic-cigarettes-report-cop7- background-papers/en/ 2 http://apps.who.int/iris/bitstream/10665/205928/1/9789241510332_eng.pdf?ua=1 32. Cigarette characteristics and design features Reinskje Talhout, Centre for Health Protection, National Institute for Public Health and the Environment, Bilthoven, The Netherlands Patricia Richter, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, USA Irina Stepanov, Associate Professor, Division of Environmental Health Sciences and Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA Christina Watson, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, USA. Clifford Watson, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, USA Contents 2.1 Introduction 2.2 Cigarette characteristics that influence perception and use 2.2.1 Overview 2.2.2 Cigarette characteristics that influence user perception 2.2.2.1 Cigarette and filter tipping paper, decorative elements 2.2.2.2 Filter ventilation 2.2.2.3 Physical dimensions, slim and “super-slim” cigarettes 2.2.2.4 Flavours 2.2.3 Cigarette characteristics that influence user behaviour 2.2.3.1 Filter ventilation 2.2.3.2 Physical dimensions 2.2.3.3 Flavours 2.3 Cigarette characteristics that affect the content of smoke emissions 2.3.1 Tobacco 2.3.2 Paper 2.3.3 Filter 2.3.3.1 Filter ventilation 2.3.3.2 Absorbent filter materials, charcoal 2.3.4 Physical dimensions 2.3.4.1 Diameter and circumference 2.4.4.2 Length 2.4.4.3 Packing density 2.3.4.4 Implications for “super-slim” cigarettes 2.4 Design features and additives that modify smoke pH and addictiveness 2.4.1 Overview 2.4.2 Ammonia, sugars and reconstituted tobacco 2.4.3 Other ingredients 2.4.4 Tobacco blend and physical characteristics 2.4.5 Measuring “smoke pH” 4W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report 2.5 Innovations that could influence either perception or delivery 2.5.1 Overview 2.5.2 Reduced-nicotine cigarettes 2.5.3 Coloured cigarette paper 2.5.4 Specialty filters 2.5.5 Tobaco industry research on delivery through special filters and with treated tobacco 2.6 Research that would inform scientific evaluation of the public health impact of design characteristics 2.7 Conclusions 2.8 Recommendations 2.8.1 Policy recommendations 2.8.2 Research recommendations 2.9 References 2.1 Introduction This report was prepared in response to a request by the COP to the WHO FCTC at its sixth session (Moscow, Russian Federation, 13–18 October 2014) to the Convention Secretariat to invite WHO to prepare a report based on scientific evidence on specific cigarette characteristics of interest, including slim and “super-slim” designs, filter ventilation and innovative filter design features, including flavour-delivering mechanisms such as capsules, to the extent that those characteristics affect the public health objectives of the WHO FCTC, for consideration by the WHO FCTC COP Working Group on Articles 9 and 10 at its meeting in February 2016. With respect to the design features of slim and super-slim cigarettes, the report should cover cigarette circumference and length in relation to nicotine delivery and exposure. The report addresses cigarette characteristics that influence user perception, user behaviour and the delivery of toxic constituents. Typical characteristics of cigarettes are the tobacco blend, additives, tobacco weight, density, cigarette paper, filter type, filter ventilation and cigarette geometry (circumference, length) (1). Recently, cigarettes have been marketed with new design features, such as filter flavour capsules, special filters and coloured paper. The main purpose of adding such characteristics to cigarettes is to increase their attractiveness and addictiveness (2), which can be achieved by reducing their negative aspects (e.g. throat irritation), increasing their positive aspects (e.g. improved draw and mouth feel), appealing to new users and target groups, increasing the convenience and ease of use and increasing perceptions of lower risk or safety. Certain ingredients may also increase the addictive potential of a product, for instance by improving nicotine delivery. Many new products have been marketed or are being investigated by the tobacco industry that are claimed to lower the concentrations of some toxicants, for instance with more efficient filters or treated tobacco. Cigarette characteristics and design features 5 This section covers the following topics: ■ the cigarette characteristics that influence user perception and behav- iour (e.g. attractiveness, risk perception, ventilation, pressure drop, flavour, design and shape, including cigarette diameter or diameter- to-length ratio) (section 2.2); ■ the cigarette characteristics that affect the delivery of toxic emissions (e.g. tobacco type, tobacco blend, amount of tobacco, ventilation, pa- per porosity, filter type) (section 2.3); ■ design features and additives that modify smoke pH and addictive- ness (section 2.4); ■ innovations that could influence perceptions and/or emissions (e.g. flavour capsules, new filter design) (section 2.5); and ■ areas of research that would inform scientific evaluation of the public health impact of design characteristics (section 2.6). The literature search was conducted mainly in the PubMed database and with the SciFinder search tool, which retrieves data from the Medline and CAplus databases. Relevant articles cited in publications and reports were also included. In addition, the Internet was used to identify websites that provide product characteristics and marketing information and to search major tobacco manufacturers’ websites, tobacco industry document repositories, blogs and news articles. 2.2 Cigarette characteristics that influence perception and use 2.2.1 Overview Cigarette characteristics can influence nicotine delivery (3) and smokers’ sensory experience, which have been shown to influence a wide range of smoking- related behaviour, from initiation, to progression, tobacco dependence and smoking satisfaction in highly dependent smokers. The combination of nicotine delivery and sensory cues is critical in determining smoking satisfaction (4, 5), psychological reward (6) and reduced craving (7). For instance, a perception of a “lighter” feel and taste of the smoke from cigarettes with highly ventilated filters may be an important factor in their wide acceptability, due to better palatability, a perception of reduced risk or both (8–10). The influence of cigarette product design on user perceptions and smoking behaviour has been investigated by academic and government researchers as well as the tobacco industry, resulting in a substantial knowledge base (11). Internal research conducted by the industry, some of which is now publically available, is of particular interest, because modifications of cigarette design to 6W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report achieve effective nicotine delivery and specific sensory characteristics have been used by manufacturers to establish brand and sub-brand identity and to enhance the consumer appeal of products. Therefore, a review of relevant internal industry research is important for designing effective policies and regulations on cigarette characteristics (12). We summarize here the most important cigarette characteristics that have been shown to affect user perception and behaviour. 2.2.2 Cigarette characteristics that influence user perception 2.2.2.1 Cigarette and filter tipping paper, decorative elements Several studies have been conducted on the effect of the appearance of cigarettes on consumers’ response. They indicate that elements such as the colour and pattern of filters and paper affect perceptions of the attractiveness and relative harm of cigarettes (13–16). Moodie et al. (14) showed that pink cigarette paper may be more appealing and give young women perceptions of a pleasant taste and less harm. In contrast, dark colours generally had little appeal and gave perceptions of strong taste and greater harm; however, a pleasant aroma from a dark-coloured cigarette could enhance its appeal and the perception of taste and decrease the perception of harm. An exploratory study by Ford et al. (16) in a group of 15-year-old participants showed that white filter tips and decorative elements on the filter tipping paper, including the font style of brand names, can generate interest, provide novelty, communicate a positive image and lead to an overall perception of attractiveness. These findings indicate that cigarette appearance can be exploited as a promotional tool. For instance, it has been suggested that white tipping paper on cigarettes with ventilated filters was designed to reinforce the perception of a safer product, in contrast to most full-flavoured cigarettes, which have cork-coloured filter tips (17). Recent innovations that include such elements of cigarette appearance as cigarette paper and filter colours are further discussed in sections 2.5.3 and 2.5.4. 2.2.2.2 Filter ventilation Perception of reduced harm The composition and ventilation of cigarette filters and the effects of these features on emission content are described in detail in section 2.3.3. Many smokers are unaware that low-yield cigarettes have ventilated filters, which dilute cigarette smoke with air (17, 18). Filter ventilation changes users’ sensory responses to cigarette smoke and affects their perception of the harm associated with low- yield cigarettes. Specifically, filter ventilation in low-yield cigarettes leads smokers to perceive that the smoke tastes lighter and is less irritating than that of regular cigarettes, which powerfully supports their belief that the tar and nicotine intakes from such cigarettes are lower (8, 10, 19). For instance, O’Connor et al. (20) found Cigarette characteristics and design features 7 that the degree of filter ventilation was consistently associated with the perceived lightness (P < 0.001) and smoothness (P = 0.005) of cigarettes. Cummings et al. (17) showed that many Marlboro Lights smokers believed incorrectly that light and ultra-light cigarettes were less harmful than higher-tar, full-flavoured cigarettes. Only 11% of Marlboro Lights smokers in that study knew that their exposure to tar and other constituents from “light” cigarettes is about the same as that from full-flavoured cigarettes. It has also been shown (10) that many smokers agree that “light” cigarettes are not less harmful in general, but they still believe that they reduce their exposure, because of their sensory experience. Sensory experiences can lead users to perceive reduced exposure when smoking low-yield cigarettes, independently of any descriptive term or colour coding on the cigarette pack (8–10, 21). Longitudinal studies show that the removal of brand descriptors such as “light”, “mild” and “low tar” has not had a sustained impact on smokers’ perceptions, as many continue to believe or rationalize that “lighter” cigarettes are less harmful (22, 23). For instance, significant proportions of smokers in Australia (55%), Canada (43%) and the United Kingdom (70%) continue to believe that low-yield cigarettes offer some health benefit as compared with regular cigarettes. While the introduction of new terms (“smooth”, “fine”) and pack colours to suggest “lightness” or “smoothness” by manufacturers contributes to sustaining this misperception (24–26), smokers are also partly encouraged by the perception that light cigarettes are “smoother” on the throat and chest than regular cigarettes (9). Perception of draw Increased filter ventilation in “lower-delivery” cigarettes and the resulting reduction in chemosensory impact can also make smokers dissatisfied because of changes in “perception of draw” or the greater perceived effort required to inhale a sufficient amount of smoke from the cigarette. Substantial research on this phenomenon has been conducted by the tobacco industry, which shows that the perception of draw from smoking cigarettes with ventilated filters can be improved by increasing the levels of nicotine, volatile aldehydes, ammonia and other constituents and additives in smoke (reviewed in (4)). The effects of ammonia and other additives on smoke characteristics are discussed in more detail in sections 2.4.2 and 2.4.3. 2.2.2.3 Physical dimensions, including slim and “super-slim” cigarettes The length and circumference of cigarettes influence their appeal and perceptions of harm. Longer, slimmer cigarettes are widely acknowledged to increase the perception of stylishness and to appeal generally to women (12, 14); and research conducted by the tobacco industry suggests that these characteristics have been exploited in targeting women. For instance, Philip Morris observed that 8W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report fashion-conscious female smokers associated slim, long, light-tasting cigarettes with increased femininity and with weight control (27). Lorillard consumer research also indicated that female smokers of slim 100-mm cigarettes perceived the style as both feminine and graceful and milder and longer lasting (27). A recent study showed that longer cigarettes were often perceived by smokers as attractive and of high quality (15). In addition, Ford et al. (16) showed that slim and super-slim cigarettes were perceived as less harmful by 15-year-olds. The draft European Commission Tobacco Products Directive proposed that cigarettes < 7.5 mm in diameter be banned to reduce the possibility that cigarette appearance will mislead consumers about the harm they cause (28). The ban was not, however, included in the final Tobacco Products Directive (29). 2.2.2.4 Flavours Flavoured tobacco products generally appeal to young adults and adolescents and are often marketed towards them (30–32). In a study of university students who smoked flavoured and unflavoured cigarettes, flavoured cigarettes elicited greater positive expectancy than unflavoured cigarettes, even among nonsmokers (33). For instance, Camel Exotics elicited greater positive expectancy than Camel Lights (F(1421) = 38.4, P < 0.001) in experimental smokers, regular smokers and nonsmokers, although only a modest effect was seen in committed nonsmokers when analysed separately (F(1249) = 5.4, P  <  0.05). Significantly less negative expectancy was observed for flavoured than for unflavoured brands. Thus, Camel Lights were rated more negatively than Camel Exotics (F(1421) = 8.2, P < 0.01), and the effect did not depend on smoking status. Logistic regression analysis showed that positive expectancy predicted “intention to try” each brand by regular smokers and by susceptible and experimental smokers. For example, study participants were 2.4 times more willing to try Camel Exotics than Camel Lights. These findings are consistent with the view that flavoured cigarettes serve as “starter” products (32). The sensory qualities of menthol, the most common flavouring additive, may result in a perception of smoothness, increasing the appeal of smoking (33). Flavours such as menthol, spearmint, peppermint, chocolate, apricot, coconut and marshmallow have been used to address concern about after-taste and the aroma preferences of women (27). Research thus shows that aromatized cigarettes are used mainly by women and young people, people who are aware of smoking-related health risks and those who perceive that some cigarettes are less harmful than others (30, 34, 35). The WHO FCTC advises countries to prohibit or restrict ingredients that may be used to increase attractiveness (36). Some countries have already promulgated legislation to decrease the attractiveness of products by regulating flavours. Brazil (RDC ANVISA No. 14) and Canada (Bill C-32) have prohibited Cigarette characteristics and design features 9 most flavours, whereas other countries restrict use in a product or package to a concentration that will not result in a strong non-tobacco flavour, such as fruit or sweets. The Food and Drug Administration in the USA has banned additives, artificial and natural flavours (other than tobacco and menthol) and herbs and spices that impart a characterizing flavour to cigarettes (37). The new European Union Tobacco Product Directive also prohibits a characterizing flavour other than one of tobacco in cigarettes and roll-your-own tobacco (38), in which a characterizing flavour is defined as a “clearly noticeable smell or taste other than one of tobacco, resulting from an additive or a combination of additives, including, but not limited to, fruit, spice, herb, alcohol, candy, menthol or vanilla, which is noticeable before or during the consumption of the tobacco product.” 2.2.3 Cigarette characteristics that influence user behaviour 2.2.3.1 Filter ventilation Filter ventilation and subsequent smoke dilution with air result in compensatory smoking, such as drawing larger puffs, inhaling more deeply and blocking filter vents to prevent smoke dilution (39), because most smokers seek to optimize their nicotine intake, with the perceived chemosensory impact, to achieve rewarding sensations and to avoid the aversive sensations associated with nicotine withdrawal (40, 41). Smokers also block filter vents with their fingers or lips, although many smokers of light and ultra-light cigarettes are unaware that they are doing so (18, 42). Such compensation is likely to be complete for most smokers who switch from higher- to lower-yield cigarettes (41). Smoking cigarettes with substantially reduced smoke nicotine yields from very-low-nicotine tobacco blends does not, as opposed to filter ventilation, lead to compensatory smoking (43). It has been demonstrated that the ratio of smoke intake to tar and nicotine delivery is nonlinear; larger, more intense puffs change the concentration of smoke constituents more drastically by reducing their retention on cigarette filters and decreasing smoke dilution (44). Smokers who believe that they are smoking a product with lower delivery of harmful emissions may actually increase their exposure by changing their behaviour, such as blocking filter vents or taking larger puffs. This is particularly relevant for smokers of highly ventilated cigarette brands. Such “brand elasticity” allows smokers to effectively regulate nicotine delivery by adjusting their puffing behaviour. It also presents a major problem for measuring the actual nicotine and tar delivery of a brand. Cigarette brands vary in elasticity, and more elastic ones appear to have the greatest market share (44). Industry researchers have long known that smokers adjust their puffing behaviour to maintain a fairly constant daily dose of nicotine when they switch to cigarettes formerly marketed as light or ultra-light (17). Furthermore, tobacco industry documents show that filter ventilation was the main approach in engineering low-yield cigarettes, with other design features such as more porous 10 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report paper (10). These features tend to encourage stronger puffing by smokers and negate any potential reduction in exposure from smoking low-yield cigarettes (39, 45–47). For instance, Strasser et al. (46) estimated that smokers who block filter vents may be increasing their exposure to cigarette smoke constituents by 30%. Hammond et al. (44) showed that smokers who switched to low-yield cigarettes increased their total smoke intake per cigarette by 40% (P = 0.007), with no significant change in their salivary cotinine levels. The compensatory changes were stable, with no observable decrease over 5 days. Self-reported smokers of “light” cigarettes also perceived themselves as less addicted, were more likely to have ever attempted to quit than regular smokers and had stronger intention to quit but less confidence in their capacity to do so. The absence of any reduction in exposure of smokers of low-yield cigarettes to nicotine and other smoke constituents has been convincingly demonstrated in many studies with biomarkers of exposure (41, 48–51). Together, these findings provide strong in- vivo evidence of behavioural compensation for filter ventilation of cigarettes. Pressure drop Resistance to draw, or “pressure drop”, is proposed as one of the major determinants of puff duration and volume (47, 52–55). As chemosensory impact defines smokers’ perception of achieving a satisfying volume of smoke, an insufficient impact in the mouth and upper respiratory tract will drive smokers to continue increasing their puff intensity until they feel an adequate draw (4). Carbon-containing filters The presence of carbon in cigarette filters may affect the levels of some smoke constituents that contribute to the perception of draw and therefore lead to changes in smoking intensity. In a study by Rees et al. (57), Marlboro Lights smokers were switched to carbon-filtered Marlboro Ultra Smooth and non- carbon Marlboro Ultra Lights cigarettes for 48 h each. Larger puff volumes were taken of the carbon-containing cigarettes than either Marlboro Lights (difference in puff volume, 2.4–13.6 mL in two study groups; overall P = 0.006) or Marlboro Ultra Lights (difference in puff volume, 2.4–3.6 mL; overall P = 0.007). 2.2.3.2 Physical dimensions Studies in which smokers smoked cigarettes of full or partial length suggest that length may affect smoking behaviour, such as puff duration and volume (52– 55). In one study, smoking full-length cigarettes was associated with more puffs and self-reported smoking “satisfaction” than smoking half-, quarter- or eighth- length cigarettes. In the same study, smokers smoked fewer cigarettes but took more puffs of full-length research cigarettes manufactured with high (2.0 mg) or low (0.2 mg) nicotine than quarter-length versions of the same cigarettes (56). In Cigarette characteristics and design features 11 a study of nationally representative data from the National Health and Nutrition Examination Survey on serum cotinine and urinary total 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanonol (NNAL) concentrations in smokers of regular-sized, king-sized and long or ultra-long cigarettes, those who smoked long or ultra- long cigarettes had higher measures of smoking intensity and addiction (e.g. time to first cigarette, number of cigarettes smoked per day) and significantly higher tobacco biomarker levels than smokers of regular- or king-sized cigarettes (geometric mean serum cotinine, 263.15 ng/mL versus 173.13 ng/mL or 213.79 ng/mL; urinary NNAL, 0.48 ng/mg creatinine versus 0.34 ng/mg or 0.33 ng/mg, respectively) (52). 2.2.3.3 Flavours Flavours in cigarettes not only have potential marketing appeal to some population groups (e.g. young people, women, certain ethnic groups) and nonsmokers but may also mask the harshness of smoke, making inhalation easier. In a pilot study of differences in puff topography and cigarette ratings among 20 university student smokers of Camel Light and Camel Exotic Blend cigarettes (with similar tar, nicotine and filter ventilation) (58), participants took smaller puffs on Exotic Blend than on Camel Light cigarettes (42 mL vs 48 mL, P < 0.001), but the difference in total smoke volume was not significant (613.9 mL vs 630.7 mL, P = 0.79), and no increase was seen in carbon monoxide (CO: 6.2 vs 6.2 ppm, P = 0.90). When participants rated each cigarette on characteristics such as strength, irritation and taste, they rated Exotic Blend cigarettes as being most different from their usual brand, but the taste ratings did not differ. These results suggest that adding flavours to cigarettes does not significantly influence how they are smoked by established smokers. One flavour that could change smoking behaviour is menthol, although the results of many studies are inconclusive or conflicting (33, 59). Some indicated that daily cigarette consumption or puffing intensity were greater with menthol cigarettes (60, 61), while others found that the puff frequency (62, 63) and volume smoked (63) were similar to those of smokers of non-mentholated cigarettes. Strasser et al. (64) found that menthol has a minimal impact on smoking behaviour, biomarkers of exposure and subjective ratings; however, smokers of mentholated cigarettes smoked their first cigarette of the day sooner than smokers of non-mentholated cigarette, implying greater dependence on nicotine with use of mentholated cigarettes (61). Smokers of mentholated cigarettes attempted to quit more often but had less successful quitting rates, which suggests that mentholated cigarettes are more addictive than non-mentholated ones (65, 66). Other studies have shown that menthol cigarettes are used disproportionately by young people, probably because of their taste, sensory properties and easier inhalation (65). While there are few, inconclusive data on the role of menthol 12 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report cigarettes in initiation of smoking (67), studies indicate that adolescents smoke more mentholated than non-mentholated cigarettes, suggesting that these cigarettes are preferred during early tobacco use (68). 2.3 Cigarette characteristics that affect the content of smoke emissions Manufacturers can introduce or manipulate many variables to affect the composition of tobacco smoke (69). Traditional, tobacco-burning cigarettes, novel products and product features (reduced ignition propensity cigarettes, potentially reduced exposure cigarette products and denicotinized tobacco) were recently addressed in a WHO technical report (70). It is difficult to determine the contribution of each cigarette characteristic to the adverse health effects of tobacco use; a general recommendation is to focus research on reducing the levels of toxicants (per cigarette or “stick” or per milligram of nicotine). WHO has recommended mandated lowering of nine toxicants in cigarette smoke – N’-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), acetaldehyde, acrolein, benzene, benzo[a]pyrene, 1,3-butadiene, CO and formaldehyde – on the basis of their toxicity and the feasibility of lowering their concentrations (71). 2.3.1 Tobacco The tobacco blend is the cigarette component that most heavily influences the delivery of various chemicals in the smoke emissions (72). The properties of each type of tobacco influence its filling power (the ability to form a firm cigarette rod with a given moisture content), burn rate, tar and nicotine deliveries, amounts of chemicals in smoke, flavour and aroma and smoulder rate (73–79). Bright tobacco, also known as flue-cured or Virginia tobacco, has a lower nitrogen content (i.e. less nicotine) and a higher sugar content than other varieties. At a given circumference, Virginia-blend tobacco cigarettes yield a higher puff count than American-blend cigarettes (80). Cigarettes with flue-cured tobacco are heavier than those made with burley tobacco, so that more puffs can be taken from a given butt length (81). As the amount of flue-cured tobacco in a blend is increased, the tar and CO yields also increase (82); more formaldehyde is delivered in smoke from bright tobacco than from burley tobacco (83). In most tobaccos, the concentration of NNN exceeds that of NNK; in bright tobacco; however, the NNK concentration exceeds that of NNN (83). Burley and Maryland tobaccos are air-cured and typically have higher nicotine contents but lower sugar contents. Burley tobacco has notably higher concentrations of nitrate and TSNAs than other tobacco types (84). Oriental tobacco is sun-cured; it is often included in blended varieties because of its aromatic properties (81). It has higher levels of phenol than flue-cured, burley Cigarette characteristics and design features 13 or Maryland tobacco (85). Maryland tobacco has lower yields of tar, nicotine, phenol and benzo[a]pyrene than burley, oriental or flue-cured tobacco (85). Expanded, puffed and freeze-dried tobaccos are processed to increase their filling power (86). They are treated with various volatile materials, which are then quickly removed, so that the tobacco cell structure greatly expands (79). These modified tobaccos are used to reduce the amount of tobacco required to “fill” a cigarette; however, they alter the levels of some smoke emissions. For example, the nicotine level in the smoke of cigarettes containing expanded tobacco leaf is lower than that in the smoke of a cigarette made without such material (86). With an increasing amount of expanded tobacco in a blend, the ratio of CO to carbon dioxide and the vapour phase aldehydes (acetaldehyde, acrolein) increases, and particulate-phase components decrease (69, 82). Smoke from cigarettes made with expanded stems has more CO, nitrogen oxides, formaldehyde, tar, benz[a] anthracene and benzo[a]pyrene than smoke from cigarettes made of puffed tobacco, expanded tobacco or freeze-dried tobacco (85). Reconstituted tobacco is made of tobacco by-products, including tobacco dust (“fines”), ribs and stems, which are extracted and then re-formed into a pulp with adhesives, fibres to provide structure, and chemicals such as humectants and flavours before being dried to various densities (81, 87, 88). Reconstituted tobacco costs less than tobacco leaf and has greater filling power, resulting in less dense tobacco filler, which contributes to a faster burn rate and fewer puffs per cigarette. These factors reduce the delivery of tar and nicotine in smoke (87, 89). The chemistry of the smoke from cigarettes made from reconstituted tobacco depends on whether it is made exclusively of stems or of a blend of stems and other tobacco-derived material. Stem-only reconstituted tobacco smoke has higher levels of nitrogen oxides, acetaldehyde and polycyclic aromatic hydrocarbons (PAHs) than reconstituted tobacco made with stems and other tobacco materials; however, the levels of tar, nicotine, CO, hydrogen cyanide and PAHs are lower in the smoke of cigarettes made from either type of reconstituted tobacco (stem only or stems and other tobacco materials) than from those that do not contain reconstituted tobacco (86). 2.3.2 Paper Cigarette paper controls combustion – free or static burn rate (i.e. the amount of cigarette consumed between puffs) and smoulder rate – and strongly affects the puff count and smoke yield of cigarettes under machine-testing conditions (79, 81). The controllable factors in cigarette paper that affect smoke emissions and composition are fibre composition; filler type, level and distribution; thickness and bulk density (standard paper or that with thicker bands used for reduced ignition propensity, “fire-safe” cigarettes); porosity (described below) and the type and level of chemicals or additives (90). 14 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report Cigarette wrapper paper may alter smoke composition by directly contributing wrapper components or combustion components to mainstream smoke; by diffusion of smoke components through the wrapper; by diffusion of air through the wrapper; by altering the linear velocity, volume and distribution of the airstream in and around the burning cone and by altering the amount of tobacco burnt per puff (69). The most common means of reducing smoke yields, after filter ventilation, is changing paper porosity (91). Porosity, which is the permeability of paper to oxygen and smoke gases when under a pressure differential, affects the burn rate, puff count and the amount of tobacco burnt per puff. The porosity of paper is controlled by the size (void volume) of the openings (pores) created by the bonded structure of cellulose fibres and calcium carbonate. Paper porosity can affect taste, delivery and variation in smoke dilution (80, 90, 92). The porosity of cigarettes in the USA typically ranges from 30 to 50 units in the system defined by the Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) (79). Paper porosity influences the burn temperature of a cigarette. As porosity increases, the coal temperature decreases (93), and the cigarette burns faster because of an increased static burn rate. The result under machine-smoking conditions is that more tobacco is consumed between puffs, fewer puffs are taken, and nicotine, tar and CO yields are reduced (82, 91, 94). Very volatile smoke constituents such as CO readily diffuse through the porous wrapper, so that they are delivered in lower concentrations than less volatile constituents (94). Delivery of benzo[a]pyrene decreases as paper porosity increases, as less tobacco is consumed during puffing and more is burnt in the interval between puffs (81). 2.3.3 Filter Most commonly used cigarette filters are made of cellulose acetate, paper or a combination of the two (81). Crimped cellulose acetate fibre (“tow”) is used in about 90% of all filters (98). Cigarette filters help to control cigarette pressure drop, absorb vapours and remove particulate matter from smoke. Filtration occurs by one of three mechanisms: mechanical trapping of particles, condensation followed by adsorption from the gas phase or transfer via the gas phase between particles and the filter (96). Acetate filters show negative selectivity for nicotine, and the average particle size of nicotine is smaller than in unfiltered cigarettes (95, 96). Consequently, more nicotine may be emitted in the mainstream smoke of cigarettes with acetate filters than in the smoke of unfiltered cigarettes, and the smaller particle size may mean that a larger percentage of the inhaled particulate matter travels further into the lungs (83). Fibrous filters significantly reduce the levels of semi-volatile and nonvolatile substances in smoke, slightly reduce the levels of vapour-phase compounds but do Cigarette characteristics and design features 15 not reduce those of gases (97). Studies of machine-smoked cigarettes indicate that the smoke constituents removed by cellulose acetate filters include: water (60–75%), cresols (70–75%), particulate matter (35–40%), volatile N-nitrosamines (≤ 75%), acrolein (reduced to “a limited extent”) and, notably, phenol (70–80%) (85, 91, 98). 2.3.3.1 Filter ventilation Filter ventilation is defined as air entering a cigarette through the portion of tipping paper that does not overlap the tobacco rod (99). Filter ventilation is achieved through a combination of a porous plug wrap and perforated or porous tipping paper. The degree of ventilation or dilution achieved depends on the porosity of the plug wrap, the perforation or porosity of the tipping-paper and the location of the perforations (81). Ventilation ranges from about 10% in some full-flavoured cigarettes to 80% in very low-delivery brands (100). The design feature of filter vent holes is easily defeated by smokers, who knowingly or unwittingly block them with their lips or fingers when they take a puff (10). The information presented here refers to the theoretical aspects of filter ventilation as a design feature and that derived from studies of machine-smoked cigarettes with unobscured filter vents. When highly ventilated cigarettes are machine smoked under more intense conditions (larger puff volume, vents blocked), their emission levels may be equal to or exceed those from less ventilated, full-flavour cigarettes machine smoked under less intense International Organization for Standardization (ISO) conditions with the filter vents unblocked (101). Filter ventilation allows more complete combustion of tobacco and greater retention of particulate matter by the cellulose acetate in the filter (85, 86). Both particulate delivery and vapour- or gas-phase delivery are reduced, generally in direct proportion to the degree of ventilation (81). The effects of ventilation are not, however, entirely due to dilution of the smoke; the emissions of some compounds are increased or decreased, while those of others, including total nicotine, are relatively unchanged (34). 2.3.3.2 Absorbent filtration materials, charcoal Cigarette filters may contain filtering aids, such as charcoal and other solid or liquid additives, for selective filtration of emissions (81). Carbon granules, silica gel and alumina are examples of solid adsorbent materials used in filters (95). Carbon effectively adsorbs chemicals with boiling-points between 0 and 100 °C (e.g. acetaldehyde, acrolein and hydrogen cyanide) and can remove some chemicals with boiling-points up to 150  °C (98). Depending on the smoking machine conditions, carbon (charcoal) filters can significantly reduce the levels of semi-volatile and vapour-phase compounds in smoke and slightly reduce the levels of non-volatile compounds (97, 102). The levels of compounds of lower molecular mass that occur in significant amounts in the vapour phase (e.g. phenol, 16 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report cresols, hydroquinone) are reduced to a greater extent by charcoal filtration than are those of compounds of higher molecular mass and significantly lower volatility (e.g. benzo[a]pyrene, TSNAs) (103). Charcoal filters usually do not reduce the levels of low-molecular-mass gases in smoke (97), although charcoal coated with a mixture of metallic oxides is reportedly effective in removing acidic gases (81). The efficiency of removal depends on the amount of charcoal, the smoking machine conditions (smoking intensity) and the age of the charcoal filter (97, 103). For example, hydrogen cyanide retention by a standard carbon filter decreases with the age of the cigarette, from about 38% at 0 weeks to about 25% at 8 weeks (97). When charcoal-filtered cigarettes (about 45 mg charcoal) were smoked under more intense smoking machine conditions, the tar, nicotine and CO emissions and the reduced emissions of volatile constituents measured under less intense ISO smoking conditions were no longer significantly lower than in the smoke of cellulose acetate-filtered cigarettes, because insufficient charcoal was present. Filters with more charcoal (120 or 180 mg) resulted in significant reductions under both intense and less intense smoking conditions (103). Synthetic high-activity carbon spheres with a different pore structure from natural carbon have been used in the filters of experimental cigarettes, alone and in various combinations with treated tobacco and alternative filter ventilation. The cigarette circumference varied from 17 mm to 24.6 mm (104). Slimmer cigarettes had less charcoal in the filter (17-mm cigarettes with a filter length of 27 or 33 mm and 20.4 or 30.6 mg charcoal, respectively, versus 24.6-mm cigarettes with a filter length of 27, 33 or 37 mm and 48, 72 or 88 mg charcoal, respectively). The smoking machine-generated tar yields of the larger cigarettes decreased as the carbon load increased; however, the tar yield of the slimmer, 17-mm cigarettes increased. The yields of many volatile constituents of smoke were significantly reduced as the carbon load increased, especially isoprene, acetaldehyde and acetone, with smaller reductions in pyridine, formaldehyde and styrene. The yields of hydrogen cyanide and 1,3-butadiene did not change significantly in the 17-mm cigarettes as carbon loading increased. The emissions of volatile smoke constituents from the slimmer cigarettes with activated carbon filters were higher than those from the wider cigarettes because of the greater smoke velocity in slimmer cigarettes and the lower activated carbon content. The reductions in volatile chemicals levelled off with the two highest charcoal loads, which the authors attributed to a limit in the amount of high-activity carbon that is effective in reducing the yields of some toxicants in a cigarette filter (104). 2.3.4 Physical dimensions 2.3.4.1 Diameter and circumference The usual diameter of a conventional cigarette is 7.5–8.0 mm, although slim vari- eties may measure 5 or 6 mm (83). The amount of tobacco consumed depends on Cigarette characteristics and design features 17 the circumference of the cigarette, and tar and CO yields increase as the circum- ference increases (105). The emissions to smokers from cigarettes with cellulose acetate filters and a smaller circumference decrease accordingly (83). 2.3.4.2 Length Cigarette length generally falls into one of four categories: “regular”, 68–70-mm unfiltered; “king size”, 79–88-mm filtered; “long”, 94–101-mm filtered and “extra- long” 110–121 mm filtered (83). Decreasing the cigarette circumference while keeping the packing density constant reduces the amount of tobacco available for burning and allows greater use of oxygen during combustion (85, 86). As the circumference of a cigarette decreases, less tobacco is available for consumption, with a corresponding decrease in some smoke emissions (106). Some chemicals are filtered through the tobacco rod as smoke is drawn through the unburnt portion of the cigarette column (98). Most smoke constituents, notably semi-volatile compounds, are formed during transit through the tobacco rod, as combustion products move from the burning zone at the lit end of the cigarette to a zone of lower temperature and lower oxygen downstream pyrolysis and distillation. For example, PAHs are formed in the lower-temperature regions of a burning cigarette. The smoke is condensated and filtered by the tobacco as it moves towards the mouth end of the cigarette (107). Filtration of nicotine by the tobacco rod decreases with decreasing rod length of filtered and unfiltered cigarettes, whereas filtration of smoke condensate by the tobacco rod is considered to be independent of the length of the rod (108). 2.3.4.3 Packing density The mediating effect of cigarette length on smoke composition depends on the packing density of the tobacco (69). Increasing packing density provides more tobacco mass to burn during puffs, with a corresponding increase in chemical emissions in mainstream smoke. As described above, however, some smoke constituents are filtered as smoke is drawn through the tobacco rod. In one study of cigarettes of different packing densities that were machine smoked to predetermined lengths, the yields of nicotine and smoke condensate were lower in cigarettes with higher packing density and higher in cigarettes with lower packing density (108). 2.3.4.4 Implications for super-slim cigarettes As the circumference of a cigarette decreases, less tobacco is available for con- sumption, with corresponding decreases in some smoke emissions (106), as noted for cigarettes with circumferences smaller than the regular 24.8–25.5 mm (e.g. ≤ 23 mm) (85). Decreasing circumference results in decreases in both total delivery and per puff delivery under machine-smoking conditions (79). 18 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report Decreasing cigarette circumference, while keeping the packing density constant, reduces the amount of tobacco available for burning and allows a larger volume of oxygen consumption during combustion. This reportedly results in reductions in the yields of some smoke emissions, including tar, nicotine, CO and several volatile smoke constituents. For example, as the circumference decreases from 26 to 21 mm, the amount of CO per puff decreases by about 20% and that of benzo[a]pyrene by about 40%; however, with the same design parameters, the level of hydrogen cyanide in mainstream smoke is relatively unchanged as the circumference decreases. Nicotine delivery in mainstream machine- generated smoke decreased from 1.56 mg from a cigarette with a circumference of 26 mm to 1.21 mg from one with a circumference of 23 mm (85, 86, 109). In a recent study of the emissions of a large number of chemical constituents from six machine-smoked, super-slim, flue-cured tobacco cigarette varieties sold in Canada (diameter, 5.3–5.4 mm; circumference, 16.7–17 mm; length, 83–99 mm; and tobacco weight, 296–371 mg), the levels of all chemicals except formaldehyde, ammonia and phenols were lower than in a standard-size research cigarette, owing to the smaller quantity of tobacco and the reduced puff count. The increase in formaldehyde emissions from the super-slim cigarettes was attributed to an increased ratio of circumference to cross-sectional area, which facilitated oxidation reactions by allowing more tobacco to come into contact with ambient air during a puff. Decreased circumference is also thought to increase the combustion temperature, which contributes to higher emissions of phenols (109). Decreasing cigarette circumference also increases flow rates, which reduces the time for the smoke to pass from the coal to the mouth end of the cigarette (residence time) and decreases the filtration achieved by the tobacco rod and retention by the filter (110). Factors that reduce filtration by the tobacco rod and retention by the filter may result in higher smoke emissions. The velocity of smoke in super-slim cigarettes is more than twice that in cigarettes of standard circumference (110). As smoke velocity increases, particulate retention decreases, and there is less time for diffusion of gas-phase chemicals through the paper. Smoke velocity negatively affects particle retention and vapour adsorption in a cigarette filter (110, 111). The effect of smoke velocity on adsorption of vapour-phase chemicals depends on the amount and the properties of the chemical (molecular mass and reactivity) and on the contact time with adsorbent materials (110). For example, filter retention of hydrogen cyanide decreases steeply as the circumference decreases and tobacco weight is held constant, suggesting that corresponding increases in air velocity with decreasing circumference influence the formation of chemicals such as hydrogen cyanide that are distributed between the particulate and the gas phases (98, 112). When experimental blended-tobacco super-slim cigarettes with unventilated carbon filters (15–90 mg per filter) were machine-smoked, about Cigarette characteristics and design features 19 twice as much carbon was required to retain about 50% of a smoke constituent when the super-slim was smoked under Canadian intense conditions than when it was smoked under ISO conditions (110). The complexity and interrelatedness of cigarette design features on smoke delivery make it difficult to propose specific design standards. More information is required on the consequences of changing design features. Furthermore, variations in the components of individual cigarettes are poorly understood, making it difficult to estimate interactions among them (79). Thus, it might be appropriate to focus on the design features and product characteristics that most influence use behaviour, such as puff volume. While it is generally recognized that some well-known design features, such as filter vents, can lead to compensatory smoking, other features, such as the porosity of the plug wrap and tipping paper and properties of the tobacco rod, also affect smoke dilution and delivery and thus allow smokers to get more nicotine and other smoke emissions for a fixed volume of smoke. Tobacco manufacturers can, however, adjust other design features in order to compensate for changes that alter emissions, such as maintaining tar and nicotine delivery levels when they switch to paper that complies with fire standards (113, 114). Consequently, product standards intended to lower the delivery of emissions should be based on delivery outcomes and not on changes in design that are anticipated to achieve such reductions. 2.4 Design features and additives that modify smoke pH and addictiveness 2.4.1 Overview Nicotine, the primary addictive substance in tobacco, determines smoker “satisfaction” and the “physiological” strength of cigarette smoke (72, 87). The addictiveness of nicotine is enhanced in various ways, such as by increasing the amount of total nicotine present in smoke, increasing uptake and controlling “smoothness” for optimal inhalation. In the tobacco leaf, nicotine is present mainly in the protonated salt form, but higher pH can increase deprotonation (115). The unprotonated (volatile) or free base form of nicotine is more “physiologically effective” than the protonated (non-volatile) form (116) and is more rapidly available, by two mechanisms: because it is present in the volatile phase of smoke, it does not have to diffuse out of the smoke particle; and the unprotonated form is more lipophilic and can therefore diffuse rapidly across cell membranes and be taken up more quickly into the bloodstream (117, 118). The unprotonated nicotine fraction – but not the total amount of deliverable nicotine – is influenced largely by the alkalinity of cigarette smoke. Cigarette smokers experienced greater electrophysiological and subjective responses to the smoke of cigarettes with nicotine as base than with nicotine 20 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report as the citrate (116). Industry documents indicate that unprotonated nicotine must be present to ensure a favourable sensory effect, termed the “impact”, of cigarette smoke (119–122). Opposing positions on the effect of smoke pH on unprotonated nicotine have been published, however, and attempts have been made to study the effect empirically. Calicutt et al. (123) found no significant difference in nicotine transfer among the test cigarettes analysed, which differed only in ammonia content. Varying the ammonia content of cigarettes would, however, affect only free nicotine and not total nicotine delivery. The total amount of nicotine absorbed is less pertinent than the rate of nicotine absorption, as the human body effectively absorbs most of the nicotine introduced by smoking. van Amsterdam et al. (124) examined nicotine uptake in venous blood samples from subjects who smoked test cigarettes with different measured levels of ammonia in the filler (0.89 and 3.43 mg/g). No difference was seen in “nicotine exposure”; however, the first sample was taken only 2.5 min after the last puff, which would not reflect absorption of free-base nicotine. 2.4.2 Ammonia, sugars and reconstituted tobacco Ammonia has been described as an “ameliorant”, an “impact booster” and a “satisfaction promoter” (125). It is an active species, capable of causing complex changes when added to a tobacco blend (126). The addition of ammonia and ammonia precursor compounds such as diammonium phosphate to tobacco increases the amount of unprotonated nicotine in both particulate matter and vapour (127). Ammonia or diammonium phosphate is used in the production of reconstituted sheets, as it reacts with pectins and forms stable complexes with nicotine. The complexes decompose at the high temperatures typically reached during smoking, thereby increasing the transfer of nicotine from the filler material to the smoke, a characteristic known as “nicotine transfer efficiency” (128). Increasing the temperature at which nicotine is released could increase the levels of unprotonated nicotine because the hydrolysis of nicotine is temperature- dependent (129, 130). Ammonia stimulates the taste receptors, olfactory endings and the trigeminal nerve, giving a sensation described as “mouth feel” (131). It reacts immediately with acids present in smoke, acting as an ameliorant. Binding of acids that could form salts with nicotine could liberate more free nicotine during pyrolysis (132). Industry documents on tobacco smoke describe the total basic fraction (pyrazines, pyridines and alkaloids) and the total acidic fraction (organic acids, phenyl acids, phenolic acids and fatty acids), the larger fraction being basic. In the manufacture of reconstituted tobacco sheet and during casing, diammonium phosphate can react with reducing sugars to produce the Maillard- reaction products deoxyfructazines (133), and pyrolysis of these products gives several pyridines and pyrazines that contribute to both the taste and the alkalinity of smoke (134, 135). Hundreds of other bases have been identified in tobacco smoke, Cigarette characteristics and design features 21 most of which are nitrogen heterocycles associated with smoke flavours, probably formed during reactions involving ammonia and sugars that may also contribute to a basic smoke pH (136, 137). Amino acids present at high levels in burley tobacco can also react with sugars to create similar, weakly basic compounds (138, 139). A major pyrolysis product of sugar is acetaldehyde, which is thought to act synergistically with nicotine and increase addiction to cigarette smoke (128, 140). 2.4.3 Other ingredients Ammonia is not the only additive capable of deprotonating nicotine and forming Maillard reaction products with sugars: several other bases present in smoke create conditions favourable for the formation of unprotonated nicotine. Industry documents indicate that the urea–urease system is also used to raise the pH of the smoke by breaking urea down to ammonia by pyrolysis (141, 142). Inorganic cations such as potassium and calcium can also raise the pH of smoke. As diammonium phosphate has been banned in some countries, other bases, such as calcium carbonate, are used to enhance nicotine delivery (142). The levels of alkali metals like potassium and calcium can be manipulated by the use of fertilizers or curing practices or added directly to a tobacco blend, so that it is difficult to differentiate between native and added amounts in routine analysis. Calcium and sodium carbonates can also be added to cigarette filters to increase smoke pH, possibly eliminating the need for adding bases to tobacco filler (143). A basic filter can liberate trapped nicotine, delivering volatile nicotine to smoke (144). If smoke is perceived as too harsh, smokers might inhale less deeply; additives like laevulinic acid and liquorice may make smoke smoother and therefore more appealing and easier to inhale (145). Additives like cocoa and menthol may not increase smoke pH but have been implicated as potential bronchodilators, thereby increasing the depth and volume of inhalation and facilitating total nicotine absorption (146). Further, combustion products of cocoa might have monoamine oxidase inhibition properties, with an anti-depressant effect, which could contribute to the addictiveness of smoking in the presence or absence of nicotine (142). 2.4.4 Tobacco blend and physical characteristics Differences in blends, inclusion of expanded tobacco and the position of tobacco leaves on the stalk can all alter the pH and chemistry of smoke, without chemical additives (147, 148). At a slightly acidic smoke pH (6.5–7.0), about 7% of nicotine is absorbed into a smoker’s system; less is absorbed at a pH < 6.6 (131). Flue-cured and American-blend cigarettes are slightly acidic, with a pH of 5.7–6.2. The pH of the smoke of cigarettes made with air-cured tobaccos is 6.5–7.8 (86), whereas that of smoke from a burley cigarette may be > 7.5. Both the total nicotine delivered and the resulting smoke pH of burley tobaccos are strongly influenced by stalk 22 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report position: leaves at higher stalk positions contain more nicotine and are more basic. Cigarettes made only with burley tobaccos may have increased unprotonated nicotine delivery; however, they may be perceived as harsh by smokers. The addition of pH-reducing sugars can mask the harshness, resulting in control of unprotonated nicotine delivery from the blend (149). Expanded tobacco produced with ammonium carbonate releases ammonia into the smoke on burning, without the addition of ammonia (142). Expanded tobacco includes stems, which have a higher nitrate content than leaves and substantially influence the smoke pH, as nitrate is partially reduced to ammonia during smoking (86). Certain characteristics of cigarettes, such as more porous paper and filter ventilation, could also raise the smoke pH. Although both smoke pH and nicotine content increase with increasing tip ventilation, the mechanisms are poorly understood. Air drawn through filter ventilation holes could act as a “drying gas”, reducing the water content of the aerosol particles and effectively increasing the pH, thereby favouring formation of unprotonated base nicotine in the gas phase (138). The burning rate of tobacco may also be affected by tip ventilation (139), or the tar:nicotine ratio could change with increased ventilation (150). Both mechanisms would raise the smoke pH and therefore the level of unprotonated nicotine. 2.4.5 Measuring “smoke pH” As pH cannot be measured in a smoke aerosol, smoke pH is usually measured in an aqueous solution (149). This measure was used to compare differences between brands by the tobacco industry for years and was useful for tracking changes made to the acidic and basic properties of cigarettes to achieve sensory effects (151). Current, non-industry methods for measuring unprotonated nicotine in mainstream smoke include headspace analysis of particulate matter collected on a Cambridge filter pad (CFP), gas chromatography (GC)–mass spectrometry (MS) of samples collected in bags (152, 153) and analysis of collected particulate matter by nuclear magnetic resonance spectroscopy (154). All the methods for analysing a dynamic reaction like the partitioning of nicotine between phases in the cigarette rod, cigarette filter and smoke aerosol have drawbacks. At best, relative differences between brands can be identified. Nonetheless, the tobacco industry has relied on such relative measurements since ammonia technology became the intense focus of industry research decades ago. 2.5 Innovations that could influence either perception or delivery 2.5.1 Overview In this section, we describe innovations that could influence perceptions or deliv- ery, which have either recently been marketed or are being developed, according Cigarette characteristics and design features 23 to publications in the scientific literature and other sources, such as websites, tobacco industry documents and patents. A background paper for the seventh meeting of WHO TobReg on the evolution of new tobacco products (155), including products that potentially “modify risk”, described notable alterations to traditional products on the market, such as menthol capsules in filters and organic cigarettes with no additives. A new line of very-low-nicotine cigarettes has been introduced, with a nicotine emission of < 0.04 mg but a “normal” level of tar when smoked under ISO machine conditions. The paper also described technologies in development, including several types of treated tobacco and novel filters. Many of these developments were claimed to result in reduced exposure, but most of the studies used as a basis for such claims were performed and published by the industry. Tobacco substitute sheet materials dilute the amount of tobacco in a blend, and treatment of the tobacco blend reduces the levels of components that are precursors of toxicants, such as proteins. Modified filters reportedly reduce the levels of toxic smoke components in mainstream smoke by reacting with or selectively filtering smoke components; examples include amine resin, which reacts with aldehydes and hydrogen cyanide, and charcoal filters. Selective reduction of some mainstream smoke toxicants was reported with most of these products, but in some cases the levels of other toxicants increased. As smokers must inhale sufficient nicotine to sustain their addiction, toxicant levels should be expressed per nicotine level; however, nicotine emission levels were often not reported. Some of these products were reported to be less toxic in vitro or to give lower levels of biomarkers of exposure (155). Consumers, however, generally found these products to be less acceptable than traditional cigarettes. It is therefore difficult to assess the net effect of these new technologies. The concerns should be kept in mind when evaluating the new tobacco industry research described in section 2.5.5. Sections 2.5.2–2.5.5 summarize innovations introduced since October 2013, when the literature search for the background paper (155) was finalized. 2.5.2 Reduced-nicotine cigarettes Unlike cigarettes that are designed (e.g. with filter ventilation) to yield less nicotine in the smoke, as measured with the ISO smoking method, reduced-nicotine cigarettes have less nicotine in the tobacco filler. “Magic” reduced-nicotine cigarettes (which emit 0.04 mg nicotine per cigarette) recently became available in tobacco shops in Spain, bearing the claim that they contain no nicotine. In accordance with European regulations that require cigarette manufacturers to list the nicotine yield directly on each pack of cigarettes and to round the yield to the nearest 1/10 place, Magic 0 packs prominently feature the words “0.0 mg nicotine” (156). Recently, use of denicotinized cigarettes was tested as a complement to standard smoking cessation treatment, consisting of behavioural support 24 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report combined with pharmacotherapy (varenicline or nicotine replacement therapy). Abstinence from cigarettes was significantly higher with nicotine-free cigarettes than with standard treatment after 1 (70% vs 53%) or 4 (58% vs 43%) weeks but not after 12 weeks (39% vs 31%) (157). In a study of 840 smokers of five or more cigarettes a day, smokers who switched to cigarettes with a lower nicotine content were smoking fewer cigarettes per day (about 16) after 6 weeks than those who smoked cigarettes with a normal nicotine content (several types were tested; about 22), and no significant compensation by smoking more intensely was observed (43). Nevertheless, the participants commonly smoked cigarettes outside the study, which probably obviated any reduction in exposure to nicotine. The researchers are conducting further studies with different approaches, such as a gradual vs an immediate reduction to very-low-nicotine cigarettes and combining such cigarettes with nicotine patches. 2.5.3 Coloured cigarette paper Some cigarette brands have coloured paper (Fig. 2.1). These include Ziganov Colours (pink, dark pink, yellow, green and purple), Ziganov Black, Sobranie Cocktails, Fantasia, Black Devil, Pink Elephant, Nat Sherman Fantasia and Vanity Fair. Coloured cigarette tubes are available for roll-your-own cigarettes (158). Fig. 2.1. Examples of coloured cigarettes A web post states that Sobranie Cocktails “… are five separate bright pastel shades with a gold foil filter, and are the same ring gauge as standard cigarettes, unlike Nat Sherman’s Fantasias, which are slimmer and use deeper, primary colours” (159). This type of cigarette is “… particularly made for ladies with its slim features and bright colours which attracted many women to this popular brand.” Cigarette characteristics and design features 25 Few studies are available on perceptions of the colour of cigarettes, in contrast to cigarette pack design. As discussed in section 2.2.2.1, brightly coloured cigarettes can create significant interest and are generally perceived as appealing, pleasant tasting and less harmful (14), whereas black cigarette paper may have low appeal and be associated with a strong taste and greater harm. The WHO FCTC advises countries to prohibit or restrict features that make tobacco products more attractive to consumers, including coloured cigarette paper. “Colouring agents are added to various components of tobacco products to make the resulting product more appealing. Attractively coloured cigarettes (e.g. pink, black, denim blue) have been marketed in some countries. Examples of colouring agents include inks (e.g. imitation cork pattern on tipping paper) and pigments (e.g. titanium dioxide in filter material)” (36). 2.5.4 Specialty filters Many filter types are available from cigarette material suppliers, suggesting a demand from the tobacco industry. For instance, the company Hauni Maschinenbau offers 18 types that differ in visual effect, filtration properties, taste enhancement and interactivity (160). Various elements and combinations can be used, such as charcoal, hollow shapes in e.g. the form of a heart and coloured filters. Tobacco, flavour capsules or herbal or botanical granules can be added to filters. Different tastes can be achieved by inserting flavoured thread or spraying flavour directly into the filter tow. Flavoured thread can be coloured “to create a more unique appearance”. Essentra Filter Products also has a wide range of filters available in different product ranges, e.g. sensory (capsules, flavour thread, direct application on filter), earth tones (faster degradation in the environment), performance (high filtration efficiency, also selectively for e.g. vapours) and visual differentiation (“…use visual appearance to indicate a flavour, a particular product attribute, a brand logo or indeed just to visually differentiate your brand”) (161). Coloured flavour threads that can be used to add ingredients such as menthol are described as a “visual indicator of taste delivery technology”. For instance, DJ Mix Flavoured Cigarettes in the USA have not only a coloured package but also the same colour applied to the filter to reflect product flavours (e.g. red for strawberry and green for apple). Marlboro Black Freeze (Mexico) has a menthol stripe running through the middle of the filter and the same symbolic stripe printed on paper. The new European Tobacco Products Directive 2014/40/EU (29), in Article 7 on regulation of ingredients, prohibits the use of flavourings, tobacco or nicotine in filters and cigarette paper: “Member States shall prohibit the placing on the market of tobacco products containing flavourings in any of their components such as filters, papers, packages, capsules or any technical features 26 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report allowing modification of the smell or taste of the tobacco products concerned or their smoke intensity. Filters, papers and capsules shall not contain tobacco or nicotine.” Flavour capsules were already described in the background paper on novel tobacco products (155). According to industry reports, flavour capsules in cigarette filters, which can be crushed to release a burst of flavour, are a significant growth segment (162). Capsules typically contain menthol or similar flavours, such as lemon mint, and are available in many different types of cigarettes; sometimes, two differently flavoured capsules are present in one filter. A study among smokers in Australia, Mexico and the USA showed that flavour capsules are most attractive to young people, use of cigarettes with flavour capsules is growing, they are associated with misperceptions of relative harm, and young people differentiate brands (162). A focus group study among young female nonsmokers and occasional smokers showed that they perceived flavour-capsule cigarettes very positively (14). They appreciated the novelty and liked the fact that the taste could be switched from “normal” to menthol. Just as research shows that cigarette packs can influence perceptions of appeal, harm and taste, this study suggests that the actual cigarettes can also do so. Two recent studies of the effects on mainstream smoke of a crushed menthol capsule in Camel Crush found no change in the yields of particle- phase constituents. Gordon et al. (163), using a real-time detector, found not only the expected increase in menthol delivery but also increased yields of several gas-phase constituents, notably five volatile organic compounds (VOCs), acetaldehyde, acrylonitrile, benzene, 1,3-butadiene and isoprene. Dolka et al. (164), at Philip Morris, however, found no such increases when using cooled impingers with methanol to sample gas-phase components. 2.5.5 Tobacco industry research on delivery through special filters and with treated tobacco Techniques are being developed for producing reduced-toxicant emission cigarettes, including filter adsorbents, blend tobacco treatments and tobacco substitute sheets. British American Tobacco examined the effects of modifying filter ventilation, varying cigarette circumference and active charcoal filter length and loading and combinations of these features (104). An air-dilution mechanism, called “split-tipping”, was developed in which a gap between two separated sections of tipping paper, exposing an area of the filter, is wrapped with a band of porous paper. This band minimizes the loss of effective filter ventilation that occurs at the high flow rates encountered during human smoking and facilitates the diffusional loss of volatile toxicants. The results showed that the ratio of these toxicants to nicotine emissions in mainstream smoke was reduced, except in the test cigarettes with 1 mg of tar. Cigarette characteristics and design features 27 Another paper from British American Tobacco described assessment of the genotoxicity and cytotoxicity in vitro of the particulate matter generated from experimental cigarettes with 50% blend tobacco, 15% tobacco substitute sheet, polymer-derived activated charcoal and split-tipping (165). In comparison with control cigarettes that had a standard cellulose acetate filter, tipping paper and typical tobacco blends (3R4F, a US-style blended product, and M4A, a flue-cured product), bacterial mutagenicity and mammalian genotoxicity were reduced with the experimental cigarette, whereas there was no significant difference in cytotoxicity. A study funded partly by Guangdong Tobacco Industrial Company (166) described use of specific filter additives and molecularly imprinted polymers with nicotinamide as the template on a silica surface for the adsorption of TSNAs in mainstream cigarette smoke. The levels of TSNAs were reduced by up to 41% as compared with those in the cigarette smoke of the control group. This study would appear to be selective, as the tar levels remained the same and nicotine levels were not reported. A study from Cultex Laboratories GmbH and Japan Tobacco Inc. showed that smoke from K3R4F cigarettes with integrated charcoal filter tips were less toxic to cilia in normal bronchial epithelial cells than regular K3R4F cigarette smoke, when machine-smoked under standard ISO conditions (167). VOCs, which were removed by the charcoal filter tip, affect cilia formation in primary bronchiolar epithelial cells. Histopathological analysis of the exposed cultures showed fewer cilia-bearing cells, shorter existing cilia and, finally, disappearance of all cilia in cells exposed to cigarette smoke. In cultures exposed to charcoal-filtered cigarette smoke, small changes in cilia length were seen after four exposures, but the effects were reversed after a 2-day recovery period. A patent issued to Philip Morris describes the development of a tobacco smoking mixture and a cigarette wrapper containing high-temperature ammonia- release agents (168). The ammonium compounds were claimed to be present “in an amount effective to reduce the cytotoxicity of gas phase or particulate matter formed during smoking of the cigarette”. Although some of these new cigarette types were found to have lower machine yields of toxicants in mainstream smoke and reduced toxicity in vitro than conventional cigarettes, substantial scientific data would be required to conclude that they represent a lower health risk. In evaluating the efficacy of design changes in reducing human risk, consideration must be given to the acceptability of a product to consumers, its effect on their smoking behaviour and whether it actually results in reduced exposure as assessed by e.g. biomarkers. 28 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report 2.6 Research that would inform scientific evaluation of the public health impact of design characteristics As discussed above, a substantial body of evidence has established that cigarette attractiveness and addictiveness and the delivery of smoke toxicants to users are strongly associated with the physical characteristics and design features of cigarettes. The effects of certain characteristics have been studied in greater detail than others. For instance, the effects of filter ventilation on consumer perceptions, machine-generated emissions and the exposure of smokers have been extensively studied and reported, while there are limited data on the effects of flavours. Similarly, more data on the potential of reduced-nicotine cigarettes (< 0.4 mg/g in tobacco filler) to facilitate smoking cessation would be helpful. A systematic review of past and pending studies would be informative; however, it should not be used in any way to promote smoking. Because of the complexity of the interplay between consumer perceptions and behaviour and smoke chemistry, however, the available data do not necessarily provide a clear understanding of how certain physical features could be modified to reduce toxicant emissions and thus protect public health. Therefore, further research would inform the scientific basis for effective regulatory measures. Given the complexity of the impact of cigarette physical characteristics, tobacco type and use of additives on human exposure and the fact that exposure is mediated by smokers’ perceptions and behaviour, studies should take a comprehensive approach to determining how specific cigarette designs influence many outcomes, including machine smoke delivery, smokers’ beliefs and smoking topography and the resulting exposure. Studies of the effect of design features on emissions should always include nicotine levels. Any effect on emissions should be reported per milligram of nicotine, as smokers inhale sufficient amounts of nicotine to sustain their addiction (2, 70). As free-base nicotine is the most bio-available form, international standards for measuring free-base nicotine or determining the ratio of free-base to protonated nicotine would be helpful. In addition, researchers should be aware that any manipulation of a product to reduce the content of one or more constituents may unintentionally increase the concentrations of other constituents. Research approaches to investigating how design features interrelate and affect mainstream smoke emissions include: ■ systematic studies of individual design features case by case. For a few selected parameters, such as filter ventilation and cigarette dimensions, this approach could be applied in many testing laboratories. For other design parameters, like filter material or paper porosity, studies would have to be done in a well-equipped testing laboratory and might re- quire the production of custom cigarettes with specific design features. Cigarette characteristics and design features 29 ■ extensive multivariate analysis of tobacco filler constituents, main- stream smoke emissions (under smoking protocols of varying inten- sity) and the physical properties of cigarette products on the market. This approach would allow identification of the design parameters that have the greatest influence on mainstream smoke emissions. ■ in-depth, detailed statistical analyses of all relevant design features, parameters and specifications, with mainstream smoke emissions provided by cigarette manufacturers. This approach could be used if there is sufficient regulatory authority and would include checking of the results by an ISO 17025-accredited government laboratory or an independent contract laboratory as part of regulatory oversight. Appropriate tools for studying the perceptions and behaviour of smokers and nonsmokers, in particular adolescents, include consumer surveys, focus group analyses and clinical (topography and biomarker analyses) investigations. Actual exposure could be estimated by measuring a relevant set of biomarkers in smokers. The results will show whether reductions in machine-measured yields of specific constituents reduce the exposure of smokers. The health effects of exposure can be assessed in clinical studies, e.g. by measuring biomarkers of (early) effects. Alternatively, a set of relevant in-vitro assays for important smoking-related diseases could be used. In-vitro tests based on air–liquid interface cell models are promising, as they model the exposure of the airways to smoke. It is important to monitor developments in the tobacco product market in order to remain informed about innovations that concern public health, by, for example, standard searches of websites, including social media, as well as field research. 2.7 Conclusions The main purpose of cigarette design is to increase the appeal of the product (i.e. to make it more palatable, attractive or less harmful), to reduce the negative aspects of the product, to ensure that smokers experience satisfaction in using the product and to attract the interest of young people and novice users. Cigarette characteristics that increase their appeal include those that influence a user’s perception of the cigarette’s appearance or whether they can “customize” it. The decorative elements of cigarettes directly and substantially affect the appeal of the cigarette by suggesting strength, novelty or reduced harm, particularly to women and young smokers. These elements are some of numerous innovations that have been introduced by manufacturers. Given that the sole purpose of such features is to attract new consumers, they can lead to misperceptions of health risk. Limiting cigarette appearance to standard features, i.e. white paper, standard tipping paper colour and standard print of cigarette brand, could be expected to protect public health. 30 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report Most of the other physical characteristics of cigarettes have complex and sometimes opposite effects on multiple outcomes. For instance, filter ventilation results in lower machine-generated emissions per cigarette and perceptions of lighter taste and greater safety by smokers. Higher filter ventilation is an example of a physical characteristic that can change smoking behaviour, resulting in similar or higher exposure to toxic and carcinogenic emissions than would result from smoking less ventilated cigarettes. Filter vents are a design feature that is easily manipulated by smokers to obtain higher nicotine and smoke emissions from a cigarette. Porous tipping paper and cigarette wrappers and the properties of tobacco blends are other design features controlled by manufacturers that allow a smoker to unwittingly take more smoke from a cigarette. Cigarette dimensions are also associated with a complex interplay of outcomes. Thus, slim cigarettes contain less tobacco for burning and can therefore result in lower overall exposure of smokers per cigarette; however, slim cigarettes appeal to women with their stylish, attractive, high-quality appearance and are perceived as being less harmful, which is a public health concern. Furthermore, the exposure of smokers of these slim cigarettes to constituents such as hydrogen cyanide and formaldehyde may not be lower than from cigarettes of standard circumference. Research has been conducted not only on ventilated filters and slim cigarettes but also to support the more general hypothesis that manufacturers use tobacco blend properties and pressure drop (paper porosity, filtration, filter retention) as a product design strategy to develop cigarettes with an “elasticity” that allows smokers to obtain the amount of nicotine they desire and sensory “satisfaction”. Most cigarettes have some elasticity, especially “ultra-low” cigarettes, whereas full- flavour brands have less. Under machine smoking conditions, elasticity appears as nonlinear increases in toxic emissions with increasingly intense puffing. Cigarette design, such as filter additives that reduce emissions of selected chemicals in smoke, can modify sensory cues, resulting in changes in smoking behaviour. It has been shown that smokers take larger puffs when smoking cigarettes with charcoal filters. Adding chemicals to the smoke as flavours can also influence sensory cues. While there is some evidence that smokers perceive a flavoured cigarette as novel and take smaller puffs, the overall design of the cigarette means that they are exposed to harmful smoke emissions like CO as much as when they are smoking a tobacco-flavoured cigarette. The interplay between perceptions, behaviour and measures of exposure is complex. Perhaps the best example is use of mentholated cigarettes, which is reported to be associated with stronger addiction and fewer successful attempts to quit smoking; however, the results of studies on the influence of mentholation on smoking behaviour are mixed. Established smokers habitually use cigarettes to obtain nicotine. The satisfaction they experience when smoking is due to the sensation of tobacco smoke entering their mouths (“impact”), followed by rapid absorption of nicotine Cigarette characteristics and design features 31 from the lungs to the brain within seconds of inhalation. The unprotonated (un- ionized) form of nicotine is reported to be taken up from smoke more effectively, and it reaches the brain more rapidly than in the protonated (ionized) state. Several design features and additives can influence the proportion of nicotine that is in the unprotonated form. Alkalinizing agents increase the amount of unprotonated nicotine while increasing “mouth feel” and improving taste by forming products from reactions with acids and reducing sugars in smoke. Mouth feel and taste act as cues to smokers to modulate their smoking behaviour in response to the physiological “strength” of the smoke. Many innovations for changing perceptions or smoke emissions have focused on tobacco blend and filter technologies, because of their roles in controlling delivery and use behaviour. Non-traditional methods of adding flavours, such as flavour capsules and flavour threads, create appeal by their novelty and brand differentiation. Flavour capsules are a significant growth segment for the tobacco industry and are particularly attractive to young people. While some new technologies have been encouraging, such as reducing selected toxicants, the gains are frequently offset by increased amounts of other toxicants or poor consumer acceptability. The combination of filter additives and treated tobacco has been explored by the tobacco industry as a means of reducing emissions of toxicants. Internal industry documents suggest that laboratory assessment of these cigarettes show reduced toxicity; however, it is not known whether any of these technologies has been reviewed by regulators or used in commercial products in unregulated markets. Recent studies of low-nicotine cigarettes in a market where standard-nicotine cigarettes were available showed that smokers’ behaviour changed (they smoked fewer cigarettes per day) and that they were significantly more likely to abstain from smoking cigarettes; however, the abstinence was no better than after standard cessation treatment 12 weeks later, and smokers frequently smoked cigarettes with standard levels of nicotine. 2.8 Recommendations The ultimate goal of research on cigarette design is to ensure that any ensuing regulatory measures simultaneously reduce the attractiveness and addictiveness of cigarettes and the harm associated with their consumption, as already recommended in the partial guidelines for implementation of articles 9 and 10 of the FCTC (36). This can be achieved by standardizing cigarette appearance; eliminating design features and ingredients that make cigarettes more appealing to new or novice smokers or more difficult for established smokers to quit; reducing the addictiveness of cigarettes by lowering their nicotine level or the biological availability of nicotine; and reducing exposure to harmful emissions by a combination of selective filtration and modifications to cigarette dimensions, packing density and tobacco blend. 32 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report On the basis of the conclusions, the following specific policy and research recommendations are proposed. 2.8.1 Policy recommendations 1. Require manufacturers to disclose information on all the design fea- tures, parameters, specifications and levels of contents and emissions levels of current and emerging products. Examples include cigarette paper, capsules in cigarettes filters and cigarette dimensions. 2. Prohibit filter ventilation and any other design characteristic that allows cigarette elasticity (increased puff volume by smokers, especially of lower- tar varieties); and prohibit filter capsules, slim cigarettes and any oth- er product attribute that increases its attractiveness, smoke emissions or addictiveness. 3. Require lowering of all toxic emissions (per mg nicotine), according to the approach set out by TobReg (71). 2.8.2 Research recommendations 1. Continue research on the design characteristics of tobacco products and innovations in that area, including their impact on: – the perceptions and behaviour of smokers, former smokers and peo- ple who have never smoked, in particular adolescents; – emissions, normalized per mg of nicotine except for reduced-nico- tine cigarettes (< 0.4 mg nicotine per g tobacco in filler); – toxicity; and – exposure. 2. Develop and validate a standard method for measuring free-base nicotine levels or determining the ratio of free base to protonated nicotine. 3. Continue research on potential use of reduced-nicotine cigarettes as a smoking cessation strategy. A systematic review on past and pending studies may be informative, although it will be important to ensure that it is not used in any way to promote smoking. Cigarette characteristics and design features 33 2.9 References 1. Podraza K. Basic principles of cigarette design and function. Bethesda, MD: Life Sciences Re- search Office; 2001 (http://www.lsro.org/presentation_files/air/m_011029/podraza_102901. pdf, accessed 23 October 2015). 2. The scientific basis of tobacco product regulation: report of a WHO study group (WHO Techn- ical Report Series No. 945). Geneva: World Health Organization; 2007. 3. WHO Study Group on Tobacco Product Regulation. Advisory note. Global nicotine reduction strategy. Geneva: World Health Organization; 2015. 4. 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Possible application of WHO Tobacco Laboratory Network standard operating procedures to evaluation of electronic nicotine delivery systems Patricia Richter, Centers for Disease Control and Prevention, USA Rima Baalbaki, American University of Beirut, Lebanon Mirjana Djordjevic, National Cancer Institute, National Institutes of Health, USA Rachel El Hage, American University of Beirut, Lebanon Bryan Hearn, Centers for Disease Control and Prevention, USA Ahmad El Hellani, American University of Beirut, Lebanon Hongwei Hou, China National Tobacco Quality Supervision and Test Centre, China Qingyan Hu, China National Tobacco Quality Supervision and Test Centre, China Walther Klerx, National Institute for Public Health and the Environment, The Netherlands Naoki Kunugita, National Institute of Public Health, Japan Joseph Lisko, Centers for Disease Control and Prevention, USA Jose Perez, Centers for Disease Control and Prevention, USA Najat A Saliba, American University of Beirut, Lebanon Shigehisa Uchiyama, National Institute of Public Health, Japan Wouter Visser, National Institute for Public Health and the Environment, The Netherlands Clifford Watson, Centers for Disease Control and Prevention, USA Liqin Zhang, Centers for Disease Control and Prevention, USA Contents 3.1 Background 3.2 General methodological considerations in evaluating electronic nicotine delivery systems (ENDS) 3.3 Nicotine 3.3.1 Nicotine in ENDS liquid 3.3.2 Nicotine in ENDS aerosol 3.4 Tobacco-specific nitrosamines 3.4.1 Tobacco-specific nitrosamines in ENDS liquid 3.4.2 Tobacco-specific nitrosamines in ENDS aerosol 3.5 Benzo[a]pyrene 3.5.1 Benzo[a]pyrene in ENDS liquid 3.5.2 Benzo[a]pyrene in ENDS aerosol 3.6 Additional analytes 42 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report 3.6.1 Carbonyls 3.6.2 Solvents 3.6.3 Volatile organic compounds 3.6.4 Phenolic compounds 3.6.5 Metals 3.6.6 Flavours 3.7 Recommendations for extension of methods 3.7.1 Nicotine 3.7.2 Tobacco-specific nitrosamines 3.7.3 Benzo[a]pyrene 3.7.4 Volatile organic compounds 3.7.5 Carbonyls 3.8 Research required for future regulatory use of data on ENDS 3.9 Conclusions 3.10 Recommendations 3.11 References 3.1 Background This section provides recommendations on the application of existing and pending WHO Tobacco Laboratory Network (TobLabNet, http://www.who. int/tobacco/global_interaction/toblabnet/en/) Standard Operating Procedures (SOPs, available at http://www.who.int/tobacco/publications/prod_regulation/ en/) to the analysis of the content and emissions of Electronic Nicotine Delivery Systems (ENDS), as requested by the seventh session of Conference of the Parties (COP) to the WHO Framework Convention on Tobacco Control (WHO FCTC, http://www.who.int/fctc/cop/cop7/FCTC_COP7_9_EN.pdf?ua=1). The recommendations in this section may also be appropriate for electronic non- nicotine delivery systems (ENNDS) after determination of suitability for that matrix (e.g., appropriate measurement range, interference, etc.). ENDS consist of a battery that heats a coil and vaporizes a liquid matrix (content) to deliver an aerosol (emission), also referred to as a vapour. For the purposes of this report, the aerosol from the mouth end of the ENDS device is referred to as “first-hand aerosol” (FHA), the liquid matrix is referred to as “e-liquid” and when the e-liquid contains nicotine the device is referred to as Electronic Nicotine Delivery Systems (ENDS). “E-cigarette” is used when referring to cigarettes-shaped ENDS or when quoting from a source that uses this word. The vaporization temperature of ENDS is a function of the battery voltage and the current through the coil (1). The e-liquid is usually a solution containing propylyene glycol alone or in combination with vegetable glycerol, nicotine, flavourings and other constituents, such as caffeine. The e-liquids and resulting first-hand aerosols usually contain nicotine in a wide range of concentrations and other chemicals added to enhance appeal. Nicotine, minor tobacco alkaloids, Possible application of WHO standard operating procedures to evaluation of electronic nicotine delivery systems 43 tobacco-specific nitrosamines (TSNAs), flavourings, metals, VOCs, phenolic compounds, and solvents have been reported in e-liquids. Carbonyls, VOCs, TSNAs and metals have been reported in ENDS aerosol (2). ENDS may be disposable or reusable and may have features (e.g. variable voltage ranges) that allow the user to “customize” the delivery and chemical composition of the aerosol. Initially, ENDS were designed to resemble cigarettes in size and shape. The new generations of ENDS are larger, have refillable tanks and may resemble cigars, pipes or hookahs (waterpipes) or not look like any tobacco product at all (2–4) (Fig. 3.1). ENDS are sold worldwide (5), sometimes regulated as tobacco products, sometimes as consumer products and sometimes as pharmaceutical products. However, other countries have banned ENDS, refills that contain nicotine and even, ENNDS (90). Fig. 3.1. Electronic nicotine delivery systems In contrast to conventional tobacco cigarettes, for which there are reference materials (e.g. CORESTA Monitor and Kentucky research cigarettes), there are currently none for ENDS, and there are no methods based on human use for machine generation of ENDS aerosol for analysis. The patterns of use (topography) of ENDS products have been examined in only a few studies (6, 7), and the issue is further complicated by the diversity of ENDS products. CORESTA (https://www.coresta.org/), an international association of tobacco product manufacturers, tobacco industry institutes and laboratories, published a recommended method for machine generation of aerosol (8) for ENDS that contain “electronic components which vaporize a liquid to generate an aerosol carried by the air drawn through the device by the user. [The device] could be 44 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report designed either as a single piece or as a modular, multiple component product for disposable, rechargeable and/or refillable use.” The products reportedly covered by the method are those that meet the above definition and also “products described as e-cigarettes, e-cigars, e-shisha, e-pipes and other related product categories’. The CORESTA method is not based on measures of human puffing topography that reflect actual use or behaviour. Several companies have begun to manufacture and sell automated machines to generate ENDS aerosol (e.g. Cerulean, Milton Keynes, United Kingdom; and Borgwaldt GmbH, Hamburg, Germany). A machine designed to generate ENDS aerosol for analytical purposes should provide a source of electrical power for the device, and research should address the requirements for the power source. The available equipment and methods are optimized for cigarette-like devices (e-cigarettes); therefore, additional equipment or modifications to the method might be required for newer designs of ENDS, notably the larger “tank’ varieties. 3.2 General methodological considerations in the evaluation of ENDS Quantitative methods for chemical analysis of any product depend on the nature of the matrix in which measurements are made. The ENDS matrices (e-liquid or first-hand aerosol) are less chemically complex and less varied in composition than conventional tobacco products (tobacco filler and mainstream tobacco smoke). Standardized measures of mainstream cigarette smoke from conventional cigarettes apply to a particular brand under standard conditions specified by ISO (10), the Federal Trade Commission in the USA (11), the Centers for Disease Control and Prevention (CDC) in the USA (12), CORESTA (13, 14), Health Canada (13), the Commonwealth of Massachusetts Department of Public Health in the USA (15) and WHO (16). The smoking regimens designed for analysing conventional tobacco cigarettes differ only slightly amongst the less intense standard methods (e.g. ISO and Federal Trade Commission); others (e.g. Canadian Intense and Massachusetts) simulate larger puff volumes and the vent- blocking behaviour of smokers, which can give widely different results than with the less intense methods. All the methods generally include specific temperature and humidity-controlled conditioning of cigarette samples, machine smoking of cigarettes in a specified regime (i.e. puff volume, duration and interval) to a butt length determined for each product (23 mm, the filter length plus 8 mm or the filter overwrap plus 3 mm) and open, partially blocked, or completely blocked filter ventilation. The findings of studies on human ENDS use topography (6, 7) raise questions about whether standard and “intense” regimes (analogous to ISO and Canada Intense machine smoking regimens for tobacco cigarettes) are appropriate and the corresponding modifications to the procedure or equipment used to analyse emissions from different ENDS products. Possible application of WHO standard operating procedures to evaluation of electronic nicotine delivery systems 45 Machine-generated mainstream tobacco smoke samples are usually analysed, after sample preparation, by GC–MS or flame ionization detection (FID), liquid chromatography with ultraviolet–visible spectrophotometry, liquid chromatography with MS or inductively coupled plasma-MS. 3.3 Nicotine ENDS products deliver nicotine, an addictive chemical, via the respiratory system. The nicotine concentration listed on the labels of ENDS cartridges and refill e-liquid may be significantly different from the values measured in the liquid (3, 18). Sleiman and colleagues recently reported the levels of nicotine in ENDS liquid, measured by headspace GC with mass-selective detection (HS-GC/MS), from commercial ENDS products purchased at retail stores in California, USA. The levels were 20.4, 25.4 and 32.1 mg/mL for e-liquids with marketed nicotine levels of 18, 24, and 18 mg/ mL, respectively (1). 3.3.1 Nicotine in ENDS liquid Two factors should be considered when measuring the nicotine content of e-liquids. The first is the insolubility of propylyene glycol and vegetable glycerol in the hexane extraction solution used in the TobLabNet method for measuring nicotine in tobacco filler. They are more soluble in the isopropanol extraction solution used in the standard ISO method for measuring tar, nicotine and CO in smoke (10). Consequently, the standard ISO method for analysis of nicotine trapped on a Cambridge Filter Pad (CFP) is more appropriate for analysis of nicotine in e-liquid than is TobLabNet SOP-04. Alternatively, WHO SOP-04 could be used with a more miscible extraction solvent. An important consideration in the analysis of nicotine in ENDS e-liquid is that the upper level may greatly exceed that in tobacco cigarette smoke extracts, even those generated under intense smoking machine conditions (e.g. about 36 mg/mL versus 0.3 mg/mL; CDC, unpublished data). Accordingly, the isopropanol extraction volume must be adjusted so that the nicotine concentrations in the e-liquid samples fall within the calibration range. Secondly, it has been noted that knowing the total amount of nicotine in a tobacco product is not sufficient to understand its effect on users (19). Nicotine can occur in either the protonated or the unprotonated (also referred to as unionized or “free” nicotine) state. Absorbed nicotine in the unprotonated state reaches the brain more quickly than that in the protonated state, which is an important factor in the addiction potential of the chemical. Addition of alkalinizing agents increases the proportion of nicotine that is in the readily absorbed unprotonated form (20). The pH of e-liquids can be measured by the procedure commonly used for measuring the pH of smokeless tobacco, with timed measurements taken with a pH meter. The pH of some e-liquids has been 46 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report reported to be greater than the pKa of nicotine (3, 21) suggesting a substantial amount of the nicotine is unprotonated. 3.3.2 Nicotine in ENDS aerosol The smoking regime (standard or intense) is usually specified in methods for measuring nicotine in tobacco smoke, while the results of both regimes capture a range of possible smoking topographies among conventional cigarette smokers. The ISO smoking regime involves smoking cigarettes with the ventilation holes unblocked, a 35-mL puff volume, a 2-s puff duration, a 60-s puff interval and enough puffs to reach a butt length equivalent to the filter length plus 8 mm or the filter overwrap plus 3 mm (whichever is longer), whereas the Canadian “intense” and the WHO smoking regimes specify a 55-mL puff volume, a 2-s puff duration, a 30-s puff interval and 100% blockage of the cigarette filter ventilation holes. The resulting mainstream smoke total particulate matter (TPM) is extracted in isopropyl alcohol and analysed by GC–FID (10). For e-cigarettes, CORESTA has recommended a “vaping” (automated machine generation of e-cigarette aerosol) regime (8) to generate aerosol, with a 55-mL puff volume, a 3-s puff duration, a 30-s puff interval and no specified puff count, although at least 50 puffs per session are considered to generate adequate TPM on a CFP for determination of nicotine (22; CDC, unpublished data). The CORESTA method reportedly covers ENDS products designed for single use, disposable units and modular, multi- component products such as rechargeable and/or refillable devices (i.e. tank systems). If the method is verified by independent laboratories, it might obviate modifications to the procedure and equipment for analysing emissions from different product designs. The puffing parameters in the WHO SOP-01 intense machine smoking method are similar to those in the CORESTA method and could be modified for aerosol generation until sufficient data on product design variables and ENDS use behaviour become available to design a protocol for generating ENDS aerosol that is more representative of how the products are used. The CORESTA method does not specify the analytical platform for quantitative measurement of nicotine in the collected aerosol. As combustion is not expected to occur when ENDS are operated under non-intense conditions, the composition of the collected aerosol resembles that of the liquid. The analytical platforms used in the WHO SOPs for analysis of tobacco smoke extract could be applied to ENDS aerosol captured on a CFP. A study in which ENDS aerosols were collected on CFPs with a downstream adsorbent trap indicated that nicotine is present in aerosol particles and that more than 98% of the nicotine was captured by the CFP (22). The conventional detection schemes could be extended for use in analysing ENDS aerosol, especially as it is significantly less complex than tobacco smoke and the chemicals are soluble in isopropyl alcohol. A preliminary comparison of the CORESTA e-cigarette regime with a standard testing protocol for tobacco cigarette mainstream smoke showed that Possible application of WHO standard operating procedures to evaluation of electronic nicotine delivery systems 47 the CORESTA method provided reliable quantification of nicotine in a limited sample of ENDS products (CDC, unpublished data); similar results are expected with WHO SOP-01. Additional ENDS configurations such as e-pipes and e-hookahs should be evaluated in the future. Nicotine in tobacco smoke is usually measured in conjunction with “tar” (TPM minus nicotine and water) and CO. As in mainstream tobacco smoke, ENDS TPM, consisting of solvents, water, nicotine and other aerosol contents, is captured on a CFP during machine generation of ENDS aerosol (22). 3.4 Tobacco-specific nitrosamines TSNAs are formed mainly during the curing, fermentation and combustion of tobacco and are found in all types of tobacco product (23). WHO has recommended mandated lowering of TSNAs, specifically NNN and NNK, which are potent human carcinogens, in tobacco and tobacco smoke (24). 3.4.1 Tobacco-specific nitrosamines in ENDS liquid While some TSNAs are formed during combustion of tobacco from alkaloid precursors, they are mainly present in cured tobacco in cigarette filler and transferred directly to mainstream smoke during the combustion of tobacco (25). As the nicotine in e-liquid is extracted from tobacco, any TSNAs in ENDS e-liquid are probably impurities introduced during nicotine extraction. Laugesen (26) analysed the liquid in Ruyan® e-cigarette cartridges and found a nicotine content that varied from 0 to 16 mg per cartridge. Of the four TSNAs, only NNK was detectable in all cartridges. NNN was found at higher levels than NNK but was detectable only in the nicotine-containing cartridges; 0.260 ng NNK was detected in the “zero nicotine” cartridge. The levels of NNN and NNK increased with increasing concentrations of nicotine. In another study, NNN and NNK were found in the refill e-liquids of brands sold by 11 companies and purchased in the Republic of Korea (27), while Westenberger (28) found no detectable levels of TSNAs in 10 varieties of cartridge e-liquids for two brands purchased in the USA. Researchers at the National Institute for Public Health and the Environment (RIVM) in the Netherlands found detectable, but low levels of TSNAs in nearly all ENDS liquids by ultra-performance liquid chromatography coupled with tandem MS. A very small fraction of ENDS liquids contained up to 150 ng/mL of individual nitrosamines and 285 ng/mL total TSNAs (29). The higher concentrations found might be due to the use of tobacco extracts as a flavour, as all the liquids in which they were found were labelled “with tobacco flavour”. A comprehensive study of NNN and NNK levels in tobacco filler from cigarettes sold in 14 countries indicated total TSNA at a concentration of 0.087– 1.9 mg/g (30). Thus, the levels of TSNAs in ENDS liquid, when present, are much lower than in cigarette tobacco filler. 48 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report 3.4.2 Tobacco-specific nitrosamines in ENDS aerosol In the WHO SOP for determination of TSNAs in mainstream cigarette smoke under ISO and intense smoking conditions (31), cigarette smoke particulate matter is collected on a CFP, extracted with ammonium acetate and analysed in a high-performance liquid chromatography (HPLC) tandem MS system. Cigarette smoke is created by combusting tobacco filler, whereas aerosol from ENNNDS is created by heating e-liquid at temperatures that depend on the device parameters. As the tobacco cigarette smoke matrix is much more complex than ENDS aerosol matrix, containing about 8000 chemicals (32), the SOP should be applicable for the analysis of TSNAs in ENDS aerosol. In one study, however, the maximum levels of TSNAs detected in ENDS aerosol were 28.3 ± 13.2 ng per 150 puffs for NNK and 4.3 ± 2.4 ng per 150 puffs for NNN. With fewer puffs (e.g. 15), the estimated levels were 2.83 ng NNK and 0.43 ng NNN. Even if the minimum volume of extraction solution (10 mL) in the TobLabNet TSNA method were used, the NNK level would be 0.28 ng/mL and that of NNN 0.043 ng/mL, which are lower than the reporting limit of the method, 0.5 ng/mL (33). Thus, a higher puff count (e.g. 50) should be used to optimize the conditions for measuring TSNAs in ENDS aerosol. Comparisons of emissions “per unit” (i.e. per stick for a tobacco cigarette and per unit for a single-use ENDS product) or “per session” might give different results but would still be expected to be substantially lower than those in the mainstream smoke of tobacco cigarettes. 3.5 Benzo[a]pyrene Polycyclic aromatic hydrocarbons (PAHs) are a diverse group of carcinogens formed during incomplete combustion of organic materials such as tobacco. Benzo[a]pyrene is a widespread environmental pollutant, a human carcinogen and the most thoroughly studied member of this class of compound (34, 35). WHO has recommended lowering of benzo[a]pyrene levels in mainstream tobacco smoke (24). 3.5.1 Benzo[a]pyrene in ENDS liquid In several studies of harmful chemicals in ENDS e-liquid, no significant quantities of PAHs were found. Kavvalakis et al. (36) found no PAHs in e-liquid samples on the Greek market, and Leondiadis found no PAHs in Nobacco brand refill e-liquids (37). The study by Laugesen (26) is one of only a few in which PAHs were found above the limit of detection. Four PAHs, anthracene, phenanthrene, 1-methyl phenanthrene and pyrene, were detected in a hexane extract of 0 mg nicotine, Ruyan® e-liquid. The authors calculated the amount of each PAH as a percentage of the amount of the PAH in the smoke of an equivalent number of tobacco cigarettes, assuming that consumption of the e-liquid was equal to Possible application of WHO standard operating procedures to evaluation of electronic nicotine delivery systems 49 smoking 20 tobacco cigarettes in one day. The levels detected, 7, 48, 5 and 36 ng per cartridge, respectively, were estimated to correspond to average deliveries of < 1% of that delivered by 20 tobacco cigarettes. The four PAHs are classified by the International Agency for Research on Cancer (IARC) in Group 3, inadequate evidence for carcinogenicity in humans and inadequate or limited evidence in animals (37). Benzo[a]pyrene was not detected. 3.5.2 Benzo[a]pyrene in ENDS aerosol In a study of environmental deposition, ENDS aerosol was introduced into a sampling bag with a large quantity of dilution air. Most PAHs, including benzo[a] pyrene, were not present above the limit of detection, and benzo[a] pyrene was found at levels similar to those in blank samples (38). Tayyarah and Long (39) found no quantifiable levels of PAHs in ENDS aerosols, and Romagna et al. (40) detected no PAHs in environmental air when comparing emissions from ENDS and conventional cigarettes. Lauterbach and Laugesen (41) reported that the level of benzo[a]pyrene in Ruyan® ENDS aerosol (more than 300 puffs of aerosol) from 16-mg nicotine cartridges was below the reporting limit. PAHs might have to be monitored if tobacco–ENDS “hybrid” products become available. The methods used for analysing PAHs in ENDS aerosol matrix in the aforementioned studies were not explicitly stated. In most studies, the methods appear to differ minimally or not at all from those used for conventional cigarette smoke. Sample preparation for analysis of benzo[a]pyrene by the CDC method (32) appeared to be similar to that in the TobLabNet method (42). Preliminary studies at CDC (unpublished data) showed minimal differences between PAH calibration curves prepared for the ENDS propylyene glycol–glycerol matrix and standard tobacco cigarettes, suggesting that the method is applicable. Most methods for preparing samples for analysis of benzo[a]pyrene include extraction with nonpolar solvents and clean-up by silica solid phase extraction. The applicability of sample generation and preparation methods to ENDS analysis should be tested before a method is considered appropriate. 3.6 Additional analytes 3.6.1 Carbonyls “Carbonyls” is a collective term for aldehydes and ketones. Studies of conventional tobacco cigarettes indicate that humectants form short-chain carbonyls and other toxic chemicals when exposed to high temperatures. For ENDS, the temperature of the heating coil, which is in contact with the e-liquid, depends on the puff duration, the puff frequency and the heat transfer properties around the coil (1). It is currently considered that thermal decomposition of solvents is the predominant source of carbonyls in ENDS aerosol. Glycerol dehydrates 50 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report at about 280 °C to form acrolein, which undergoes reactions to formaldehyde and acetaldehyde. Contact of liquids such as propylyene glycol and vegetable glycerol in e-liquid with heated atomizer nichrome wire has been proposed as a source of carbonyls (43). Carbonyls are of public health concern, as some have been evaluated as known or probable human carcinogens, and propylyene glycol is thermally degraded to propylene oxide, which is carcinogenic in laboratory animals (23, 25, 44, 45). Formaldehyde, acetaldehyde and acrolein are present at notable levels under certain conditions, especially in later puffs, in ENDS aerosol when glycerol and propylene glycol are heated and in the absence of other e-liquid constituents, such as nicotine or flavourings (1). In a recent study of ENDS aerosol from flavoured and unflavoured e-liquids, however, Khlystov and Samburova showed that carbonyl formation also depends on the concentration of flavourings, independently of e-liquid solvents (46). Some carbonyls (e.g. formaldehyde) have been detected in the particulate and gas phases of ENDS aerosol (N. Kunugita, personal communication). Recently, Sleiman and colleagues (1) found trace levels (ng/mL) of formaldehyde, acetaldehyde and acrolein in e-liquid. When the liquid was aerosolized, there was a notable, voltage-dependent increase in the levels of formaldehyde, acetaldehyde and acrolein. Between the first five puffs (“initial”) and the puffs captured between the 30th and 40th puffs (“steady state”), the level of formaldehyde increased from 2900 ng/mg of liquid consumed to 8950 ng/mg at 3.8 V and from 7250 ng/mg of liquid consumed to 48 200 ng/mg at 4.8 V. Larger increases with increased puff count were observed for acetaldehyde and acrolein (from 230 ng/mg of liquid consumed to 1820 ng/mg at 3.8 V and 740 ng/ mg of liquid consumed to 19 080 ng at 4.8 V, and from 90 ng/mg of liquid consumed to 1700 ng/mg at 3.8 V and 400 ng/ mg of liquid consumed to 10 060 ng at 4.8 V, respectively). Other carbonyls found at levels above the limits of detection were crotonaldehyde, methacrolein, butrylaldehyde, benzaldehyde, valeraldehyde, p-tolualdehyde and hexaldehyde. Formaldehyde, acetaldehyde, acrolein, propionaldehyde, benzaldehyde and glyoxal were measured in ENDS aerosol at levels of micrograms per gram of e-liquid with flavourings. In contrast, in the same ENDS devices, unflavoured e-liquid produced detectable levels of only glyoxal and benzaldehyde (46). The parameters chosen for generating aerosol from ENDS strongly determine the amounts of carbonyls found. Independent variables including the battery voltage, puff volume, puff duration, coil number, placement, resistance, wick design and length, solvent, e-liquid viscosity and air flow resistance may affect the rate at which carbonyls are formed. An SOP for carbonyls in mainstream tobacco smoke is being validated by TobLabNet. Briefly, it is based on trapping the carbonyls in smoke with a combination of an absorbent and a filter, followed by extraction, derivatization and analysis by HPLC with photodiode array detection. Acrolein cannot be Possible application of WHO standard operating procedures to evaluation of electronic nicotine delivery systems 51 analysed with a standard 2,4-dinitrophenylhydrazine cartridge because the derivative is unstable and decomposes in the cartridge during sample collection (47–51). In the hydro-quinone–2,4-dinitrophenylhydrazine method (52) and the CX-572 methods (45, 52), acrolein does not appear to decompose, because carbonyls, including acrolein, are collected on the sorbent hydroquinone or the CX-572-cartridge. When it is validated, the WHO SOP for carbonyls can be expected to be applicable to the analysis of carbonyls in ENDS aerosol. Because other ingredients such as flavourings can contribute interference (53), steps should be taken to ensure analytical validity and suitability. Extreme testing conditions (e.g. very high battery voltage) might yield amounts of carbonyls that exceed the levels to which a user would usually be exposed (54). Additional investigation is required to standardize the device parameters during aerosol generation. 3.6.2 Solvents Although propylene glycol and vegetable glycerol are commonly termed “humectants”, these compounds function in e-liquids as solvents and form droplets during aerosolization that, when existing in the e-liquid, carry nicotine and flavour compounds in the aerosol to facilitate inhalation (55). The solvents may be used alone or a mixture of the two (56). A few e-liquids contain low- molecular-mass polyethylene glycols, either pure or in a mixture with propylene glycol or glycerol. Polyethylene glycol-400 is used because it is liquid at room temperature and because it is readily available in high purity, as it is used as an excipient in pharmaceutical products (57). Rainey et al. (58) demonstrated that GC–FID and GC–MS can be used to measure these chemicals in tobacco, although GC-MS was recommended for full chromatographic resolution of glycerol and triethylene glycol. However, the suitability of GC–FID for comprehensive analysis of solvents in e-liquids is supported by a report from the RIVM, in which this method was used to quantify propylene glycol, glycerol, polyethylene glycol, diethylene glycol and nicotine (29). The method was shown to be suitable for e-liquids and offers the advantage of including nicotine. It is recommended as a starting point for a comprehensive method that includes the solvents of interest, chemically related contaminants such as ethylene glycol and diethylene glycol and, additionally, nicotine. A standard containing polyethylene glycol molecules in the molecular mass range of interest is required for quantification of polyethylene glycol. Such standards are commercially available (e.g. Sigma Aldrich 81396). Solvents in ENDS aerosol can be collected on a standard 44-mm CFP. Staff at R.J. Reynolds observed that more than 98% of the glycerol and propylene glycol in ENDS aerosol is captured on a CFP (22). Experiments with a wide range of e-liquids (RIVM, personal communication) indicate that the amount 52 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report of TPM collected on the filters corresponds closely to the amount of liquid lost. These finding have been replicated and confirmed in the CDC tobacco laboratory (unpublished data). They are important because they indicate that the glass- fibre filters used for conventional cigarette analysis efficiently retain solvents, which are the most abundant chemicals present in aerosol TPM generated from e-liquids. The solvents can be extracted from the filter with methanol and the extract directly injected onto a GC by the same method used for the analysis of e-liquids. This approach is recommended as a basis for a detailed protocol for the quantification of solvents in ENDS aerosol. One concern is that e-liquids often contain a large number of flavour components (53), which may co-elute with the solvents and interfere with their quantification. As many different flavour components could be present in e-liquids, it would be time-consuming to optimize the chromatographic method to ensure complete separation of solvents. Use of a more selective approach, GC– MS, instead of GC–FID would be advantageous in this respect. Any method to be validated should be applicable to a wide range of product types, including those that are highly flavoured. Several authors have reported GC–FID or GC–MS methods for the quantification of humectants in tobacco (58). TobLabNet SOP-06 for the determination of vegetable glycerol, propylene glycol and triethylene glycol in tobacco filler has been validated (59) and provides both GC–FID and GC–MS variants of the method. The SOP is expected to be applicable to the analysis of solvents in ENDS e-liquid and aerosol. It has been suggested that the method for measuring nicotine in ENDS e-liquids could be adapted for simultaneous determination of solvents (glycerol and propylene glycol) and nicotine. Further method development is required to optimize the determination of solvents in ENDS e-liquid and aerosol, taking into account possible interferences. Adjustment of existing methods to determine glycerol, propylene glycol and e-liquid contaminants simultaneously should be considered. 3.6.3 Volatile organic compounds Some VOCs are potent carcinogens and therefore potential targets of policy and regulation to mitigate the toxicity of tobacco products. For example, benzene and 1,3-butadiene in mainstream tobacco smoke are included as priorities in Articles 9 and 10 of the WHO FCTC (24). A few reports have been published on the analysis of VOCs in ENDS refill e-liquids, cartridges and aerosols. Laugesen (26) found xylene and styrene in ENDS e-liquid cartridges, and the China National Tobacco Quality Supervision and Test Centre found several VOCs at levels of parts per million in refill e-liquids for ENDS, including benzene, styrene, ethylbenzene and toluene (60), some of which are classified as carcinogenic or possible carcinogenic to humans by IARC (23, 35, 61). Goniewicz et al. (33) Possible application of WHO standard operating procedures to evaluation of electronic nicotine delivery systems 53 detected toluene and m- and p-xylene in ENDS aerosol, the content of toluene being 0.2–6.3 mg per ENDS (150 puffs). VOCs may originate from tobacco extracts, solvents or other sources. The differences in the levels found may be due to the different nature of the samples (aerosol or e-liquids) or differences in the sensitivity of analytical methods used. The SOP for VOCs in mainstream tobacco smoke is being validated in TobLabNet. It is expected to be applicable to the analysis of VOCs in ENDS e-liquid and aerosol. 3.6.4 Phenolic compounds Phenolic compounds are on the initial WHO list of 18 priority toxicants and also on the non-exhaustive priority list of 39 toxic contents and emissions of tobacco products (24). Most analytical studies on phenolic compounds have focused on cigarette smoke, and few studies are available on their presence in e-liquids or ENDS aerosol. p- and o-dihydroxybenzene, phenol and m-, p- and o-cresol were detected in refill e-liquids at a total amount of 0.5–5 µg/g. No relation was found between the amount of nicotine and the amount of phenols, implying that phenolic compounds originate from ingredients other than the nicotine source (56). HPLC with fluorescence detection is the most commonly used method for determining phenolic compounds in cigarette smoke. Both Health Canada (62) and CORESTA (63) have recommended methods for analysing selected phenolic compounds in mainstream cigarette smoke with this method. Generally, it is expected that the methods commonly used to determine phenolic compounds in tobacco emissions could be extended to the less chemically complex e-liquids and ENDS aerosols. The corresponding SOPs should be established. 3.6.5 Metals Metals were not included in the original priorities for tobacco and mainstream tobacco smoke in Articles 9 and 10 of the WHO FCTC. ENDS devices, however, contain several metallic parts, including wiring, a heating element, solder connections and structural components. The metallic elements commonly found in the alloys used in ENDS devices include chromium, nickel, aluminum, iron, lead, tin and gold. Metals could also be introduced into e-liquids during manufacture, as contaminants during extraction of nicotine from tobacco plants. Metals in e-liquid and aerosol have been identifed by several independent laboratories using inductively coupled plasma-MS and scanning electron microscopy (29, 33, 64). Williams et al. (64) used this method to identify amorphous and fibrous particles in e-liquid. Inductively coupled plasma-MS is a highly sensitive, versatile technique for the analysis of metals in a variety of matrices, and its suitability for the analysis of e-liquids has been demonstrated (29, 33, 64). Metals may occur in e-liquids in 54 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report the form of small metallic particles or dissolved as ions (64). As different forms vary widely in their bioavailability and toxicity, any method should distinguish between the forms. The different species can be separated by HPLC, and it is recommended that an HPLC-inductively coupled plasma-MS method be developed for analysis of metals in e-liquids. An additional sample preparation step may be necessary to dissolve metallic particles. For safety reasons, it should be noted that the reaction between nitric acid (often used for dissolving metals) and glycerol (a common component of e-liquids) may yield nitroglycerine, which is an impact- and friction-sensitive explosive. A safe, efficient sample preparation procedure is therefore imperative. WHO has not prepared a SOP for metals in tobacco or mainstream tobacco smoke. Several researchers reported using quartz CRFs to collect metals in e-liquids (29, 64); however, others have noted that CFPs already contain significant amounts of metals, which could contribute to a high baseline level (65). Quartz CFPs can be leached with dilute hydrochloric acid and nitric acid before use to reduce background levels of metals (65). A possible alternative to CFPs for collecting aerosol for the analysis of metals is Whatman 47 mm QMA grade filters (catalogue No. 1851-047), which have been found to contain low background levels of metals (22). Their slightly greater diameter will require manufacture of appropriately sized filter holders. It is recommended that precautions be taken to ensure accurate measurements of metals in e-liquid and in the collection and analysis of ENDS aerosol. 3.6.6 Flavours Flavourings in tobacco or tobacco smoke were not included in the original priorities for tobacco and mainstream tobacco smoke in Articles 9 and 10 of the WHO FCTC. E-liquids are available in over 7500 unique flavours, and new flavours are being introduced daily (66). Most e-liquid flavourings have been found to be “generally regarded as safe” (GRAS) when ingested, but GRAS certification does not apply to chemicals that are heated at high temperatures and inhaled; therefore, certification has not been issued for flavourings inhaled with ENDS aerosol (67). Although evidence is emerging of health effects resulting from inhaling ENDS first-hand aerosol (68, 69), the role of flavourings is largely unknown. Nevertheless, some classes of flavour compounds reported in e-liquids pose potential health risks (70). Farsalinos et al. (71) found diacetyl and acetyl propionyl, chemicals which impart a characteristic buttery flavour, in 69% of the refill e-liquids and aerosols of sweet-flavoured varieties. Voltage had no apparent effect on the levels of diacetyl in ENDS aerosol, with 438 ng/mg of ENDS liquid consumed at 3.8 V versus 433 ng/ mg of e-liquid consumed at 4.8 V) (1). While the measured concentrations of diketones were significantly lower than in conventional cigarettes, a number of Possible application of WHO standard operating procedures to evaluation of electronic nicotine delivery systems 55 products tested contained acetyl propionyl and diacetyl at concentrations greater than occupational exposure limits. Exposure to diacetyl is associated with severe respiratory illness, including bronchiolitis obliterans, or “popcorn lung”. The first documented case of “popcorn lung” due to use of flavoured e-liquid was reported recently (72). Other common flavour additives are also of concern. For example, cinnamon-flavoured e-liquids contain cinnamaldehyde and 2-methoxycin- namaldehyde at concentrations that are toxic to cultured cells (73), and a direct correlation was found between the number and concentration of cinnamon flavour chemicals in the e-liquids and toxicity (74). A number of e-liquids list pyrazines as additives. These compounds have been used to make inhalation easier and to reduce the harshness associated with nicotine in conventional cigarettes (75, 76). It is possible that they also ease the use of ENDS by novice smokers (77). Sweet- or “candy”-like flavours may make ENDS products attractive to children or novice users (78). The literature on measurement of flavour additives in e-liquids and aerosol is limited. A recent survey of 18 flavourings in three commercial e-liquids found that one marketed as “classic tobacco” contained detectable levels of vanillin, while two others (“bubblicious” and “mojito mix”) had detectable levels of seven flavour compounds (1). The most commonly used techniques for analysing products are HPLC, GC–MS and GC–MS/MS. Diketone compounds like diacetyl and acetyl propionyl were determined on an HPLC–MS platform (71). Other flavours in e-liquids, including menthol, vanillin, methyl anthranilate, benzaldehyde and piperonal, were quantified by GC–MS and GC–MS/MS (3, 79). Most of the methods used to analyse ingredients and toxicants in tobacco can be extended to e-liquids and aerosols. For the analysis of e-liquids with large numbers of different flavourings, chromatographic separation must be assured for methods with non-specific detectors. 3.7 Recommendations for extension of methods A matrix for considering extension of the WHO SOPs based on the reported and observed presence of toxicants in ENDS e-liquid or aerosol was presented at a meeting of the WHO collaborating centres for tobacco product testing and research in Manila, Philippines, in September 2015. An updated version of the matrix is presented in Table 3.1. 56 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report Table 3.1. Proposed decision matrix for extension of current and pending WHO standard operating procedures Current method Applicability of current TobLabNet SOP to proposed matrices E-liquid Aerosol Nicotine in tobacco (filler) ? No Tobacco-specific nitrosamines in mainstream smoke ? ? Benzo[a]pyrene in mainstream smoke No No Nicotine in mainstream smoke ? Yes Humectants in tobacco (filler) Probably Probably Volatile organic compounds in mainstream smokea No Probably Carbonyls in mainstream smokea No Probably a Method under development Many factors, notably voltage and e-liquid composition, can influence the chemical composition of first-hand aerosol. For example, the coil temperature, which is a major factor in formation of emissions, can reportedly vary widely between devices for a given battery and vaping behaviour (1). CORESTA Recommended Method No. 81 (8) and WHO SOP-01 with a fixed number of puffs (≥ 50 to ensure adequate TPM on the CFP) are adequate as standardized, publicly available regimes for machine generation of ENDS aerosol for the limited purpose of evaluating application of the WHO SOPs to analysis of nicotine, TSNAs, and benzo[a]pyrene in samples of disposable and refillable ENDS. Reports generated during method extension should contain a statement that “The [CORESTA/ WHO] method was used for convenience; the method is not based on how ENDS are used by consumers and its use does not constitute an endorsement of the method as appropriate for all current or future ENDS product configurations.” The puffing regime of CORESTA method No. 81 includes a puff volume of 55 ± 0.3 mL, a puff frequency of one every 30 ± 0.5 s, a flow rate of 18.5 mL/s, a puff profile of rectangular (or “square”) shape, a puff duration of 3 ± 0.1 s and a counted, recorded puff count. The CORESTA-recommended method states that it is applicable to a variety of single-use and refillable ENDS (e-cigarettes, e-cigars, e-shisha, e-pipes); therefore, it could be used for the “cigalike” products that are used in method extension studies. The WHO SOP for intense smoking of cigarettes specifies a puff volume of 55 ± 0.1 mL, a puff frequency of 30 s and a puff duration of 2 s. As the WHO SOP was developed for tobacco cigarettes, it does not specify a flow rate. The analytical smoking machine used in method verification should be capable of drawing a fixed volume of air, contain devices to control the puff volume, puff duration and puff frequency, be mechanically and electrically reliable, be capable of sufficient compensation, be able to produce a rectangular puff profile and be capable of taking clearing puffs after termination of smoking. Possible application of WHO standard operating procedures to evaluation of electronic nicotine delivery systems 57 The machine should count puffs at each port. Depending on the product, actuation should start no later than 0.1 s or when the flow rate rises to > 50% of peak flow 0.1 s after starting the puff and shall not be stopped later than 0.1 s after the puff is finished and terminated by the operator or a sensor. The ENDS device holder should be leak-free and impermeable to air and aerosol. The pressure drop in the instrument should not exceed 300 Pa, and the temperature and the relative humidity in the room should be maintained constant ± 2 °C and ± 5%, respectively, throughout the session, as for tobacco cigarette machine smoking. The aerosol trap holders should be airtight, with non-hygroscopic, chemically inert end caps; the retaining efficiency of the filter should be 99.9% of all particles with a diameter ≥ 0.3 µm of a dioctyl phthalate aerosol at 140 mm/s velocity; the content of the binder should not exceed 5% as mass fraction; and the pressure drop should not exceed 250 Pa after completion of aerosol collection. For extension of any new method or cross-matrix method, recovery of the targeted analyte should be measured at low (e.g. 25%), medium (e.g. 50%) and high (e.g. 75%) spike levels, corresponding to the reportable analytical range (e.g. acceptable at 100 ± 10% recovery), to determine whether flavourings or liquid formulations are biasing the results for the target analyte. Only new products should be used for testing purposes. 3.7.1 Nicotine The ENDS conditioning method in ISO 3402 and a modification of ISO standard procedures specified in SOP-04 should be modified, as the nicotine in ENDS is in liquid form in a closed container. In cigarettes, tobacco leaves are wrapped in paper and are easily affected by the medium and the conditions in which they are stored. It should be determined whether conditioning of ENDS or e-liquid cartridges is required. The content of nicotine in a sample will depend on the ENDS brand and model; therefore, extraction and the range of the calibration curve will have to be optimized in terms of the liquid volume and nicotine concentration to be analysed. The concentration of nicotine in e-liquid usually ranges from 0 to about 36 mg/mL, the upper range being much higher than in cigarette smoke extracts generated under intense smoking machine conditions (0.3 mg/mL) (CDC, unpublished data). The analyte volume spiked into the extraction solution should be adjusted (e.g. approximately 0.25–0.5 mL) to be within the existing calibration range. As propylene glycol and glycerine are present as nicotine solvents, the recovery of nicotine after extraction should be assessed, as these compounds are not soluble in some solvents (e.g. hexane). Nicotine can be analysed in e-liquid after extraction with isopropanol, as in the standard ISO method for tar, nicotine and CO in smoke or with an appropriate modification of WHO SOP-04. The analytical specifications should be recalculated for the new matrix containing propylene glycol and/or glycerol. 58 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report The range of nicotine levels observed in ENDS aerosol are comparable to those reported in tobacco smoke. Adapters or special holders may be required to accommodate diverse ENDS designs and configurations. A study of a variety of ENDS products (tank, refillable, disposable) in the United Kingdom (71) found no statistical relation between the concentration of nicotine in the liquid and that in the aerosol; however, voltage was not measured, although it has been shown to affect nicotine levels in machine-generated aerosol (80, 81). Voltage settings are under discussion; they should account for the maximum delivery to consumers (analogous to the Canadian “intense” cigarette smoking machine regimes for conventional tobacco cigarettes) and use of “pre-heating” options recommended by the manufacturer. 3.7.2 Tobacco-specific nitrosamines Very low levels of TSNAs have been found in e-liquid and aerosol, and they varied widely by brand (33, 39, 82). For example, Goniewicz and colleagues (33) found NNN at levels of 0.8–4.3 ng and NNK at 1.1–28.3 ng in the total aerosol of 10 of 12 ENDS purchased in Poland. As there was no tobacco in the ENDS tested in the Goniewicz study, the appreciable levels of TSNAs in aerosol may be due to direct transfer from the e-liquid. It has been hypothesized that TSNAs in e-liquid are contaminants of nicotine extraction. A review of WHO SOP-03 for TSNAs in mainstream tobacco smoke suggests that the chemistry of ENDS aerosol is compatible with the WHO SOP and that either aerosol TPM collected on CFPs or e-liquid could be analysed. As noted above, however, the reported levels of TSNAs in ENDS aerosol are below the reporting limit of the WHO SOP for TSNAs in tobacco smoke. If “hybrid” ENDS product designs that incorporate tobacco are introduced, TSNA levels in aerosol may be higher. 3.7.3 Benzo[a]pyrene WHO TobLabNet SOP-05 for the analysis of benzo[a]pyrene in tobacco smoke could be adapted for ENDS aerosols, although the benzo[a]pyrene concentration in aerosol is expected to be much lower than that in cigarette smoke. Consequently, the number of CFPs to be analysed in one flask, the volume of the extraction solvent as well as the range of the calibration curve would have to be adjusted accordingly. As there are high concentrations of propylene glycol and glycerol in ENDS aerosols, the recovery of benzo[a]pyrene from the propylene glycol– glycerol matrix should be assessed when cyclohexane is used as the extraction solvent. If recovery in cyclohexane is low, other extraction solvents should be tested to determine the solubility of propylene glycol and glycerol. The analytical calibrations should be recalculated for the new matrix. Possible application of WHO standard operating procedures to evaluation of electronic nicotine delivery systems 59 As there are few or no PAHs in e-liquid or ENDS aerosol (26, 36–39), it is recommended that liquid or aerosol from ENDS not be analysed for benzo[a] pyrene, as the results will not significantly inform public health or regulatory decision-making. 3.7.4 Volatile organic chemicals The SOP for VOCs in mainstream tobacco smoke is being validated in TobLabNet and could be adapted to the analysis of ENDS aerosols. Besides 1,3-butadiene and benzene, other harmful VOCs may be present in e-liquids and aerosols, such as toluene, styrene and ethylbenzene. The concentrations of VOCs in aerosols may, however, be much lower than in tobacco mainstream smoke (33). Therefore, the number of puffs, the type of carbon molecular sieve, the volume of the extraction solvent and the range of the calibration curve should be adjusted accordingly. 3.7.5 Carbonyls Carbonyls are generated during vaporization of e-liquids and have been widely reported in ENDS aerosol. In most studies, carbonyls were found in trace amounts or much lower levels than in tobacco cigarette smoke (43). The choice of solvent, device design (e.g. refillable, single-use) and voltage should be considered. “Dry puffing”, when the wick is not in contact with sufficient liquid because the cartridge is empty or the coil is overheating, can lead to the formation of toxic chemicals (83); however, this phenomenon is not thought to represent common ENDS consumer use patterns (84). The analytical specifications in the pending WHO SOP for carbonyls in mainstream tobacco smoke should be evaluated and modified as necessary to account for potential ENDS-specific emissions and their concentrations in ENDS aerosol. These include glyoxal and methyl glyoxal, which have been reported in ENDS aerosols but not in tobacco cigarette smoke (85). Toxic and carcinogenic carbonyls have been detected in aerosols (86) and are thus potential targets of policy and regulation to mitigate the toxicity of tobacco products. Consequently, extension of the pending SOP for analysis of carbonyls in tobacco smoke to analysis of carbonyls in ENDS aerosol is recommended. 3.8 Research that will inform future regulatory use of data on ENDS ■ Identify or develop standard ENDS research products. ■ Identify or develop standardized research materials for testing ENDS batteries. 60 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report ■ Review and refine the specifications of commercial ENDS aerosol generating machines. ■ Develop ENDS device holders and trapping system(s) for a variety of ENDS. ■ Determine whether current analytical methods are applicable for a variety of ENDS and how they should be modified to provide accu- rate, reproducible, robust measurements. ■ Define the critical aspects of ENDS use topography, including puff duration, frequency, volume and count. ■ Determine which product design variables (e.g. variable voltage, bat- tery power, heating coil temperature settings) should be specified in an aerosol generating regime. ■ Determine “standard” and “intense” aerosol generation methods that reflect ENDS use behaviour and, for the “intense” method, adjust- ments of products design variables, which will inform regulatory decision-making. ■ Assess whether separate regulatory limits are appropriate for early- versus later-generation products or for different kinds of ENDS (e.g. e-cigars, e-waterpipes). ■ Survey the extent of impurities in solvents and nicotine extracts to determine whether routine testing of impurities is warranted. ■ Determine whether the pH of ENDS aerosol can be derived or in- ferred from that of e-liquid with procedures similar to those devel- oped for smokeless tobacco. ■ Assess interference, recovery, matrix comparisons and the appropri- ate range of the calibration curve for all analytical methods. 3.9 Conclusions A series of chemicals have been detected in ENDS e-liquids and aerosols. Considering the prevalence of ENDS use and the evolving nature of these products the application of existing and pending WHO TobLabNet SOPs to the analysis of ENDS e-liquid and aerosol is justified. Whereas carbonyls are generated when e-liquid solvents and flavourings are exposed to elevated temperatures, benzo[a]pyrene and TSNAs in current products are attributed to impurities in nicotine extracts; they are therefore not routinely detected and, when present, are found at very low levels. TSNA levels increased with increasing nicotine level in a study of cartridges sold for an ENDS brand by one manufacturer, and there are several independent reports Possible application of WHO standard operating procedures to evaluation of electronic nicotine delivery systems 61 that the TSNA levels in machine-generated aerosol are much lower than those in the smoke of tobacco cigarettes and below the reporting limit of the WHO SOP for TSNAs in mainstream cigarette smoke. Requiring manufacturers to use nicotine that is certified free of contaminants should eliminate TSNAs in e-liquid and aerosol. Routine testing for TSNAs and benzo[a]pyrene in ENDS is not warranted because they do not contain tobacco. However, validated methods will allow regulators and researchers to screen e-liquid at their discretion and new “hybrid” products as they emerge. Extension of the WHO SOPs for the analysis of TSNAs and benzo[a]pyrene in mainstream tobacco smoke to ENDS will provide researchers and regulators with analytical methods suitable for future configurations and design variations in which tobacco is included, which could result in higher levels of these toxicants. Major transnational tobacco companies have launched such “hybrid” products called heat-not-burn products. Examples are IQOS which releases a nicotine-containing vapor [2], Vype which passes a nicotine-containing vapor through tobacco [3], and Ploom which delivers a vapor that passes through a capsule of granulated tobacco [4]. Examples are iQOS which releases a nicotine-containing vapor (91), Vype which passes a nicotine- containing vapor through tobacco (92), and Ploom which delivers a vapor that passes through a capsule of granulated tobacco (93). It would be advisable to measure nicotine and toxicants (e.g. metals) of public health or regulatory significance that are either frequently detected or present at more than trace levels in e-liquid and aerosol to better characterize potential exposure. Many aspects of the design of ENDS devices affect the composition of the aerosol, including the heating coil resistance, wick design and material, reservoir design and airflow openings. For example, the level of nicotine in ENDS aerosol under initial (during the first five puffs) and steady-state conditions (30th to 40th puffs) and at two voltage settings ranged from 13.1 µg/mg to 23.9 µg/mg of e-liquid consumed with a battery setting of 3.8 V, and 7.6 µg/mg to 22.7 µg/mg of e-liquid consumed with a battery setting of 4.8 V (1). These features should be fully specified in any standardized ENDS device used to validate analytical methods. In addition, the pH of e-liquid, which can be expected to influence the amount of nicotine present as rapidly absorbed unionized (free) nicotine, has not been fully characterized. In developing an “intense” aerosol generation method that approximates an upper limit of the device, research should be conducted on product design variables. The availability of standardized ENDS devices with well-documented critical design parameters would facilitate the development of additional analytical methods for ENDS. Research should address which aspects of device design are the most important. “Hybrids” products such as Heat-Not-Burn products may be associated with substantially different use behaviour and reach 62 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report higher heating temperatures, which can qualitatively and quantitatively influence smoke emissions, including possible generation of CO. Extension of the SOP for humectants in tobacco filler to detect and quantify impurities in propylyene glycol, glycerol and polyethylenes (e.g. ethylene glycol and diethylene glycol) will assist investigations into the prevalence of such impurities, which raise concern about toxicity that is not present with propylyene glycol or glycerol alone. Analysis of nicotine could be combined with analysis of solvents, so that both can be determined in a single GC–FID run. Sample preparation in this case would consist of dilution of the liquid with a suitable solvent such as methanol (29). The Flavor and Extract Manufacturers Association (FEMA) in the USA issued the statements (67) that: “FEMA GRASTM status for the use of a flavor ingredient in food does not provide regulatory authority to use the flavor ingredient in e-cigarettes in the US” and “E-cigarette and flavor manufacturers and marketers should not represent or suggest that the flavor ingredients used in e-cigarettes are safe because they have FEMA GRASTM status for use in food because such statements are false and misleading.” New and existing methods for the analysis of flavourings in e-liquids and aerosols should be evaluated individually to ensure data quality. As many flavourings contain a ketone or aldehyde moiety, the large amounts of flavourings in e-liquids could interfere with the analysis of carbonyl compounds when a non-specific detector is used. This could be an advantage if certain flavourings and short-chain carbonyls are quantified in a single run. If interference proves problematic for routine analysis, a compound-specific detector (MS) could be used. In view of the highly variable nicotine yield of different devices and because users adjust their behaviour to modify the nicotine yield, a different machine method for generating aerosol should be developed that reflects changing ENDS use behaviour. ENDS use behaviour varies among users. It has been reported as two to four puffs per minute, a puff volume of about 50 mL, puff durations of 2–8 s, inter-puff intervals of 18–30 s and a puff flow rate of about 20 mL (1). Reports of variations in puffing regimes (88, 89) indicate that several parameters should be assessed, including puff duration, frequency, volume and count as well as battery power, to better approximate use behaviour. The procedure or equipment for assessing different product designs might have to be modified in accordance with the results of studies on ENDS use and of discussions on possible standard and “intense” aerosol generation regimes. Further, the requirements of the ENDS power source should perhaps be specified to set voltage, power or temperature settings for the heating coil. Various batteries are available that can be combined with different devices. They may be unregulated (DC, with lower voltage as the battery runs down) or regulated. Regulated batteries can be designed to provide a fixed voltage, fixed power or even a fixed temperature of the heating element; Possible application of WHO standard operating procedures to evaluation of electronic nicotine delivery systems 63 recent high-end models of ENDS batteries measure and regulate the temperature of heating coils made of certain metals (e.g. titanium). A laboratory power supply that imitates the battery could be used. Research should be conducted to define the electrical specifications of the power source, including regulation of the voltage, power and temperature, the voltage, power and temperature for maximum output, the allowable ripple current and voltage and ripple frequency. Additional research and consensus are therefore needed on aerosol generation regimens and instrumentation for future testing of aerosols. Several scientists (78, 90) have found that the patterns of ENDS use differ widely from those for conventional tobacco cigarettes. Users are thought to adjust their behaviour to maximize nicotine yield, achieving plasma levels of nicotine and cotinine similar to those of tobacco cigarette smokers (78). It has been observed that the puff duration from ENDS is significantly longer than that from ordinary tobacco cigarettes. It is not clear to what extent use behaviour affects the chemical composition of the vapour; however, puff duration and puff frequency are reported to influence the temperature of the coil (1). The operating temperature of ENDS cannot be predicted from battery and coil characteristics alone (1), and more research is needed to inform this area as ENDS devices evolve. In summary, the marketing and promotion of ENDS and their subsequent popularity and availability to consumers through retail sales in most countries and over the Internet warrant monitoring of the chemical composition of e-liquids and aerosols, including measurements of nicotine, solvents and carbonyls, with current methods. Metals should be measured to determine whether they represent a health risk, and validated methods should be developed for routine analysis if a risk is identified. Routine measurement of TSNAs is not warranted for current products if policy-makers and regulators require certification of the quality of the nicotine extract. Measurement of benzo[a]pyrene is also not warranted at this time. Introduction of “hybrid” products such as heat-not-burn products, however, might warrant additional testing of tobacco-derived toxicants like TSNAs and PAHs. Flavour compounds, phenolics and VOCs should be considered in future discussions, as they are present in e-liquid and aerosol and may influence their potential toxicity. 3.10 Recommendations ■ Sufficient data are available from independent laboratories to support extension of existing and pending WHO SOPs for nicotine, humec- tants (solvents), carbonyls, benzo[a]pyrene and TSNAs in ENDS e- liquid and aerosol. ■ Routine measurement of TSNAs is not warranted for current prod- ucts if policy-makers and regulators require certification of the qual- 64 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report ity of the nicotine extract. Measurement of benzo[a]pyrene is also not warranted at this time. ■ Emergence of “hybrid” products such as heat-not-burn products may require additional testing of tobacco-derived toxicants like TSNAs and PAHs. ■ It is recommended that the pH of the e-liquid be measured to estab- lish the range of pH in e-liquids, as this information may contribute to investigations of the addictive potential of the nicotine delivered to ENDS users (21). ■ Metals should be measured to determine whether they represent a potential health risk; if so, validated methods should be developed for their routine analysis. ■ Flavour compounds, phenolics and VOCs should be considered in future discussions, as they are present in e-liquid and aerosol and may influence the toxicity of the products. ■ Development of an “intense” aerosol generation method to approxi- mate an upper limit of the device should systematically include the relative importance of product variables and should establish a stand- ardized ENDS device that can be used to compare aerosols. ■ The applicability of the CORESTA method or a SOP (e.g. SOP 01) for an intense smoking machine method for tobacco cigarettes to all cur- rent and future ENDS product designs remains to be determined. As use data are established and as products evolve, the choice of regime for generating aerosol must be reevaluated. ■ For cross-matrix verification of a method, the slopes of the calibra- tion curves for each analyte in each matrix should be compared to evaluate the equivalence of the method for each applicable matrix. ■ As part of method development or extension to new sample matrices, a recovery study with low medium and high spike levels is recom- mended to ensure applicability. ■ Sample preparation techniques should be investigated to ensure their compatibility with e-liquid solvent matrices (propylene glycol and glycerol). In some instances, the miscibility of the extraction solvent and the matrix solvent may cause insufficient extraction. ■ Testing procedures should require the use of new, unused products and follow any actuation or pre-heating recommendations provided by the manufacturer. 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Waterpipe toxicant content and emissions Marielle Brinkman, Battelle Public Health Center for Tobacco Research, USA Alan Shihadeh, Center for the Study of Tobacco Products, American University of Beirut, Lebanon Contents 4.1 Introduction 4.2 Puff topography and emissions testing regimens 4.3 Toxicant content and emissions 4.4 Influence of testing protocols on measurements of toxicant emissions from waterpipes 4.4.1 Puffing regimen 4.4.2 Heat source 4.4.3 Temperature of tobacco 4.4.4 Effect of water 4.5 Influence of waterpipe design on levels of emissions of waterpipe tobacco products 4.5.1 Components and accessories 4.5.2 “Real-world” and research-grade waterpipes 4.5.3 Waterpipe hose 4.5.4 Waterpipe tray versus foil 4.6 Conclusions 4.7 Recommendations for regulators 4.8 References 4.1 Introduction Broadly defined, a “waterpipe” is an instrument commonly used to smoke tobacco, characterized by a container in which smoke bubbles through a column of water. Use of variants of the waterpipe has been reported in indigenous cultures in the Americas, Africa and Asia, even before the introduction of tobacco (1). In recent years, a variant of the waterpipe used in south-west Asia and North Africa – often referred to as “narghile”, “shisha” or “hookah” – has become widely popular, attracting young and new tobacco users around the globe. Fig. 4.1 illustrates the main features of this type of waterpipe. The head (fired clay), body (metal), water bowl (glass) and corrugated hose (leather or nylon stretched over a wound flexible wire coil, or more recently, plastic tubing) are the primary elements from which it is typically assembled, and each is manufactured in a variety of sizes. The overall height of a common waterpipe can vary from approximately 40 cm to more than 1 m and the length of the hose from 75 to 150 cm. 72 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report Fig. 4.1. Narghile waterpipe Source: reference 2 Because the tobacco preparation has high moisture and humectant contents, it does not burn in a self-sustaining manner, and lumps of burning charcoal are placed on top of the tobacco to keep it alight. The charcoal is periodically replenished or adjusted to maintain the smoke strength desired by the smoker. Usually, a pile of burning charcoal is kept in a nearby firebox for this purpose, particularly in restaurants and cafés where waterpipes are provided. Waterpipe users may also use quick-lighting charcoal briquettes to avoid preparing and maintaining a firebox every time they smoke. Interestingly, the weights of charcoal and maassel, a heavily flavoured tobacco mixture, consumed during a session are comparable (3). When a smoker sucks from the hose, air is drawn over and heated by the charcoal in the head. The hot air and charcoal combustion products then pass through the tobacco, from which smoke is produced. The smoke thus contains wood charcoal fumes in addition to the fumes emanating from the tobacco preparation. The smoke continues from the head through the central conduit in the body and then bubbles through the water before entering the hose. Thus, by the time the smoke reaches the mouthpiece, it has been humidified and cooled to room temperature. Adding to the sensory experience of inhaling a cool, humid, sweet aerosol, users feel and hear the action of the bubbler as they smoke the waterpipe. Waterpipe toxicant content and emissions 73 The two most common waterpipe configurations are referred to as maassel and ajami. In the maassel configuration, a relatively deep (approximately 3 cm) head is filled with 10–20 g of maassel (“honeyed” in Arabic), which consists by weight of up to 65% humectant (4) (mainly glycerol), the balance consisting of tobacco, water, flavourings and other additives. Hundreds of flavours are available on the market, mimicking an array of fruits, sweets, beverages, spices, flowers and herbs. The maassel is covered with an aluminium foil sheet perforated for the passage of air (Fig. 4.2a), and burning coals are placed on top of the aluminium foil. In the second configuration, that of the more traditional “unflavoured” ajami tobacco (commonly referred to as tombac, or “tobacco” in Arabic), smokers mix a small amount of water with dry, shredded tobacco to make a mouldable matrix, which they shape into a mound on top of a shallow clay head (Fig. 4.2b); the coal is placed directly on the moistened tobacco. The maassel configuration is the most prevalent worldwide. Fig. 4.2. Waterpipe heads: (a) maassel configuration with tobacco underneath foil; (b) ajami configuration with tobacco on top of the head and no foil to separate charcoal from tobacco (a) (b) Tobacco-free versions of maassel have appeared in shops and on-line recently, which are commonly marketed as a “healthy” option. The toxicant delivery profile and biological activity of the smoke produced with these products was, however, found to be essentially identical to the tobacco-containing versions, apart from the absence of nicotine (see section 4.3). 4.2 Puff topography and emissions testing regimens Unlike cigarettes, narghiles allow relatively high puff volumes, largely because of their low resistance to draw, which is very similar to free inhalation. Puff volumes of the order of 1000 mL are common, in contrast to the volumes of 30–50 mL for cigarettes. Thus, a single narghile puff may displace as much smoke as is drawn during the consumption of an entire cigarette. A typical smoking session consists of hundreds of puffs over about 1 h, for a cumulative inhaled volume of about 100 L (5). In addition, unlike cigarettes, waterpipes are smoked to no well-defined end point until they are considered to have been “consumed”; in general, a smoker simply stops when smoking is no longer appealing, whether because of a change in flavour, a sense of satiation or a change in social circumstances (e.g. the end of a dinner during which a narghile was used). 74 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report Laboratory characterization of toxicant emissions produced in a smoking machine requires specification of puff topography parameters, such as puff number, volume and duration and interpuff interval, because toxicant emissions are strongly influenced by the puffing parameters used to smoke a given product (3, 6, 7). Several studies of waterpipe puff topography have been reported, in various populations and in both clinical laboratory and natural environments. These studies are summarized in Table 4.1, which shows a mean puff volume of 500–1000 mL, a puff duration of 2–3 s and an interpuff interval of 10–35 s. The variations among studies shown in Table 4.1 probably reflect the influence on puff topography of factors such as years of experience, smoking frequency and setting. Some experimental data suggest that waterpipe puff topography is influenced by the nicotine content of the product smoked; in a blinded experiment, experienced waterpipe users were found to puff more intensively when they were given a nicotine-free waterpipe product (14). Experimental data also show that puff topography is affected by the degree of nicotine dependence (11). Such variations notwithstanding, it is noteworthy that the puff volumes taken during waterpipe smoking are more than 10 times greater than those taken during cigarette smoking. It is clear, therefore, that cigarette puff topography parameters cannot be used in waterpipe machine smoking tests. Table 4.1. Reported measurements of waterpipe puff topography Waterpipes Cigarettes Study Shihadeh et al. (5) Maziak et al. (8) Katurji et al. (9) Cobb et al. (10) Alzoubi et al. (11) Pulcu & McNeil (12) Brinkman et al. (13) Djordjevic et al. (6) Location Beirut, Lebanon Aleppo, Syrian Arab Republic Beirut, Lebanon Richmond, VA, USA Irbid, Jordan Istanbul, Turkey Columbus, OH, USA Westches- ter, NY, USA Setting Café Laboratory (30 min) Café Laboratory (45 min) Laboratory Laboratory (30 min) Laboratory Laboratory No. of par- ticipants 52 61 61 54 59 ´ 2 20 35 77 Inter- puff interval (s) 17.0 12.6 15.2 35.4 12.4/8.0 11.7 26.2 18.5 Puff volume (mL) 530 511 590 834 520/480 1040 640 44.1 Puff dura- tion (s) 2.6 3.2 2.8 Not reported 2.3/2.7 3.5 4.5 1.5 Total no. of puffs 171 169 169 75 157/199 120 71 12.1 Total vol- ume (L) 90.6 79.1 130 61.6 82.6/91.8 114 45.4 0.523 Cigarette topography from Djordjevic et al. (6) shown for comparison Waterpipe toxicant content and emissions 75 To date, the most commonly used puff topography regimen for analytical studies of waterpipe tobacco smoke is that of the Beirut method (9), which specifies 171 puffs of 2.6-s duration, 530-mL volume and 17-s inter-puff interval. This method is based on two field campaigns in cafés in the Beirut area where waterpipes were provided and was validated by measuring “tar”, nicotine and CO in smoke sampled in real time from waterpipes as they were smoked by café patrons (5, 9). 4.3 Toxicant content and emissions Laboratory studies during the past decade have begun to elucidate the chemistry of waterpipe smoke with modern analytical methods, reliable machine smoke generation and sampling protocols. A recent review of the scientific literature showed that approximately 300 chemical species have been identified and 82 quantified in waterpipe smoke (15). In addition to the addictive drug nicotine, the quantified species include carcinogens such as TSNAs, PAHs, benzene, furans and heavy metals, as well as other important toxicants such as volatile aldehydes, nitric oxide and CO. Like cigarette smoke, waterpipe smoke includes constituents that are simply transferred from the raw material (e.g. heavy metals, nicotine, TSNAs), constituents that are chemically synthesized during smoking (e.g. CO, nitric oxide) and constituents that are both transferred and synthesized in situ (e.g. PAHs) (16). Furthermore, because burning charcoal is usually used as the heat source during waterpipe smoking, the smoke contains toxicants emitted from the charcoal in addition to those from the tobacco product itself. Thus, the composition of both the charcoal and the tobacco preparation can influence smoke constituents. A large fraction of the PAHs and heavy metal content of waterpipe smoke may be accounted for by the PAH content of raw charcoal (16) and the metal content of the maassel products (17, 18), respectively. These constituents were found to vary by product, suggesting that regulation to limit the toxicant content might be feasible. Because published reports on waterpipe toxicant yields are specific to particular combinations of charcoal and tobacco product, puffing protocol and waterpipe design, the reported toxicant contents vary widely. Nonetheless, as noted in an extensive review of the toxicants and biological activity of waterpipe tobacco smoke (15), all studies to date point to the same conclusion, that, during a typical waterpipe use session, the user will draw large doses of toxicants, ranging from less than one to tens of cigarette equivalents, depending on the toxicant (see Fig. 4.3). These toxicants are linked to addiction, heart and lung diseases and cancer in cigarette smokers and can result in similar outcomes in waterpipe users. 76 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report Fig. 4.3. Reported levels of mainstream smoke toxicants produced during a single 1-h waterpipe use session and during smoking of a single cigarette Source: reference 2; data on cigarettes from references 18 and 19 and on waterpipes from references 3, 20 and 21 Reports of toxicant emissions in waterpipe smoke have been corroborated by biomarker assays in smokers, which show that users are systemically exposed to CO, nicotine, PAHs and TSNAs (22–27). In addition, differences in systemic exposure patterns to toxicants between cigarette and waterpipe smokers mimic the differences found in measured toxicant emissions with these smoking methods; e.g., on a nicotine-normalized basis, waterpipe smokers have greater exposure to CO and PAHs and lower exposure to TSNAs than cigarette smokers. Such agreement between markers of exposure and measured toxicant yields gives confidence in the findings to date that waterpipe smoke contains and delivers large doses of toxicants. Waterpipe toxicant content and emissions 77 Increasing awareness of such findings may be a factor in the appearance of tobacco-free maassel preparations that are marketed as products “for the health- conscious user”. Except for nicotine, the smoke produced by use of tobacco-free maassel products has essentially the same toxicant profile and biological activity as that of conventional tobacco-based products (Table 4.2 and 17, 28, 29). Table 4.2. Direct comparisons of mainstream smoke toxicant yields from tobacco-based and tobacco-free waterpipe products Toxicant Waterpipe preparation (mean ± 95% confidence interval) PTobacco Non-tobacco “Tar” (mg) 464 ± 159 513 ± 115 NS Nicotine (mg) 1.04 ± 0.30 < 0.01 < 0.001 CO (mg) 155 ± 49 159 ± 42 NS Nitric oxide (mg) 437 ± 207 386 ± 116 NS Polyaromatic hydrocarbons (ng) Fluoranthene 385 ± 74 448 ± 132 NS Pyrene 356 ± 70 444 ± 125 NS Benz[a] anthracene 86.4 ± 15.2 113 ± 46 NS Chrysene 106 ± 16 124 ± 36 NS Benzo[b+k] fluoranthenes 64.7 ± 11.3 72.9 ± 12.6 NS Benzo[a]pyrene 51.8 ± 12.9 66.1 ±17.8 NS Benzo[ghi] perylene 33.6 ± 10.2 39.6 ± 10.7 NS Indeno[1,2,3-cd] pyrene 47.3 ± 10.7 44.3 ± 10.4 NS Carbonylic compounds (µg) Formaldehyde 58.7 ± 21.6 117.6 ± 78.7 NS Acetaldehyde 383 ± 121 566 ± 370 NS Acetone 118 ± 36 163± 68 NS Propionaldehyde 51.7 ± 15.3 98.4 ± 65.0 NS Methacrolein 12.2 ± 4.4 20.4 ± 9.7 NS Adapted from Shihadeh et al. (29) NS, not significant Smoke was generated by reproducing human puffing with machine smoking during 62 ad libitum smoking sessions by 31 waterpipe users, each of whom completed two sessions in a controlled clinical setting: one with their preferred tobacco-based product and one with a flavour-matched tobacco-free product. Since maassel, the heavily flavoured form of waterpipe tobacco, was introduced in the early 1990s (43), very little research has been conducted to identify and quantify the flavourings in these tobacco products. The number of manufacturers and the number and variety of flavours available have increased steadily in the past 20 years, in conjunction with the popularity of this form of tobacco smoking (44). Several flavourings were identified in the mainstream smoke from maassel 78 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report in quantities up to 1000 times greater than in mainstream cigarette smoke, including vanillin, ethyl vanillin and benzyl alcohol (45). Using a non-targets analysis approach, Schubert et al. (33) tentatively identified 79 volatile flavourings, and quantitatively confirmed the presence of 11, in the headspace of a variety of waterpipe tobaccos from Egypt, India, Jordan and the United Arab Emirates. Flavours in tobacco products can be directly harmful by increasing the toxicity of the inhaled smoke. One example is cinnamon flavoured e-liquids. Behar et al. (47) showed that the cytotoxicity of e-cigarette emissions correlated strongly with the concentration of cinnamaldehyde in the e-liquid that was vaped. Another example is sweet flavour additives such as fructose and glucose. Soussy et al. (48) showed that these sugars decompose thermally during e-cigarette vaping to form 5-hydroxymethylfurural and furfural. Although not yet rigorously investigated, the same decomposition pathways are plausible for waterpipe tobacco, as it can contain up to 70% by weight of sugars, and both 5-hydroxymethylfurural and furfural were measured by Schubert et al. (46) in mainstream waterpipe tobacco smoke. Perhaps the greater potential contribution of flavours to adverse health effects in new and established tobacco smokers is increasing the appeal of smoking. Flavours may cause harm indirectly by lowering the barrier to initiation of use of tobacco products, by smoothing or sweetening the harshness of tobacco smoke, making it easier to inhale. Cross-sectional data on a nationally representative sample of young people (≤ 17 years) in the USA indicated a positive correlation between reporting that one’s first tobacco product was flavoured and current tobacco use (49). Recent preliminary  longitudinal data from the same study show  that young people who first use  a flavoured tobacco  product  are significantly more likely to be tobacco users at one-year follow up than if their first-use product was unflavoured.3 While second-hand smoke is not a focus of this report, it should be noted that environmental exposure to waterpipe smoking also poses a significant health hazard. In controlled laboratory experiments, large quantities of volatile aldehyde species, CO, PAH and nanoparticles are emitted directly into the environment from the waterpipe head during smoking (50). It has been estimated that during the course of a one-hour use session, a single waterpipe user will generate toxicant emissions equivalent to 2 to 10 cigarette smokers during the same one hour period, depending on the toxicant in question. Reports of observations in natural settings where waterpipes are used also show that waterpipe smoking results in high ambient concentrations of fine particulate matter (PM2.5) (18, 51–53). 3 Villanti AC. Are youth and young adults who first try a flavored tobacco product more likely to continue using tobacco? Findings from the PATH study. Presented at the annual meeting of the Society for Research on Nicotine and Tobacco, Florence, Italy, 9 March 2017. Waterpipe toxicant content and emissions 79 4.4 Influence of testing protocols on measurements of toxicant emissions from waterpipes Protocols for testing tobacco product emissions should include procedures for sampling and preparing waterpipe tobacco products and generating, collecting and quantifying toxicants in the mainstream smoke from these products. Such protocols, the activities that they should include and the questions they should address are summarized in Table 4.3. Table 4.3. Procedures, activities and variables that require specification in tobacco product testing protocols Testing protocol Activity Specific testing variables Tobacco sampling and waterpipe preparation Homogenization Number of purchased units? Sticks or twigs removed? Storage conditions? Stability in storage? Conditioning Conditioned tobacco or as is from newly opened manufacturer’s packaging? If conditioned, for how long and at what temperature and humidity? Tobacco packing Clean head with solvents or water? Pack tobacco loosely or tightly? Aluminium foil perforation pattern? Quantity of tobacco? Pipe and hose cleaning Clean with organic solvents and/or water? Use fresh hose each time? Check air infiltration rate? Sample generation Puffing regimen Single- or multi-stage puffing? High-resolution reproduction of human puffing? Puff volume, duration and frequency? Heat source Charcoal or electric? Amount and timing of charcoal application? Type of charcoal? Smoking machine Puffing mechanism? Puff waveform? Sample collection Particulates and semivolatiles Type and size of filter? How many filters are sufficient? Gas phase and volatiles Impingers, sorbents, canister or bag collection? Toxicant quantification Extraction Which solvent? Which clean-up method? Which surrogate standard? Quantification Which internal standard? Which instrumental method? As there are no protocols for testing waterpipe emissions, however, there have been no studies on the influence of such protocols on toxicant emissions. In the absence of standard protocols, research groups have used a wide variety of equipment and procedures to study waterpipe emissions. While studies of toxicant emissions cannot be compared, data collected by research groups are available for estimating the influence of certain variables. In particular, the influence of puffing regimen, heat source, tobacco temperature and bowl water have been investigated and are discussed briefly below. The effects of other variables such as tobacco conditioning (loose versus tight packing into the bowl), sample generation (e.g. heat source ignition timing, puffing mechanism), smoke collection conditions (e.g. filter type, number and diameter for the particle phase; impingers, sorbent or real- time collection) and toxicant quantification methods still require investigation. Some methods for quantifying toxicants in cigarette tobacco and emissions have been modified for waterpipe emissions, but none has been validated with reference materials or inter-laboratory studies, and thus more work is required. 80 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report Mainstream constituents of waterpipe tobacco smoke have been ana- lysed with machine smoking and various equipment, including commercially available waterpipes (e.g. 3), a specially designed waterpipe smoking machine equipped with a laboratory waterpipe (e.g. 21) and a research-grade waterpipe (13), as shown in Table 4.4. Commercially available waterpipes were generally smoked with radial positive displacement vacuum pumps, such as rotary vane, diaphragm, piston or scroll pumps, operating at a constant flow rate. Computer- controlled solenoid or manual valve switching control was used for puffing (e.g. 3, 17, 44). In the “shisha smoker” and the research-grade waterpipe, smooth ac- tion, single-stroke piston displacement is used to generate the puff, which more closely approximates the human diaphragm than vacuum pumps. “Playback” smoking machines to reproduce human puffing behaviour in fine detail have also been used to study waterpipe emissions (e.g. 45). How different waterpipe de- signs and puffing mechanisms affect the level of toxicants in mainstream smoke is not well understood. Table 4.4. Waterpipe machine smoking regimens Waterpipe Pump mechanism Pu ff v ol um e (L ) Pu ff du ra ti on (s ) In te r- pu ff in te rv al (s ) To ta l n o. of p uff s To ta l p uff vo lu m e (L ) Sm ok in g du ra ti on (m in ) Co al n o. / t yp e/ di am et er (m m ) A m ou nt o f to ba cc o (g ) Tr ay o r f oi l ( no . of h ol es ) H os e m at er ia l Beirut method, Black Single Pearl, Khalil Mamoon (6, 42) Mechanical pump with digital solenoid control 0.53 2.6 17 171 90.6 55.6 1.5C / 3 Kings / 33 mm 10 Foil (18) Leather and plastic (43) Modified Beirut method (21) Pneumatic single-stroke cylinder 0.53 2.6 17 171 90.6 55.6 1C / 3 Kings / 40 mm 10 Foil (18) Plastic Super shisha (18) Vacuum pump, 6 L/min 0.3 3 15 100 30 30 1C / Swift- Lite / 33 mm 10 Foil (19) Not re- ported Clay bowl (43)a Mechanical pump and manual syringe every 10th breath 1.0 5 25 100 100 50 1C / Swift- Lite / 33 mm 8 Tray (nor report- ed) Not re- ported Research-grade waterpipeb Single-stroke glass syringes 0.72 0.46 4.6 3.6 16.4 28.7 32 42 74 23.0 19.1 42.1 11.2 22.6 33.8 1C / 3 Kings / 40 mm Electric heat source 10 Foil (18) Tray (30) Plastic a Waterpipe smoked for 3-min “warm-up” period before mainstream smoke sampling b Kroeger RR, Brinkman MC, Buehler SS, Gordon SM, Kim H, Cross KM, et al. The impact of variation of hookah components on chemical and physical emissions. Presented at the annual conference of the Society for Research on Nicotine and Tobacco, Seattle, WA, USA, 6 February 2014. Waterpipe toxicant content and emissions 81 4.4.1 Puffing regimen Most testing of waterpipe emissions has been conducted with one of three types of puffing regimen: steady, periodic summary data modelling of human puffing behaviour in a waterpipe café (46); multi-stage, steady, periodic summary data modelling of human puffing behaviour in a laboratory setting;4 and high- resolution, time-resolved (10 Hz) “playback” puffing to mimic each person’s behaviour precisely (29, 45, 47). In the first case, with the Beirut method (5, 48), the smoking machine is programmed with the average puff in a rectangular waveform and a fixed puff volume and duration, repeated at a fixed frequency. In the second case, a smooth parabolic waveform is used, with two waveform and frequency stages – one used for the first third of the smoking session (stage 1) and the other for the remainder (stage 2) – to account for the observation that smokers take larger, more frequent, intense puffs at the beginning of a waterpipe smoking session (5).2 In the third case, the puffing topography collected during a participant’s smoking session is “played back” or uploaded to the smoking machine to replicate the session exactly. To compare the emissions in the first and third types of puffing regimen, Shihadeh and Azar (45) compared the tobacco consumed, tar, smoke temperature and CO yields. The periodic regimen resulted in 20% less CO in mainstream smoke, indicating that CO data generated during these regimens may be underestimates of actual exposure. Using a single-stage periodic regimen, Shihadeh (3) also explored the influence of puff volume and frequency on waterpipe tobacco consumption and mainstream tar and nicotine delivery. Larger puff volumes resulted in increased consumption of tobacco, probably because of greater airflow through the coal and head and the resulting higher tobacco temperature. Larger puff volumes also resulted in more TPM (wet and dry) in mainstream emissions, even when normalized by the mass of tobacco consumed and total puff volume. Doubling the puffing frequency (by halving the inter-puff interval from 30 to 15 s) while holding the puff volume constant resulted in about 1.5 times more tar in mainstream emissions, even when tar was normalized by the mass of tobacco consumed; however, doubling the puffing frequency did not significantly change nicotine delivery. 4.4.2 Heat source Several researchers have examined how the heating source influences the delivery of toxicants including furans, VOCs and PAHs. To better understand whether the primary source of specific toxic emissions is charcoal or maassel, researchers have conducted machine smoking with electric and charcoal heat sources and also 4 Kroeger RR, Brinkman MC, Buehler SS, Gordon SM, Kim H, Cross KM, et al. The impact of variation of hookah components on chemical and physical emissions. Presented at the annual conference of the Society for Research on Nicotine and Tobacco, Seattle, WA, USA, 6 February 2014. 82 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report with charcoal alone (no maassel). Most furans were not detectable in emissions generated with charcoal alone, indicating that maassel may be the dominant source (36). This is not the case for VOCs such as benzene and toluene, which are present in waterpipe smoke at similar levels whether the waterpipe head contains maassel or not (49). To identify the dominant source of CO and PAHs in mainstream waterpipe smoke, Monzer et al. (20) used an electric heat source designed to match the spatial and temporal temperature distribution of quick- light charcoal. Emissions collected individually with each heat source showed that charcoal contributed most of the CO (90%) and benzo[a]pyrene (95%). In a comparison of a commercially available electric heater with quick-light charcoal, a research-grade waterpipe and a two-stage puffing regimen, Kroeger et al.2 reported a reduction in the yields of fine-particle PAHs (50 time less) and nicotine (about four times less) in mainstream smoke and of CO (about 2000 times less) and benzene (about 1200 times less) in sidestream smoke. 4.4.3 Temperature of tobacco The concentrations of carbonyls such as acetaldehyde, formaldehyde, acetone and acrolein are strongly influenced by the peak temperature reached in tobacco, higher temperatures resulting in greater yields (4). In turn, the peak temperature reached in tobacco is influenced by the concentrations of glycerol and propylene glycol, the primary humectants in waterpipe tobacco, greater humectant content resulting in lower temperatures (36). 4.4.4 Effect of water Several studies have indirectly and directly addressed whether toxicants dissolve in the bowl water during puffing and are thus effectively “filtered” from mainstream waterpipe smoke. Indirect measures indicate that the concentrations of toxicants in mainstream waterpipe smoke depend on the presence of water in the bowl. The presence of water reduced the level of nicotine by 4.4 times (3) and the level of carbonyls by 3.7 times (4). Schubert et al. (49) measured the phenol content of the bowl water directly and found that it contained detectable levels of two phenols: phenol and guaiacol (7.9 and 3.3 times more, respectively, in water than in smoke). Shihadeh (3) reported, however, that the level of tar was not significantly different when water was removed from the bowl. 4.5 Influence of waterpipe design on levels of emissions of waterpipe tobacco products 4.5.1 Components and accessories In developing a testing protocol for waterpipe emissions, it is useful to distinguish between waterpipe components and accessories. Components are defined as Waterpipe toxicant content and emissions 83 necessary elements of the apparatus required for smoking tobacco in a waterpipe, whereas accessories are optional elements that may be incorporated into the apparatus but are not strictly required. The components and examples of some of the many accessories available, and the physical and chemical attributes that may affect waterpipe emissions, are shown in Table 4.5. The influence of overall waterpipe design and some components and accessories such as the hose, tray and foil on emissions is discussed briefly below. The influence of other components and popularly used accessories is unknown and requires investigation. Table 4.5. Waterpipe components and accessories that may affect emissions Component Purpose Physical attributes Head Holds the tobacco Construction material; geometry; connection or joint; location, diameter and number of airway holes; weight Body Transfers smoke from head to mouth of bowl Construction material, geometry, connection or joint, immersion depth Stem Transfers smoke from mouth of bowl into water Construction material, geometry, connection or joint, immersion depth Bowl Holds water Construction material, shape (dimensions), volume Water Bubble formation Volume, purity, pH Hose Transfers smoke from bowl to user Construction materials, length, inner and outer diameter Charcoal tray or foil Barrier to reduce burning of tobacco Fabrication material; thickness; shape (dimensions); area for holding charcoal; location, diameter and number of airway holes; weight Hookah cream Increases the amount of smoke or aerosol generated Ingredients, mass used per mass of waterpipe tobacco, preparation method (layered or “stacked” or mixed evenly with the tobacco) Bubble diffuser Produces smaller bubbles, quieter puffing, reduced smoke harshness Fabrication material; shape (dimensions); location, diameter and number of airway holes; length of stem covered when installed; type of sealing joint to stem Mouthpiece To prevent spread of germs during group smoking Fabrication material, surface smoothness, length, inner and outer diameter Wind cover Shields charcoal from wind Fabrication material; thickness; shape (dimensions); area for holding charcoal; location, diameter and number of airway holes; weight; type of sealing joint to head 4.5.2 “Real-world” and research-grade waterpipes Waterpipe emissions have been tested with either commercially available waterpipes (e.g. 3, 46) or waterpipes especially designed for use in an analytical laboratory (e.g. 13, 21). Commercially available waterpipes and their components vary widely in design and durability, including in the materials used to fabricate stems, bases, bowls and hoses, sealing joint designs and degree of leak-tight fit and the diameter of the flow path. All the variables can affect the net thermal energy transferred from the heat source to the tobacco, which in turn can affect the nature and concentration of the mainstream smoke particle phase (4, 36) and the smoker’s puffing behaviour. The fabrication materials and design of any commercial waterpipe may change without notice, which could confound emissions testing. 84 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report Research-grade waterpipes, such as that shown in Fig. 4.4, were designed to address these issues. They are fabricated from inert materials that should minimize chemical adsorption to and desorption from surfaces and eliminate stray sources of chemicals from the waterpipe itself (e.g. metal solder and thermal degradation products). Research-grade waterpipes have benchmarked performance metrics for precision and accuracy (13) and inter- and intra-subject variability (50) and have been well accepted in terms of satisfaction and reward by experienced smokers in clinical studies (13). Fig. 4.4. Standardized research-grade waterpipe equipped with human puff topography data collection and acquisition 4.5.3 Waterpipe hose In most studies of machine smoking, the waterpipe hoses were made of leather or plastic. Plastic hoses resulted in more than twice the amount of TPM and CO in mainstream smoke, largely because leather hoses infiltrate air (43) and result in water loss (51). The nicotine levels were not significantly different. 4.5.4 Waterpipe tray versus foil In most studies of machine smoking, either foil or a metal tray was used as the interface between charcoal and tobacco. Kroeger et al.5 compared mainstream and sidestream emissions generated in a research-grade waterpipe equipped with a metal tray or foil and a two-stage puffing regimen. The concentrations of some 5 Kroeger RR, Brinkman MC, Buehler SS, Gordon SM, Kim H, Cross KM, et al. The impact of variation of hookah components on chemical and physical emissions. Presented at the annual conference of the Society for Research on Nicotine and Tobacco, Seattle, WA, USA, 6 February 2014. Waterpipe toxicant content and emissions 85 toxicants in the mainstream fine-particle phase were significantly lower, including those of the TSNAs NNN and NNK (two to three times lower) and the PAHs benzo[a]pyrene and pyrene (two to three times lower), when the metal tray was used; however, the concentrations of some sidestream gas-phase toxicants were significantly higher, including those of acetaldehyde, acetonitrile, acrylonitrile, benzene, 1,3-butadiene and isoprene (one to three times higher). In summary, the testing protocols for waterpipe tobacco smoke emissions and waterpipe components and accessories can influence tobacco consumption and the identity and concentration of the resulting mainstream and sidestream emissions. A preliminary list, based on current knowledge, of protocol conditions and their effects on priority toxicants is shown in Table 4.6. Overall, the heat source has the greatest influence on mainstream and sidestream waterpipe smoke emissions. Table 4.6. Waterpipe testing protocol conditions and influence on resulting toxic emissions Condition 1 Condition 2 Toxicants in MS, SS and BW Toxicant level in condition 1 Periodic puffing (45) Playback puffing MS CO 1.2 times greater MS TPM (dry) Not significantly different Tobacco consumption 1.2 times greater 300 mL puff volume (3) 150 mL puff volume Tobacco consumption 1.4 times greater MS TPM (wet)a 3.8 times greater MS TPM (dry)a 3.2 times greater MS nicotine Not significantly different 1 puff every 30 s (3) 1 puff every 15 s MS tar 1.5 times greater MS nicotine Not significantly different With tobacco No tobacco MS furans (48) Contains ~100% of the furans MS benzene (36) Not significantly different MS toluene (36) Not significantly different Quick-light charcoal Electric heat source (20) MS CO Contains 90% of the CO MS benzo[a]pyrene Contains 95% of the benzo[a] pyrene Commercial electric coalb MS nicotine (3) 4 times higher SS CO 2000 times higher SS benzene 1200 times higher Peak temperature reached in tobacco, 277 °C Peak temperature reached in tobacco, 203 °C (4) MS acetaldehyde 3.3 times higher MS acrolein 1.3 times lower MS formaldehyde 1.2 times higher With bowl water Without bowl water MS nicotine (3) 4.4 times lower MS acetaldehyde (4) 3.9 times lower MS acrolein (4) 3.5 times lower MS formaldehyde (4) 2.8 times lower In bowl waterb In MS waterpipe smoke BW phenol 7.9 times higher BW guaiacol 3.7 times higher 86 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report Plastic hose (43) Leather hose Tobacco consumption 1.4 times higher MS TPM (wet) 2.4 times higher MS CO 2.4 times higher Foilc Tray MS NNN 3.2 times higher MS NNK 1.8 times higher MS pyrene 1.9 times higher MS benzo[a]pyrene 2.6 times higher SS acetaldehyde 1.5 times lower SS acetonitrile 1.4 times lower SS acrylonitrile 1.5 times lower SS benzene Not significantly different SS 1,3-butadiene 1.9 times lower SS isoprene 1.6 times lower MS, mainstream (active); SS, sidestream (passive); BW, bowl water after machine smoking a TPM normalized by mass of tobacco consumed b Measured directly in bowl water c Kroeger RR, Brinkman MC, Buehler SS, Gordon SM, Kim H, Cross KM, et al. The impact of variation of hookah components on chemical and physical emissions. Presented at the annual conference of the Society for Research on Nicotine and Tobacco, Seattle, WA, USA, 6 February 2014. 4.6 Conclusions Waterpipe puff topography varies by population and setting; however, too few studies have been conducted to draw conclusions about the extent of the variation. In all studies to date, the puff volume, flow rate and puff number were much larger than during cigarette smoking, and machine testing regimens must be adjusted accordingly. Waterpipe tobacco smoke contains and delivers high concentrations of the toxicants associated with tobacco-related diseases, including nicotine addiction, lung disease, heart disease and cancer. Waterpipe smoke generated from tobacco- free products also contains and probably delivers high concentrations of the toxicants associated with tobacco-related diseases, including lung disease, heart disease and cancer. Toxicant emissions depend not only on the tobacco product smoked but also on the combination of tobacco product, charcoal type, waterpipe design, waterpipe preparation method, puff topography and their interactions. In the current state of knowledge, protection of public health requires regulation of the characteristics and contents of tobacco products and charcoal. The global resurgence of waterpipe smoking and the high exposure to toxicants associated with waterpipe use indicate that waterpipe smoking should be included in all tobacco control programmes and policies, including banning flavouring additives and indoor smoking. Waterpipe toxicant content and emissions 87 4.7 Recommendations for regulators ■ Require that manufacturers disclose the ingredients and contaminants (specified in Table 4.2) of tobacco and charcoal products marketed for waterpipe use (including maassel, herbal maassel, waterpipe stones and other products intended for mixing with tobacco or charcoal). ■ Require manufacturers of products intended for waterpipe smoking, including tobacco and tobacco-free products, charcoal, waterpipe components (e.g. hose infiltration) and accessories (e.g. aluminium foil), to disclose to regulators their intent to market such products. ■ Require points of sale of waterpipe products to maintain records of compliance of product with regulations, once regulations are adopted. ■ Ban the use of flavour compounds in tobacco-based and tobacco-free waterpipe products. ■ Include all forms of waterpipe use in indoor smoking bans. ■ Communicate to users that used waterpipe water is hazardous be- cause of its chemical and microbial content. 4.8 References 1. 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Applicability and adaptability of the WHO Tobacco Laboratory Network standard operating procedures for cigarettes to waterpipe tobacco Marielle Brinkman, Battelle Public Health Center for Tobacco Research, USA Walther Klerx, Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands Alan Shihadeh, Center for the Study of Tobacco Products, American University of Beirut, Lebanon Reinskje Talhout, Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands Ghazi Zaatari, Department of Pathology and Laboratory Medicine, American University of Beirut, Lebanon Contents 5.1 Introduction 5.2 Smoking methods 5.2.1 Heat sources 5.2.2 Head 5.2.3 Head covering 5.2.4 Water 5.2.5 Hose 5.2.6 Filter 5.3 Smoking machines 5.4 Sampling of waterpipe tobacco 5.5 Sample preparation 5.6 Determination of contents and emissions 5.6.1 Contents of waterpipe tobacco 5.6.2 Emissions of tar, nicotine and carbon monoxide 5.7 Discussion 5.8 Conclusions and recommendations 5.8.1 Recommendations for regulators 5.8.2 Recommendation for researchers 5.9 References 5.1 Introduction This section includes recommendations on the application of existing and pending TobLabNet SOPs for waterpipe tobacco smoking, which were considered by the WHO FCTC COP Working Group on Articles 9 and 10 at its meeting in February 2016. The features of waterpipes used globally are described in section 4. 92 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report Laboratory characterization of toxicant emissions by use of a smoking machine requires specification of puff topography parameters such as puff volume, duration and inter-puff interval. Specification is necessary because toxicant emissions are strongly influenced by the puffing parameters used to smoke a given product (1–3). Several studies of waterpipe puff topography have been reported in the scientific literature, covering various populations in clinical laboratory and natural environments. These studies are summarized in section 4, Table 4.1, which shows mean puff volumes of 500–1000 mL, puff durations of 2–3 s and inter-puff intervals of approximately 10–35 s. The variations among studies seen in the Table probably reflect the influence on puff topography of factors such as years of experience, smoking frequency and setting. Some experimental data suggest that waterpipe puff topography can be influenced by the nicotine content of the product smoked; in a blinded experiment, experienced waterpipe users puffed more intensively when they were provided with a nicotine-free waterpipe product (4). Experimental data also show that puff topography is affected by the level of nicotine dependence (5). Such variations notwithstanding, it is noteworthy that waterpipe smoking involves puff volumes more than 10 times greater than those of a cigarette and that a single waterpipe puff displaces approximately the same smoke volume as an entire cigarette. It is clear, therefore, that cigarette puff topography parameters should not be used in waterpipe machine smoking tests. To date, the most commonly used puff topography regimen for analytical studies of waterpipe tobacco smoke is that of the Beirut method (6), which specifies 171 puffs of 2.6 s duration, 530 mL volume and 17 s inter-puff interval, in addition to waterpipe design, preparation and charcoal addition procedures. This method was based on two field campaigns in cafés in the Beirut area in which waterpipes were served (6, 7) and was validated by measuring “tar”, nicotine and CO in smoke sampled in real time from waterpipes as they were smoked by café patrons (6). It is the only method to date that has been validated against human data. 5.2 Smoking methods As noted in the previous section, methods designed for cigarette testing are not applicable to quantification of waterpipe emissions. Numerous factors unique to waterpipe smoking have been considered in investigations of emissions, which are discussed below. 5.2.1 Heat sources Quick-lighting charcoal is the most popular heat source described in published research. After the charcoal has been lit with an open flame, it is placed on the head for 60 (8) to 100 s (1) before machine smoking is started. Applicability and adaptability of the WHO standard operating procedures for cigarettes to waterpipe tobacco 93 Researchers have investigated two electric heat sources, one fabricated in the laboratory (9) and the other purchased commercially.6 Temperature measurements in both studies at two locations, just under the heat sources and in the tobacco in the head, indicate that an electric heat source can mimic the behaviour of charcoal. The results with both devices indicate that the most the CO and PAHs come from the charcoal (9). Kroeger et al.2 also showed that most benzene comes from burning charcoal, and, with a different experimental approach, Schubert et al. (10) confirmed this result. The constituents of mainstream waterpipe tobacco smoke should be tested with both electric and charcoal heating sources so that toxicity can be properly attributed. We recommend that protocols for charcoal and electric heating sources be included in the adapted SOP or that separate SOPs be developed for charcoal emissions. 5.2.2 Head Levels of constituents have been reported mainly for waterpipes with heads made of ceramic (e.g. 1) or metal (e.g. 8), but some research has been conducted with a waterpipe with a glass head (11)1. Each of these materials has different thermal conductivity, which will probably affect the temperature of the tobacco, which in turn may influence the variety and concentration of constituents in mainstream smoke, although this has not yet been rigorously proven. We recommend that the type and thickness of the head material and its dimensions, including the number and diameter of the holes in the head, be specified in the adapted SOP. The emissions also depend on the amount of tobacco used. A head of standardized dimensions is therefore required, and the emissions per gram of tobacco used should be calculated. To be certain that the distance between the heating device and the tobacco does not vary, the head should be completely filled, with a special cover on which the heating device is placed. 5.2.3 Head covering In most studies with machine-smoking, aluminium foil or a metal tray with holes was used to cover the head of the pipe so that the charcoal or other heat source dis not touch the tobacco. These two materials are likely to transfer heat to the tobacco with different efficiency, thereby affecting the toxic content of mainstream smoke. We recommend that the thickness and size of the foil or tray and the number and diameter of the holes in these coverings be specified in the adapted SOP. Depending on the heat source used, covering the head might reduce heat transfer too much, which might imply that tests should be performed without covering the head. 6 Kroeger RR, Brinkman MC, Buehler SS, Gordon SM, Kim H, Cross KM, et al. The impact of variation of hookah components on chemical and physical emissions. Presented at the annual conference of the Society for Research on Nicotine and Tobacco, Seattle, WA, USA, 6 February 2014. 94 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report 5.2.4 Water The amount of water in the bowl should be specified and measured, because it is directly related to the pressure drop, or resistance to flow, that the smoker must overcome to inhale smoke through the hose. The smoker must, by sucking on the hose, create a vacuum in the bowl that is greater than that in the static head (1). This is directly related to the size of the bowl and the distance between the bottom end of the stem and the water level. We recommend that the bowl dimensions, the length of the stem and the length of the stem that is covered by the bowl water be specified in the adapted SOP. 5.2.5 Hose In most studies of machine-smoking, waterpipe hoses made of leather or plastic were used. Researchers have shown that, owing to air infiltration through (12) and water loss to (13) leather hoses, plastic hoses result in more than twice the amounts of TPM and CO generated in mainstream smoke, although the level of nicotine was not significantly different (12). We recommend that the adapted SOP specify use of a plastic hose in order to reduce variation arising from the different porosity and humidity of leather and that the length and diameter of the hose also be specified, as these factors affect both flow resistance and particle deposition. 5.2.6 Filter In mainstream cigarette smoke, the majority of nicotine (90–99%) is in the protonated form and thus attached to the smoke aerosol (14, 15). Standard analyses of cigarette constituents involve collection of TPM onto a glass-fibre filter, which is extracted with a solvent and quantified by GC (16). The mass of TPM generated during waterpipe tobacco smoking may be 10–100 times more than that from cigarette smoking (17). Therefore, during waterpipe machine smoking, the filter must not be overloaded, as this will create too high a pressure drop, which may result in poor sample retention, damage to the filter and/or pump overload. In routine testing, filter pads should not be changed during a machine smoking run, as this may jeopardize the integrity of the puff volume. The system cannot be checked for leaks after a filter change without modifying the machine smoking regimen, and a leak-tight system is critical for reproducible constituent analyses. Researchers reported 1–2.7 g of TPM in the mainstream smoke from a single waterpipe tobacco smoking session (1, 8), of which about 60% is attributable to water. Nicotine is soluble in water, and comparison of machine smoking with and without water in the bowl indicates that approximately 75% of the nicotine is retained in the water (1). The high water content of the waterpipe aerosol requires that hydrophobic filter media such as Teflon be avoided for Applicability and adaptability of the WHO standard operating procedures for cigarettes to waterpipe tobacco 95 smoke sampling in order to avoid blockage; in a hydrophilic medium such as a glass-fibre wick, the moisture travels along the filter fibre. To the extent that nicotine is in the particle phase, filter sampling will be effective in trapping it, provided that the filter is not overloaded during machine smoking (i.e. that it becomes saturated such that liquid droplets are found on the back of the filter). The degree to which semi-volatile analytes that can partition between the gas and particle phases are retained on the filter may be affected by such variables as the particle size distribution, the hygroscopicity of the analyte and the duration of the smoking session (18). For the adapted SOP, it is recommended that mainstream smoke be split into a minimum of two equivalent streams and that two filter cartridges (92 mm in diameter) be installed for the duration of the waterpipe smoking session to ensure that particle loading remains within the carrying capacity of the filters. Breakthrough of semi-volatile chemicals due to different particle sizes and combinations of filters require more rigorous testing. 5.3 Smoking machines In view of the differences in puffing parameters and mechanical design of waterpipes and cigarettes, analytical smoking machines designed for testing cigarette emissions cannot be used for testing those from waterpipe tobacco. To determine emissions from waterpipe tobacco, the smoking machine must consist of the same principal components as waterpipes; head, body, bottle and suction device. While the exact parameters of waterpipe smoking topography to be used in machine smoking of waterpipe tobacco remain to be specified, it is essential that the machines fulfil at least the following requirements: ■ applicable for testing various types and amounts of waterpipe tobacco or molasses; ■ accommodate different types of heating device (e.g. charcoal, electri- cal heating); ■ have components that are chemically resistant, inactive and free of contamination, including all tubes, hoses and connectors; ■ suitable for different bottle types and sizes; ■ capable of drawing puffs up to a volume of at least 1000 mL; ■ capable of connection to different trapping systems for particulate matter as well as for gaseous phase components; ■ have pipes, hoses, collection devices and other components of de- fined length, diameter and position; and ■ include device(s) for setting parameters, controlling the equipment and storing and printing data. 96 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report A working group within ISO/TC 126 is establishing definitions and standard conditions for a waterpipe tobacco smoking machine, and a smoking machine for generating waterpipe smoke has been made commercially available by Borgwaldt GmbH (Fig. 5.1). An analytical waterpipe smoking machine, the heating devices and the settings to be used for determining the emissions of waterpipe tobacco should all be adjusted according to future demands and regulations. The specific requirements of an analytical smoking machine for testing waterpipe emissions and their possible influence on emissions are described in section 5.4. It is recommended that a standard waterpipe design and puff profile be adapted for emission testing purposes. Fig. 5.1. Analytical waterpipe smoking machine developed by Borgwaldt GmbH 5.4 Sampling of waterpipe tobacco Currently, cigarettes are sampled for regulatory purposes mainly according to ISO 8243 (19). This ISO standard describes sampling of cigarettes at one time or over a period of time, both for sampling at a point of sale and at the premises of the manufacturer or importer. This standard also establishes the confidence intervals for the amounts of tar, nicotine and CO emitted when cigarettes are smoked according the ISO regime. Sampling of roll-your-own or make-your-own tobacco products is described in ISO 15592 part 1 (20), by the same procedures as for cigarette sampling. In sampling for (regulatory) testing of tobacco products (including waterpipe tobacco), a representative sample of a specific product must be obtained, either at one time or over a period of time. When all the products available to consumers comply with regulations, sampling (and testing) should be done at one time. If the purpose is to check whether the product in general complies with the regulations, sampling over time is advisable, although each set of samples should still be tested. Applicability and adaptability of the WHO standard operating procedures for cigarettes to waterpipe tobacco 97 The location of sampling (point of sale or premises of the manufacturer or importer) depends on the purpose: to determine whether products to be used by consumers are in compliance with regulations or whether the manufacturer or importer produces or imports waterpipe tobacco that is in compliance with regulations. As the intention of regulation is to protect consumers, sampling at points of sale is the best option, although a possible disadvantage is that a manufacturer or importer might claim that they are not responsible for the product after it leaves their premises. To avoid manipulation by the manufacturer or importer (preselection of samples that are in compliance), it is advisable to arrange sampling by a government agency or an independent organization. These recommendations for sampling are also applicable to related prod- ucts to be tested, such as charcoal. 5.5 Sample preparation Sample preparation as described in this section means handling of a waterpipe tobacco sample from the moment it enters a test facility until the start of the test procedure(s). Additional preparation required for a specific test should be included in the procedures of that test. The main goal of sample preparation is to create a homogeneous, stable, representative sample for testing from the laboratory sample. Important procedures are mixing and conditioning of waterpipe tobacco. As all individual sales units must comply with regulatory limits, each package of the product should be homogenized separately. Waterpipe tobacco may not be homogeneous and may contain components that might be discarded by consumers before smoking, such as large tobacco plant stems. Further investigation is needed to determine the influence of these components on the contents and emissions of waterpipe tobacco and how consumers deal with these components. The results will indicate whether these components should be included in or excluded from the test procedures. The number of tests required for verification depends on the variation in the product that is allowed and the confidence intervals (CIs). For cigarettes, ISO 8243 (19) specifies that the average result requires the average of 20 test results, in each of which each 20 cigarettes are smoked on an analytical smoking machine for verification of their regulatory compliance. The number of homogenized packages of waterpipe tobacco to be tested remains be defined, taking into account variation among packages and acceptable CIs. The CIs of measurements can be determined in inter-laboratory validation studies. The CIs at one time in ISO 8243 (19) are 20% for tar and nicotine and 25% for CO. Depending on the variation among packages and the analytical variability of specific determination(s), the number of packages to be tested can be limited by setting a maximum acceptable CI. 98 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report As determination of waterpipe tobacco contents and emissions starts with weighing a certain amount, the moisture content is an important variable, as more water corresponds to less tobacco in the same product weight. In other tobacco products (cigarettes and roll-your-own tobacco), the relative humidity depends on the desired moisture content, with an average of about 13% in cigarettes and about 20% in roll-your-own tobacco (20), corresponding to a relative humidity for conditioning of 60% and 75%, respectively, as specified in CORESTA-recommended method 42 (21). As both product types are conditioned at 22 °C, there is no need to adjust the temperature according to the moisture content. For waterpipe tobacco, conditioning for stabilization might interfere in the determination of some components. For regulatory purposes, it can be decided that waterpipe tobacco should be analysed as sold to consumers. Waterpipe tobacco is usually sold in sealed containers, which might increase the variation in results over time and between laboratories due to differences in water content and therefore different amounts of tobacco used in the determination. To minimize variation, regulatory limits can be set for the dry product, such that the water content must be determined or the waterpipe tobacco must be dried before analysis. Both options will require additional testing, increasing the cost of regulatory measurement of components of waterpipe tobacco. Alternatively, the water content of waterpipe tobacco might be determined at the same time as nicotine, as both components are soluble in isopropanol. The applicability of combined measurement should be investigated further. The moisture content of waterpipe tobacco influences its emissions during smoking. To minimize variation over time and between laboratories in the emissions of waterpipe tobacco, the products should be be stable and smoked under defined conditions. Laboratory testing of a few waterpipe tobacco samples for water extractable with isopropanol showed a moisture content of 10–30%. Differences in relative humidity would require several conditioning steps; as this would be difficult to apply in practice, it is advisable to use one setting for relative humidity. In comparison with the moisture content of cigarettes and roll- your-own tobacco, a relative humidity of 75% would be suitable for conditioning waterpipe tobacco, rather than 60%. As only a few laboratories have access to conditioning equipment suitable for 75% relative humidity, waterpipe tobacco could be conditioned at 60% relative humidity and 22°C, as described in ISO 3402 (22). The minimum and maximum duration of conditioning waterpipe tobacco should be investigated further and included in the SOP. Currently, the influence of the temperature and the humidity of the environment during (machine) smoking of waterpipe tobacco is unknown. As both cigarettes and roll-your-own tobacco are smoked at the same temperature (22 °C) and humidity (60%), despite different moisture contents, it is recommended that waterpipe tobacco be (machine) smoked in the same conditions. Applicability and adaptability of the WHO standard operating procedures for cigarettes to waterpipe tobacco 99 5.6 Determination of contents and emissions TobLabNet has validated analytical methods for the determination of contents (three methods) and emissions (four methods) of cigarettes. Below, the applica- bility of these methods to waterpipe tobacco is discussed. 5.6.1 Contents of waterpipe tobacco Of the three validated TobLabNet SOPs for determination of the contents of ciga- rette tobacco filler, those for humectants and nicotine are discussed in relation to their applicability to waterpipe tobacco. 5.6.1.1 Humectants TobLabNet SOP-06 for the determination of humectants in cigarette tobacco filler is validated for glycerol, propylene glycol and triethylene glycol. Glycerol and propylene glycol are present in cigarette tobacco at 0.5–4.0%, while triethylene glycol is present only occasionally in cigarette tobacco as a possible contaminant of the humectants used during production. In contrast, triethylene glycol was identified in 6 of 44 waterpipe tobacco products tested, and nearly all the products contained much higher levels of glycerol than cigarette tobacco (23). A similar extraction procedure for humectants in waterpipe tobacco was tested by Rainey et al. (23), as described in TobLabNet SOP-06. This implies that there is no need to adapt the extraction procedure of TobLabNet SOP-06 for the determination of humectants in waterpipe tobacco. Because of the much higher levels of glycerol in waterpipe tobacco, precautions should be taken in GC settings to avoid co-elution of glycerol and triethylene glycol. The calibration range of glycerol and propylene glycol should also be adjusted for the higher levels of these compounds in waterpipe tobacco. 5.6.1.2 Nicotine The determination of nicotine in cigarette tobacco filler is described and validated in TobLabNet SOP-04. In this method, nicotine is extracted from cigarette tobacco with water, a sodium hydroxide solution and hexane. During extraction, nicotine is transferred to hexane and is analysed by GC–FID. The high levels of humectants in waterpipe tobacco might result in incomplete extraction of nicotine. This should be investigated by testing recovery of added nicotine dissolved in glycerol or propylene glycol or with different extraction solutions. InTobLabNet SOP-04, nicotine is analysed by GC–FID. This technique is widely used for the analysis of nicotine and is applicable for the determination of nicotine in various matrices. Waterpipe tobacco, however, contains not only high levels of humectants but also various types and amounts of flavours, which might 100 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report contain chemical components that interfere with nicotine analysis (Fig. 5.1). Changing the chromatographic parameters to avoid co-elution of interfering flavours would be very time-consuming or almost impossible because of the huge number of different flavours used in waterpipe tobacco. A more practical approach would be to use GC–MS to achieve more reliable identification of nicotine and more reliable quantitative results. Fig. 5.1. Chromatogram of nicotine determination in waterpipe tobacco with different flavours Source: unpublished measurements by RIVM 5.6.2 Emissions of tar, nicotine and carbon monoxide The determination of emission components depends on the type of smoking machine, the smoking protocol, trapping of components, extraction and preparation of the sample solution and measurement of specific components. In this section, the applicability of procedures for trapping components, preparing sample solutions and measuring components are discussed, with possible adjustments of the waterpipe smoking machine or protocol to avoid loss or interference. For the determination of cigarette emissions, the trapping systems prescribed in the TobLabNet SOPs are: ■ a CFP for tar, nicotine, benzo[a]pyrene and TSNAs; ■ a gas sampling bag for CO; and ■ a Carboxen cartridge for aldehydes and VOCs. In general, the applicability of the TobLabNet SOPs for the determination of cigarette emissions to waterpipe tobacco emissions depends on the level of each component, the sensitivity of the equipment and the presence of components that interfere with trapping efficiency or instrument measurements. The CFPs used for collecting the particulate phase of cigarette smoke, as described in ISO 3308 (24), can collect particles with a diameter ≥ 0.3 μm with an Applicability and adaptability of the WHO standard operating procedures for cigarettes to waterpipe tobacco 101 efficiency > 99.9%. Depending on the type of smoking machine used for smoking cigarettes (linear or rotary), ISO 4387 (16) notes that breakthrough of the filter pads might occur when more than 150 mg (linear) or 600 mg (rotary) particles per filter are trapped. The composition of waterpipe smoke will influence the collection of particles on the CFP. If the amount of total particulate matter from waterpipes is approximately the same as that from cigarettes, the same CFPs can be used for trapping. More research is required to determine whether the CFPs used for collecting cigarette smoke particles can also be used for collecting the particulate phase of waterpipe smoke. The efficiency of the trapping devices to be used for collecting components of waterpipe smoke should be investigated with respect to the levels of the components in waterpipe smoke and machine smoking topography. If the trapping devices cannot collect all the components in one smoke run, methods will be required for replacing the trapping device during a run. This might include adjustment to the smoking machine by, for instance, by introduction of a multi-trapping system or by introducing pressure drop monitoring during smoking to determine when the trapping devices must be replaced. In the latter case, special precautions must be taken to prevent leakage when traps are replaced. Special CFP holders will be required, because at least two traps will have to be attached simultaneously. If it is assumed that all the nicotine in waterpipe smoke is present in the particulate phase, the nicotine will be trapped on the CFPs. The composition of the TPM of waterpipe smoke will probably not interfere in the extraction of nicotine from the CFPs with isopropanol. The number of CFPs and the extraction volume should be further investigated to define the optimal conditions and quantifiable levels of nicotine. When waterpipe tobacco has large amounts of flavours, they might also be present in the smoke. Further investigation is required to determine whether flavours are trapped on CFPs and thus interfere with the determination of nicotine or whether they remain in the gaseous phase of waterpipe smoke. CO levels in waterpipe smoke are substantially higher than those in cigarette smoke (25, 26). CO in cigarette smoke is collected in gas sampling bags provided by the manufacturers of smoking machines, which can hold 3 L (linear smoking machine) or 10 L (rotary smoking machine) of gas. The size of the gas collection bag should be adjusted to the machine smoking topography. Another option is to define the number of puffs to be collected in one bag. Precautions must be taken in measurement procedures because of the harmful effects of CO. To protect laboratory staff from exposure to CO, it is advisable that the waterpipe smoking machine be placed under an exhaust system and the gas collection bags be deflated in a safe environment by staff wearing personal alarm systems. Laboratory tests show that the CO level in waterpipe tobacco emissions depends on the device used to heat waterpipe tobacco (9). Almost no CO is 102 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report emitted when an electrical heating device is used. Thus, CO is produced from charcoal used to heat waterpipe tobacco and not from the tobacco itself. There is no standardized method for determining CO production and emission from charcoal. 5.7 Discussion The WHO FCTC recognizes that regulation of tobacco products is required to prevent initiation and promote cessation of tobacco product use and to protect the public from secondhand exposure (27). In 2003, the Scientific Advisory Committee on Tobacco Product Regulation (28) addressed tobacco product contents and emissions and recommended that upper limits be set for known toxic chemicals in the ingredients and emissions. Progress has been made in implementing this recommendation through collaboration between IARC and the WHO Tobacco Free Initiative. The aim of collaboration with IARC was to restrict emissions on the basis of their toxicity. Although the yields of chemicals from machine-smoked cigarettes generated with standardized puffing regimens (ISO/FTC (16), Massachusetts Benchmark (29), Canadian Intense (30)) do not provide valid estimates of human exposure (31), they do provide a framework for establishing and monitoring mandated thresholds for chemical yields. In this approach, products that exceed emission limits for the selected toxicants will not be permitted for sale. The allowable emission levels can be lowered regularly over time, and additional toxicants can be added, resulting in cigarettes with lower intrinsic toxicity. This performance-based paradigm is particularly suitable for cigarettes, because, unlike most tobacco products, cigarettes are presented to the consumer ready to use. As a result, cigarettes have intrinsic emissions that can be measured reproducibly, generally within a variation of 15% relative to the standard deviation, by a given method (32), and the responsibility for meeting regulatory performance standards rests on a clearly identifiable party: the manufacturer. These two characteristics are not applicable to the many tobacco products that are not standardized, such as bidis, roll-your-own cigarettes and waterpipes. While each component that is part of the final product is manufactured to clear specifications, the combination of components is controlled by the user or in a cottage industry under less rigid quality control. This combination of components for non-standard products involves selection and preparation of consumables and hardware. Waterpipe users select the hardware and accessories, the tobacco product, the charcoal and the aluminium foil, each of which is usually of a different origin and each of which can influence toxicant emissions, either as a source or by interacting with the other components. For example, while most of the carcinogenic PAH emissions in waterpipe smoke derive from the burning charcoal, PAHs survive the tortuous path through the waterpipe only by coalescing with particles emitted by the tobacco mixture. Thus, without Applicability and adaptability of the WHO standard operating procedures for cigarettes to waterpipe tobacco 103 the particulate matter generated by the tobacco mixture, PAHs deposit on the interior surfaces of the waterpipe and do not exit the mouthpiece in significant quantities (9). Other examples of interactions that influence toxicant emissions are the porosity of the waterpipe hose and the combustion conditions in the waterpipe head. Hose porosity, a property of the material of manufacture and construction quality, affects the amount of air passing through the charcoal and into the waterpipe head. The more porous the hose, the less air is drawn through the head, affecting both the combustion conditions in the charcoal and the heat transfer rate to the waterpipe tobacco preparation, which in turn affects both “tar” and CO emissions (12). Therefore, waterpipe emissions are the net outcome of the combination of selections made by the consumer, and responsibility for meeting emission standards cannot readily be assigned to an entity that sells one or another product for use in waterpipe smoking.7 Furthermore, except for nicotine emissions, the smoke emitted from tobacco-free products, which are commonly advertised for health-conscious users, has essentially the same toxicant profile and biological activity as that of conventional tobacco-containing products (33–35). In view of the lack of a demonstrated method for individually characterizing the emissions from the various consumables (charcoal, tobacco preparation, aluminium foil) and hardware options, setting product emission standards for regulating waterpipe products could be complicated. Thus, a simpler approach – regulation of product contents – may be feasible, such as setting limits on ingredients that are known to result in high toxicant emissions and on harmful contaminants in waterpipe products that are not essential to their intended use (e.g. heavy metals in tobacco leaf). In accordance with the emissions standards paradigm advanced by TobReg, when systematic differences in contaminants are found in products available on the market, regulations can be promulgated to limit their concentrations to the minimum observed values. One component to which this approach could be applied immediately is waterpipe charcoal. The PAH content of waterpipe charcoal varies systematically by product type (36) and accounts for a significant fraction of the PAHs found in smoke. Similarly, the heavy metal content (e.g. lead, chromium, arsenic, nickel) varies systematically by tobacco preparation, so that it would be possible to require that their concentrations not exceed the lowest concentrations currently found in marketed products. Interestingly, emissions of furans and aldehydes have been found to be inversely related to the humectant content of tobacco preparations (37, 38), probably because of the lower temperatures attained in the mixture when the humectant content is high. 7 The scope of this report as commissioned by the COP does not include considering these complex inter- actions in developing an SOP. The scientific literature base is not yet sufficient to support development of an SOP that considers these factors 104 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report Thus, in the short term, regulation could be focused on the harmful contaminants that have been found in products marketed for waterpipe use – both tobacco and charcoal – such as inorganic metals and elements (39, 40), nicotine (41), TNSAs (26) and PAHs (9, 36), as summarized in Table 5.2. In addition, the pH of the tobacco–humectant mixture may affect the fraction of total nicotine in mainstream smoke that is in the more biologically available unprotonated or “free-base” form (14, 42). Table 5.2. Candidate chemicals for regulation in tobacco and charcoal products marketed for waterpipe use Waterpipe sample matrix Monitored chemical class Target chemicals and metric Tobacco Alkalinity pH Humectants Diethylene glycol, ethylene glycol, glycerol, propylene glycol Inorganic metals and elements Arsenic, cadmium, chromium, cobalt, lead, mercury, nickel, selenium Nicotine Nicotine TSNAs NNN, NNK, N-nitrosoanatabine, N-nitrosoanabasine Charcoal Inorganic metals and elements Arsenic, cadmium, chromium, cobalt, lead, mercury, nickel, selenium PAHs Naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[b+k] fluoranthene, benzo[a]pyrene, benzo[ghi]perylene, dibenz[a,h]anthracene, indeno[1,2,3-cd]pyrene In the long term, as evidence and standardized measurement methods become available, the list of regulated waterpipe product constituents may be extended to include constituents that are found to contribute to the toxicant emissions, listed in Table 5.3. Table 5.3. Chemicals recommended for measurement in mainstream smoke from waterpipe tobacco and charcoal brands Monitored chemical class Target chemicals Aldehydes Acetaldehyde, acrolein, crotonaldehyde, formaldehyde Aromatic amines 1-Aminonaphthalene, 2-aminonaphthalene, 4-aminobiphenyl Flavours Acetylpropionyl, diacetyl Furans 5-(Hydroxymethyl)-2-furaldehyde, 3-furan methanol, furfuryl alcohol, 2-furoic acid, 2-furaldehyde, 3-furaldehyde, 2-furyl methyl ketone, 5-methyl-2-furaldehyde, methyl- 2-furoate Humectants Diethylene glycol, ethylene glycol, glycerol, propylene glycol Inorganic metals and elements Arsenic, cadmium, chromium, cobalt, lead, mercury, nickel, selenium Nicotine Nicotine PAHs Naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[b+k]fluoranthene, benzo[a] pyrene, benzo[ghi]perylene, dibenz[a,h]anthracene, indeno[1,2,3-cd]pyrene Phenols Catechol, m-cresol, o-cresol, p-cresol, phenol TSNAs NNN, NNK, N-nitrosoanatabine, N-nitrosoanabasine VOCs Acrylonitrile, benzene, 1,3-butadiene, CO, isoprene Applicability and adaptability of the WHO standard operating procedures for cigarettes to waterpipe tobacco 105 5.8 Conclusions and recommendations The body of evidence on the toxicity, addictiveness and appeal of waterpipe tobacco smoke indicates the need for urgent public health intervention (17). Measurement of toxicant yields in mainstream waterpipe tobacco smoke is, however, in its infancy, and there are no standardized methods for waterpipe smoke analysis that could be used as a basis for regulating emissions. Given the complexity of the interactions among the waterpipe, accessories, tobacco, heat source and human puffing behaviour and the myriad products available, a product regulation approach that focuses on measuring and reporting the content of chemicals known to contribute to the toxicity, addictiveness and appeal of waterpipe tobacco smoking might be more effective than regulating emissions from various combinations of heating source, tobacco product, puff topography and waterpipe design. The data reviewed in the previous sections lead to the following conclusions. ■ Waterpipe puff topography is characterized by a much larger puff vol- ume, flow rate and puff number than cigarette smoking. ■ Machine-generated waterpipe toxicant emissions are sensitive to puff topography. ■ Waterpipe-specific smoking machines are required to test emissions. One such machine is commercially available. ■ Toxicant emissions do not depend only on a particular waterpipe, charcoal or tobacco product but rather on combinations of these var- iables and puff topography. ■ Standard TobLabNet operating procedures for measuring the con- tents and emissions of cigarette tobacco products would have to be modified for use to test waterpipe products. ■ Standard TobLabNet operating procedures are not suitable for meas- uring charcoal constituents. ■ For research purposes, the Beirut method can be used to generate waterpipe smoke. 5.8.1 Recommendations for regulators 1. Regulations should focus primarily on the chemical composition of waterpipe tobacco products and charcoal. 2. Standard TobLabNet operating procedures should be adapted for the measurement of nicotine, TSNAs and humectants in the contents of waterpipe tobacco products. 106 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report 3. Analytical methods should be adapted to determine the pH and the heavy metal content of waterpipe tobacco (and tobacco-free) prod- ucts. 4. Analytical methods should be adapted for measuring metals and PAHs in emissions from waterpipes heated with charcoal products. 5. The priority of regulation should be to reduce the levels of TSNAs, PAHs and heavy metals in waterpipe products, in accordance with the approach recommended by TobReg (43). The list of regulated constituents should evolve as knowledge becomes available on toxi- cant emissions and/or health effects. 5.8.2 Recommendation for researchers 1. The effects on toxicant emissions of waterpipe tobacco product com- position, charcoal composition, puff regimen, waterpipe design and waterpipe use conditions should be elucidated to facilitate product regulation. 5.9 References 1. Shihadeh, A. Investigation of mainstream smoke aerosol of the argileh water pipe. Food Chem Toxicol 2003;41:143–52. 2. Djordjevic MV, Stellman SD, Zang E. Doses of nicotine and lung carcinogens delivered to ci- garette smokers. J Natl Cancer Inst 2000;92:106–11. 3. Ramoâ C, Shihadeh A, Salman R, Eissenberg T. Group waterpipe tobacco smoking increases smoke toxicant concentration. Nicotine Tob Res 2016;18:770–6. 4. Cobb C, Blank M, Morlett A, Shihadeh A, Jaroudi E, Karaoghlanian N, et al. Comparison of puff topography, toxicant exposure, and subjective effects in low-and high-frequency waterpipe users: a double-blind, placebo-control study. Nicotine Tob Res 2015;17:667–74. 5. Alzoubi K, Khabour O, Azab M, Shqair D, Shihadeh A, Primack B, et al. Carbon monoxide exposure and puff topography are associated with Lebanese waterpipe dependence scale score. Nicotine Tob Res 2013;15:1782–6. 6. Katurji M, Daher N, Sheheitli H, Saleh R, Shihadeh A. Direct measurement of toxicants delive- red to waterpipe users in the natural environment using a real-time in-situ smoke sampling (RINS) technique. J Inhal Toxicol 2010;22:1101–9. 7. Shihadeh A, Azar S, Antonios C, Haddad A. Towards a topographical model of narghile wa- ter-pipe café smoking: a pilot study in a high socioeconomic status neighborhood of Beirut, Lebanon. Pharmacol Biochem Behav 2004;79:75–82. 8. Schubert J, Hahn J, Dettbarn G, Seidel A, Luch A, Schulz TG. Mainstream smoke of the water- pipe: Does this environmental matrix reveal a significant source of toxic compounds? Toxicol Lett 2011;205:279–84. 9. Monzer B, Sepetdjian E, Saliba N, Shihadeh A. Charcoal emissions as a source of CO and carci- nogenic PAH in mainstream narghile waterpipe smoke. Food Chem Toxicol 2008;46:2991–5. 10. Schubert J, Müller FD, Schmidt R, Luch A, Schulz TG. Waterpipe smoke: source of toxic and carcinogenic VOCs, phenols and heavy metals? Arch Toxicol 2015;89:2129–39. Applicability and adaptability of the WHO standard operating procedures for cigarettes to waterpipe tobacco 107 11. Brinkman MC, Kim H, Gordon SM, Kroeger RR, Reyes IL, Deojay DM, et al. Design and validati- on of a research-grade waterpipe equipped with puff topography analyzer. Nicotine Tob Res 2016;18:785–93. 12. Saleh R, Shihadeh A. Elevated toxicant yields with narghile waterpipes smoked using a plastic hose. Food Chem Toxicol 2008;46:1461–6. 13. Haddad AN. 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Toxic contents and emissions of smokeless tobacco products Stephen Stanfill, Centers for Disease Control and Prevention, USA Contents 6.1 Introduction 6.1.1 Global prevalence 6.1.2 Diversity in the manufacture and physical properties of smokeless tobacco products 6.2 Product composition 6.2.1 Tobacco 6.2.2 Additives 6.3 Smokeless tobacco product emissions 6.3.1 Nicotine 6.3.2 Toxic and carcinogenic agents 6.3.2.1 Tobacco-specific nitrosamines 6.3.2.2 Volatile nitrosamines 6.3.2.3 Aldehydes 6.3.2.4 Polycyclic aromatic hydrocarbons 6.3.2.5 Areca nut 6.3.2.6 Metals 6.3.2.7 Nitrate and nitrite 6.3.3 Microbes and their constituents 6.4 Reducing the concentrations of toxicants in smokeless tobacco products 6.5 Conclusions and recommendations 6.6 References 6.1 Introduction This report was prepared in response to the request made by the COP at its sixth session (Moscow, Russian Federation, 13–18 October 2014) to the Convention Secretariat, to invite WHO to prepare a report on the toxic contents and emissions of smokeless tobacco products. Smokeless tobacco globally consists of a diverse array of manufactured products (moist snuff, dry snuff, dissolvables, gutkha, khaini, snus, chewing tobacco, zarda) and hand-made preparations (betel quid, dohra, tombol, toombak, iq’mik) (Table 6.1). Most smokeless tobaccos are used orally, although some drier products are used nasally. Oral smokeless tobacco products and preparations can be chewed, sucked, held against oral mucosa (“dipped”) or applied to the teeth and gums. Addictive and toxic chemicals are liberated from the products during use, absorbed across the mucosa (1) and enter the bloodstream (2, 3). 110 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report Smokeless tobacco use causes cancer (4). The adverse health consequences of smokeless tobacco use were reviewed recently (5). Table 6.1. Types of smokeless tobacco products used globally Product WHO region African Americas Eastern Medi- terranean European South-East Asian Western Pacific Afzal (Oman) √ Betel quid (paan) √ √ √ Caffeinated moist snuff √ Chimó √ Creamy snuff √ Dissolvables √ √ Dohra √ Dry snuff √ √ √ Ghana traditional snuff (tawa) √ Gudakhu or gudakha √ Gul √ Gundi (kadapan) √ Gutka √ √ Hnat hsey √ Hogesoppu (leaf tobacco) √ Iq’mik √ Kadapan √ Kaddipudi √ Khaini √ Kharra √ Kiwam (qiwam, kimam) √ √ Kuberi √ Loose leaf √ Mainpuri (kapoori) √ Mawa √ Mishri (masheri, misri) √ Moist snuff √ √ Nass (naswar) √ √ √ Nasway (nasvay) √ √ √ Neffa √ √ √ Nicotine chewing gum √ Nigerian traditional snuff (taaba) √ NuNu √ Pattiwalla without lime √ Plug (chewing tobacco) √ √ Rapé √ Red toothpowder (lal dant manjan) √ Sada pata √ Toxic contents and emissions of smokeless tobacco products 111 Shammah √ √ Snus √ √ √ Surti √ Taaba √ Tapkeer (bajjar, dry snuff) √ Thinso √ Tobacco leaf √ √ Tobacco water (tuiber) √ Tombol √ Tombol with khat √ Toombak √ Traditional South African snuff (snuif) √ Tumbaco √ Twist √ Ugoro √ Zarda √ √ 6.1.1 Global prevalence It is estimated that more than 300 million people in the six WHO regions use some form of smokeless tobacco (5). Adult use is highly prevalent in countries from Kazakhstan to the Lao People’s Democratic Republic. The prevalence is also high in certain Pacific Islands, Norway, Sweden and other parts of western Europe, several African countries, Mongolia, South America and the USA (6, 7). Globally, 89% of all use by adults is in South-East Asia (mainly Bangladesh and India), where 268 million adults use smokeless tobacco products (5). Smokeless tobacco use represents a substantial global health problem, with an estimated 1.7 million disability-adjusted life years (DALYs) lost due to cancers related to smokeless tobacco use (8). In India, where the prevalence of smokeless tobacco use is high, an estimated 368 000 deaths are attributable to smokeless tobacco use among nonsmokers (9). Globally, 652 494 deaths are estimated to be due to smokeless tobacco use (10). 6.1.2 Diversity in the manufacture and physical properties of smokeless tobacco products Smokeless tobacco products differ in appearance, scale of production, ingredients and formulation (4, 5, 11, 12). They include products that are manufactured commercially and those that are made in traditional environments, such as homes, shops, market stalls and street vending sites. The products range from those containing only tobacco to elaborate hand-made preparations that consist of tobacco mixed with a wide spectrum of non-tobacco plant materials and chemicals. The products come in various forms, including entire tobacco leaves, finely cut tobacco, pulverized tobacco powder, pressed cakes, pellets, pastes, tars 112 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report and mixtures of tobacco with chemicals and plant materials (4, 5, 11, 12). Ground, loose tobacco may be enclosed in teabag-like pouches for discreet, convenient use (e.g. snus and moist snuff). Products known as “dissolvables” consist of finely ground tobacco pressed into tablets, thin cylindrical rods (sticks) or thin wafers or strips that dissolve in the mouth when used (13). Tobacco sticks are essentially dry snuff coated onto a toothpick that can be sucked to liberate the contents (4, 11, 12, 14). A new product, Verve®, is a flavoured cellulose polymer disc impregnated with nicotine extracted from tobacco, which boosts blood nicotine concentrations when chewed, is physiologically active (i.e. raises heart rate and blood pressure) and reportedly satisfies the nicotine cravings of some users (15). 6.2 Product composition 6.2.1 Tobacco Tobacco (Nicotiana spp.) of one or more species is used in the manufacture of most smokeless tobaccos. Exceptions are products like Verve® that contain nicotine extracted from tobacco but no ground or loose tobacco. Although numerous Nicotiana species exist worldwide, N. tabacum is that most often used in commercially manufactured products, whereas N. rustica, which has higher concentrations of nicotine, minor alkaloids and TSNAs than N. tabacum, is commonly used in products in Africa, the Middle East, South America and South Asia (4, 16). For example, in India, an estimated 35–40% of smokeless tobacco products contain N. rustica (17, 18). Infrared analysis confirmed the presence of N. rustica in products sold in several countries, such as gul and some forms of toombak, zarda and rapé (19, 20). Toombak and gul may also contain another tobacco species, N. glauca (4, 21), which has no nicotine but contains a high level of N-nitrosoanabasine (22). Despite the absence of nicotine, N. glauca is consider highly toxic, and ingestion of this species has been lethal in some cases (17, 22). Use of high-nicotine tobacco (N. rustica) or a more toxic species (N. glauca) should be strongly discouraged. 6.2.2 Additives In addition to tobacco, smokeless tobacco products often contain sweeteners, humectants, flavourings, salt and alkaline agents. In 1994, 10 manufacturers of smokeless tobacco products in the USA released a list of more than 560 additives used in the manufacture of their products (4). In products made by hand or in “cottage industries”, it is common to mix tobacco with other plant materials. In South Asia, smokeless tobacco preparations such as paan (betel quid) and dohra contain tobacco, areca nut (Areca catechu), alkaline agents, catechu (Acacia catechu) and spices (e.g. ginger, clove, camphor, saffron) and may be wrapped in a betel leaf (Piper betle). Areca nut is also used Toxic contents and emissions of smokeless tobacco products 113 in mainpuri, mawa, guthka, kharra and some forms of zarda (in South Asia), tombol (in the Middle East) and thinso (in Africa) (5, 10). In Yemen, some types of tombol are made by wrapping a mixture of tobacco and the psychoactive plant khat (Catha edulis) in a betel leaf (5, 17). A smokeless tobacco product in South America, called rapé, can contain a considerable amount of tonka bean (Dipteryx odorata), which has high coumarin levels and is on the list of “harmful and potentially harmful constituents in tobacco products and tobacco smoke” of the Food and Drug Administration in the USA and is banned for use in food (24). Other non-tobacco plant materials include coriander seeds, aniseed, musk, black pepper, vanilla, garlic, mustard, turmeric and ginseng (5). The sweeteners added include simple sugars, molasses, honey and xylitol. Commercial products like loose leaf tobacco and gutkha and cottage industry products such as gul are manufactured with sweeteners (4). An early study of smokeless tobaccos sold in the USA (25) found that the sugar content of pouch and plug forms of tobacco (13.5–65.7%) was much higher than that of snuff (1.9%), and the sugar content of pouch and plug smokeless tobaccos was higher than that of pipe, cigarette or cigar tobacco. Humectants, usually propylene glycol and glycerol, are added to maintain moisture. Research on loose-leaf chewing tobacco products at North Carolina State University in the USA revealed glycerol concentrations of 3.2% (CRP4) and 3.75% (STRP 1S1) and 3.0% propylene glycol (CRP1, snus) (26). In snus manufactured to GothiaTek® standards (described in section 6.4), humectants are added at 1.5–3.5% (27) to reduce microbial growth in order to prevent the formation of TSNAs (28). Flavourings include individual flavour compounds, fruit juices, cocoa, rum, spice powders, extracts and more than 60 essential oils (11, 29, 30). In a survey of the chemistry of smokeless tobacco products, methyl salicylate, ethyl salicylate, benzaldehyde, citronellol and menthol were the flavours found most frequently (31). Other researchers have detected methyl salicylate, ethyl salicylate and menthol in moist snuff products with wintergreen and mint flavouring (32). Further ingredients may include caffeine, coconut, liquorice, herbal medicines, vegetable dyes, colourings, edible oils, butter, soil, saltpetre (potassium nitrate) and flecks of silver metal. Dissolvable smokeless tobaccos may also contain adhesives, binders and whiteners (5, Appendix 1). Alkaline agents added to manufactured smokeless tobacco products include carbonates, bicarbonates and slaked lime (calcium hydroxide) (5, 12, 29), whereas cottage industry products (toombak, shammah) and hand-made preparations (iq’mik, nass, betel quid) generally include slaked lime, sodium bicarbonate or ashes from certain plants or fungi (4, 33, 34). Iq’mik, a product used by native populations of the North American Arctic contains tobacco in twist or leaf form mixed with fungus or ash (35). 114 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report 6.3 Emissions from smokeless tobacco products 6.3.1 Nicotine Nicotine, the principal addictive chemical in tobacco, is present at a wide range of concentrations in smokeless tobacco products and plays a key role in repetitive use, resulting in continuous exposure to toxicants and carcinogens. Total nicotine – the entire amount of nicotine in a product, regardless of its ionic form – is an important consideration, but pH also plays a role in nicotine chemistry. In unprocessed tobacco, which is usually acidic (pH 5.0−6.5) (36), very little nicotine is present in the un-ionized form (<  5%). Un-ionized nicotine, which is readily absorbed, is also called “un-protonated” or “free” nicotine. Oral absorption of nicotine usually requires added alkaline agents to raise the pH and convert a sufficient percentage of nicotine into free nicotine (5). Products with similar total nicotine content but different pH have widely different concentrations of free nicotine (5). Free nicotine, which increases as the pH rises, is readily released from tobacco and crosses biological membranes. Thus, alkaline agents play a key role in releasing nicotine, contributing (in conjunction with the total nicotine of a product) to higher blood nicotine concentrations, which are thought to contribute to the addictiveness of smokeless tobacco (2–4, 37). Nicotine itself is toxic and has health effects, causing, e.g. cardiovascular diseases and diabetes; therefore, increasing its absorption by means of alkaline agents makes the products more addictive and potentially more toxic. The pH values reported for smokeless tobacco products range from 4.6 to 11.8, which result in 0.02–99.9% of nicotine in the free form. Iq’mik and nass, which contain alkaline ash, have extremely high pH (11.0–11.8) (38, 39). Gul powder, naswar, khaini, South African dry snuff (19) and afzal (in Oman) (40) also have high pH values (9–10.5). A survey of zarda products showed alkaline pH values of 8.1–9.0 (41). Other smokeless tobacco products, such as toombak, chimó, rapé and snus, range from acidic to very alkaline (5, 19). Chewing tobaccos (twist, chew, plug and loose leaf) are generally acidic (pH < 7) (42), and the pH of moist snuff generally ranges from 5.5 to 8.6 (43, 44). The total nicotine concentration (on a wet weight basis) in about 700 products ranged from 0.39 to 95 mg/g. The best-characterized smokeless tobacco product is moist snuff made in the USA (226 products). In these products, the total nicotine concentration ranged from 4.15 to 25.0 mg/g and that of free nicotine from 0.01 to 15.2 mg/g (43, 44). The total nicotine concentration in less commonly used chewing tobaccos (twist, chew, plug and loose leaf) was 2.92–40.1 mg/g, but they contained less free nicotine (0.01–0.47 mg/g). The total nicotine concentrations in dry snuff products made in the USA, which are acidic to mildly basic, ranged from 0.30 to 28.0 mg/g and those of free nicotine from 0.05 to 3.12 mg/g (42). Conversely, manufactured dry snuff in South Africa had lower Toxic contents and emissions of smokeless tobacco products 115 total nicotine concentrations (1.17–14.9 mg/g) but more free nicotine (1.16–13.8 mg/g) because of higher alkalinity (19). Although very limited data were available, the pH of Nigerian traditional and medicated snuff and South Africa traditional snuff was 9.0–9.5, and the concentrations of total nicotine (2.49–7.41 mg/g) and free nicotine (2.39–6.72 mg/g) were similar. One very alkaline product (pH 10.5) from Oman called azfal had very high total nicotine (48.8 mg/g) and free nicotine (48.6 mg/g) concentrations (40). Snus products purchased in South Africa were mildly acidic and had moderate total nicotine concentrations (13.4–17.2 mg/g) but less free nicotine (0.47–1.19 mg/g) (18). Swedish snus products had a wide range of concentrations of total nicotine (6.83–20.6 mg/g) and free-base nicotine (0.71–15.5 mg/g) (45), some of which were higher than those reported in moist snuff products in the USA (44). Moderate concentrations of total nicotine (3.0–20.5 mg/g) and free nicotine (0.37–2.47 mg/g) were found in 124 dissolvable products (46). A product similar to dissolvables, called Verve®, had low total nicotine (1.68 mg/g) and free nicotine (0.37 mg/g) concentrations (15). Sudanese toombak, which contains the tobacco species N. rustica, had the highest reported concentration of total nicotine (95 mg/g) (47). Smokeless tobacco products in South Asia include red toothpowder, glycerine-based creamy snuff (both used as a dentifrice), gutkha and zarda. It is common in South-East Asia to mix tobacco with supari packets, which can include areca nut, spices, sweeteners and alkaline agents. Gupta and Sankar (48) found that red tooth powder is mildly acidic, with concentrations of total nicotine of 4.47–5.09 mg/g and of free nicotine of 0.03–0.23 mg/g, whereas creamy snuff, which is more alkaline, had higher concentrations of total nicotine (5.62–10.0 mg/g) and free nicotine (0.71–3.39 mg/g). They also found that gutkha is alkaline (pH 8.6–9.2), with a total nicotine concentration of 0.71–3.39 mg/g and free nicotine at 0.03–0.25 mg/g. Zarda products in India were slightly acidic and had total nicotine concentrations of 2.61–9.5 mg/g but very little free nicotine (0.01–0.02 mg/g). Zarda products in Pakistan were more alkaline and had higher total nicotine concentrations (7.35–26.7 mg/g) and free nicotine (5.52–21.4 mg/g) (41). As some products are intentionally combined with alkaline agents before use, the pH and free nicotine concentrations may be higher in the resulting smokeless tobacco preparation. Gupta and Sankar (48) reported that five mixtures of tobacco with supari were alkaline (pH 8.6–10.1) and had total nicotine concentrations of 1.77–4.96 mg/g and free nicotine of 1.56–4.06 mg/g. Alkaline agents may be added to hand-made preparations (e.g. betel quid) to suit the user’s preference for a certain product “strength”. 116 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report 6.3.2 Toxic and carcinogenic agents Because of the presence of cancer-causing agents in smokeless tobacco, it has been classified in IARC Group 1 (known human carcinogen) (4). More than 40 compounds or agents that have been identified as carcinogens by working groups convened by the IARC (4, 11) have been found in smokeless tobacco products (5), including reactive inorganic ions (nitrate and nitrite), TSNAs, N-nitrosamino acids, volatile N-nitrosamines, mycotoxins, PAHs, volatile aldehydes, metals and metalloids and areca nut. The most abundant carcinogens in smokeless tobacco are TSNAs, N-nitrosoamino acids, volatile N-nitrosamines and aldehydes (4). The groups concluded that there is sufficient evidence that use of smokeless tobacco causes precancerous oral lesions and also oral, oesophageal and pancreatic cancers (5). 6.3.2.1 Tobacco-specific nitrosamines TSNAs are formed during the curing, processing, fermentation and combustion of tobacco (49, 50). In most tobaccos, the concentrations of NNN exceed those of NNK, except in bright tobacco, where those of NNK exceed those of NNN (51). Consequently, the blend of the tobacco determines the amounts of NNN and NNK. Of the seven known TSNAs, NNN and NNK generally occur in larger quantities in tobacco products and are clearly the most carcinogenic (52). NNN and NNK are classified as Group 1 human carcinogens (4) and are quantitatively the most prevalent “strong” carcinogens in smokeless tobacco (53). NNN in particular is thought to play a role in oral cancer in smokeless tobacco users and has been found to occur at levels as high as 79 µg/g (4, 53, 54). Table 6.2 provides a summary of the concentrations of TSNAs in commercial and hand-made smokeless tobacco products in various regions of the world. Table 6.2. Concentrations of tobacco-specific nitrosamines in commercial and hand-made smokeless tobacco products Product Reference Concentration (μg/g product wet weight) NNK NNN All TSNAs Toombak 47 578–7300 395–2860 1500–12 630 Toombak 19 147–516 115–368 295–992 Snuff Moist snuff 44 0.38–9.95 2.20–42.6 5.11–90.0 Dry snuff 42 1.34–14.6 6.12–31.3 10.3–76.5 Dry snuff (pouch) 42 0.08–0.12 0.93–0.97 1.52–1.85 Chewing tobacco Plug 42 0.34–0.94 2.92–4.64 4.09–7.75 Loose leaf 42 0.24–0.31 0.94–2.83 1.55–4.10 Twist 42 0.31–0.56 0.83–2.46 2.59–4.95 Snus 19, 42 0.084–1.34 0.27–5.57 0.60–5.85 Dissolvables 42, 46 0.31 0.06–0.26 0.31–0.74 Toxic contents and emissions of smokeless tobacco products 117 Products in the Americas Iq’mik 39 0.19–0.54 1.99–4.00 5.64–8.84 Rapé 20 0.04–3.30 0.013–14.5 0.04–24.2 Chimó 19 0.31–2.60 0.32–4.62 0.95–9.39 South Asian products Gul 19 5.19–8.02 1.33–1.37 13.4–17.1 Khaini 19 0.29–0.50 16.8–17.5 21.6–23.5 Zarda 19 0.46–3.84 2.91–28.6 5.49–53.7 Gutha (handmade) 19 0.007–0.38 0.21–18.6 0.26–23.9 Gutkha 19 0.057–0.46 0.17–1.28 0.37–2.25 Central Asian products Naswar 19 0.029–0.31 0.36–0.54 0.48–1.38 African products Nigerian traditional snuff 19 0.28 0.71 1.52 Medicated dry snuff 19 0.36 1.46 2.42 Dry snuff 19 0.13–0.35 0.89–3.40 1.71–4.67 Traditional snuff 19 1.61 5.57 20.5 Source: reference 4 Smokeless products with higher TSNA concentration tend to be those with microbial contamination. The TSNA levels in products such as dissolvables (0.31–0.61 μg/g), which are solid, low-moisture products (46), and Swedish snus (0.60–5.85 μg/g), which is often pasteurized (19,42,55) are usually lower than typical products. Higher TSNA concentrations are found in fermented products such as Indian zarda (5.5–53.7 µg/g) (19), moist snuff (5.11–90.0 µg/g) (44) and dry snuff made in the USA (10.3–76.5 µg/g) (42). Traditional snuffs in Nigeria and South Africa had total TSNA concentrations of 1.52 and 20.5 µg/g, respectively (19). Interestingly, dry snuff in Africa had lower TSNA concentrations (1.71– 4.67 µg/g) than that made in the USA. Other products with high total TSNA concentrations include khiani, naswar, iq’mik, rapé and chimó. Chewing tobacco has very low TSNA concentrations (1.55–7.75 µg/g) (42). In best-selling brands of moist snuff in the USA, the concentrations of NNN (2.2–42.6 µg/g) were higher than those of NNK (0.38–9.95 µg/g) (44). The TSNA concentrations in Sweden-made snus decreased by approximately 85% between 1983 and 2002, to very low average concentrations of NNN (0.49 µg/g) and NNK (0.19 µg/g) in 27 products in 2002 (56, 57), which are among the lowest reported in commercial smokeless tobacco products. A product known as chaini khaini, labelled and marketed in India as “snus”, had very high levels of NNN ((22.9 ± 4.9 µg/g) and NNK (2.6 ± 1.0 µg/g) (58). In a study of 117 “spit-free” and dissolvable smokeless tobacco products, the concentration of total TSNA (the sum of NNN, NNK, N-nitrosoanatabine and N-nitrosoanabasine) was slightly lower in Camel Strips (0.53 µg/g) than in Camel Snus (1.19 µg/g) (46). In a study of 53 products from nine countries (19), the highest NNK concentrations were found in toombak from Sudan and dry 118 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report zarda from Bangladesh, whereas the highest NNN concentrations were found in toombak, dry zarda and khaini from India. Handmade gutkha and mawa from Pakistan had the lowest NNK concentrations among these products. The highest TSNA concentrations ever reported in smokeless tobacco products were in Sudanese toombak, a highly fermented product, with total TSNA concentrations reaching 12 600 µg/g, perhaps due to the extremely high concentrations of alkaloids, which are important reactants in TSNA formation. The NNN concentrations in toombak were as high as 2860 µg/g and those of NNK were up to 7300 μg/g (47). TSNAs were also found at extremely high concentrations in saliva from toombak users (47, 59, 60). Over 50% of oral cancers in Sudanese men are attributed to use of toombak or other oral tobacco products, probably due to the high concentrations and carcinogenicity of TSNAs (10, 60, 61). 6.3.2.2 Volatile nitrosamines Accumulation of nitrite is thought to lead to formation of carcinogenic volatile N-nitrosamines during curing through the same microbial reactions that lead to formation of TSNAs (5). Analysis of Swedish snuff and chewing tobacco in the early 1980s demonstrated the presence of volatile N-nitrosamines (N-nitrosdimethylamine, N-nitrosopyrrolidine, N-nitrosopiperidine and N-nitrosomorpholine) at levels ranging from 0.5 to 145.9 μg/kg wet weight (56). A reduction in the use of the agricultural chemical maleic hydrazide diethanolamine and of the manufacturing chemical morpholine have reduced the levels of N-nitrosodiethanolamine and N-nitrosmorpholine in commercial tobacco products (62). Nass (also called nasswar), a mixture of tobacco, alkaline agents and cotton oil used in Afghanistan, India, the Islamic Republic of Iran, Pakistan, the Russian Federation and Central Asia (63) was also found to contain volatile N-nitrosamines but at lower levels than in chewing tobacco or snuff. The difference in levels of volatile N-nitrosamine has been attributed to shorter ageing in nass manufacture (64). 6.3.2.3 Volatile aldehydes Carcinogenic aldehydes (formaldehyde, acrolein, crotonaldehyde, acetaldehyde) have been shown to be present at levels of parts per million in smokeless tobaccos, including snus products. The levels tend to be higher in fire-cured tobacco than in air-cured tobacco (5, 55). 6.3.2.4 Polycyclic aromatic hydrocarbons PAHs may be present in smokeless tobaccos that contain tobacco cured with wood and sawdust burnt during fire-curing, and the concentrations are higher Toxic contents and emissions of smokeless tobacco products 119 in fire-cured than air-cured tobacco (5). Moist snuff produced with fire-cured tobacco has a higher concentration of PAHs (including IARC Group 1 and 2 carcinogens) than snus, which does not contain fire-cured tobacco (55, 65). Ten PAHs in IARC groups 1 (benzo[a]pyrene), 2A (dibenz[a,h]anthracene) and 2B (benzo[b]fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene, dibenzo[a,i]pyrene, indeno[1,2,3-cd]pyrene, 5-methylchrysene, naphthalene and benz[a]anthracene) (66) have been found in smokeless products (65). The total concentration of PAHs in 23 products made in the USA ranged from 921 to 9070 ng/g in moist snuff and 660 to 1100 ng/g in snus. The concentrations of benzo[a]pyrene in moist snuff (9.7–44.6 ng/g) were higher than those in snus (3.0–12.3 ng/g), and about 40% of the snus brands analysed had levels below the detectable limit (1.6 ng/g). The concentrations of naphthalene in moist snuff (409–1110 ng/g) were similar to those in snus (636–1065 ng/g). When the values for naphthalene were excluded from the total PAH concentration, those of the remaining PAHs in moist snuff (145–8120 ng/g) exceeded those in snus (21–213 ng/g). One brand, often viewed as a “starter”, contained only 145 ng/g of PAHs other than naphthalene (776 ng/g). Marlboro snus products contained seven PAHs at detectable levels of 1.1–13.5 ng/g individually; when naphthalene was excluded, the summed concentration of PAHs was 20–70 ng/g. Camel snus brands contained detectable levels of 14 PAHs at 3.1–79.4 ng/g and 110–320 ng/g when naphthalene was excluded (65). Very low PAH concentrations can be attained when fire-cured tobacco content is decreased or eliminated from smokeless tobaccos. 6.3.2.5 Areca nut Unripe areca nuts have extremely high alkaloid levels, and they are preferred in certain cultures because they “generate a better buzz” (5). IARC working groups have placed areca nut in Group 1 (66). Arecoline is thought to be the most important alkaloid. Extracts of areca nut are highly cytotoxic and genotoxic, including to human oral mucosal cells and fibroblasts. Betel quid alone, not mixed with tobacco, has also been shown to be genotoxic (5) and carcinogenic (11). 6.3.2.6 Metals Metals and metalloids may accumulate in tobacco plants or on leaf surfaces, depending on the soil composition, pH and environmental contamination (67). Metals found in various smokeless tobacco products include some in IARC Group 1 (human) carcinogens (arsenic, beryllium, chromium VI, cadmium, polonium-210) and also Group 2A probable carcinogens (nickel compounds) and Group 2B possible carcinogens (lead, cobalt). Arsenic, which is technically a metalloid, is a Group 1 human carcinogen. Mercury and aluminium have also been detected. Detectable concentrations of arsenic (0.1–14.0 μg/g), beryllium 120 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report (0.01–0.038 μg/g), chromium (0.71–54.0 μg/g), cadmium (0.25–9.2 μg/g), nickel (0.84–64.8 μg/g), lead (0.23–111 μg/g) and cobalt (0.056–1.22 μg/g) have been found in smokeless tobacco products from Canada, Ghana, India, Pakistan and the USA (67). In a study of smokeless tobacco products from India (zarda, creamy snuff, khaini, gutkha), higher concentrations of copper were found in four gutka products (237–656 μg/g) than in the other products (0.012–36.1 μg/g) (68). Arsenic, cadmium and lead were found in components such as slaked lime, betel leaves and flavoured tobacco (zarda) used to make betel quid (69). 6.3.2.7 Nitrate and nitrite Plants take up fertilizer-derived nitrate from soil, which is used by plant cells. When tobacco dries during curing, the cells rupture, releasing nitrate (70–72). Microbes are present as endophytes in plants (73). If viable nitrate-reducing microorganisms are present, nitrite is produced and released. Several genera of bacteria and fungi identified in tobacco and tobacco products (71, 74, 75) can convert nitrate to nitrite. Nitrite released by microbes can react with tobacco alkaloids to form TSNAs and can also contribute to the formation of volatile nitrosamines and nitrosamino acids (70, 76). Nitrite and TSNA concentrations increase during tobacco fermentation (71, 72) and tobacco storage, especially at elevated temperature and moisture (77). If nitrate-reducing microorganisms are not eliminated during processing, they can affect the chemistry of tobacco products (70–72). 6.3.3 Microbes and their constituents Microorganisms such as bacteria and fungi are often present in tobacco and tobacco products (71, 75, 78, 79). In studies with microbial DNA sequencing methods (75, 80), 33 bacterial families were identified in various smokeless tobacco products. Genes for respiratory nitrate reductases and, to a lesser extent, periplasmic nitrate reductases were predicted to be involved in the production and extracellular release of nitrite. Some bacterial families include known anaerobes, which use nitrate as an electron acceptor instead of oxygen (81), which may account for the accumulation of extracellular nitrite in the conditions of low oxygen that are likely to occur in processes such as fermentation, ageing and storage of tobacco (71, 72, 77). Bacterial genera that contain genes for respiratory nitrate reductases include Corynebacterium, Lactobacillus and Staphylococcus species and certain bacteria in the Enterobacteriaceae family (75).  Bacteria and fungi may proliferate more rapidly and form harmful or reactive by-products during tobacco fermentation (70–72). Accordingly, the concentrations of both nitrite and TSNAs are higher in fermented products such as khaini (82), dry snuff (82), moist snuff (65) and Sudanese toombak (19, 47) than in products such as snus, which is pasteurized (19, 46). Toxic contents and emissions of smokeless tobacco products 121 A few fungal species (e.g. Fusarium, Alternaria and Candida) have also been identified in tobacco and tobacco products (71, 78, 79, 83). Aflatoxin B1, a mycotoxin produced by Aspergillus fungi, was reported in six dry snuff products made in the USA (0.01–0.27 μg/g) but not in 16 moist snuff or 3 snus products (84). 6.4 Reducing the concentrations of toxicants in smokeless tobacco products Reduction of the concentrations of toxicants in tobacco products requires understanding of the agricultural practices and manufacturing processes that result in their formation and accumulation. Table 6.3 lists the toxic and carcinogenic substances found in tobacco and their potential sources during tobacco processing. Table 6.3. Possible sources of IARC carcinogens, toxicants and biologically active compounds in smokeless tobacco products Agent class IARC carcinogens (groups 1, 2A, 2B), toxicants or biologically active compounds Possible source Metals and metalloids Group 1: Arsenic, beryllium, cadmium, nickel compounds, polonium-210 Group 2A: Inorganic lead compounds Group 2B: Cobalt sensitization: aluminum, chromium, cobalt, nickel Dermal irritants: barium, mercury May contribute to oral submucosal fibrosis: copper (in areca nut) Soil absorption or present in soil particles deposited on tobacco; potentially present in other ingredients (betel leaf, areca nut, slaked lime, etc.) used in conjunction with tobacco Nitrosation agents Group 2B: Nitrate Group 2B: Nitrite Soil absorption Generated by microorganisms Mycotoxins Group 1: Aflatoxins (mixtures of ) Group 2B: Aflatoxin M1, ochratoxin A Formed by fungi (Aspergillus) Nitrosamines TSNAs Group 1: NNN, NNK, NNAL Formed by nitrosation during curing, fermentation and ageing (nitrite reacts with alkaloids) Volatile N’-nitrosoamines Group 2A: N-Nitrosodimethylamine Group 2B: N-Nitrosopyrrolidine, N-nitrosopiperidine, N-nitrosomorpholine, N-nitrosodiethanolamine Formed by nitrosation during curing, fermentation and ageing (nitrite reacts with secondary and tertiary amines) Nitrosoacids Group 2B: N-Nitrososarcosine Carbamates Group 2A: Ethyl carbamate Formed during fermentation (reaction of urea and ethanol) PAHs Group 1: Benzo[a]pyrene Group 2A: Dibenz[a,h]anthracene Group 2B: Benz[a]anthracene, benzo[b] fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene, dibenzo[a,i] pyrene, dibenzo[a,i]pyrene, indeno[1,2,3- cd]pyrene, 5-methylchrysene, naphthalene Deposited on tobacco during fire-curing Volatile aldehydes Group 1: Formaldehyde Group 2B: Acetaldehyde Deposited on tobacco during fire curing Non-tobacco plant materials Group 1: Areca nut Liver toxicant: Tonka bean Stimulant: Khat Additives Source: reference 85 122 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report During cultivation, plants such as tobacco absorb metals, metalloids and dissolved ions (e.g. nitrate and ammonium) from the soil (86), and soil particles (including metals), agricultural chemicals and microorganisms in the soil and other constituents of the environment can deposit and remain on tobacco leaves. The levels of metals in tobacco are affected by soil pH, soil composition and environmental contaminants (67). Because deposited materials may remain on the leaf throughout processing, removing soil and microbes, including those that produce nitrite, from tobacco could help to decrease the formation of TSNAs and other nitrosamines and lower the levels of metals and agrochemicals deposited on the leaves. Nitrate, commonly found in soils and certain fertilizers, increases plant biomass but remains in tobacco after harvesting (5). When microbes capable of converting nitrate to nitrite are present, nitrite can be generated. Nitrite expelled from microbial cells can react with tobacco alkaloids to form TSNAs. TSNA production can be minimized by washing tobacco at harvest (88), heat treatment in a closed system (pasteurization) (28), cleaning of fermentation equipment and addition of non-nitrite-producing microbes during fermentation (75). Refrigerated storage can also slow the growth of microbial populations and reduce formation of nitrosamine compounds; at least one manufacturer encourages retailers to refrigerate products to prevent formation of TSNAs during storage (4). Eliminating or reducing the use of nitrate-containing fertilizers or employing other strategies (e.g., using urea or other non-nitrate fertilizers late in the growing season) could also limit the formation of nitrosamines by decreasing the accumulation of nitrate present at harvest (5). Use of air-cured rather than fire-cured tobacco could reduce the levels of PAHs and volatile aldehydes. In Sweden, the GothiaTek® standard established maximum levels for contents of public health concern in snus, which are nitrite, NNN, NNK, N-nitrosodimethylamine, benzo[a]pyrene, aflatoxin, cadmium, lead, arsenic, nickel, chromium and agrochemicals. The constituents of the starting materials must be carefully controlled to minimize their levels in the final snus product. In addition, the flavour additives used in these products must comply with the Swedish Food Act (28). The results of adherence to these standards suggest that integrated agrochemical policies, specification of raw material and process controls can result in lower concentrations of targeted toxicants in the snus (moist snuff) variety of smokeless tobacco. Such rigorous attention to the constituents of products might decrease the levels of harmful constituents in other tobacco product types. WHO has recommended (88) that, when feasible, the upper limit of TSNAs in smokeless tobacco be reduced to 2 µg/g; when this is not immediately feasible, the level should be gradually reduced to 2 µg/g. Toxic contents and emissions of smokeless tobacco products 123 6.5 Conclusions and recommendations Smokeless tobaccos include a wide range of products, ranging from those that contain only tobacco to those consisting of tobacco combined with chemicals and non-tobacco plant materials. The products differ in appearance, production methods, contents and ingredients, and the ways in which the products are used. Many of the harmful chemicals present in these products and preparations result from organic, inorganic and microbiologic components and the interactions among them as tobacco is processed into the final product. Plant materials and other additives used with tobacco can effect product appeal (taste or appearance), absorption of nicotine, addictive potential, toxicity and, most notably, their cancer- and disease-causing properties (4, 5, 11, 12). As 89% of all smokeless tobacco users are in South Asia, ingredients unique to South Asian products, particularly areca nut, should be given priority in assessing the health risks associated with smokeless tobacco products. Areca nut is an IARC Group 1 carcinogen (66) and is used both with and without tobacco by an estimated 600 million people worldwide (89). Areca nut use is a global health concern because of its carcinogenicity, addictiveness and prevalent global use (89) and its continuing spread in some form (90). Some of the concerns associated with use of smokeless tobacco products worldwide are: ■ inclusion of high-nicotine (N. rustica) or toxic (N. glauca) tobacco species; ■ presence of toxic metals in tobacco due to soil uptake or leaf surface deposition from contaminated soil; ■ soil fertilization practices that result in elevated levels of nitrate in tobacco at harvest; ■ presence of harmful agricultural chemical residues remaining on the tobacco at harvest; ■ presence of microbial contamination on tobacco leaves that promotes the formation of nitrosamines, particularly TSNAs; ■ fermentation or ageing, which provides an anaerobic environment that contributes to rapid nitrite and TSNA formation; ■ fire-curing, which can introduce chemicals from smoke, such as PAHs and volatile aldehydes; ■ alkaline agents that raise the pH and increase the free nicotine con- centration; and ■ presence of areca nut (IARC Group 1 human carcinogen) and other additives with recognized toxicity. 124 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report Worldwide, only GothiaTek® snus products manufactured by Swedish Match are tested for certain pesticides, metals and nitrosamines and also for nitrite and benzo[a]pyrene (a PAH) to ensure that the concentrations do not exceed certain thresholds. Although the testing does not result in a risk-free product, maintenance of these concentrations shows that they can be decreased and maintained for some toxicants (28). Manufacturers of smokeless tobacco products can control a number of factors, including the type of and quality of the tobacco used, processes and ingredients used or omitted from their products. Unfortunately, although techniques are available to reduce the levels of carcinogens and other toxicants, manufacturers use the techniques selectively. Newer products often have lower levels of TSNAs, while older and traditional products that continue to be sold have higher levels of TSNAs (91). Regulators have the opportunity to monitor and regulate pH and the nicotine, metal, PAH, TSNA and nitrite contents. An integrated process consisting of specifications for raw materials and process controls could reduce the levels of toxicants, especially those attributed to the curing of tobacco and microbial reactions responsible for the formation of TSNAs and volatile N-nitrosamines. The technology required to test pH (pH paper, pH probe), nitrate/nitrite (indicators, handheld probe) and microbial contamination (culture plates) is not expensive and could be implemented in most countries. Hand-held infrared scanners could be used to identify harmful tobacco species (N. rustica, N. glauca), non-tobacco plant materials (areca nut, tonka bean, khat), and alkaline agents (magnesium carbonate, slake lime). Regulators should also consider requiring better storage conditions, such as refrigerating product before sale, affixing the date of manufacture and regulating packaging material. Manufacturers should also be required to inform retailers about the effect of storage conditions on smokeless tobacco products. The information summarized in this section supports the WHO TobReg recommendation that smokeless tobacco should be subjected to comprehensive regulatory control by an independent, scientific government agency (92). In view of the diversity of the composition and concentrations of toxicants in smokeless tobacco products, the serious adverse health outcomes and the extremely high prevalence of use in regions of the world with disproportionately high rates of oral cancer and other health effects (92), it may not be appropriate to considering these products as a homogeneous class of tobacco products in a generalized policy or regulatory decision. Use of the term “snus,” which connotes a Swedish moist snuff product, to denote products manufactured by different processes and with different characteristics (93) is an example of marketing that can create confusion among consumers and others. Careful review of the design, composition and content of smokeless tobacco products and process controls is warranted for regulation to reduce the harm due to their use throughout the world. Toxic contents and emissions of smokeless tobacco products 125 6.6 References 1. Boffetta P, Hecht S, Gray N, Gupta P, Straif K. Smokeless tobacco and cancer. Lancet Oncol 2008;9:667–75. 2. Fant RV, Henningfield JE, Nelson RA, Pickworth WB. Pharmacokinetics and pharmacodyna- mics of moist snuff in humans. Tob Control 1999;8:387–92. 3. Lunell E, Lunell M. Steady-state nicotine plasma levels following use of four different types of Swedish snus compared with 2-mg Nicorette chewing gum: a crossover study. Nicotine Tob Res 2005;7:397–403. 4. 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Applicability or adaptability of standard operating procedures for nicotine, tobacco-specific N-nitrosamines and benzo[a]pyrene in cigarette contents and emissions to tobacco products other than cigarettes, particularly smokeless tobacco products Nuan Ping Cheah, Cigarette Testing Laboratory, Health Sciences Authority, Singapore Patricia Richter, Centers for Disease Control and Prevention, Atlanta, GA, USA Hongwei Hou, China National Tobacco Quality Supervision and Test Centre Qingyuan Hu, China National Tobacco Quality Supervision and Test Centre Clifford Watson, Centers for Disease Control and Prevention, Atlanta, GA, USA Contents 7.1 Introduction 7.2 Nicotine, tobacco-specific N-nitrosamines and benzo[a]pyrene in smokeless tobacco products 7.2.1 Nicotine 7.2.2 Tobacco-specific N-nitrosamines 7.2.3 Benzo[a]pyrene 7.3 Evaluation of applicability of WHO standard operating procedures for analysis of smokeless tobacco products 7.3.1 Analytical considerations 7.3.1 Determination of nicotine 7.3.2 Determination of tobacco-specific N-nitrosamines 7.3.3 Determination of benzo[a]pyrene 7.4 Discussion and recommendations 7.5 References 7.1 Introduction The COP to the WHO FCTC at its fifth session (1) asked WHO to identify options to regulate chemicals in smokeless tobacco products. At its sixth session, the COP asked the Secretariat to invite WHO to assess, within two years, whether the SOPs for nicotine, TSNAs and benzo[a]pyrene in cigarette contents and emissions are applicable or adaptable, as appropriate, to tobacco products other than cigarettes, including smokeless tobacco. The China National Tobacco Quality Supervision and Test Centre, the CDC (USA) and the Health Sciences Authority (Singapore) agreed to undertake the task, to determine whether the WHO SOPs for nicotine in tobacco filler, TSNAs in mainstream tobacco smoke and benzo[a]pyrene in 132 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report mainstream tobacco smoke could be adapted for use in analysing smokeless tobaccos. Commercial and research smokeless tobaccos representing snus, moist snuff, dry snuff and loose leaf chewing tobacco were selected for testing. To meet WHO’s deadline, the testing laboratories agreed to use test materials that had been characterized chemically to some extent, represented common forms of smokeless tobacco and differed in physical and chemical properties. The assessment of the applicability and adaptability of validated WHO SOPs to smokeless tobacco products and the recommended approach are presented in this section. 7.2 Nicotine, tobacco-specific N-nitrosamines and benzo[a] pyrene in smokeless tobacco products 7.2.1 Nicotine As discussed in section 6, nicotine is considered to be the primary addictive agent in smokeless tobaccos. It is present in an ionized or an un-ionized (also referred to as unprotonated or free) state. The un-ionized form is of particular public health and regulatory interest because it is the form in which nicotine is absorbed most rapidly across the mucous membranes of the mouth (2). Products may have similar levels of total nicotine yet provide different amounts of un-ionized nicotine according to their pH. The total and the percentage of un-ionized nicotine can be calculated from the measured pH and total nicotine content, from the pKa of nicotine and Henderson-Hasselbalch equations (2). Consequently, measurement of nicotine levels and pH is important for informing policy and regulation. Table 7.1 lists nicotine levels reported in the published literature. Table 7.1. Concentrations of nicotine, un-ionized nicotine, pH, moisture, tobacco-specific N-nitrosamines and benzo[a]pyrene in various smokeless tobacco products Type Total nicotine, wet weight (mg/g) Calculated un-ionized nicotine (mg/g) pH Moisture (%) Total TSNAs (µg/g) Benzo[a] pyrene (ng/g) Gul powder, tobacco leaf, zarda 9.55–65.0 0.05–31.0 5.22–9.22 7.47–25.23 (wet weight) 3–38.2 Khaini and gutkha 0.16–21.3 0.12–4.68 7.43–9.65 0.14–127.93 (dry weight) Mawa, mainpuri, naswar, toombak 0.16–40.6 0.11–13.2 7.38–11.0 6–60 0.10–7870 Moist snuff (snus) 7.76–26.92 (dry weight) < 0.01–13.8 5.54–10.1 35–60 2.0–7870 ≤ 940 Dry snuff < 0.01–71.4 6–7 ≤ 1219 > 0.1–90 From references 3–12 Applicability of standard operating procedures for nicotine, tobacco-specific N-nitrosamines and benzo[a]pyrene 133 7.2.2 Tobacco-specific N-nitrosamines TSNAs are strong carcinogens (13) formed from tobacco alkaloids and nitrosating agents during curing, fermentation, ageing and storage at high temperature and high relative humidity (3). The TSNA concentrations in smokeless tobaccos are 500-fold higher than in mainstream cigarette smoke (Table 7.2), although they vary widely by product and country (6, 14). The highest concentration of total TSNAs (992 000 ng/g) was reported in toombak, a smokeless tobacco used in Sudan (5). Table 7.2. Concentrations of nicotine, tobacco-specific N-nitrosamines and benzo[a]pyrene in smokeless tobacco and cigarette tobacco and emissions Analyte Smokeless tobacco product Cigarette tobacco filler Cigarette mainstream smoke (ng/cigarette) Fold difference between concentration in smokeless tobacco and in cigarettes Nicotine ≤ 71.4 mg/g 23.18 mg/g – > 2.5 TSNAs ≤ 992 000 ng/g – 1068.8 Benzo[a]pyrene ≤ 940 ng/g – 29.93 From references 3, 15, 16 7.2.3 Benzo[a]pyrene Benzo[a]pyrene emitted in the mainstream smoke of cigarettes is the result of tobacco combustion, while that in smokeless tobacco is due to use of fire-cured tobacco, which contains detectable levels of PAHs (17). Benzo[a]pyrene was present in smokeless tobaccos that contain fire-cured tobacco, at levels from not detected to 940 ng/g, which is significantly higher than the yields from mainstream cigarette tobacco (Table 7.2). Benzo[a]pyrene is an IARC Group I human carcinogen. It is frequently measured as a surrogate for exposure to PAHs (18). 7.3 Evaluation of applicability of WHO standard operating procedures for analysis of smokeless tobacco products 7.3.1 Analytical considerations Numerous methods have been published for the analysis of nicotine, including determination of pH and moisture content. Techniques based on GC-FID (2) are the most widely used; they have been adopted by the Commonwealth of Massachusetts in the USA (19) and validated by TobLabNet for the analysis of cigarette tobacco filler. Other published procedures include use of MS for detection (4, 5). GC coupled with a thermal energy analyser (20) or MS (7) are commonly used in the determination of TSNAs in smokeless tobaccos. A modification of a GC–MS method (21) for analysis of PAHs in cigarette mainstream smoke (20) was adapted for their analysis in smokeless tobaccos. 134 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report 7.3.2 Determination of nicotine Determination of nicotine in smokeless tobaccos can be based on WHO SOP-04 (22). The values of both total and un-ionized nicotine are important for evaluating the addiction potential of smokeless tobaccos (20, 23). In SOP-04, nicotine is extracted from cigarette filler with an aqueous solution of sodium hydroxide and hexane, during which, all the nicotine is transferred to hexane. The extract is analysed by GC–FID, which is commonly used for analysis of nicotine in mainstream cigarette smoke and in e-liquid. The equipment is generally available in analytical laboratories. The concentrations of nicotine in smokeless tobacco products are comparable to or slightly higher than those reported in cigarette tobacco filler (Table 7.2). pH and moisture content (up to 50% in moist snuff) should also be measured so that the results can be reported on both a dry and a wet weight basis. Measurements of moisture and pH are not included in TobLabNet SOP-04. Gravimetric methods for measuring volatile compounds in smokeless tobacco and the pH of a mixture of tobacco and water have been described (2, 5, 20, 24). These additional measurements are not complex but require equipment for processing tobacco samples (e.g. grinding samples that contain large pieces of tobacco leaf, such as loose-leaf tobacco) and a drying oven capable of maintaining a temperature of 99–100 °C for several hours. One method for measuring the moisture content of smokeless tobacco is a modification of AOAC Method 966.02 (25), referred to as “total moisture determination”, for determining water and tobacco constituents that are volatile at 99 ± 1.0 °C (2). The pH of smokeless tobacco should be determined with a standard pH meter. Usually, 2 g of smokeless tobacco are mixed with 20 mL of analytical- grade water to create a slurry, and the pH is measured with a calibrated pH meter within 60 min of shaking or stirring at room temperature (20–25 °C). The pH meter is calibrated with certified standard buffers. It is important to confirm that there is no systematic drift in pH values (2). Depending on the type of sample, an additional 10 mL of water are added to dilute the mixture to facilitate measurement. The total and un-ionized nicotine content of smokeless tobacco are measured from the pH and total nicotine, with the Henderson-Hasselbalch equation based on total measured nicotine, pH and a pKa value of 8.02 (2, 20). 7.3.3 Determination of tobacco-specific N-nitrosamines The current CDC methods for determining TSNAs in smoke emissions and tobacco are similar to WHO SOP-03 (26), with a few exceptions. The sample preparation and analytical sections of WHO SOP-03 could be adapted for smokeless tobacco, and extension of the TobLabNet method for determining TSNAs in cigarette filler to analysis of smokeless tobacco should be Applicability of standard operating procedures for nicotine, tobacco-specific N-nitrosamines and benzo[a]pyrene 135 relatively straightforward. The NNN and NNK contents of smokeless tobacco can vary from 20 to 10 000 ng/g, whereas those in mainstream tobacco smoke (Table 7.2) are comparable at the lower calibration end. Thus, the upper calibration range would have to be extended to cover smokeless tobacco products with concentrations of NNN and NNK anticipated to be higher. This should not be problematic for linearity or detector saturation. Adaptations to SOP-03 should be based on a comparison with the current CDC method for TSNAs in mainstream smoke emissions and tobacco content (15). Specifically, as noted above, the calibration curve for smokeless tobaccos should be extended (and remain linear), and smokeless tobacco samples might have to be ground and filtered so that tobacco “fines” do not clog the injection system. Sample preparation should be identical to those in the WHO TobLabNet method and the current CDC method, including extraction procedures. The sample size for extraction will have to be optimized, and other modifications, such as grinding tobacco to improve extraction efficiency, should be considered. Thus, the TobLabNet method for measuring TSNAs in cigarette emissions, with appropriate modifications, could be used for measuring NNN and NNK in smokeless tobacco. 7.3.4 Determination of benzo[a]pyrene Determination of benzo[a]pyrene in smokeless tobaccos could be based on WHO SOP-05 for the determination of benzo[a]pyrene in mainstream cigarette smoke (27). In the WHO method, mainstream cigarette smoke is trapped on a CFP made of 1-µm glass fibre. After smoking, the filter pad is extracted with a cyclohexane solution containing an isotopically labelled internal standard, deuterated benzo[a]pyrene-D12. The cyclohexane extract is eluted through a silica solid-phase extraction cartridge, and the eluent is collected and analysed by GC–MS in electron ionization mode. Samples of 0.2–1.0 g of smokeless tobacco product (amount to be optimized during verification) should be extracted with cyclohexane (10 mL at room temperature) and shaken for 1 h and the extract centrifuged at 200 rpm for 60–80 min. A 5-mL aliquot of the extract should be spiked with benzo[a]pyrene-D12 internal standard and mixed well. Sample clean-up indicated in SOP-05 includes solid-phase extraction on a silica cartridge, followed by rotary evaporation. Laboratories should investigate whether rotary evaporation is required. For sample clean-up with solid-phase extraction, the mixture should be loaded onto a pre-cleaned cartridge (Sep-pak Vac silica cartridge from Waters or equivalent), which will be washed and eluted with cyclohexane. The eluent from both the load and the wash should be combined and dried. The residue will then be reconstituted with 1 mL cyclohexane and a reconstituted aliquot used for GC–MS analysis. Optional steps, which should be investigated during method 136 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report verification, include sample clean-up with solid-phase extraction, followed by rotary evaporation, as specified in reference 27. The aliquot should be analysed by GC-MS. Seven smokeless tobacco products (snus, moist snuff, dry snuff and loose leaf) were selected for this study by CDC (Table 7.3). Four were reference products obtained from CORESTA, and three were obtained from a commercial vendor (Lab Depot, Atlanta, GA, USA). CDC shipped the seven smokeless products to the China National Tobacco Quality Supervision and Test Centre and the Health Sciences Authority in Singapore for method verification. Table 7.3. Smokeless tobacco test materials selected for method verification Smokeless tobacco product Type Reference or com- mercial Total nico- tine pH Moisture (%) TSNAs Benzo[a] pyrene CRP1 Snus Reference 0.8% (wet weight) 8.5 52 ~1.46 ppm To be determined CRP2 Moist snuff Reference 1.2% (wet weight) 7.7 54.6 ~4.40 ppm To be determined CRP3 Dry snuff Reference 1.2% (wet weight) 7.7 54.6 18–19 ppm To be determined CRP4 Loose leaf Reference 1.9% (wet weight) 6.9 8.0 ~3.70 To be determined Silvercreek Wintergreen (7) Moist snuff Commercial 8.2 to 11.96 mg/g (wet weight) 6.29–7.08 51.9–52.6 15.86 mg/g (wet weight) To be determined Skoal Original (14, 28) Moist snuff Commercial 11.4 mg/g (dry weight) 7.27 59 ? To be determined Red Seal Wintergreen Moist snuff Commercial 14.9 mg/g (wet weight) 7.55 53.3 4.87–5.27 mg/g (wet weight) To be determined ppm, parts per million 7.4 Discussion and recommendations The objective of this section is to recommend quantitative analytical procedures for adapting and applying existing TobLabNet-validated methods for cigarettes to the analysis of smokeless tobaccos. Numerous methods have been published for the analysis of nicotine and TSNA, including pH determination and moisture content. GC–FID is the method of choice, as the equipment is commonly available in analytical laboratories globally. The conclusion of this review of the TobLabNet SOPs for nicotine, benzo[a] pyrene and TSNAs by knowledgeable experts is that these methods should be applicable for smokeless tobacco products. Cross-matrix studies will have to be performed on representative samples for confirmation. Although a variety of research and commercial smokeless tobacco test materials were selected in order to cover a range of physical and chemical properties (Table 7.3), this sample Applicability of standard operating procedures for nicotine, tobacco-specific N-nitrosamines and benzo[a]pyrene 137 does not cover all the varieties of this diverse type of tobacco product. Limited method optimization will be required for sample preparation, and, for NNN and NNK, the calibration range will have to be extended to cover the higher contents typically present in smokeless tobacco (Table 7.1). In addition, the methods for determining pH and moisture should be discussed and consensus reached. We recommend that cross-matrix validation be conducted for nicotine, pH, benzo[a] pyrene, NNN and NNK in smokeless tobacco products with adapted versions of the TobLabNet SOPs. Conclusions ■ TobLabNet methods for TSNAs and nicotine could be applied or adapted for determination of smokeless tobacco products. ■ The applicability of the TobLabNet method for determining benzo[a] pyrene should be validated, as the matrix is different from that speci- fied in the SOP. ■ The specific, selective TobLabNet methods, with clean up steps, should allow extraction of toxicants. ■ Extension of the calibration range or dilution of samples should be considered to cover the higher values found in smokeless tobacco products. Recommendations ■ Require manufacturers to disclose the pH of products and the levels of the toxicants TSNAs, benzo[a]pyrene and nicotine, measured with WHO-verified methods or country’s official methods, by an inde- pendent laboratory ■ Compliance can be tested in any analytical laboratory designated by a government authority Further work ■ Analyse metals, humectants and aldehydes in smokeless tobacco products by published methods for tobacco, food, plants and envi- ronmental matrices with available laboratory resources. 138 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report 7.5 References 1. Report of the sixth session of the Conference of the Parties to the WHO Framework Conven- tion on Tobacco Control. Geneva: World Health Organization; 2014. 2. Richter P, Spierto FW. Surveillance of smokeless tobacco nicotine, pH, moisture, and unproto- nated nicotine content. Nicotine Tob Res 2003;5:885–9. 3. Smokeless tobacco and some tobacco-specific N-nitrosamines (IARC Monographs on the Evaluation of the Carcinogenicity of Chemicals to Humans, Vol. 89). Lyon: International Agen- cy for Research on Cancer; 2007: 641. 4. Stepanov I, Hecht SS, Ramakrishnan S, Gupta PC. Tobacco-specific nitrosamines in smokeless tobacco products marketed in India. Int J Cancer 2005;116:16–19. 5. Personal habits and indoor combustions. A review of human carcinogens (IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 100E). Lyon: International Agency for Research on Cancer; 2012: 598. 6. Stanfill SB, Connolly GN, Zhang L, Jia LT, Henningfield JE, Richter P, et al. Global surveillance of oral tobacco products: total nicotine, unionised nicotine and tobacco-specific N-nitrosa- mines. Tob Control 2011;20:e2. 7. Richter P, Hodge K, Stanfill S, Zhang L, Watson C. Surveillance of moist snuff: total nicoti- ne, moisture, pH, un-ionized nicotine, and tobacco-specific nitrosamines. Nicotine Tob Res 2008;10:1645–52. 8. Hoffmann D, Harley NH, Fisenne I, Adams JD, Brunnemann KD. Carcinogenic agents in snuff. J Natl Cancer Inst 1986;76:435–7. 9. Idris AM, Ibrahim SO, Vasstrand EN, Johannessen AC, Lillehaug JR, Magnusson B, et al. The Swedish snus and the Sudanese toombak: are they different? Oral Oncol 1998;34:558–66. 10. Rodu B, Jansson C. Smokeless tobacco and oral cancer: a review of the risks and determinants. Crit Rev Oral Biol Med 2004;15:252–63. 11. Caraway JW, Chen PX. Assessment of mouth-level exposure to tobacco constituents in US snus consumers. Nicotine Tob Res 2013;15:670–7. 12. Sharma P, Murthy P, Shivhare P. Nicotine quantity and packaging disclosure in smoked and smokeless tobacco products in India. Indian J Pharmacol 2015;47:440–3. 13. Stepanov I, Biener L, Knezevich A, Nyman AL, Bliss R, Jensen J, et al. Monitoring tobacco-spe- cific N-nitrosamines and nicotine in novel Marlboro and Camel smokeless tobacco products: findings from round 1 of the New Product Watch.” Nicotine Tob Res 2012;14:274–81. 14. Smokeless tobacco and public health: a global perspective. Bethesda, MD, Centers for Disease Control and Prevention and National Institutes of Health, National Cancer Institute; 2014: 558. 15. Ashley DL, Beeson MD, Johnson DR, McCraw JM, Richter P, Pirkle JL, et al. Tobacco-speci- fic nitrosamines in tobacco from US brand and non-US brand cigarettes. Nicotine Tob Res 2013;5:323–31. 16. Counts M, Morton M, Laffoon S, Cox R, Lipowicz P. Smoke composition and predicting relati- onships for international commercial cigarettes smoked with three machine-smoking condi- tions. Regul Toxicol Pharmacol 2005;41:185–227. 17. McAdam K, Faizi A, Kimpton H, Porter A, Rodu B. Polycyclic aromatic hydrocarbons in US and Swedish smokeless tobacco products. Chem Cent J 2013;7:18. 18. Evaluation of certain food contaminants. Sixty-fourth report of the Joint FAO/WHO Expert Committee on Food Additives (WHO Technical Report Series 930). Geneva: World Health Or- ganization: 2006:109. 19. 105 CMR 660.000 Cigarette and smokeless tobacco products: reports of added constituents and nicotine ratings. Boston, MA: Commonwealth of Massachusetts; 1999 (www.mass.gov/ eohhs/docs/dph/regs/105cmr660.pdf). Applicability of standard operating procedures for nicotine, tobacco-specific N-nitrosamines and benzo[a]pyrene 139 20. Stepanov I, Jensen J, Hatsukami D, Hecht S. New and traditional smokeless tobacco: compa- rison of toxicant and carcinogen levels. Nicotine Tob Res 2008;10:1773–82. 21. Ding YS, Ashley DL, Watson CH. Determination of 10 carcinogenic polycyclic aromatic hydro- carbons in mainstream cigarette smoke. J Agric Food Chem 2007;55:5966–73. 22. Standard operating procedure. Determination of nicotine in cigarette tobacco filler (WHO SOP-04). Geneva: World Health Organization; 2014. 23. Hatsukami DK, Severson HH. Oral spit tobacco: addiction, prevention and treatment. Nicotine Tob Res 1999;1:21–44. 24. Determination of pH of smokeless tobacco products method No. 69. Paris: Cooperation Cen- tre for Scientific Research Relative to Tobacco; 2010. 25. AOAC method 966.02. Rockville, MD: Association of Official Analytical Chemists; 1990. 26. Standard operating procedure. Determination of tobacco-specific nitrosamines in main- stream cigarette smoke under ISO and intense smoking conditions (WHO SOP-03). Geneva: World Health Organization; 2014. 27. Standard operating procedure. Determination of benzo[a]pyrene in mainstream cigarette smoke (WHO SOP-05). Geneva: World Health Organization; 2015. 28. Hoffmann D, Djordjevic MV, Fan J, Zang E, Glynn T, Connolly GN. Five leading US commercial brands of moist snuff in 1994: assessment of carcinogenic N-nitrosamines. J Natl Cancer Inst 1995;87:1862–9. 140 141 8. Overall recommendations The WHO Study Group on Tobacco Product Regulation (TobReg) publishes a series of reports to provide a scientific foundation for tobacco product regulation. In line with Articles 9 and 10 of the WHO Framework Convention on Tobacco Control (WHO FCTC),1 these reports identify evidence-based approaches to the regulation of tobacco products. The eighth meeting focused on issues critical to advancing the regulation of tobacco products, particularly as outlined at the sixth session of the Conference of the Parties to the WHO FCTC.2 The topics discussed included: (1) cigarette characteristics and design features; (2) toxicants in waterpipe tobacco and smokeless tobacco; and (3) applicability of WHO Tobacco Laboratory Network (TobLabNet) standard operating procedures (SOPs) of measuring selected content and emission chemicals in cigarette tobacco products to ENDS, waterpipe tobacco and smokeless tobacco products. Main recommendations 1. This report provides relevant guidance regarding specific cigarette design features, as well as testing and disclosure of the contents and emissions of a wide array of smokeless tobacco products, waterpipe tobacco products, and other devices like ENDS. – Design features: Member States should require that manufacturers and importers of tobacco products disclose information on design features listed in Appendix 2 of the Partial Guidelines of the WHO FCTC to governmental authorities at specified intervals, including the results of tests conducted by the tobacco industry. Member States should also consider restricting or prohibiting other design features that may increase the attractiveness of tobacco products such as fla- vours and capsules. Lastly, should there be any change to the design fea- tures of a particular brand of tobacco product, Member States should require that manufacturers notify governmental authorities of the change and provide the updated information when the change is made. – Smokeless tobacco: Manufacturers could be required to disclose the levels of tobacco-specific nitrosamines (TSNAs), benzo[a]pyr- ene (B[a]P) and nicotine, as well as pH levels in SLT products, as 1 For more information, see: http://apps.who.int/iris/bitstream/10665/42811/1/9241591013.pdf?ua=1 (accessed 20 September 2016) 2 For more information on the Conference of the Parties of the WHO Framework Convention on Tobacco Control, see decision FCTC/COP6(10), paragraph 2(a) and decision FCTC/COP6(12) paragraph 2(b) at http://apps.who.int/gb/fctc/E/E_cop6.htm (accessed 20 September 2016). 142 W H O T ec hn ic al R ep or t S er ie s N o. 1 00 1, 2 01 7 WHO Study Group on Tobacco Product Regulation Sixth report the WHO TobLabNet methods can be adapted or applied to these specific toxicants. Furthermore, since there are existing technologies which can reduce levels of SLT carcinogens, manufacturers should be required to use these in order to reduce the toxicity of these prod- ucts. Regulators should also consider requiring improved storage conditions such as refrigerating product before sale, affixing date of manufacture, and regulating packaging material. Lastly, manufactur- ers should also be required to educate retailers on the effect of storage conditions on the SLT product. – Waterpipe tobacco: Waterpipe smoking normally utilizes burning charcoal as the heat source, thus, waterpipe smoke includes toxicants emitted from the charcoal in addition to those from the tobacco product itself. Because of this complexity, regulators should consid- er an approach which focuses initially on measuring and reporting the chemical contents in the waterpipe tobacco products which are known to contribute to their toxicity, addictiveness and appeal, and expand this to selected chemicals and toxicants in emissions as the assessment and analytical methods are validated. – ENDS: Sufficient data exist to support extension of existing and pending WHO SOPs for nicotine, humectants (solvents), carbonyls, B[a]P and TSNAs in ENDS liquid and aerosol. It is recommended to measure the pH of the liquid to establish the range of pH across ENDS liquids, as this will assist with investigations into the addictive potential of the nicotine delivered to the user. Metals should be exam- ined to determine if there is the potential for associated health risk. Significance for public health policies 2. One of the challenges in developing a comprehensive and effective tobacco control policy is the wide range and heterogeneity of com- mercially available tobacco products. TobReg’s report provides help- ful guidance in understanding the contents, emissions and design features of selected products such as cigarettes, smokeless tobacco, and waterpipes. The report highlights the impact of their toxicants or features on public health. In addition, the report expounds on how the WHO TobLabNet SOPs can serve as reliable methods by which to test these products. The current state of knowledge dictates the need to keep active surveys of use of the diverse tobacco products and also monitoring novel new tobacco products. 143 Overall recommendations Significance for the Organization’s programmes 3. This report fulfils TobReg’s mandate to provide the WHO Director- General with scientifically sound, evidence-based recommendations for Member States about tobacco product regulation. In line with the provisions of Articles 9 and 10 of the WHO FCTC, TobReg has identified evidence-based approaches to regulating the vast array of tobacco products which concern Member States. TobReg’s report also lists areas for future research which will expand the knowledge base with respect to tobacco product regulation. This report presents the conclusions reached and recommen- dations made by the members of the WHO Study Group on Tobacco Product Regulation at its eighth meeting, where the group reviewed background papers specially commissioned for the meeting and considered the following topics: 1. Cigarette characteristics and design features 2. Possible application of WHO Tobacco Laboratory Network standard operating procedures to evaluation of electronic nicotine delivery systems 3. Waterpipe toxicant content and emissions 4. Possible application of WHO Tobacco Laboratory Network standard operating procedures for cigarettes to waterpipe tobacco 5. Toxic contents and emissions of smokeless tobacco products 6. Possible application or adaptation of standard operating procedures for nicotine, tobacco-specific N-nitro-samines and benzo[a]pyrene in cigarette contents and emissions to tobacco products other than cigarettes, particularly smokeless tobacco products The Study Group’s recommendations in relation to each theme are set out at the end of the relevant chapter, and overall recommendations are summarized in the final chapter of the report. WHO study group on tobacco product regulation ISBN 978-92-4-121001-0

定　价：98.00元 胡清源 侯宏卫 等　译 　　本报告介绍了WHO烟草制品管制研究小组在第八次会议上 得出的结论和提出的建议，小组审查了会议专门委托的背景文 件，并审议了以下主题： 　　　卷烟的特征和设计特色； 　　　WHO烟草实验室网络标准操作规程对电子烟碱传输系统 　　　评估的潜在应用； 　　　水烟的有害内容物及释放物； 　　　针对卷烟的WHO烟草实验室网络标准操作规程对水烟的 　　　适用性； 　　　无烟烟草制品的有害内容物和释放物； 　　　卷烟内容物和释放物中烟碱、烟草特有亚硝胺和苯并[a] 　　　芘的标准操作规程在无烟烟草制品中的应用。　　　 　　研究组关于每个主题的建议在相关章节末尾列出，最后一 章为总体建议。 （TS-0528.31） W H O 研究组第六份报告 WHO研究组第六份报告 1001 本书英文版于 2017年由世界卫生组织（World Health Organization）出版，书名为： WHO Study Group on Tobacco Product Regulation: Report on the Scientific Basis of Tobacco Product Regulation: Sixth Report of a WHO Study Group (WHO Technical Report Series; No. 1001) © World Health Organization 2017 世界卫生组织（World Health Organization）授权中国科技出版传媒股份有限公 司（科学出版社）翻译出版本书中文版。中文版的翻译质量和对原文的忠实性 完全由科学出版社负责。当出现中文版与英文版不一致的情况时，应将英文版 视作可靠和有约束力的版本。 中文版《烟草制品管制科学基础报告：WHO研究组第六份报告》 © 中国科技出版传媒股份有限公司（科学出版社） 2018 WHO 烟草制品管制研究小组 烟草制品管制科学基础报告 WHO研究组第六份报告 胡清源  侯宏卫  等  译 WHO技术报告系列  1001  北京教图印刷有限公司 印刷 图书在版编目(CIP)数据 烟草制品管制科学基础报告：WHO研究组第六份报告/WHO烟草制品 管制研究小组著；胡清源等译. —北京：科学出版社，2018.12 （WHO技术报告系列 1001） 书名原文：WHO Study Group on Tobacco Product Regulation: Report on the Scientific Basis of Tobacco Product Regulation: Sixth Report of a WHO Study Group (WHO Technical Report Series; No. 1001) ISBN 978-7-03-059712-0 I. ①烟… II. ①W… ②胡… III. ①烟草制品 – 科学研究 – 研究报告 IV. ①TS45 中国版本图书馆CIP数据核字(2018)第263065号 责任编辑：刘　冉 / 责任校对：张小霞 责任印制：张  伟 / 封面设计：铭轩堂 科学出版社发行 各地新华书店经销 * 2018年12月第 一 版 开本：890 × 1240 A5 2018年12月第一次印刷 印张：7 1/4 字数：220 000 定价：98.00元 (如有印装质量问题，我社负责调换 ) 内  容  简  介 本报告介绍了WHO烟草制品管制研究小组在第八次会议上得出的 结论和提出的建议，小组审查了会议专门委托的背景文件，并审议了 以下主题：①卷烟的特征和设计特色；②WHO烟草实验室网络标准 操作规程对电子烟碱传输系统评估的潜在应用；③水烟的有害内容物 及释放物；④针对卷烟的WHO烟草实验室网络标准操作规程对水烟 的适用性；⑤无烟烟草制品的有害内容物和释放物；⑥卷烟内容物和 释放物中烟碱、烟草特有亚硝胺和苯并 [a]芘的标准操作规程在无烟烟 草制品中的应用。研究组关于每个主题的建议在相关章节末尾列出，最 后一章为总体建议。 本书会引起吸烟与健康、烟草化学和公共卫生学等诸多应用领域科 学家的兴趣，为客观评价烟草制品的管制和披露措施提供必要的参考。 ·i· 译  者  序 2003年 5月，第 56届世界卫生大会 *通过了《烟草控制框架公约》 (FCTC)，迄今已有包括我国在内的 180个缔约方。根据 FCTC第 9 条和第 10条的规定，授权世界卫生组织 (WHO)烟草制品管制研究 小组 (TobReg)对可能造成重要公众健康问题的烟草制品管制措施进 行鉴别，提供科学合理的、有根据的建议，用于指导成员国进行烟 草制品管制。 自 2007年起，WHO陆续出版了六份烟草制品管制科学基础报 告，分别是 945，951，955，967，989和 1001。WHO烟草制品管制 科学基础系列报告阐述了降低烟草制品的吸引力、致瘾性和毒性等 烟草制品管制相关主题的科学依据，内容涉及烟草化学、代谢组学、 毒理学、吸烟与健康等烟草制品管制的多学科交叉领域，是一系列 以科学研究为依据、对烟草管制发展和决策有重大影响意义的技术 报告。将其引进并翻译出版，可以为相关烟草科学研究的科技工作 者提供科学性参考。希望引起吸烟与健康、烟草化学和公共卫生学 等诸多应用领域科学家的兴趣，为客观评价烟草制品的管制和披露 措施提供必要的参考。 第一份报告 (945)由胡清源、侯宏卫、韩书磊、陈欢、刘彤、付 亚宁翻译，全书由韩书磊负责统稿； 第二份报告 (951)由胡清源、侯宏卫、刘彤、付亚宁、陈欢、韩 * 世界卫生大会 (World Health Assembly，WHA)是世界卫生组织的最高决策机构， 每年召开一次。 ·ii· 烟草制品管制科学基础报告： WHO研究组第六份报告 书磊翻译，全书由刘彤负责统稿； 第三份报告 (955)由胡清源、侯宏卫、付亚宁、陈欢、韩书磊、 刘彤翻译，全书由付亚宁负责统稿； 第四份报告 (967)由胡清源、侯宏卫、陈欢、刘彤、韩书磊、付 亚宁翻译，全书由陈欢负责统稿； 第五份报告 (989)由胡清源、侯宏卫、陈欢、刘彤、韩书磊、付 亚宁翻译，全书由陈欢负责统稿。 第六份报告 (1001)由胡清源、侯宏卫、韩书磊、陈欢、刘彤、 付亚宁、王红娟翻译，全书由韩书磊统稿。 由于译者学识水平有限，本中文版难免有错漏和不当之处，敬 请读者批评指正。 2018年 11月 ·iii· 目    录 WHO 烟草制品管制研究小组第八次会议 ··········································· ix 致谢 ······································································································· xiii 缩略语表 ································································································ xv 1. 前言 ······································································································ 1 2. 卷烟特征和设计特色 ·········································································· 3 2.1 引言 ············································································································5 2.2 影响感观和行为的卷烟特征 ································································6 2.2.1 概述 ································································································6 2.2.2 影响使用者感观的卷烟特征 ···················································7 2.2.3 影响使用者行为的卷烟特征 ················································ 11 2.3 影响烟气释放物的卷烟特征 ····························································· 15 2.3.1 烟草 ····························································································· 15 2.3.2 卷烟纸 ························································································ 17 2.3.3 滤嘴 ····························································································· 18 2.3.4 物理尺寸 ···················································································· 21 2.4 可改变烟气 pH和成瘾性的设计特色和添加剂 ··························· 24 2.4.1 概述 ····························································································· 24 2.4.2 氨、糖和再造烟叶 ·································································· 25 2.4.3 其他成分 ···················································································· 26 2.4.4 烟丝配方和物理特征 ······························································ 27 2.4.5 测量“烟气 pH” ···································································· 28 2.5 可能影响感观和传送的创新 ····························································· 29 ·iv· 烟草制品管制科学基础报告： WHO研究组第六份报告 2.5.1 概述 ····························································································· 29 2.5.2 低烟碱卷烟 ··············································································· 30 2.5.3 彩色卷烟纸 ··············································································· 31 2.5.4 特色滤嘴 ···················································································· 32 2.5.5 烟草行业对特殊滤嘴和再造烟叶的研究 ·························· 34 2.6 对设计特色的公众健康影响进行科学评价的研究 ····················· 35 2.7 结论 ········································································································· 37 2.8 建议 ········································································································· 40 2.8.1 政策建议 ···················································································· 40 2.8.2 研究建议 ···················································································· 41 2.9 参考文献 ································································································· 42 3. WHO 烟草实验室网络标准操作规程对电子烟碱传输系统评估的 潜在应用 ··············································································································63 3.1 背景 ········································································································· 65 3.2 电子烟碱传输系统（ENDS）的一般方法学评价························ 67 3.3 烟碱 ········································································································· 68 3.3.1 ENDS 烟液中的烟碱 ······························································· 69 3.3.2 ENDS 气溶胶中的烟碱 ·························································· 69 3.4 烟草特有亚硝胺 ··················································································· 71 3.4.1 ENDS 烟液中的烟草特有亚硝胺········································· 71 3.4.2 ENDS 气溶胶中的烟草特有亚硝胺 ···································· 72 3.5 苯并 [a]芘 ······························································································ 73 3.5.1 ENDS 烟液中的苯并 [a] 芘 ··················································· 73 3.5.2 ENDS 气溶胶中的苯并 [a] 芘 ·············································· 74 3.6 其他分析物 ···························································································· 75 3.6.1 羰基化合物 ··············································································· 75 3.6.2 溶剂 ····························································································· 77 ·v· 目 录 3.6.3 挥发性有机化合物 ·································································· 79 3.6.4 酚类化合物 ··············································································· 79 3.6.5 金属 ····························································································· 80 3.6.6 香精 ····························································································· 81 3.7 关于扩展方法的建议··········································································· 82 3.7.1 烟碱 ····························································································· 84 3.7.2 烟草特有亚硝胺 ······································································ 85 3.7.3 苯并 [a] 芘 ················································································· 86 3.7.4 挥发性有机化合物 ·································································· 87 3.7.5 羰基化合物 ··············································································· 87 3.8 为未来监管 ENDS提供数据所需的研究 ······································· 88 3.9 结论 ········································································································· 89 3.10 建议 ······································································································· 93 3.11 参考文献······························································································· 94 4. 水烟的有害内容物和释放物 ·························································· 107 4.1 引言 ······································································································· 108 4.2 抽吸方式和释放物测试方案 ··························································· 110 4.3 有害物质的含量及释放量 ································································ 112 4.4 测试方法对水烟有害物质释放量的影响 ····································· 116 4.4.1 抽吸模式 ·················································································· 120 4.4.2 热源 ··························································································· 121 4.4.3 烟草温度 ·················································································· 121 4.4.4 水的影响 ·················································································· 122 4.5 水烟设计对水烟烟草制品释放物的影响 ····································· 122 4.5.1 组件和配件 ············································································· 122 4.5.2 实际水烟和研究级水烟 ······················································· 123 4.5.3 水烟软管 ·················································································· 124 4.5.4 水烟托和铝箔 ········································································· 125 ·vi· 烟草制品管制科学基础报告： WHO研究组第六份报告 4.6 结论 ······································································································· 127 4.7 对监管部门的建议 ············································································· 128 4.8 参考文献 ······························································································· 128 5. 针对卷烟的 WHO 烟草实验室网络标准操作规程对水烟的 适用性 ····························································································· 137 5.1 引言 ······································································································· 138 5.2 抽吸方法 ······························································································· 139 5.2.1 热源 ··························································································· 139 5.2.2 水烟头 ······················································································ 140 5.2.3 水烟头覆盖物 ········································································· 140 5.2.4 水 ······························································································· 141 5.2.5 软管 ··························································································· 141 5.2.6 滤嘴 ··························································································· 141 5.3 吸烟机 ··································································································· 142 5.4 水烟烟草取样 ······················································································ 144 5.5 样品制备 ······························································································· 145 5.6 内容物和释放物的测定 ···································································· 147 5.6.1 水烟烟草的内容物 ································································ 147 5.6.2 焦油、烟碱和一氧化碳的释放 ·········································· 149 5.7 讨论 ······································································································· 151 5.8 结论和建议 ·························································································· 154 5.8.1 对监管机构的建议 ································································ 155 5.8.2 对研究人员的建议 ································································ 156 5.9 参考文献 ······························································································· 156 6. 无烟烟草制品的有害内容物和释放物 ··········································· 163 6.1 引言 ······································································································· 164 6.1.1 全球流行情况 ········································································· 166 ·vii· 目 录 6.1.2 无烟烟草制品在制造和物理特性上的多样性 ··············· 166 6.2 产品构成 ······························································································· 167 6.2.1 烟草 ··························································································· 167 6.2.2 添加剂 ······················································································ 168 6.3 无烟烟草制品的释放物 ···································································· 169 6.3.1 烟碱 ··························································································· 169 6.3.2 有害物质和致癌物 ································································ 172 6.3.3 微生物及其组成 ···································································· 178 6.4 降低无烟烟草制品中的有害物质浓度·········································· 179 6.5 结论和建议 ·························································································· 181 6.6 参考文献 ······························································································· 184 7. 卷烟内容物和释放物中烟碱、烟草特有亚硝胺和苯并 [a] 芘的 标准操作规程在无烟烟草制品中的应用 ······································ 197 7.1 引言 ······································································································· 198 7.2 无烟烟草制品中的烟碱、烟草特有亚硝胺和苯并 [a]芘 ········ 198 7.2.1 烟碱 ··························································································· 198 7.2.2 烟草特有亚硝胺 ···································································· 199 7.2.3 苯并 [a] 芘 ··············································································· 200 7.3 WHO标准操作规程对无烟烟草制品分析的适用性评价 ······ 200 7.3.1 分析方法评价 ········································································· 200 7.3.2 烟碱的测定 ············································································· 201 7.3.3 烟草特有亚硝胺的测定 ······················································· 202 7.3.4 苯并 [a] 芘的测定 ································································· 202 7.4 讨论和建议 ·························································································· 204 7.5 参考文献 ······························································································· 206 8. 总体建议 ·························································································· 210

·ix· WHO烟草制品管制研究小组第八次会议 巴西里约热内卢，2015年 12月 9~11日 参加者 D. L. Ashley博士，美国食品药品监督管理局（马里兰州罗克维尔） 烟草制品中心科学办公室主任 O. A. Ayo-Yusuf教授，Sefako Makgatho卫生科学大学（南非比勒陀 利亚）口腔卫生科学院院长 A. R. Boobis教授，英国伦敦帝国学院医学系药理学和治疗中心生化 药物学专业；伦敦帝国学院公共卫生英格兰毒理学课题组组长 Mike Daube教授，科廷大学（澳大利亚西澳大利亚州珀斯）公共卫 生咨询研究所主任，卫生政策学教授 M. V. Djordjevic博士，美国国家癌症研究所（美国马里兰州贝塞斯达） 癌症控制与人口科学部行为研究处烟草控制研究项目主任 /项目 负责人 P. Gupta博士，Healis Sekhsaria 公共卫生研究所（印度孟买）所长 S. K. Hammond博士，加利福尼亚大学伯克利分校（美国加利福尼亚 州伯克利）公共卫生学院环境卫生学教授 D. Hatsukami博士，美国明尼苏达大学（美国明尼苏达州明尼阿波 利斯）精神病学教授 A. Opperhuizen博士，荷兰乌得勒支风险评估和研究办公室主任 G. Zaatari博士，WHO烟草制品管制研究小组主席；贝鲁特美国大 ·x· 烟草制品管制科学基础报告： WHO研究组第六份报告 学（黎巴嫩贝鲁特）病理学与实验医学教授 发言人 Nuan Ping Cheah博士，新加坡卫生科学局应用科学组药物学分部化 妆品和卷烟测试实验室主任 Gregory Connolly博士，美国东北大学（美国马萨诸塞州波士顿）教 授 omas Eissenberg博士，美国弗吉尼亚州联邦大学（美国弗吉尼亚 州里士满）烟草制品研究中心副主任，心理学教授 Esteve Fernández博士，Bellvitge生物医学研究所加泰罗尼亚肿瘤研 究机构烟草控制负责人；巴塞罗那大学（西班牙巴塞罗那）流行 病学与公共卫生专业副教授 Patricia Richter博士，美国疾病控制与预防中心（佐治亚州亚特兰大） 国家环境卫生中心烟草和挥发性组分分会副主任 Alan Shihadeh博士，贝鲁特美国大学（黎巴嫩贝鲁特）建筑与工程 学院机械工程教授 Reinskje Talhout博士，荷兰国家公共卫生与环境研究所（荷兰比特 欧文）卫生防护中心 Georey Ferris Wayne先生，美国加利福尼亚州塞瓦斯托波尔研究所 顾问 Ana Claudia Bastos de Andrade女士，巴西国家卫生监督管理局（巴 西里约热内卢）烟草制品控制司司长 Katja Bromen博士，欧盟健康与消费者理事会（比利时布鲁塞尔） D4单元人类起源物质与烟草控制组政策官员 Denis Chonière先生，加拿大卫生部（加拿大安大略省渥太华）控制 ·xi· WHO烟草制品管制研究小组第八次会议 物质与烟草理事会烟草制品管制办公室主任 Nalan Yazicioğlu女士，土耳其烟草和酒精市场监管局（土耳其安卡拉） 工程师 WHO FCTC 秘书处 Carmen Audera-Lopez博士，世界卫生组织技术官员，瑞士日内瓦 WHO 秘书处（非传染性疾病预防部，瑞士日内瓦） M. Aryee-Quansah女士，无烟草行动组行政助理 A. Peruga博士，无烟草行动组项目理事 G. Vestal女士，无烟草行动组技术官员（法定）

·xiii· 致    谢 世界卫生组织烟草制品管制研究小组（TobReg）对提供本报告 基础背景文件的作者表示感谢。 本报告是在 Vinayak Prasad 博士和 Douglas Bettcher博士的监督 和支持下，由 Sarah Emami女士协调出版。Armando Peruga博士和 Gemma Vestal 女士负责协助组织会议。以下WHO工作人员提供行 政支持：Miriamjoy Aryee-Quansah 女士、Gareth Burns 先生、Luis Madge 先生、Rosane Serrao 女士、Moira Sy 女士、Elizabeth Tecson 女士和 Angeli Vigo女士。 TobReg对世界卫生组织《烟草控制框架公约》（WHO FCTC） 第 9条和第 10条工作组的协调人员表示感谢，他们帮助确保了 WHO和 TobReg能充分响应缔约方会议的要求。他们是：Ana Clau- dia Bastos de Andrade 女 士（ 巴 西）、Katja Bromen 博 士、Denis Chonière先生（加拿大）和 Nalan Yazicioğlu女士（土耳其）。 TobReg谨对主办此次会议的巴西国家卫生监督管理局（ANVI- SA）Ana Claudia Bastos de Andrade女士以及 Adriana Blanco 博士（世 界卫生组织美洲地区办事处烟草控制地区顾问）表示感谢，他们确 保了巴西 TobReg会议的顺利举办。 TobReg感谢世界卫生组织《烟草控制框架公约》秘书处的同 事们协助编写本报告，他们包括：Carmen Audera-Lopez博士、 Guangyuan Liu女士、Tibor Szilagyi博士（技术官员）和 Vera da Cos- tae Silva博士（现任WHO FCTC秘书处负责人）。

·xv· 缩略语表 CDC 美国疾病控制与预防中心 CFP 剑桥滤片 CI 置信区间 CO 一氧化碳 COP 缔约方会议 CORESTA 烟草科学研究合作中心 ENDS 电子烟碱传输系统 FCTC 烟草控制框架公约 FEMA 美国香精和提取物制造商协会 FID 火焰离子化检测器 GC 气相色谱 GRAS 一般认为安全 HPLC 高效液相色谱 IARC 国际癌症研究机构 ISO 国际标准化组织 MS 质谱 NNAL 4-(甲基亚硝基氨基 )-1-(3-吡啶基 )-1-丁醇 NNK 4-(甲基亚硝基氨基 )-1-(3-吡啶基 )-1-丁酮 NNN N′-亚硝基降烟碱 PAH 多环芳烃 ppm 百万分之一 RIVM 荷兰国家公共卫生与环境研究所 SOP 标准操作规程 TobLabNet 烟草实验室网络 TobReg 世界卫生组织烟草制品管制研究小组 TPM 总粒相物 TSNA 烟草特有亚硝胺 VOC 挥发性有机化合物

·1· 1. 前    言 有效的烟草制品管制是综合烟草控制规划中必不可少的组成部 分。烟草制品管制包括对内容物和释放物进行管制，管制的方法包 括强制测试、公布测试结果、适当设定限值、对包装和标识设置限 制条件等。世界卫生组织《烟草控制框架公约》（WHO FCTC）的 第 9、10和 11条以及实施第 9、10条的部分指导原则中涵盖了烟草 制品管制的内容。 为了填补烟草管制空白，世界卫生组织在 2013年正式成立了烟 草制品管制研究小组（TobReg）。它的主要职责是向WHO总干事 提供有关烟草制品管制的循证政策建议。TobReg由产品管制、烟草 依赖治疗、烟草成分和释放物实验室分析等领域的国际科学专家组 成。这些专家来自于WHO六大地区的国家。 作为WHO的正式实体组织，TobReg通过总干事向WHO执行 委员会提交技术报告，提醒成员国注意WHO在烟草制品管制中所 做的工作。技术报告主要是在未发表的背景文件的基础上由 TobReg 讨论得出的。 TobReg第八次会议于 2015年 12月 9~11日在巴西里约热内卢举 行。讨论的内容包括烟草制品的优先管制清单和WHO FCTC第六次 缔约方会议上提出的请求，具体如下： • 编写有关烟草特征的科学证据报告，具体包括细支和超细支 卷烟的设计，滤嘴通风及创新滤嘴设计特点（如胶囊的香味 传递机制），在某种程度上，这些特征将影响 TobReg 在第 ·2· 烟草制品管制科学基础报告： WHO研究组第六份报告 六次缔约方会议后第一次会议上审议通过的WHO FCTC公 众健康目标。 • 对电子烟碱传输系统和电子非烟碱传输系统的管制进行评估 以达到 FCTC第六次缔约方会议的要求，并考察测量这些产 品中内容物和释放物的方法。 • 在两年内评估标准操作规程（SOP）是否适用于卷烟内容物 和释放物中的烟碱、烟草特有亚硝胺（TSNA）和苯并 [a]芘 的检测，以及是否适用于除卷烟外的烟草制品，包括水烟、 无烟烟草的检测。 • 编写有关水烟及无烟烟草中有害内容物和释放物的报告。 在该会议上，还对一篇有关电子烟碱传输系统（ENDS）的气溶 胶的背景文件进行了讨论，这份文件已经单独出版①。TobReg还对 烟草制品中薄荷醇的使用和流行情况进行了讨论，会议之后，针对 决策者和监管者的基于证据支持的结论和建议出版了一份报告②，其 中提到禁止在卷烟中使用薄荷醇（及其类似物、衍生物和前体物）。 TobReg希望本报告和咨询说明中的结论、建议能够有益于各国 实施WHO FCTC产品管制条约。 ① http://www.who.int/tobacco/industry/product_regulation/eletronic-cigarettes-report- cop7- background-papers/en/ ② http://apps.who.int/iris/bitstream/10665/205928/1/9789241510332_eng.pdf?ua=1 ·3· 2. 卷烟特征和设计特色 Reinskje Talhout，荷兰国家公共卫生与环境研究所（荷兰比特欧文）卫 生防护中心 Patricia Richter，美国疾病控制与预防中心（佐治亚州亚特兰大）国家环 境卫生中心实验室科学处 Irina Stepanov，美国明尼苏达大学（明尼苏达州明尼阿波利斯）环境卫 生科学与共济会癌症中心副教授 Christina Watson，美国疾病控制与预防中心（佐治亚州亚特兰大）国家 环境卫生中心实验室科学处 Cliord Watson，美国疾病控制与预防中心（佐治亚州亚特兰大）国家 环境卫生中心实验室科学处 目录 2.1 引言 2.2 影响感观和行为的卷烟特征 2.2.1 概述 2.2.2 影响使用者感观的卷烟特征 2.2.2.1 卷烟纸和滤嘴纸——装饰性元素 2.2.2.2 滤嘴通风 2.2.2.3 物理尺寸（细支和超细支卷烟） 2.2.2.4 香精 2.2.3 影响使用者行为的卷烟特征 2.2.3.1 滤嘴通风 2.2.3.2 物理尺寸 2.2.3.3 香精 ·4· 烟草制品管制科学基础报告： WHO研究组第六份报告 2.3 影响烟气释放物的卷烟特征 2.3.1 烟草 2.3.2 卷烟纸 2.3.3 滤嘴 2.3.3.1 滤嘴通风 2.3.3.2 吸附过滤材料——炭 2.3.4 物理尺寸 2.3.4.1 直径和周长 2.3.4.2 长度 2.3.4.3 填充密度 2.3.4.4 超细支卷烟 2.4 可改变烟气 pH和成瘾性的设计特色和添加剂 2.4.1 概述 2.4.2 氨、糖和再造烟叶 2.4.3 其他成分 2.4.4 烟丝配方和物理特征 2.4.5 测量“烟气 pH” 2.5 可能影响感观和传送的创新 2.5.1 概述 2.5.2 低烟碱卷烟 2.5.3 彩色卷烟纸 2.5.4 特色滤嘴 2.5.5 烟草行业对特殊滤嘴和再造烟叶的研究 2.6 对设计特色的公众健康影响进行科学评价的研究 2.7 结论 2.8 建议 ·5· 2. 卷烟特征和设计特色 2.8.1 政策建议 2.8.2 研究建议 2.9 参考文献 2.1  引    言 本章是根据世界卫生组织《烟草控制框架公约》（WHO FCTC）第六次缔约方会议（俄罗斯莫斯科，2014年 10月 13~18日） 向公约秘书处提出的一项请求编写的。该请求是编写有关烟草特征 的科学证据报告，具体包括细支和超细支卷烟的设计、滤嘴通风及 创新滤嘴设计特点（如胶囊的香味传递机制）。在某种程度上，这些 特征将影响WHO FCTC COP在 2016年 2月会议上审议的第 9条和第 10条：WHO FCTC公众健康目标。关于细支和超细支卷烟的设计特点， 本章包含了卷烟的周长和长度与烟碱传输和暴露的关系。 本报告表明卷烟的特征可以影响使用者的主观感受、使用者行 为和有害成分的传输。卷烟的一般特征包括烟丝、添加剂、质量、密度、 卷烟纸、滤嘴类型、滤嘴通风、卷烟几何形状（圆周、长度）[1]。最 近，市售卷烟有了一些新的设计特征，如滤嘴中的香味胶囊、特殊 滤嘴和彩色卷烟纸。为卷烟设计这些特征主要是为了减少负面影响 （如咽喉刺激），增加积极影响（如改善抽吸和口感），吸引新的 使用者和目标群体，增加使用的便利性，以及降低风险和提高安全性， 从而增加卷烟的吸引力和成瘾性 [2]。其中某些成分也可以增加成瘾 性，例如通过提高烟碱传输等。目前许多声称可以降低有害物质含 量（如更有效的滤嘴或对烟草进行处理）的新产品已经上市或者正 ·6· 烟草制品管制科学基础报告： WHO研究组第六份报告 在由烟草行业进行调查。 本章包含以下主题： • 影响使用者感观和行为的卷烟特征（如吸引力、风险认知、 通风、压降、香味、设计和形状，包括卷烟直径长度比）（2.2 节）； • 影响有害物质释放的卷烟特征（如烟草类型、烟丝、烟草用量、 通风、纸张孔隙度、滤嘴类型）（2.3节）； • 影响烟气 pH和成瘾性的设计特色和添加剂（2.4节）； • 可能影响感观和 /或释放的创新（如香味胶囊、新的滤嘴设计） （2.5节）； • 可以揭示设计特色对公众健康影响的科学评估研究领域（2.6 节）。 文献检索主要在 PubMed数据库 SciFinder搜索工具中进行，其 中 SciFinder搜索工具主要检索Medline和 CAplus数据库中的文献。 出版物及报告中引用的相关文献也在查找范围内。此外，我们使用 互联网来查找提供产品特征和市场信息的网站，并搜索主要烟草制 造商的网站、烟草行业文档库、博客和新闻文章。 2.2  影响感观和行为的卷烟特征 2.2.1  概述 卷烟的特征可以影响烟碱传送 [3]和吸烟者的感官体验，对吸烟 相关行为有重要的影响。对于高度依赖吸烟者，从开始吸烟到成瘾 ·7· 2. 卷烟特征和设计特色 的过程中，这些特征影响了他们对烟草的依赖和吸烟满意度。烟碱 传送和主观感受对吸烟满意度 [4, 5]、心理奖励 [6]及减少渴望 [7]有着 重大的影响。例如使用高通风滤嘴的卷烟给使用者带来了一种“更淡” 的感觉，由于有着更好的适口性、主观感受可以减少健康风险或两 者兼而有之，这种卷烟得到了广泛的接受 [8-10]。 学术界和政府研究人员已经对卷烟的设计对用户感知和吸烟行 为的影响进行了研究，并获得了很多该方面的基础知识 [11]。目前， 行业内部研究的部分结果已经公开，为了达到有效的烟碱传送和特 殊的感观特性，制造商对卷烟的设计进行了改良，建立了品牌及子 品牌形象，提升产品对消费者的吸引力。回顾相关内部行业研究对 于制定有效的卷烟特征政策和法规十分重要 [12]。本章我们对影响使 用者感观体验和行为的卷烟特征进行了总结。 2.2.2  影响使用者感观的卷烟特征 2.2.2.1 卷烟纸和滤嘴纸——装饰性元素 一些研究对卷烟外观与对应的消费者反馈进行了分析，结果表 明某些元素，如滤嘴和卷烟纸的颜色及图案影响了卷烟吸引力和人 们对它危害的认识 [13-16]。Moodie等 [14]的研究表明，粉红色的卷烟 纸可能更具吸引力，并给年轻女性带来了愉快的感觉和更小的危害 感。相反地，深色外观的吸引力较小，给人一种有强烈味道和更大 危害的感觉。然而，宜人的香气会增加深色卷烟的吸引力，减少人 们对其危害的主观认识。Ford等 [16]进行了一项探索性研究：在一组 15岁的受试者中发现当在滤嘴和卷烟纸上有装饰性元素（包括品牌 名称的字体风格）时，能够让受试者对它产生兴趣并感到新颖，传 ·8· 烟草制品管制科学基础报告： WHO研究组第六份报告 达积极的形象并使人受到吸引。这些研究表明，卷烟外观可以被当 作一种促销工具。例如，与带有软木色过滤嘴的卷烟 [17]相比，设计 有白色且通风的滤嘴会增强人们对于产品安全的感知。有关卷烟中卷 烟纸和滤嘴颜色的最新款式将在 2.5.3小节和 2.5.4小节中进行讨论。 2.2.2.2 滤嘴通风 主观感受可以减少危害 滤嘴成分和通风对释放物的影响将在 2.3.3小节进行详细描述。 许多吸烟者不知道低害卷烟中含有通风滤嘴（可以用空气对卷烟烟 气进行稀释）[17, 18]。滤嘴通风不仅改变了使用者对卷烟烟气的感觉， 并且影响了使用者对吸烟危害的主观感受，尤其是低害卷烟中的滤 嘴通风使吸烟者感到烟味比普通卷烟更淡、刺激性更小，这使他们 相信使用这种卷烟焦油和烟碱摄入量更低 [8, 10, 19]。例如，O’Connor 等 [20]研究发现滤嘴通风的程度与感觉烟味轻（P<0.001）和平滑 度（P=0.005）有关。Cummings等 [17]的研究表明，许多 Marlboro Lights吸烟者错误地认为烟味轻和超轻的卷烟比高焦油和全香型卷烟 危害更小。该研究中，Marlboro Lights吸烟者只有 11%的人知道“清 香型”卷烟与全香型卷烟中的焦油及其他成分含量差不多。另有研 究表明 [10]，许多吸烟者认为，一般来说，使用“清香型”卷烟并不 会减少危害，但是由于主观感受不同，他们还是认为“清香型”卷 烟会减少有害物质的暴露。 与其他卷烟相比，在吸低害卷烟时，吸烟者会主观感受到有 害物质的暴露更低，这与卷烟包装纸上的描述性语言及彩色标识 无关 [8-10, 21]。纵向研究表明，去除品牌描述，如“清香”、“柔和” 和“低焦油”等，对吸烟者的主观感受没有产生持续影响，原因是 ·9· 2. 卷烟特征和设计特色 许多吸烟者仍然相信烟味轻的卷烟危害较小 [22, 23]。例如，55%的澳 大利亚吸烟者、43%的加拿大吸烟者和 70%的美国吸烟者仍然相信， 与普通卷烟相比，低害卷烟对健康的危害较小。尽管制造商推出了 新的术语（如“温和”、“良好”）和包装的颜色来表明卷烟的“更淡” 或“更温和”[24-26]，仍有部分吸烟者受主观感受的影响，认为烟味轻 的卷烟比普通卷烟更“温和”[9]。 吸烟时的感知 增加“低传送”卷烟的滤嘴通风、减少化学感应有时会使吸烟 者感到不满，其原因是吸烟者“抽吸时的感觉”改变或者说使吸烟 者感觉从卷烟中吸入足够量的烟气需要付出更大的努力。针对这一 现象，烟草行业进行了大量的研究，研究结果表明，通过增加烟气 中烟碱、挥发性醛、氨及其他成分和添加剂的含量，可以改善滤嘴 通风对吸烟者感觉的影响 [4]。氨和其他添加剂对卷烟特征的影响将 在 2.4.2小节和 2.4.3小节进行详细描述。 2.2.2.3 物理尺寸（细支和超细支卷烟） 卷烟的长度和周长影响卷烟对吸烟者的吸引力，以及对于吸烟 危害健康的主观感知。人们普遍认为细支卷烟可以增加时髦感，对 女性更具有吸引力 [12, 14]；烟草行业进行的相关研究表明这些特征已 经被用于针对女性吸烟者。例如 Philip Morris发现赶时髦的女性吸 烟者将细支、长而轻的卷烟与女性气质和体重控制相联系 [27]。Loril- lard的消费者调查也表明 100 mm细支卷烟的吸烟者认为这种烟的风 格既女性化又优雅，且温和持久 [27]。近期的一项研究显示，吸烟者常 常会认为长度较长的卷烟更有吸引力，并且给人感觉其质量更好 [15]。 此外，Ford等 [16]表明 15岁以上的人认为细支和超细支卷烟的危害 ·10· 烟草制品管制科学基础报告： WHO研究组第六份报告 较小。欧盟委员会烟草制品指令草案提议禁止直径小于 7.5 mm的卷 烟，以减小由于烟草外观使消费者对其危害产生误解 [28]，然而，该 项禁令并未包含在《欧盟烟草制品指令》中 [29]。 2.2.2.4 香精 有香味的卷烟通常会吸引年轻人和青少年，年轻人和青少年也 是它的主要消费群体 [30-32]。一项关于大学生吸烟者的研究发现，吸 烟者对香味卷烟有着更高的积极期望，即使在非吸烟者中也如此 [33]。 例如尝试吸烟者、日常吸烟者及非吸烟者对 Camel Exotics的积极期 望高于 Camel Lights（F(1421) = 38.4，P<0.001），但在单独分析时， 在非吸烟者中仅观察到了较小的影响（F(1249) = 5.4，P<0.05）。此外， 还观察到香味卷烟的消极期望低于非香味卷烟，Camel Lights的消极 期望高于 Camel Exotics（F(1421) = 8.2，P<0.01），并且效果与吸烟 状态无关。Logistic回归结果显示日常吸烟者、易受影响的吸烟者和 尝试吸烟者对香味卷烟有着积极期望，打算试一试这些品牌。例如 受试者中打算尝试 Camel Exotics的人是 Camel Lights的 2.4倍。这些 研究结果与认为香味卷烟是“入门产品”的观点是一致的 [32]。 薄荷醇是卷烟中最常见的香味添加剂，可能让人产生“温和” 的感觉，从而增加吸烟的吸引力 [33]。诸如薄荷醇、留兰香、薄荷、 巧克力、杏、椰子和棉花糖等香精已用于解决女性关于后味和喜欢 香味的问题 [27]。 研究表明，香味卷烟的使用者主要是女性和年轻人，他们了解 吸烟相关的健康风险并认为这些卷烟的危害比其他卷烟小 [30, 34, 35]。 WHO FCTC建议国家禁止或限制添加剂的使用，因为它们会增 加卷烟的吸引力 [36]。一些国家已经立法对添加剂的使用进行管制以 减少相关产品的吸引力。 ·11· 2. 卷烟特征和设计特色 巴西（RDC ANVISA第 14号）和加拿大（第 C-32号法案）已 经禁止大多数香精的使用，而其他国家限制了产品或包装中香精的 使用，使其不会有强烈的非烟草味道（如水果或甜食）。美国食品 药品监督管理局已禁止了添加剂、人造香精和天然香精（烟草和薄 荷醇除外）、草药、影响卷烟味道特征的香精的使用 [37]。《欧盟烟 草制品指令》还禁止除卷烟烟草及手卷烟烟草 [38]以外的香精的使用， 这些特征气味被定义为：由一种添加剂或多种添加剂共同产生的明显 的除烟草以外的气味，包括水果、香精、药草、醇、糖果、薄荷醇或 香草等，这些气味可以在消费烟草制品之前和过程中明显感受到。 2.2.3  影响使用者行为的卷烟特征 2.2.3.1 滤嘴通风 大多数吸烟者通过化学感应来优化烟碱的摄入量，以达到舒服 的感觉并避免与烟碱戒断相关的厌恶感觉。因此滤嘴通风与随后的 空气稀释将导致补偿性吸烟，如吸更多的卷烟、吸入更深和堵塞滤 嘴通风口防止烟气稀释等 [39-41]。吸烟者也会用手指或嘴堵塞滤嘴通风口， 尽管许多淡烟和超淡烟的吸烟者并没意识到自己这样做了 [18, 42]。在刚 从普通卷烟换到抽吸低害卷烟的吸烟者中，大多数都存在这种补偿 性吸烟的行为 [41]。与滤嘴通风相反，减少卷烟的烟碱含量，如低烟 碱卷烟，不会导致这种补偿性吸烟 [43]。 研究证实，烟气摄入量与焦油和烟碱传送的比例之间呈非线性 关系，更大、更强烈的吸烟行为会减少卷烟滤嘴的滞留物并减少烟 气稀释而在更大程度上改变烟气成分的浓度 [44]。吸烟者认为他们所 使用的卷烟产品有害物质传送较弱，实际上可以通过改变吸烟行为 ·12· 烟草制品管制科学基础报告： WHO研究组第六份报告 增加他们的暴露，如堵塞滤嘴通风口或吸入更大口，这对于高度通 风卷烟品牌的吸烟者尤其重要，这种“品牌弹性”允许吸烟者通过 调整其吸烟行为来有效调节烟碱的传递。这也是测量品牌具体烟碱 和焦油释放量中存在的一个主要问题。不同的卷烟品牌有不同的弹 性，且具有较大弹性的卷烟品牌有更大的市场份额 [44]。 行业研究人 员早就发现吸烟者在改抽淡味或超淡味卷烟时，会通过改变吸烟行 为来维持相当恒定的每日剂量的烟碱摄入 [17]。此外，烟草行业文件 显示，与其他设计特色（如多孔纸）相比，改变滤嘴通风是低害卷 烟中常用的一种方式 [10]，这些特色趋向于鼓励吸烟者吸更多的烟并 减少低害卷烟的使用 [39,45-47]。例如 Strasser等 [46]估算得到堵塞滤嘴 通风的吸烟者的烟气成分暴露增加了 30%。Hammond等 [44]研究显 示，转为使用低害卷烟的吸烟者每天的吸烟量增加了 40%（P=0.007）， 唾液中可替宁水平没有显著变化。这种补偿性吸烟的行为很稳定， 在 5天内没有观察到明显的减少。自我报告的“淡味”卷烟吸烟者 认为自己的嗜烟程度较低，戒烟可能性比日常吸烟者更高，有强烈 的戒烟意愿，但是缺乏对自己戒烟能力的信心。许多研究 [41,48-51]通 过检测暴露生物标志物，证明了低害卷烟吸烟者的烟碱和其他烟气 成分的暴露并没有减少。总之，这些发现为吸烟行为可以补偿滤嘴 通风提供了强有力的人群实验证据。 压降 “压降”是抽吸持续时间和抽吸容量的主要决定因素 [47,52-56]。 化学感应决定了吸烟者对达到满意烟量的感知，在口腔和上呼吸道 感觉吸烟不足的情况下，促使吸烟者继续增加吸烟量直到他们感到 吸烟量足够 [4]。 ·13· 2. 卷烟特征和设计特色 含碳滤嘴 卷烟滤嘴中碳的存在可能会影响某些对吸烟者主观感受有影 响的烟气成分的含量，从而导致吸烟强度的变化。Rees等 [57]将 Marlboro Lights卷烟替换成含碳滤嘴的 Marlboro Ultra Smooth和非 碳Marlboro Ultra Lights卷烟各 48 h，发现含碳滤嘴的吸烟量明显多 于Marlboro Lights卷烟（两组抽吸容量相差 2.4~13.6 mL；P=0.006） 和非碳Marlboro Ultra Lights卷烟（两组抽吸容量相差 2.4~3.6 mL； P=0.007）。 2.2.3.2 物理尺寸 有关吸烟者吸入全长或部分长度卷烟的研究表明，卷烟长短可 能会影响吸烟行为，如抽吸持续时间和抽吸容量 [52-55]。一项研究报道， 吸入整支卷烟与吸入半支、四分之一支或八分之一支卷烟相比，自 我报告的吸烟“满意度”更高。在同一项研究中，吸烟者在抽吸正 常长度的高烟碱（2.0 mg）或低烟碱（0.2 mg）的研究型卷烟时，往 往比抽吸四分之一长度相同卷烟时的抽烟指数更少，但抽吸口数更 多 [56]。美国国家健康和营养调查研究对常规大小、特大号、长、超 长等卷烟吸烟者的血清可替宁和尿总 4-(甲基亚硝基氨基 )-1-(3-吡 啶基 )-1-丁醇（NNAL）的浓度进行检测。结果显示，与常规大小、 特大号卷烟吸烟者相比，长卷烟或超长卷烟吸烟者的吸烟强度及上 瘾程度更高（如吸第一支烟的时间，每天吸烟的数量），烟草生物 标志物水平也更高（血清可替宁的几何平均数分别为 263.15 ng/mL 与 173.13 ng/mL或 213.79 ng/mL；尿NNAL为 0.48 ng/mL肌酐与 0.34 ng/mL或 0.33 ng/mL）[52]。 ·14· 烟草制品管制科学基础报告： WHO研究组第六份报告 2.2.3.3 香精 卷烟中的香精不仅对一些群体（如年轻人、妇女、特定种族群体） 和非吸烟者具有潜在的市场吸引力，而且可能掩盖烟气的刺激性气 味，使吸入更容易。 一项对 20名抽 Camel Light和 Camel Exotic Blend卷烟（类似 焦油、烟碱和滤嘴通风）的大学生的初步研究，主要研究了吸烟 行为和卷烟等级差异 [58]，结果显示，与吸 Camel Light卷烟相比， Camel Exotic Blend卷烟吸烟者每口的吸烟量较小（42 mL vs. 48 mL， P<0.001），但是两组的吸烟总量没有显著差异（613.9 mL vs. 630.7 mL，P=0.79），此外，两组的一氧化碳含量无明显差异（CO：6.2 ppm③ vs. 6.2 ppm，P=0.90）。当受试者对每一支卷烟的强度、刺激 性和口味等特征进行评价时，他们认为与其他卷烟品牌相比，Camel Exotic Blend卷烟是最特别的，但口味等级没有明显差异。这些结果 表明，在卷烟中添加香精不会对吸烟者吸烟产生显著影响。 尽管许多研究的结果是不确定的或相互矛盾的，薄荷醇仍被认 为是一种可以改变吸烟行为的香精 [33, 59]。一些研究表明，卷烟中添 加薄荷醇与每日吸烟量或每口吸烟强度明显相关 [60, 61]，而另一些研 究则发现含薄荷醇卷烟吸烟者的抽吸频率 [62, 63]和抽吸容量 [63]与非薄 荷醇卷烟吸烟者相似。Strasser等 [64]发现薄荷醇对吸烟行为、暴露 生物标志物和主观评分的影响较小；然而，含薄荷醇卷烟吸烟者比 非薄荷醇卷烟吸烟者每天抽第一支烟的时间早，这意味着使用薄荷 卷烟的吸烟者对烟碱有更大的依赖性 [61]。 研究表明，含薄荷醇卷烟吸烟者尝试戒烟更频繁，但戒烟成功 ③ ppm, parts per million, 10–6。 ·15· 2. 卷烟特征和设计特色 率低，这表明抽含薄荷醇卷烟比非薄荷醇卷烟更容易上瘾 [65, 66]。另 有研究表明，含薄荷醇卷烟在年轻人中被不成比例地使用，这可能 与它们的口味、感观特性和更容易吸入有关 [65]。尽管关于薄荷醇卷 烟在初始吸烟中作用的研究较少 [67]，研究表明在青少年中吸薄荷醇 卷烟比非薄荷醇卷烟数量多，这说明薄荷醇卷烟在初始吸烟中是优 选的 [68]。 2.3  影响烟气释放物的卷烟特征 制造商可以通过多种方法来改变烟草烟气的组分 [69]。世界卫生 组织最近组织的一项技术报告 [70]中提到了传统的燃烧烟草的卷烟， 以及新型产品和产品特征（低引燃卷烟，潜在减少暴露的卷烟产品 和低烟碱的烟草）。要确定每种卷烟特征对吸烟者的不利健康影响 的程度是很困难的，因此一般的研究方法主要集中在降低有害物质 的含量（每支或每“根”卷烟或每毫克烟碱）。基于有害物质的毒 性和降低其浓度的可行性，世界卫生组织建议降低卷烟烟气中的 9 种物质含量——N′-亚硝基降烟碱（NNN）、4-(甲基亚硝基氨基 )-1-(3- 吡啶基 )-1-丁酮（NNK）、乙醛、丙烯醛、苯、苯并 [a]芘、1,3-丁 二烯、一氧化碳和甲醛 [71]。 2.3.1  烟草 烟草混合物是对烟气排放中各种化学物质的传递影响最大的卷 烟成分 [72]。烟草的每种特性都会影响它的膨松度（形成具有一定水 分含量的卷烟条的能力）、燃烧速率、焦油和烟碱的传送、烟气中 ·16· 烟草制品管制科学基础报告： WHO研究组第六份报告 化学物质的含量、口味和香气以及阻燃率 [73-79]。金黄烟草，又称烤 烟或弗吉尼亚烟草，与其他品种相比，它具有含氮量低（即烟碱含 量低）和含糖量高的特点。在周长一定时，弗吉尼亚混合烟草卷烟 的产烟量比美式混合卷烟多 [80]。含烤烟叶的卷烟比用白肋烟制成的 卷烟烟气重，因此在卷烟的长度一定时，含烤烟烟叶的卷烟可以吸 更多次 [81]。随着烤烟烟叶在混合物中含量的增加，焦油和 CO的产 量也随之增加 [82]；金黄烟草烟气中甲醛的含量比白肋烟中多 [83]。在 大多数卷烟中，NNN的浓度比 NNK的浓度高；但是在金黄烟草中 NNK浓度高于 NNN[83]。 白肋烟和马里兰烟通常是风干的，烟碱含量较高，而糖的含量较 低。白肋烟中硝酸盐和 TSNA的浓度明显高于其他类型烟草 [84]。香料 烟是通过晾晒制成的，由于其芳香性质，通常被划分到混合品种中 [81]， 它的苯酚含量比烤烟、白肋烟和马里兰烟更高 [85]。与香料烟、烤烟相比， 马里兰烟的焦油、烟碱、苯酚和苯并 [a]芘的含量更低 [85]。 为了提高膨松度，对烟草进行膨胀、膨化和冷冻干燥等处理 [86]。 主要的处理物质是各种挥发性的物质，这些物质可以在处理烟草后 迅速挥发除去，从而使烟草细胞结构得到膨胀 [79]。这些处理后的烟 草，也就是膨胀烟丝，可以用于填充卷烟，从而减少卷烟中未处理 烟丝的使用量。但是，膨胀烟丝的使用也会改变卷烟烟气的释放。 例如，与不含膨胀烟丝的卷烟相比，含有膨胀烟丝的卷烟烟气中的 烟碱含量较低 [86]。随着卷烟中膨胀烟丝使用量的增加，一氧化碳与 二氧化碳的比值以及气相醛（乙醛、丙烯醛）增加，颗粒相组分减 少 [69, 82]。含有膨胀茎的卷烟烟气中一氧化碳、氮氧化物、甲醛、焦油、 苯并 [a]蒽和苯并 [a]芘的含量比膨化烟草、膨胀烟草或冻干烟草制 成的卷烟烟气高 [85]。 ·17· 2. 卷烟特征和设计特色 再造烟叶由烟叶副产品制成，这些烟叶副产品包括烟末（“细 粉”）、烟梗和茎等，制造过程为首先进行提取分离，然后再用黏合剂、 纤维将其制成匀浆，形成结构，再加入化学物质如保润剂和香精， 最后干燥成不同密度 [81, 87, 88]。再造烟叶成本低于烟叶，并且具有更 强的填充能力，用它填充的卷烟烟料填充密度较小，这些卷烟的燃 烧速率更快，每支卷烟能吸的口数较少，这些因素使得烟气中焦油 和烟碱的传送减少 [87, 89]。由再造烟叶制成的卷烟的烟气化学成分取 决于再造烟丝是由茎还是由茎和其他烟草衍生材料混合制成的。与 用茎和其他烟草材料混合制成的再造烟叶相比，单纯由茎制得的再 造烟叶的烟气中氮氧化物、乙醛和多环芳烃的含量更高。但是，未 使用再造烟叶的卷烟烟气中焦油、烟碱、CO、氰化氢和多环芳烃的 含量比再造烟叶（由茎或茎和其他烟草材料制得）制得的卷烟要低 [86]。 2.3.2  卷烟纸 卷烟纸不仅可以影响卷烟的无燃烧或静态燃烧率（即抽吸中所 消耗的卷烟量）和阴燃率，而且对吸烟机测量的卷烟能吸的口数及 烟气产量有很大的影响 [79, 81]。 卷烟纸中影响烟气释放量和成分的可控因素包括：纤维组成， 填充物类型、等级和分布，厚度和体积密度（标准纸或用于减少引 燃倾向的较厚条带，“防火”卷烟），孔隙度（如下所述）以及化 学物质或添加剂的类型及含量 [90]。 卷烟包装纸可以通过以下几种方式来影响烟气组成：直接将包 装纸或燃烧成分引入主流烟气；烟气成分通过包装纸扩散；空气通 过包装纸进行扩散；改变燃烧锥和周围气流的速度、体积以及分布； 改变每口吸烟的燃烧量 [69]。 ·18· 烟草制品管制科学基础报告： WHO研究组第六份报告 除了滤嘴通风以外，降低烟气产量的最常见手段是改变卷烟 纸的孔隙率 [91]。孔隙率是纸张在压差下对氧气和烟气的渗透率， 它会影响燃烧速度、抽烟的口数和每口吸烟的燃烧量。纸张的孔隙 率是由纤维素纤维和碳酸钙的黏合结构产生的开口（孔隙）的大小 （孔隙容积）决定的，它会影响口感、烟气传送和稀释度 [80, 90, 92]。 烟草科学研究合作中心（CORESTA）规定美国卷烟的孔隙率通常在 30~50个单位范围内 [79]。 卷烟纸的孔隙率会影响卷烟的燃烧温度，随着孔隙率的增加， 燃烧温度降低 [93]，并且因为静态燃烧速率增加，卷烟消耗速度更快。 在吸烟机吸烟条件下，得到的结果是吸烟口与口之间消耗的烟草更多， 吸入的烟气量更少，且烟碱、焦油和 CO的产量更低 [82, 91, 94]。极易挥 发烟气成分（如 CO）很容易通过多孔包装纸扩散出去，与低挥发性 成分相比，它们可以在较低浓度时挥发 [94]。此外，随着纸张孔隙率 的增加，苯并 [a]芘的输送量减少，其原因是在抽吸过程中消耗的烟 草量较少，而在抽吸之间的间歇中消耗的烟草量较多 [81]。 2.3.3  滤嘴 最常用的卷烟滤嘴是由醋酸纤维素、纸或两者混合制成 [81]，大 约 90%的滤嘴使用了卷曲醋酸纤维素纤维（“丝束”）。 卷烟滤嘴有助于控制压降，吸收烟气以及去除烟气中的微粒物 质。它的过滤机制有以下三种：对颗粒物进行机械捕获，冷凝然后吸 附或以气相的形式在颗粒与滤嘴之间传递。醋酸纤维滤嘴对烟碱有负 选择性，滤过后卷烟的烟碱平均粒径比未过滤的卷烟小 [95, 96]。因此， 含有醋酸纤维滤嘴卷烟的主流烟气中烟碱量可能高于未经过滤的卷 烟烟气中的含量。颗粒较小可能意味着在吸入颗粒中有更大比例进 ·19· 2. 卷烟特征和设计特色 一步传播至肺部 [83]。 纤维滤嘴的使用可以使烟气中半挥发性和非挥发性物质的含量显 著降低，气相化合物的含量轻微降低，但不会降低总的气体含量 [97]。 对吸烟机抽吸卷烟的研究表明，醋酸纤维素滤嘴能去除的成分包 括水（60%~75%）、甲酚（70%~75%）、颗粒物（35%~40%）、 挥发性 N- 亚硝胺（≤75%）、丙烯醛（减少到“有限程度”）、 苯酚（70%~80%）[85, 91, 98]。 2.3.3.1 滤嘴通风 滤嘴通风指的是空气通过不与烟丝重叠的接装纸再进入卷烟 [99]， 它是通过一个多孔滤棒成型体和穿孔 /多孔接装纸来实现的，通风 或稀释的程度取决于滤棒成型体的孔隙率、接装纸的穿孔 /孔隙率 和穿孔的位置 [81]。卷烟的通风率范围为 10%（一些全香卷烟）~80%（低 传送品牌）[100]。但是当吸烟者吸烟时，他们的嘴或手常会有意或无 意地堵住通风口，这使得通风滤嘴的设计无法发挥作用 [10]。这里所 叙述的有关滤嘴通风这个设计特征的信息均是理论上，信息源自于 吸烟机吸未遮蔽通风口卷烟的相关研究。当吸烟机在高强度吸烟（更 大的抽烟体积、通风口被阻塞）下吸高度通风卷烟时，烟气的排放 水平可能等于或超过国际标准化组织（ISO）中全香卷烟吸烟机在吸 较低通风卷烟且通风口未被堵塞时的排放量 [101]。 滤嘴通风使得烟草燃烧更充分，并且滤嘴中的醋酸纤维素可以 更好地保留颗粒物 [85,86]。颗粒物的传送和蒸汽 /气相传送都会减少， 它们通常与通风程度成正比 [81]。然而，通风的效果并非完全是由于 烟气被稀释了，因为某些化合物的排放量出现了增加或减少，而另 外一些物质，包括总烟碱的排放量保持相对不变 [34]。 ·20· 烟草制品管制科学基础报告： WHO研究组第六份报告 2.3.3.2 吸附过滤材料——炭 卷烟滤嘴中可包含过滤助剂，如炭和其他固体或液体添加剂， 主要用于选择性过滤排放物 [81]。炭颗粒、硅胶和氧化铝是滤嘴中常 使用的固体吸附材料 [95]。 炭可以有效地吸附沸点介于 0~100℃的化学物质（如乙醛、丙 烯醛和氰化氢），并能除去沸点高达 150℃的一些化学物质 [98]。根 据吸烟机的条件，含碳（炭）滤嘴可以显著降低烟气中的半挥发性 和气相化合物的含量，并能降低非挥发性化合物的水平 [97, 102]。通过 炭的过滤，气相中分子量较低的一些化合物（例如苯酚、甲酚、对 苯二酚）的含量水平降低的比大分子量和低挥发性化合物多（例如 苯并 [a]芘、TSNA）[103]。但尽管据说涂有金属氧化物混合物的炭能 有效去除酸性气体 [81]，含炭滤嘴通常不会降低烟气中的低分子量气 体的含量 [97]。 去除效率取决于炭的含量、吸烟机吸烟的条件（吸烟强度）和 含炭滤嘴的使用年限 [97, 103]。例如，标准含炭滤嘴的氰化氢保留量随 卷烟的使用时间增长从 0周的约 38%降至 8周的约 25%[97]。吸烟机 在深度抽吸含滤嘴卷烟（约 45 mg炭）时，焦油、烟碱和 CO的释 放量以及吸烟机在较低强度的 ISO吸烟条件下测得的挥发性成分的 减少量与醋酸纤维素过滤卷烟相比没有显著降低，其原因是炭的含 量不足。使用含炭量更高的滤嘴（120 mg或 180 mg）后，无论是在高 强度吸烟还是低强度吸烟的条件下，这些物质的含量均显著减少 [103]。 与天然炭孔隙结构不同的合成高活性炭目前已经应用于实验卷 烟的滤嘴中，它的使用方式为单独使用或与处理过的烟草和替代滤嘴 通风组合在一起使用。卷烟周长的范围为 17~24.6 mm[104]，其中细支卷 烟的滤嘴中含炭量较低（17 mm卷烟，滤嘴长度为 27 mm或 33 mm， ·21· 2. 卷烟特征和设计特色 含炭量为 20.4 mg或 30.6 mg，对照组为 24.6 mm卷烟，滤嘴长度为 27 mm、33 mm或 37 mm，含炭量为 48 mg、72 mg或 88 mg）。当含 炭量增加时，较大卷烟产生的焦油量降低，但 17 mm细支卷烟产生 的焦油量反而增加。随着含炭量的增加，烟叶中许多挥发性组分的 产率显著降低，特别是异戊二烯、乙醛和丙酮，另外吡啶、甲醛和 苯乙烯的减少量较小。当含炭量增加时，17 mm卷烟中的氰化氢和1,3- 丁二烯的产率没有显著变化。由于细支卷烟的燃烧速度快，含炭量 较低，因此使用含炭滤嘴的细支卷烟的挥发性烟气成分的释放量高 于较粗的卷烟。挥发性化学物质的减少与炭的含量水平一致，该研 究的作者认为原因是高活性炭有效地降低了卷烟滤嘴中一些有害物 质的产量 [104]。 2.3.4  物理尺寸 2.3.4.1 直径和周长 传统卷烟的直径通常为 7.5~8 mm，细支卷烟的直径约为 5 mm 或 6 mm[83]。烟草的消耗量取决于卷烟的周长，焦油和 CO的产量也 随着卷烟周长的增加而增加 [105]。有醋酸纤维素滤嘴且周长较小的卷 烟的烟气排放量相应减少 [83]。 2.3.4.2 长度 卷烟长度一般可分为四类：“常规”，68~70 mm，无滤嘴；“特 大号”，79~88 mm，有滤嘴；“长”，94~101 mm，有滤嘴；“超长”， 110~121 mm，有滤嘴 [83]。研究表明，当在保持填充密度不变的条件 下减小卷烟周长时，可用于燃烧的烟草量减少，燃烧过程中相对的 氧气使用量则会增多 [85,86]。此外，当卷烟的周长减少时，可供使用 ·22· 烟草制品管制科学基础报告： WHO研究组第六份报告 的烟草量减少，烟气排放量也相应减少 [106]。 当烟气通过未燃烧的卷烟被吸入时，在卷烟条的过滤下，一些 化学物质会被滤过 [98]。此外，烟气中的大多数成分都是在燃烧产物 从卷烟燃烧区移动到较低温度和低氧气区的过程中产生的，特别是 半挥发性化合物。例如，多环芳烃主要产生于点燃卷烟的较低温度 区域。在烟气向卷烟的嘴端移动的过程中，烟气会被烟丝冷凝和过 滤 [107]，随着卷烟长度的减少，卷烟条对烟碱的过滤量也会相应减少， 但是大家普遍认为卷烟条对烟气凝结物的过滤量与卷烟条的长度无 关 [108]。 2.3.4.3 填充密度 卷烟长度对卷烟组分的中介作用取决于烟草的填充密度 [69]。增 加填充密度会使吸烟过程中燃烧更多的烟草，从而使得主流烟气中 化学物质的排放量增多，然而，如上文所述，烟气中的一些成分也 会经卷烟条滤过。一项研究用吸烟机抽吸长度一定但填充密度不等 的卷烟，结果显示，与低填充密度的卷烟相比，填充密度高的卷烟 的烟碱和烟气冷凝物的产量都较低 [108]。 2.3.4.4 超细支卷烟 当卷烟的周长减小时，可以消耗的烟草量随之较少，相应的烟 气释放量也会减少 [106]，如周长小于常规卷烟 24.8~25.5 mm的卷烟（例 如 ≤23 mm）[85]。研究显示在机械吸烟条件下， 减少周长会引起总 输送量和每次吸烟输送量的减少 [79]。 在填充密度不变的条件下，减少卷烟周长将会使可用于燃烧的 烟草总量减少，并增大燃烧期间消耗氧气的相对量。据报道，这会 引起烟气中某些物质的释放量减少，具体包括焦油、烟碱、CO和一 ·23· 2. 卷烟特征和设计特色 些挥发性烟气成分。例如，当卷烟周长从 26 mm减少到 21 mm时， 每口烟中的 CO量减少约 20%，苯并 [a]芘减少约 40%。但是，在相同 的吸烟条件下，随着卷烟周长的减小，主流烟气中的氰化氢水平变化 不明显。另有研究表明，随着卷烟周长从 26 mm变为 23 mm，吸烟机 产生的烟气中烟碱的排放量从 1.56 mg减少到了 1.21 mg[85, 86, 109]。 最近一项研究对加拿大销售的六种超细支烤烟（直径 5.3~5.4 mm；周长 16.7~17 mm；长度 83~99 mm；烟草质量 296~371 mg） 的释放物成分进行了分析，结果显示除甲醛、氨和酚外，超细支卷 烟释放物中有害物质的含量均比常规尺寸的卷烟低，其原因可能是 烟草含量较低和抽烟次数较少。超细支卷烟甲醛释放量增加的原因 是周长与横截面面积的比值增加，使得在吸烟过程中有更多的烟草 与周围空气接触发生氧化反应。减少卷烟周长会增加燃烧温度，这 会使得酚类的释放量增多 [109]。此外，减少卷烟周长也增加了流速， 这将会减少烟气从点燃区域到烟嘴端的时间，从而影响卷烟条和滤 嘴的过滤效果 [110]。 使卷烟条过滤和滤嘴保留减少的因素可能会引起烟气释放量增 加。超细支卷烟的烟气流速是标准圆周卷烟的两倍以上 [110]，随着烟 气流速的增加，颗粒截留减少，气相化学物质在卷烟纸中扩散的时 间也减少。烟气流速会负向影响卷烟滤嘴对颗粒物的保留和对蒸气 的吸附 [110, 111]。它对气相化学物质吸附的影响大小取决于该化学物质 的量和性质（分子量和反应性）以及与吸附材料的接触时间 [110]。例 如，在烟草质量保持不变的条件下，氰化物的过滤保留随着卷烟周 长的减小而急剧减小，表明随着卷烟周长的减小，空气流速相应增加， 进而影响了化学物质如氰化氢的形成 [98, 112]。用吸烟机吸有含炭滤嘴 （每个滤嘴15~90 mg）但没有通风口的实验性混合烟草超细支卷烟时， ·24· 烟草制品管制科学基础报告： WHO研究组第六份报告 若要吸附大约 50%的烟气组分，吸烟强度为加拿大深度抽吸时需要 的炭的用量为 ISO条件时的两倍多 [110]。 跟烟气输送有关的卷烟设计特征具有复杂性和相关性，故很难 提出具体的设计标准。要明确改变卷烟设计特征所导致的结果，还 需要有更多的信息来支持。此外，由于对个别卷烟组分的变化知之 甚少，因此难以估计它们之间的相互作用 [79]。综上所述，将研究重 点放在影响吸烟行为（如每口吸入量）的卷烟设计特征和产品特性 上可能是一个合适的方法。虽然人们普遍认识到的一些众所周知的 卷烟设计特征如滤嘴通风可以导致补偿性吸烟，但是其他一些特征， 如卷烟纸的孔隙率和烟支的性能也会影响烟气稀释和传送，在烟气 量固定的条件下，会导致吸烟者吸入更多的烟碱和其他烟气成分。 然而，烟草制造商可以调整其他设计特征来补偿释放量的变化，例 如更换符合防火标准的卷烟纸同时可以保持焦油和烟碱的传送水平 不变 [113,114]。因此，旨在降低释放量的产品标准应以释放结果为基准， 而不应基于预期可以降低释放的产品设计改变。 2.4  可改变烟气 pH和成瘾性的设计特色和添加剂 2.4.1  概述 烟碱是烟草中主要的致瘾性物质，它决定了吸烟者的“满意度” 和吸烟的“生理”劲头 [72, 87]。用多种方式可以增强烟碱的成瘾性， 例如增加烟气中总烟碱的含量，增加吸入量和控制吸入的“温和度”。 在烟叶中，烟碱主要以质子化盐的形式存在，较高的 pH可以增加 ·25· 2. 卷烟特征和设计特色 烟碱的去质子化 [115]。非质子化（挥发性）或游离态形式存在的烟碱 比质子化（非挥发性）形式的烟碱 [116]不仅更具有“生理效应”，且 可以更迅速地吸入，其机制主要有以下两种：因为它存在于烟气的 挥发相中，所以不必从烟气中扩散出去；非质子化形式的烟碱具有 更强的亲脂性，可以迅速扩散穿过细胞膜进入血液 [117, 118]。 烟碱的非质子化（不是可传送烟碱的总量）主要受卷烟烟气酸 碱度的影响。当吸烟者吸入含有游离烟碱的烟气时，与吸入柠檬酸 盐化的烟碱相比，有更大的电生理和主观反应 [116]。工业文献表明， 非质子化烟碱的存在可以确保卷烟烟气有良好的感官效应（称为“影 响”）[119-122]。然而，关于 pH对非质子化烟碱的影响也存在着一些 争议，目前已经有相关的文章发表，且有学者正在尝试对它进行实 证研究。Calicutt等 [123]对实验卷烟进行了分析，结果发现氨含量有 差异，而烟碱的含量没有显著差异。改变卷烟的氨含量只会影响游 离烟碱，而不会影响传送的总烟碱量。此外，由于人体能有效吸收 吸烟气中的大部分烟碱，所以烟碱的吸收量与烟碱吸收率几乎无关。 Van Amsterdam等 [124]让受试者吸含氨量不同的实验卷烟（0.89 mg/g 和 3.43 mg/g），然后采集受试者的静脉血进行分析，发现烟碱暴露 无明显差异，但是由于第一个样品是在吸烟后 2.5 min时采集的，故 无法反映游离烟碱的吸收情况。 2.4.2  氨、糖和再造烟叶 氨，又称 “改良剂”、“增效剂”和“满意促进剂”[125]。它是 一种活性物质，将其添加到烟草混合物中会引起复杂的变化 [126]。向 烟草中添加氨和氨前体化合物如磷酸二铵可增加颗粒物和蒸气中非 质子化烟碱的含量 [127]。氨或磷酸二铵常用来生产重组片，因为它们 ·26· 烟草制品管制科学基础报告： WHO研究组第六份报告 能与果胶反应，与烟碱形成稳定的络合物。吸烟过程中产生的高温 会使该络合物发生分解，从而增加了烟碱向烟气中的转移，也就是“烟 碱转移率”[128]。烟碱的水解依赖于温度，因此增加烟碱释放时的温 度可以增加非质子烟碱的水平 [129, 130]。氨可以刺激味觉感受器、嗅觉 末梢和三叉神经，产生刺激性感觉 [131]，但是它可以与烟气中存在的 酸迅速反应，从而减小它的刺激性作用。酸与烟碱反应形成盐后， 在热解过程中会释放更多的游离烟碱 [132]。工业文献中对烟草烟气中 的总碱性组分（吡嗪类、吡啶类和生物碱类）和总酸性组分（有机酸、 苯基酸、酚酸类和脂肪酸）进行了总结，发现其中大部分成分是碱 性的。在再造烟叶的制造过程中，磷酸二铵可以与还原糖反应生成 Maillard反应产物脱氧果糖 [133]，这些产物热解后可以产生几种吡啶 和吡嗪，从而对烟气的口感和碱度产生影响 [134, 135]。在烟草烟气中已 经发现了数百种碱性物质，其中大部分是与烟味有关的氮杂环，它 们可能是在氨和糖的反应中生成的，它们的存在也可能会影响烟气 pH[136, 137]。白肋烟中存在的高水平的氨基酸也能与糖反应生成类似的 弱碱性化合物 [138, 139]。糖的主要热解产物是乙醛，它能与烟碱发生协 同作用增加卷烟的成瘾性 [128, 140]。 2.4.3  其他成分 氨不是烟气中唯一能够使烟碱脱质子化和与糖形成Maillard反 应产物的物质：烟气中存在其他几种物质也有利于非质子化烟碱的 形成。行业文献表明，尿素 -脲酶系统可以通过热解将尿素分解成 氨提高烟气 pH[141, 142]。此外，无机阳离子如钾和钙也能提高烟气的 pH。由于磷酸二铵在一些国家已经禁止使用，因此，其他一些物质 如碳酸钙被用来增强烟碱传递 [142]。由于烟气中碱性金属如钾和钙的 ·27· 2. 卷烟特征和设计特色 含量可以通过使用肥料固化或者直接添加的方式来调节，因此在常 规分析中很难区分它们是天然的还是后期添加的。此外，还可以通 过在卷烟滤嘴中添加钙和碳酸钠的方式来提高烟气 pH，采用这种方 式不需要向烟草填料中添加碱性物质 [143]。这种碱性滤嘴有利于烟碱 的释放，可以将挥发性烟碱释放到烟气中 [144]。当吸烟者感觉到烟气 较重时，他们会减少吸烟的深度。使用乙酰丙酸和甘草等添加剂可 以使烟气更平滑，从而更具有吸引力 [145]。尽管可可和薄荷醇等添加 剂的使用不会增加烟气 pH，但是会使支气管扩张，从而增加烟气吸 入的深度和体积，促进总烟碱吸收 [146]。此外，可可的燃烧产物可能 具有单胺氧化酶抑制特性，有抗抑郁作用，可以在烟碱存在或不存 在的条件下促成吸烟成瘾 [142]。 2.4.4  烟丝配方和物理特征 在没有化学添加剂的情况下，烟丝配方的差异，包括膨化烟草、 烟叶在茎上的位置都可以改变烟气的 pH和化学性质 [147,148]。当 pH 为弱酸性（6.5~7）时，吸烟者会吸收约 7%的烟碱；当 pH＜ 6.6时， 吸收的烟碱更少 [131]。烤烟和美式混合卷烟的酸性较弱，pH为 5.7~6.2。 通过空气晾干烟叶制得的卷烟的烟气 pH为 6.5~7.8[86]，而白肋烟的 烟气 pH>7.5。制造白肋烟所用烟叶在烟株中的位置对烟气中的总烟 碱量及 pH有很大的影响：位置较高的烟叶中烟碱含量较高，碱性 更强；仅用白肋烟烟草制成的卷烟的非质子化烟碱传送更加有效， 但是吸烟者可能会感觉烟味较重。降 pH糖的添加可以掩盖烟气的 粗糙程度，从而控制混合物中非质子化烟碱的传送 [149]。添加了碳酸 铵的膨化烟草在燃烧时可以将氨释放到烟气中，因此不需要额外添 加氨 [142]。包含茎的膨化烟草不仅硝酸盐含量比只含有叶的膨化烟草 ·28· 烟草制品管制科学基础报告： WHO研究组第六份报告 高，而且在吸烟期间硝酸盐被部分地还原成氨，因此它还会对烟气 的 pH产生影响 [86]。此外，卷烟的某些特征，如卷烟纸的孔隙较多 和滤嘴通风，也能使烟气 pH升高。尽管烟气 pH和烟碱含量随着滤 嘴通风量的增加而增加，但其机制尚不明确。通过滤嘴通风口的空 气可以充当“气体干燥剂”，使气溶胶中的水分减少，pH增加，从 而使气相中的非质子化烟碱的含量增加 [138]。通风量也会影响烟草的 燃烧速率 [139]，增加通风可以改变焦油与烟碱的比值 [150]，这两种机 制都会使烟气 pH和非质子化烟碱含量升高。 2.4.5  测量“烟气 pH” 由于气溶胶中的 pH无法测量，因此，烟气 pH常在水溶液中测 量 [149]。测量烟气 pH时，乙腈常被用于比较不同品牌卷烟之间的差 异，它有助于追踪卷烟酸性和碱性特性的变化对感官效果的影响 [151]。 目前，用于测量烟气中非质子化烟碱的非工业方法有：对剑桥滤片 （CFP）上收集的颗粒物进行顶空分析；对收集的样品进行气相色 谱（GC）-质谱（MS）分析 [152, 153]；对收集的颗粒物质进行核磁共 振波谱分析 [154]。 所有分析动态反应（如卷烟条、卷烟滤嘴和烟气气溶胶之间的 烟碱分配）的方法都存在一定的缺陷，它们充其量只能够反映出不 同品牌卷烟之间的相对差异。然而，由于氨技术在几十年前已经成 为工业研究的热点，因此烟草工业依赖于这样的相对测量。 ·29· 2. 卷烟特征和设计特色 2.5  可能影响感观和传送的创新 2.5.1  概述 在本节中，根据科学文献和其他来源的出版物，如网站、烟草 行业文献和专利，描述了可以影响感知或传送的创新，这些创新或 最近已经被市场化，或正在开发。 世界卫生组织 TobReg第七次会议关于新型烟草制品（包括潜在 的“改变危害”的产品）演变的背景文件中 [155]对市场上传统卷烟产 品的变化进行了描述，如滤嘴中的薄荷醇胶囊和无添加的有机卷烟。 目前，一种新型的烟碱含量很低的卷烟已经开始市场化，在 ISO吸 烟条件下，它的烟碱释放量小于 0.04 mg，但是焦油含量为一般水平。 该文件还介绍了技术的发展过程，包括几种新类型的再造烟草和新 型过滤器。据称这些新技术的发展可以减少暴露，但是支撑这些结 果的大部分研究都是由业界进行并发表的。烟草替代薄片材料的使 用可以减少混合物中的烟草用量，且对烟草混合物进行一些处理 ,降 低诸如蛋白质之类的有害物质前体成分的水平。据报道，改良滤嘴 可以通过与烟气成分反应或选择性滤过烟气的方式，降低主流烟气 中有害成分的含量，如在滤嘴中加入可以与醛和氰化氢反应的氨基 树脂以及炭。大部分报道的可以选择性降低主流烟气中有害物质水 平的产品都采用了这种方式，但在某些情况下增加了其他有害物质 的水平。由于吸烟者必须吸入足够量的烟碱，因此卷烟中有害物质 的水平应表示为单位烟碱含量，但是很多文献中都没有报道具体的 ·30· 烟草制品管制科学基础报告： WHO研究组第六份报告 烟碱释放量。有报道称部分产品的体外毒性较低或暴露生物标志物 的含量较低 [155]。但是，消费者普遍认为，这些产品与传统卷烟相比 更令人难以接受。因此，很难评估这些新技术的净效应。在对 2.5.5 小节所述的新型烟草研究进行评估时应考虑这些问题。2.5.2~2.5.5小 节总结了自 2013年 10月以来的创新，并且对背景文件中提到的文 献进行了检索 [155]。 2.5.2  低烟碱卷烟 与含有减少烟气烟碱含量设计的卷烟（例如有滤嘴通风）相 比，在 ISO吸烟条件下，测得低烟碱卷烟烟草填料中的烟碱含量较 低。近期，“Magic”牌低烟碱卷烟（每根卷烟含烟碱 0.04 g）开始 在西班牙的烟草店出售，并且声称其不含烟碱。根据欧洲法规所要 求的卷烟制造商在每包卷烟上最接近 1/10的地方标出烟碱释放量， “Magic”牌低烟碱卷烟在包装上突出标注了“0 mg烟碱”[156]。 标准戒烟治疗包含两部分，分别是行为支持和药物疗法（伐尼 克兰或烟碱替代疗法）。近期以来，无烟碱卷烟常被用来辅助戒烟。 研究表明，使用无烟碱卷烟辅助戒烟组在治疗 1周和 4周后戒烟率 比标准戒烟治疗组较高，分别是 70% vs. 53%和 58% vs. 3%。但是在 治疗 12周后，两组的戒烟率无明显差异，为 39% vs. 31%[157]。另一 项研究对 840名每天吸烟量为 5支及以上的吸烟者进行了研究，研 究结果表明在进行实验 6周后，低烟碱卷烟组的每天吸烟量（约 16支） 少于正常卷烟组（对好几种不同类型卷烟进行测试，约 22支），并 且在低烟碱卷烟组未观察到明显的补偿性吸烟 [43]。然而，由于受试 者常常在非研究时段吸烟，因此他们的烟碱暴露水平可能并未减少。 此外，研究人员还对吸烟方法进行了研究，如逐渐过渡至直接过渡 ·31· 2. 卷烟特征和设计特色 到低烟碱卷烟，或将卷烟与烟碱贴片相结合。 2.5.3  彩色卷烟纸 一些卷烟品牌的卷烟纸为彩色的（图 2.1），包括 Ziganov Co- lours（粉红色、深粉色、黄色、绿色和紫色）、Ziganov Black、So- broui Cocktails、Fantasia、Black Devil、Pink Elephant、Nat Sherman Fantasia以及 Vanity Fair等。彩色卷烟管可用于手卷烟 [158]。 图 2.1 彩色卷烟纸 一篇网站文章描述 Sobranie Cocktails“有 5种柔和的颜色和 5 个黄金箔滤嘴，环规与标准卷烟相同。与 Nat Sherman Fantasias不同 的是，它更纤细且使用了更深的原色”[159]。这种类型的卷烟“特别 适用于女性，它纤细的特征和明亮的颜色吸引了很多女性”。 与卷烟包装设计相比，有关卷烟颜色的研究很少。如 2.2.2.1所 讨论的，颜色鲜艳的卷烟可以使人产生兴趣，人们通常认为它们有 吸引力、口味淡且危害小 [14]，而黑色卷烟纸对人的吸引力较小，并 且人们常认为它们烟味重且危害大。 ·32· 烟草制品管制科学基础报告： WHO研究组第六份报告 世界卫生组织 FCTC建议各国禁止或限制烟草制品使用能对消 费者产生吸引的特征，包括彩色卷烟纸。“为了使产品更具吸引力， 在烟草制品的各种成分中加入着色剂。在一些国家，有吸引力的彩 色卷烟（例如粉红、黑色、牛仔蓝）已经上市。着色剂包括油墨（例如， 在接装纸上的仿软木图案）和颜料（例如过滤材料中的二氧化钛）。”[36] 2.5.4  特色滤嘴 卷烟材料供应商供应的滤嘴类型有很多，表明烟草行业在这方 面有需求。例如，Hauni Maschannbau公司有 18种不同的视觉效果、 滤过特性、味觉增强和交互性的滤嘴 [160]。在制造滤嘴时，可以使用 很多元素及其组合，如木炭、中空形状（如形状像心脏一样）及彩 色滤嘴等。此外，还能在滤嘴中添加烟草、口味胶囊、草药和植物 颗粒等。可以通过在滤嘴丝束中直接添加香精线或喷洒香精的方式 使卷烟产生不同的口味。并且可以对香精线进行上色从而“创造更 独特的外观”。 Essentra Filter Products公司也生产了各种各样的滤嘴，如感官 滤嘴（在滤嘴中直接使用胶囊，香精线），土调滤嘴（在环境中可 以更快降解），性能滤嘴（高滤过效率、选择性），视觉差异滤嘴（“使 用视觉外观来表明口味，特定的产品属性，商标标识，或者区分 品牌”）[161]。还可以在滤嘴中添加含有薄荷醇等成分的有色香精线， 被称为“味觉传递技术的视觉指示器”。例如，美国 DJ混合香味 卷烟不仅有彩色包装，而且用了不同颜色的滤嘴，通过这种方式来 反映产品风味（例如，草莓红和苹果绿）。Marlboro Black Freeze （墨西哥）卷烟滤嘴中有薄荷醇条，在它的卷烟纸上也有相同的 条纹符号。 ·33· 2. 卷烟特征和设计特色 新的《欧盟烟草制品指令》2014/40/EU[29]在第七节成分管制中 提到禁止在滤嘴和卷烟纸中使用调味品、烟草或烟碱：“成员国应 当禁止在滤嘴、卷烟纸、包装、胶囊等任何组件中含有香料或是通 过技术手段改善烟草制品吸味、吃味或烟气度的烟草制品投放市场。 滤嘴、卷烟纸和胶囊中不能含有烟草或烟碱。” 新型烟草制品的背景文件对香味胶囊进行了描述 [155]。据业内报 道，卷烟滤嘴中的香味胶囊可以破碎，从而释放出一阵香味 [162]。胶 囊通常含有薄荷醇或类似味道的香精，如柠檬薄荷，这种胶囊可以 用于许多不同类型的卷烟；有时，在一个滤嘴中会含有两种不同口 味的胶囊。一项有关澳大利亚、墨西哥和美国吸烟者的研究表明， 香味胶囊对年轻人最有吸引力，含有香味胶囊的卷烟的使用呈上升 趋势，这与年轻人对它危害的误解有关 [162]。一项关于不吸烟和偶尔 吸烟的年轻女性的研究表明，她们认为含有香味胶囊的卷烟很有吸 引力 [14]。他们欣赏新奇，喜欢味道从“一般”转变为薄荷醇这个过程。 正如一项研究结果所示：卷烟包装不仅会影响它对消费者的吸引力， 而且会影响消费者对其危害和口味的看法，该结果表明，实际的卷 烟也可以这样做。 最近两项有关 Camel Crush的研究结果表明，压碎薄荷脑胶囊 对主流烟气中的颗粒相组分没有明显影响。Gordon等 [163]使用实时 检测器检测不仅发现了薄荷醇递送增加，而且发现几个气相组分的 产量也增加了，特别是五种挥发性有机化合物（VOC）乙醛、丙烯腈、 苯、1,3-丁二烯和异戊二烯。但是 Dolka等 [164]在菲利普·莫里斯使 用甲醛冷却冲击器采集气相成分，分析并未发现这几种物质产量的 增加。 ·34· 烟草制品管制科学基础报告： WHO研究组第六份报告 2.5.5  烟草行业对特殊滤嘴和再造烟叶的研究 许多以减少卷烟中有害物质释放为目的的技术不断被开发出来， 这类技术具体包括：滤嘴吸附剂、混合烟草处理和烟草替代薄片。 英美烟草公司研究了改良滤嘴通风、改变卷烟圆周、活性炭滤嘴 长度和负荷及这些特征的组合对减少有害物质释放的效果 [104]。有 一种名称为“分流翻转”的空气稀释机制，在两个独立的接装纸之 间有一个间隙，可以露出过滤器，并用一个多孔纸带包裹，可以最大 限度地减少人吸烟期间高流速下发生的有效滤嘴通风的损失，并有助 于挥发性有害物质的扩散。对它的研究结果表明，除了含有 1 mg焦 油的卷烟外，有害物质与主流烟气中烟碱的比例降低。 英美烟草公司的另一篇文章中描述了由 50%混合烟草、15%烟 草替代薄片、聚合物衍生活性炭和分离式倾翻组成的实验卷烟产生 的颗粒物质的遗传毒性和体外细胞毒性评估 [165]。结果发现与具有标 准醋酸纤维素过滤器、接装纸的混合烟草（3R4F，美国风格的混合 产品；M4A，一种烤烟）的对照卷烟相比，实验卷烟组的细菌致 突变性和哺乳动物遗传毒性降低，而两组卷烟的细胞毒性无显著 性差异。 一项由广东烟草工业公司资助的研究描述了在二氧化硅表面使 用特定的滤过添加剂和以烟碱为模板的分子印迹聚合物吸附主流烟 气中的 TSNA[166]，与对照组卷烟烟气相比，TSNA水平降低了 41%。 这项研究中的结果表明可能发生了选择性吸附，因为焦油水平保持 不变，烟碱水平没有报道。 Cultex Laboratories GmbH和日本烟草公司的一项研究表明，在 标准 ISO条件下吸烟时，具有集成木炭滤嘴的 K3R4F卷烟的烟气对 ·35· 2. 卷烟特征和设计特色 正常支气管上皮细胞纤毛的毒性低于常规 K3R4F卷烟烟气 [167]。木炭 滤嘴去除的 VOC会影响原发性细支气管上皮细胞纤毛的形成。病理 组织学结果显示暴露会使纤毛细胞较少，纤毛长度变短，最后，暴 露于卷烟烟气的细胞纤毛消失。对于暴露于木炭过滤卷烟烟气细胞， 在第 4次暴露后可以观察到纤毛长度的微小变化，但在 2天恢复期 后则未观察到该改变。 在 Philip Morris的一项专利中介绍了一种含有高温氨释放剂的 烟草混合物和卷烟包装材料的研制 [168]。所描述的铵化合物被称为“可 有效减少吸烟过程中形成的气相或颗粒物质的细胞毒性”。 虽然研发出的一些新型卷烟的主流烟气中有害物质的吸烟机产 率比传统卷烟低，且体外毒性较低，但是要得出它们健康风险较低 的结论仍需要大量的科学数据来支持。在评估设计变更对降低人类 风险方面的作用时，必须考虑消费者对产品的接受性，以及它对吸 烟行为的影响和是否实际降低了暴露量如评估生物标志物。 2.6  对设计特色的公众健康影响进行科学评价的研究 如上所述，大量的证据已经证实，卷烟对人的吸引力、成瘾性 以及烟气中有害物质的传送都与卷烟的物理特性和设计特征密切相 关。其中对某些特征的研究明显多于其他特征。如有大量有关滤嘴 通风对消费者感知的影响，以及吸烟机产生的释放物和吸烟者的暴 露等的研究和报道。此外，有关低烟碱卷烟（0.4 mg/g烟碱的烟草填 充物）在促进戒烟方面的研究数据也有很多。一项对过去和待开展 研究的系统综述提供了很多信息。但是，在任何条件下都不应该把 ·36· 烟草制品管制科学基础报告： WHO研究组第六份报告 它用于促进吸烟。由于消费者感知、行为和烟气化学之间相互作用 的复杂性，现有的数据不一定能够清楚地反映出物理特征减少有害 物质释放、保护健康的机制。因此，还需要进一步研究，为卷烟监 管控制提供科学依据。 卷烟物理特性、烟草类型和添加剂对人体暴露的影响具有复杂 性，且暴露与吸烟者的感知和行为有关，因此，研究应采取综合措 施来明确具体的卷烟设计如何影响结果，包括吸烟机烟气传送，吸 烟者的信念、吸烟方式以及暴露情况。 有关设计特色对烟气排放的影响应该始终包含烟碱水平。因为 吸烟者需要吸入足够量的烟碱来满足他们的烟瘾，因此设计特色对 释放的影响应该以每毫克烟碱的形式报告 [2, 70]。游离碱烟是生物利 用率最高的烟碱形式，因此国际标准的定量检测游离烟碱或确定游 离烟碱与质子化烟碱的比值是非常有用的。此外，研究人员应该意 识到，任何减少卷烟中一种或多种成分含量的操作，都可能会无意 地增加其他成分的浓度。研究设计特征如何相互关联并影响主流烟 气排放的研究方法包括： • 对个别设计特征逐一地进行系统研究。对于一些选定的参数 如滤嘴通风和卷烟尺寸，这些方法可以应用于许多实验室。 对于其他设计参数如滤嘴材料和纸张孔隙率，相关研究必须 要在装备良好的测试实验室中完成，并且可能需要定制具有 特定设计特色的卷烟。 • 对市场上的卷烟产品的烟草填料成分、主流烟气排放（不同 吸烟强度下）和物理性质进行多变量分析。通过这种方法可 以找出对主流烟气排放影响最大的设计参数。 • 对与卷烟制造商提供的主流烟气排放的相关设计特色、参数 ·37· 2. 卷烟特征和设计特色 和规格进行深入、详细的统计分析。在有足够监管机构的条 件下，可以使用这种方法，并且由 ISO 17025认证的政府实 验室或独立实验室对结果进行监督。 使用适当的方法研究吸烟者和非吸烟者尤其是青少年的认知和 行为，包括消费者调查、焦点小组分析和临床调查（吸烟行为和生 物标志物分析）。实际的暴露量可以通过测量吸烟者的相关生物标 志物来估计。结果将揭示减少吸烟机测试的特定成分释放量是否会 减少吸烟者的暴露。 暴露的健康效应可以在临床研究中进行评估，例如通过测量（早 期）效应生物标志物。此外，可以进行吸烟相关疾病的体外试验。 基于气 /液界面细胞模型的体外试验有较大的应用前景，因为它们 模拟了气道在烟气中的暴露。 监测烟草制品市场发展情况有重要的意义，有利于通过标准搜 索网站包括社交媒体和实地研究等方法，继续获得有关公众健康的 信息。 2.7  结    论 卷烟设计的主要目的是提高产品的吸引力（即使其更可口、更 有吸引力或减少其危害）、减少产品的负面影响、确保吸烟者在使 用该产品时感到满意、吸引年轻人以及新吸烟者。能增加卷烟吸引 力的卷烟特性包括影响使用者对卷烟外观的感知或是否能够“定制” 的卷烟特征。卷烟的装饰元素可以直接或间接地通过暗示强度、新 颖性或较少危害来影响卷烟的吸引力，特别是对女性和年轻吸烟者。 ·38· 烟草制品管制科学基础报告： WHO研究组第六份报告 这些元素是由制造商推出的众多创新中的一部分。鉴于这些特征的 唯一目的是吸引新的消费者，其可能会导致人们对它的健康风险产 生误解。将卷烟外观限制为标准特征，即白色卷烟纸、标准接装纸 颜色和标准卷烟品牌印刷方式有望可以保护公众健康。 卷烟的其他大多数物理特征的研究结果比较复杂，甚至有些研 究结果是相反的。例如，滤嘴通风会影响每根卷烟的吸烟机释放量， 使吸烟者感到烟味较低且更安全。滤嘴通风是改变吸烟行为的物理 特性之一。与低通风的卷烟相比，较高的滤嘴通风会导致相似或更 高的有害物质及致癌物暴露。吸烟者可以很容易地操控滤过通风口， 从而获得较高的烟碱和烟气释放量。制造商可以控制的另外一些卷 烟特征包括多孔的接装纸和卷烟包装纸及烟草混合物的特性，它会 使吸烟者不知不觉地从卷烟中吸入更多的烟气。 卷烟尺寸也会对结果产生复杂的影响。细支卷烟中可用于燃烧 的烟草含量较少，吸烟者吸一支烟的总暴露也会较少。然而，细支 卷烟的时尚、有吸引力、高质量的外观以及人们认为它危害较小等 会对女性有较大吸引力，这是一个公共卫生问题。此外，细支卷 烟吸烟者的氰化氢和甲醛等成分的暴露量可能并不低于标准圆周 卷烟。 研究除了对滤嘴通风、细支卷烟进行验证外，还对另外一些假 设进行了验证，如制造商使用的混合烟草的性能、压降（纸孔隙率、 过滤、过滤器保持）等，这些设计可以使卷烟具有“弹性”，可以 让吸烟者获得他们想要的烟碱量并且使他们感到“满意”。大多数 卷烟都有弹性，特别是“超低”卷烟，全味卷烟的弹性较少。在吸 烟机吸烟的条件下，弹性表现为随着吸烟强度的增加，有害物质的 释放量呈非线性增加。 ·39· 2. 卷烟特征和设计特色 卷烟设计，如可以减少烟气中特定化学物质含量的滤嘴添加， 可以改变感官的设计等，都会导致吸烟行为的变化。有研究表明， 吸烟者在吸含有木炭过滤器的卷烟时每口吸烟量更多。在卷烟中添 加有香味的化学物质也会影响吸烟者的主观感受。 有证据表明，虽然吸烟者认为有香味的卷烟是新颖的且吸入的 烟量较小，但研究结果表明吸烟者接触到有害的烟气释放如 CO等 与吸烟草风味卷烟一样。主观感受、行为及暴露之间的相互作用很 复杂。最好的例子也许是使用薄荷醇卷烟，据报道，它与更强的成 瘾和减少戒烟成功率有关。然而，有关薄荷醇对吸烟行为的影响的 研究结果确是很混乱的。 吸烟者会习惯性地使用卷烟来获得烟碱。吸烟者吸烟时感受到 的满足感是由卷烟烟气通过口腔（“冲击”）后烟碱迅速经肺吸收 最后进入大脑来实现的。据报道，与烟气中质子化（离子化）状态 的烟碱相比，未质子化（未离子化）的烟碱更易被吸收，且能更迅 速地到达大脑。一些设计特色和添加剂可以影响烟气中未质子化 烟碱的比例。碱化剂在增加非质子化烟碱含量的同时增加了“口 感”，并通过与烟气中的酸和还原糖反应形成产物来改善味道。 吸烟者会根据口感和味道来调节他们的吸烟行为使烟气达到生理 “强度”。 许多有关改变感观或烟气释放的创新都集中在烟草混合和滤过 技术方面，因为它们在控制传送和使用行为方面有重要作用。添加 卷烟口味的非传统方法如香精胶囊和香精线等，可以通过新颖性和 品牌差异来吸引消费者。 香精胶囊是烟草行业的一个重要发展部分，它对年轻人尤其具 有吸引力。虽然一些新的技术令人鼓舞，例如减少特定有害物质的 ·40· 烟草制品管制科学基础报告： WHO研究组第六份报告 含量，但是它带来的收益经常被其他有害物质含量的增加或消费者 可接受性差所抵消。 烟草行业已经探索了将滤嘴添加剂和处理过的烟草进行组合作 为减少有害物质释放的手段。内部行业文件表明，这些卷烟的实验 室评估结果显示有害性降低；然而，目前尚不清楚监管机构是否对 这些技术进行过审查，或在无监管的市场中进行出售。最近的一项 有关市场上的低烟碱卷烟（市场上也有标准烟碱含量卷烟）的研究 表明吸烟者的行为发生了变化（每天吸烟量减少），而且他们更易 戒烟。但是戒烟效果并不比标准戒烟治疗 12周后的戒烟效果好，而 且吸烟者会经常吸标准烟碱含量的卷烟。 2.8  建    议 正如 FCTC的第 9条和 10条所提到的，卷烟设计研究的最终目 标是确保随后的监管措施可同时降低卷烟的吸引力和成瘾性，以及 与吸烟相关的危害 [36]。可以通过以下方法来实现这个目标：标准化 卷烟外观；消除卷烟中可以吸引新吸烟者或新手吸烟者及增加戒烟 难度的设计特征和成分；降低烟碱水平或生物利用度从而降低卷烟 成瘾性；通过综合选择性滤过、卷烟尺寸、包装密度和混合烟草等 来降低有害物质的暴露。 在此基础上，提出了以下的具体政策建议和研究建议。 2.8.1  政策建议 （1）要求制造商披露当前产品和新兴产品的所有设计特点、参 ·41· 2. 卷烟特征和设计特色 数、规格和含量水平及释放水平。示例包括卷烟纸、卷烟滤嘴中的 胶囊和卷烟尺寸。 （2）禁止滤嘴通风和任何其他使卷烟有弹性的设计特性（增加 吸烟者每口吸入体积，尤其是低焦油品种）；禁止滤嘴胶囊、细支 卷烟或其他任何能增加卷烟吸引力、烟气释放及成瘾的产品属性。 （3）根据 TobReg所述的方法，降低所有有害物质释放量（每 毫克烟碱）[71]。 2.8.2  研究建议 （1）继续研究烟草产品的设计特点和该领域的创新，包括它们 对以下方面的影响： • 吸烟者、前吸烟者和从未吸烟的人特别是青少年的认知和行 为； • 释放量，标准化每毫克卷烟中的烟碱含量，除低烟碱卷烟（每 克烟草中烟碱含量 <0.4 mg）外； • 有害性和暴露量。 （2）开发测定游离烟碱水平或确定游离碱与质子化烟碱比值的 标准方法。 （3）继续研究低烟碱卷烟在戒烟中的潜在用途。确保低烟碱卷 烟在任何情况下不被用于促进吸烟很重要，因此对过去和未决研究 进行系统综述可能会提供很多信息。 ·42· 烟草制品管制科学基础报告： WHO研究组第六份报告 2.9  参  考 文 献 [1] Podraza K. 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Patent US 20150122280 A1-Synthesis and incorporation of high-temperature ammonia-release agent in lit-end cigarettes. 2015 (https://www.google.com/patents/ US20150122280?dq= tobacco&hl=en&sa=X&ved=0CDwQ6AEwB- Dg8ahUKEwjU7P_ht9rHAhWMVRQKHTlWBiY, accessed 4 Sep- tember 2015). ·63· 3. WHO烟草实验室网络标准操作规程对 电子烟碱传输系统评估的潜在应用 Patricia Richter，美国疾病控制与预防中心 Rima Baalbaki，贝鲁特美国大学（黎巴嫩贝鲁特） Mirjana Djordjevic，美国国立卫生研究院国家癌症研究所 Rachel El Hage，贝鲁特美国大学（黎巴嫩贝鲁特） Bryan Hearn，美国疾病控制与预防中心 Ahmad El Hellani，贝鲁特美国大学（黎巴嫩贝鲁特） 侯宏卫 Hongwei Hou，中国国家烟草质量监督检验中心 胡清源 Qingyuan Hu，中国国家烟草质量监督检验中心 Walther Klerx，荷兰国家公共卫生与环境研究所 Naoki Kunugita，日本国立卫生研究院 Joseph Lisko，美国疾病控制与预防中心 Jose Perez，美国疾病控制与预防中心 Najat A Saliba，贝鲁特美国大学（黎巴嫩贝鲁特） Shigehisa Uchiyama，日本国立卫生研究院 Wouter Visser，荷兰国家公共卫生与环境研究所 Cliord Watson，美国疾病控制与预防中心 Liqin Zhang，美国疾病控制与预防中心 目录 3.1 背景 3.2 电子烟碱传输系统（ENDS）的一般方法学评价 3.3 烟碱 3.3.1 ENDS烟液中的烟碱 3.3.2 ENDS气溶胶中的烟碱 ·64· 烟草制品管制科学基础报告： WHO研究组第六份报告 3.4 烟草特有亚硝胺 3.4.1 ENDS烟液中的烟草特有亚硝胺 3.4.2 ENDS气溶胶中的烟草特有亚硝胺 3.5 苯并 [a]芘 3.5.1 ENDS烟液中的苯并 [a]芘 3.5.2 ENDS气溶胶中的苯并 [a]芘 3.6 其他分析物 3.6.1 羰基化合物 3.6.2 溶剂 3.6.3 挥发性有机化合物 3.6.4 酚类化合物 3.6.5 金属 3.6.6 香精 3.7 关于扩展方法的建议 3.7.1 烟碱 3.7.2 烟草特有亚硝胺 3.7.3 苯并 [a]芘 3.7.4 挥发性有机化合物 3.7.5 羰基化合物 3.8 为未来监管 ENDS提供数据所需的研究 3.9 结论 3.10 建议 3.11 参考文献 ·65· 3. WHO烟草实验室网络标准操作规程对电子烟碱传输系统评估的潜在应用 3.1  背    景 本部分按照第七次缔约方会议的要求（WHO FCTC，http:// www.who.int/fctc/cop/cop7/FCTC COP7_9_EN.pdf?ua=1）， 就 现 行 和未完成的WHO烟草实验室网络（TobLabNet，http://www.who. int/ tobacco/global_interaction/toblabnet/en/）标准操作规程（SOP， http://www.who.int/tobacco/ publications /prod_regulation/en/）的应用 情况提出建议，以分析电子烟碱传输系统（ENDS）的内容物和释放 情况。在确定该矩阵（例如适当的测量范围、干扰等）的适用性后， 本章中的建议也适用于电子非烟碱传输系统（ENNDS）。 ENDS包括一个电池，用来加热线圈和汽化液体基质（内容物）， 以提供气溶胶（释放物），也被称为雾化器。本报告中将来自 ENDS 装置嘴端的气溶胶称为“第一手气溶胶”（FHA），液体基质称为“电 子烟液”，当电子烟液含有烟碱时，该装置被称为电子烟碱传输系 统（ENDS）。卷烟形状的 ENDS用“电子烟”表示。 ENDS的汽化温度是由电池电压和通过线圈的电流决定的 [1]。 电子烟液通常是单独的丙二醇或是与植物甘油、烟碱、香精和其他 成分如咖啡因混合的溶液。电子烟液和产生的第一手气溶胶通常含 有不同浓度的烟碱和其他化学物质以增强其吸引力。在电子烟液中 已经报道了烟碱、小烟草生物碱、烟草特有亚硝胺（TSNA）、香 料、金属、VOC、酚类化合物和溶剂。ENDS气溶胶中包含羰基类、 VOC、TSNA和金属 [2]。 ENDS可以是一次性的或可重复使用的，并且允许用户“定制” ·66· 烟草制品管制科学基础报告： WHO研究组第六份报告 气溶胶的传送和化学组成特征（例如可调节电压范围）。最初， ENDS在尺寸和形状上与卷烟相似。新一代的 ENDS体积较大，有 可充液储液腔，可能与雪茄、烟斗或水烟袋（水烟筒）类似，或者 根本不像任何烟草产品 [2-4]（图 3.1）。ENDS在世界范围内销售 [5]， 有时作为烟草制品管理，有时当作普通消费品管理，有时当作药品 管理。然而，其他国家已经禁止含烟碱的 ENDS，甚至 ENNDS[90]。 图 3.1 电子烟碱传输系统 与传统的含有参考物质的卷烟（例如 CORESTA Monitor和 Ken- tucky研究卷烟）相比，目前没有针对 ENDS，也没有任何方法可以 用于吸烟机生成 ENDS气溶胶的分析。ENDS产品的使用模式（拓 扑图）仅在少数几项研究中得到检验 [6, 7]，而且 ENDS产品的多样性 使问题更加复杂。CORESTA（https://www.coresta.org/）是烟草产品 制造商、烟草工业研究所和实验室的国际协会，它发布了一个检测 ENDS气溶胶的推荐方法 [8]。ENDS包含的电子组件，可以蒸发液体 ·67· 3. WHO烟草实验室网络标准操作规程对电子烟碱传输系统评估的潜在应用 产生使用者可吸入的气溶胶。ENDS可以设计成单件一次性、可充 电和 /或可重复使用的产品。据报道，该方法涵盖的产品是符合上 述定义的产品，也是“电子卷烟、电子雪茄、电子烟、电子烟管和 其他相关产品类别”中描述的产品。CORESTA方法不是基于反映实 际使用或行为的人类吸烟模式采取的措施。 有几家公司已经开始制造和销售可以生成 ENDS气溶胶的自 动化机器（例如，英国的 Cerulean, Milton Keynes，以及德国的 Borgwaldt GmbH, Hamburg）。应该为设计用于生成分析目的的 ENDS气溶胶的机器提供电源，研究应该解决电源的供应问题。现有 设备和方法针对卷烟类装置（电子卷烟）进行了优化，因此，ENDS 的新品种可能需要新设备或方法的调整，特别是对于有较大“储液仓” 的品种。 3.2  电子烟碱传输系统（ENDS）的一般 方法学评价 产品化学分析的定量方法取决于测量基质的性质。ENDS基质 （电子烟液或第一手气溶胶）与传统烟草制品（烟草填料和主流烟气） 相比化学成分复杂，成分变化较小。传统卷烟主流烟气的标准化测 量适用于 ISO[10]、美国联邦贸易委员会 [11]、美国疾病控制与预防中 心（CDC）[12]、CORESTA[13, 14]、加拿大卫生部 [13]、美国马萨诸塞州 公共卫生部 [15]和世界卫生组织 [16]。用于分析常规烟草卷烟的吸烟方 案在较不严苛的标准方法（如 ISO和美国联邦贸易委员会）中仅有 ·68· 烟草制品管制科学基础报告： WHO研究组第六份报告 略微差异。模拟较大的抽吸量和吸烟者的通气阻塞行为（如加拿大 的 Intense和Massachusetts）与较不严苛的方法相比，可能产生完全 不同的结果。所有的方法都包括对卷烟样品进行温湿度控制调节， 在特定状态（如抽吸容量、持续时间和时间间隔）下，吸烟机吸烟 至每个产品所确定的烟头长度（23 mm，滤网长度加 8 mm或滤网外 包层加 3 mm），并打开，部分堵塞或完全堵塞滤嘴通风。关于人们 对 ENDS使用方式的研究 [6, 7]结果引发了关于标准和“深度”模式 （类似于 ISO和加拿大深度抽吸模式）是否合适以及是否对分析不 同 ENDS产品释放的程序或设备进行相应的修改的问题。 吸烟机生成的主流烟气样品通常在样品制备后，通过 GC-MS或 火焰离子化检测器（FID）、配紫外 -可见分光光度仪的液相色谱、 液相色谱 -质谱法或电感耦合等离子体质谱法进行分析。 3.3  烟    碱 ENDS产品通过呼吸系统提供一种令人上瘾的化学物质——烟 碱。ENDS烟弹和再充填电子烟液标签上列出的烟碱浓度可能与烟 液中测得的数值有显著差异 [3, 18]。Sleiman及其同事最近报告了美国加 利福尼亚州零售商店购买的 ENDS产品中烟碱的含量。通过顶空气相 色谱 -质谱法（HS-GC/MS）测定市售浓度为 20.4 mg/mL、25.4 mg/mL 和 32.1 mg/mL的电子烟液中，烟碱的含量分别为 18 mg/mL、24 mg/mL 和 18 mg/mL[1]。 ·69· 3. WHO烟草实验室网络标准操作规程对电子烟碱传输系统评估的潜在应用 3.3.1  ENDS烟液中的烟碱 在测定电子烟液中烟碱含量时应考虑两个因素。首先是在 To- bLabNet方法检测烟草烟丝中使用正己烷萃取溶液，而丙二醇和植物 甘油不溶于正己烷，它们更易溶于标准 ISO方法中用于测量烟气中 焦油、烟碱和一氧化碳的异丙醇萃取溶液 [10]。因此，用于分析附着 在 CFP上的烟碱的标准 ISO方法比 TobLabNet SOP-04法更适合分析 电子烟液中的烟碱。另外，WHO SOP-04可以与更易混溶的萃取溶 剂一起使用。在 ENDS电子烟液中分析烟碱的另一个重要考虑因素 是：ENDS中烟碱的含量 可能远远超过烟草烟气提取物，即使是在 深度抽吸吸烟机条件下产生的烟气（例如 CDC未发表的数据显示约 36 mg/mL vs. 0.3 mg/mL）。因此，必须调整异丙醇的萃取体积，以 使电子烟液样品中的烟碱浓度落在校准曲线范围内。 其次，了解烟草制品中烟碱的总量并不足以了解其对使用者的 影响 [19]。烟碱存在质子化和非质子化（也称为未离子化或“游离” 烟碱）状态。在非质子化状态下吸收的烟碱比在质子化状态下吸收 的烟碱更快地到达大脑，这是化学品成瘾性的重要原因。碱化剂的 添加增加了未质子化形式的烟碱比例 [20]。电子烟液的 pH可以通过 常用测量无烟烟草的 pH的步骤来测量，使用 pH计进行定时测量。 据报道，某些电子烟液的 pH远大于烟碱的 pKa[3, 21]，表明大量的烟 碱未被质子化。 3.3.2  ENDS气溶胶中的烟碱 通常，不同吸烟方式（标准或深度）下测量烟草烟气中烟碱的 ·70· 烟草制品管制科学基础报告： WHO研究组第六份报告 方法是不同的，这两种方式都可以在普通吸烟者中获得一系列可能 的吸烟行为。ISO抽吸模式要求通气孔畅通、35 mL抽吸量、2 s抽 吸持续时间、60 s抽吸间隔和足够的抽吸数使烟蒂长度等于滤嘴长 度加上 8 mm或过滤器外层包裹物加上 3 mm（取较长者），而加拿 大的“深度”抽吸方式和WHO抽吸方式规定为 55 mL的抽吸量、2 s 的抽吸持续时间、30 s的抽吸间隔和 100%堵塞通气孔。得到的主流 烟气总颗粒物（TPM）用异丙醇萃取并通过 GC-FID[10]分析。对于 电子烟，CORESTA推荐使用“vaping”（自动生成电子烟气溶胶的 机器）方案 [8]来产生气溶胶，抽吸体积为 55 mL、抽吸持续时间为 3 s、 时间间隔为 30 s，没有规定抽吸次数，尽管被认为每次至少有 50次 抽吸才能够在 CFP上产生足够的 TPM来测定烟碱 [22]（CDC未发表 的数据）。据报道，CORESTA方法涵盖了为 ENDS产品设计的单独 使用、一次性使用的配件和多组分产品如可充电和 /或可充液装置（即 储液腔系统）。如果该方法由独立实验室进行验证，则可以避免分 析不同设计产品释放量时对方法和装置的修改。WHO SOP-01深度 抽吸中的抽吸参数与 CORESTA方法中的抽吸参数类似，并可以对 气溶胶释放量进行修改，直到有足够多的产品设计参数和 ENDS使 用行为的数据可用于设计产生 ENDS气溶胶的方案，该方案更具有 代表性。 CORESTA方法没有详细说明定量测量收集的气溶胶中烟碱的分 析平台。预计 ENDS在非深度条件下运行时不会发生燃烧，所以收 集的气溶胶的组成与烟油的组成相似。WHO SOP中用于分析烟草 烟气提取物的分析平台可应用于 CFP捕获的 ENDS气溶胶。采用下 游吸附阱 CFP收集 ENDS气溶胶的一项研究表明气溶胶颗粒中含有 烟碱，且超过 98%的烟碱被 CFP捕获 [22]。常规检测方案可以扩展用 ·71· 3. WHO烟草实验室网络标准操作规程对电子烟碱传输系统评估的潜在应用 于分析 ENDS气溶胶，其较烟草烟气成分复杂且化学物可溶于异丙 醇。CORESTA电子烟抽吸模式与烟草卷烟主流烟气标准测试方案的 初步比较表明，CORESTA方法在有限的 ENDS产品样本（CDC未 发表的数据）中提供了可靠的烟碱定量分析结果——预计与WHO SOP-01的结果类似。未来应评估诸如电子烟管和电子烟水烟袋等其 他 ENDS形态。 烟草烟气中的烟碱通常与“焦油”（TPM除去烟碱和水）和 CO一起测量。与主流烟草烟气一样，ENDS TPM由溶剂、水、烟 碱和其他气溶胶成分组成，并在 ENDS气溶胶生成过程中被 CFP 捕获 [22]。 3.4  烟草特有亚硝胺 TNSA主要是在烟草的固化、发酵和燃烧过程中形成的，并且 存在于所有类型的烟草制品中 [23]。WHO建议在烟草和烟草烟气中 强制降低TNSA，特别是NNN和NNK，它们是有效的人体致癌物 [24]。 3.4.1  ENDS烟液中的烟草特有亚硝胺 虽然有些 TNSA是在生物碱前体燃烧烟草过程中形成的，但它 们主要存在于卷烟填充物中的烤烟，并在烟草燃烧过程中直接转移 到主流烟气中 [25]。由于电子烟液中的烟碱是从烟草中提取的，因此 ENDS电子烟液中的任何TSNA都可能是烟碱提取过程中引入的杂质。 Laugesen[26]分析了 Ruyan®电子烟烟弹中的液体，发现烟碱含量 从 0到 16 mg不等。在四个 TSNA中，只有 NNK在所有烟弹中检 ·72· 烟草制品管制科学基础报告： WHO研究组第六份报告 出。NNN的含量高于 NNK，但仅在含烟碱的烟弹中可检测到，在 “零烟碱”烟弹中检测到 0.260 ng NNK。NNN和 NNK的水平随着 烟碱浓度的增加而增加。另一项研究报道，在 11家公司出售并在 韩国购买的品牌的补充电子烟液中发现 NNN和 NNK[27]，而West- enberger[28]在美国购买的两种品牌 10种盒式电子烟烟弹的烟液中未 检测出 TSNA。荷兰国家公共卫生与环境研究所（RIVM）的研究人 员通过超高效液相色谱和串联质谱联用方法，在几乎所有 ENDS烟 液中发现了可检出但低含量的 TSNA。非常小部分的 ENDS烟液含 有高达 150 ng/mL的单个亚硝胺和 285 ng/mL的总 TSNA[29]。较高浓 度的 TSNA可能是由于使用烟草提取物作为香味，因为所有发现它 们的烟液都被标记为“烟草味”。 对来自 14个国家销售的卷烟烟叶的 NNN和 NNK水平进行的 综合研究表明，TSNA总浓度为 0.087~1.9 μg/g[30]。因此，即使 ENDS 烟液中存在 TSNA，其水平也远低于卷烟烟草。 3.4.2  ENDS气溶胶中的烟草特有亚硝胺 在 ISO和深度抽吸条件下，确定主流卷烟烟气中 TSNA的WHO SOP[31]中，将卷烟烟气颗粒物收集在 CFP上，用乙酸铵提取并在高 效液相色谱（HPLC）串联MS系统中分析。卷烟烟气是通过燃烧烟 丝而产生的，而 ENDS的烟气是通过加热电子烟液而产生的，加热 温度取决于装置参数。由于烟草烟气基质比 ENDS气溶胶基质复杂 得多，含有约 8000种化学物 [32]，SOP应适用于分析 ENDS气溶胶 中的 TSNA。然而，在一项研究中，ENDS气溶胶中检测到的 TSNA 的最高水平为每 150次吸入 NNK量为 (28.3±13.2) ng，NNN量为 (4.3±2.4) ng。在较少的抽吸口数下（例如 15口），估计释放约 ·73· 3. WHO烟草实验室网络标准操作规程对电子烟碱传输系统评估的潜在应用 2.83 ng NNK和 0.43 ng NNN。即使使用 TobLabNet TSNA方法中的 最小体积的提取溶液（10 mL），NNK水平为 0.28 ng/mL，NNN水 平为 0.043 ng/mL，低于该方法的报告限值 0.5 ng/mL[33]。因此，应 该使用较高的抽吸口数（例如 50口）来优化 ENDS气溶胶中测量 TSNA的条件。单位释放量（即每一支卷烟和每单位一次性使用的 ENDS产品）或“每个环节”的释放量比较可能会得出不同的结果， 但仍然预计将远远低于卷烟主流烟气中的释放量。 3.5  苯并 [a] 芘 多环芳烃（PAH）是在有机物质如烟草不完全燃烧期间形成的 多种致癌物质。苯并 [a]芘是一种广泛的环境污染物，是人体致癌物， 也是这类化合物中研究最深入的物质 [34, 35]。WHO建议在主流烟草 烟气中降低苯并 [a]芘含量 [24]。 3.5.1  ENDS烟液中的苯并 [a] 芘 在一些关于 ENDS电子烟液中有害化学物质的研究中，没有发 现大量的 PAH。Kavvalakis等 [36]在希腊市场上的电子烟液样品中 未发现 PAH，Leondiadis在 Nobacco品牌可填充电子烟液中未发现 PAH[37]。Laugesen[26]的研究是少数几个检测 PAH超出极限的研究 之一。在 0 mg烟碱 Ruyan®电子烟液的正己烷提取物中检测到四种 PAH：蒽、菲、1-甲基菲和芘。作者假设电子烟液的消费量等于一 天内吸 20支卷烟，计算了每种 PAH的量占相同数量烟草卷烟烟气中 PAH量的百分比。检测到的含量分别为 7 ng、48 ng、5 ng和 36 ng， ·74· 烟草制品管制科学基础报告： WHO研究组第六份报告 低于20支卷烟中含量的1%。这四种PAH被国际癌症研究机构（IARC） 分类在第 3组中，即人体致癌性证据不足、动物证据不足或有限 [37]。 未检测到苯并 [a]芘。 3.5.2  ENDS气溶胶中的苯并 [a] 芘 在环境沉积研究中，将 ENDS气溶胶引入具有大量稀释空气的 取样袋中。大多数 PAH，包括苯并 [a]芘，都没有达到检测限，苯并 [a] 芘的含量与空白对照相似 [38]。Tayyarah和 Long[39]在 ENDS气溶胶中 未发现可定量的 PAH，Romagna等 [40]在比较 ENDS和传统卷烟的排 放时，未检测到环境空气中的 PAH。Lauterbach和 Laugesen[41]研究 报告，来自 16 mg烟碱的 Ruyan® ENDS气溶胶（超过 300抽吸次数 的气溶胶）中的苯并 [a]芘水平低于报告限值。如果将烟草与 ENDS 产品混合使用，则必须监测 PAH。 上述研究对于分析 ENDS气溶胶基质中 PAH的方法没有明确说 明。在大多数研究中，这些方法看起来与常规卷烟烟气中使用的方 法有所不同或完全不相同。用 CDC方法 [32]分析苯并 [a]芘的样品制 备似乎与 TobLabNet方法 [42]相似。CDC的初步研究（未发表的数据） 显示，ENDS丙二醇 -甘油基质和标准卷烟制备的 PAHs校准曲线之 间的差异很小，表明该方法是适用的。制备用于分析苯并 [a]芘的样 品的大多数方法包括用非极性溶剂萃取并通过硅胶固相萃取净化。 样品生成和制备方法对 ENDS分析的适用性应在方法被认为合适之 前进行测试。 ·75· 3. WHO烟草实验室网络标准操作规程对电子烟碱传输系统评估的潜在应用 3.6  其他分析物 3.6.1  羰基化合物 “羰基”是醛和酮的统称。传统烟草卷烟的研究表明，保润 剂暴露于高温时会形成短链羰基化合物和其他有害化学物质。对于 ENDS，与电子烟液接触的加热线圈的温度取决于线圈周围的抽吸持 续时间、抽吸频率和传热特性 [1]。目前认为溶剂的热分解是 ENDS 气溶胶中羰基化合物的主要来源。甘油在约 280℃脱水形成丙烯醛， 并进一步反应生成甲醛和乙醛。电子烟烟液中的丙二醇和植物甘油与 加热的雾化镍铬合金丝接触被认为是羰基化合物的来源 [43]。羰基化合 物具有公共卫生意义，因为一些化合物已被评估为已知或可能的 人体致癌物质，丙二醇被热降解为环氧丙烷，其在实验室动物中 是致癌的 [23, 25, 44, 45]。在甘油和丙二醇加热且没有其他电子烟液成分 （如烟碱或香味剂）的情况下，甲醛、乙醛和丙烯醛在某些条件下（特 别是在后来的抽吸中）在 ENDS气溶胶含量较高 [1]。然而，在最近一 项关于来自有味和无味电子烟液的 ENDS气溶胶的研究中，Khlystov 和 Samburova指出，羰基化合物的形成还取决于香味物质浓度，而 不仅仅取决于电子烟液溶剂 [46]。 在 ENDS气溶胶的粒相和气相中检测到一些羰基化合物（例如 甲醛）（N. Kunugita，个人通信）。最近，Sleiman及其同事 [1]在电 子烟液中发现含有微量甲醛、乙醛和丙烯醛（ng/mL）。当烟液雾化时， 甲醛、乙醛和丙烯醛的水平随着电压升高而显著升高。在前 5口抽 ·76· 烟草制品管制科学基础报告： WHO研究组第六份报告 吸（初始）和第 30口到第 40口抽吸（稳定状态）之间，甲醛水平在 3.8 V时消耗的烟液从 2900 ng/mg增加到 8950 ng/mg，在 4.8 V时消 耗的烟液从 4850 ng/mg到 7250 ng/mg。随着抽吸口数的增加，乙醛 和丙烯醛的浓度增加（乙醛：在 3.8 V时从 230 ng/mg消耗的烟液增加 到 1820 ng/mg，在 4.8 V时消耗的烟液从 740 ng/mg到 19080 ng/mg； 丙烯醛：在 3.8 V时从 90 ng/mg消耗的烟液量至 1700 ng/mg，在 4.8 V 时为 400 ng/mg消耗的烟液量至 10060 ng/mg）。其他发现的高于检 测限的羰基化合物是巴豆醛、甲基丙烯醛、丁醛、苯甲醛、戊醛、 对甲基苯甲醛和己醛。在 ENDS气溶胶中测量甲醛、乙醛、丙烯醛、 丙醛、苯甲醛和乙二醛，其浓度为微克每克含香精的电子烟液 [46]。 相反，在相同的 ENDS装置中，没有添加香味物质的电子烟液仅可 检测到乙二醛和苯甲醛。 ENDS产生气溶胶的参数决定了羰基化物的释放量。电池电压、 抽吸量、抽吸持续时间、线圈数量、位置、电阻、芯吸设计和长度、 溶剂、电子烟液黏度和空气流动阻力等变量可能会影响羰基化合物 形成的速率。 TobLabNet正在验证主流烟草烟气中羰基化合物的 SOP。简而 言之，它用吸收剂和过滤器的组合吸收烟气中的羰基化合物，随后 进行萃取、衍生化，最后用光电二极管阵列进行 HPLC分析。丙烯 醛不能用标准的 2,4-二硝基苯肼盒进行分析，因为衍生物在样品收 集过程中不稳定并且在分析盒中分解 [47-51]。在氢醌 -2,4-二硝基苯肼 方法 [52]和 CX-572方法 [45, 52]中，丙烯醛不分解，因为包括丙烯醛在 内的羰基化合物被收集在吸附剂氢醌或 CX-572中。 经过验证，WHO SOP羰基化合物可用于 ENDS气溶胶中羰基 化合物的分析。因为香味等其他成分可能会造成干扰 [53]，应采取措 ·77· 3. WHO烟草实验室网络标准操作规程对电子烟碱传输系统评估的潜在应用 施确保分析的有效性和适用性。极端的测试条件（例如非常高的电 池电压）可能会产生超过用户通常暴露水平的羰基化合物含量 [54]。 在气溶胶产生过程中需要进行进一步的调查以对设备参数进行标准 化设置。 3.6.2  溶剂 尽管丙二醇和植物甘油通常被称为“保润剂”，但这些化合物 在电子烟液中作为溶剂并在雾化过程中形成液滴，当存在于电子烟 液中时，在气溶胶中携带烟碱和香味化合物以促进吸入 [55]。溶剂可 以单独使用或者两种混合使用 [56]。一些电子烟液含有低分子量聚乙 二醇，它可能是纯的也可能与丙二醇或甘油形成混合物。使用聚乙 二醇 -400是因为它在室温下为液体，且在药物产品中用作赋形剂 [57]， 所以容易获得高纯度。 Rainey等 [58]证明 GC-FID和 GC-MS可用于测量烟草中的化学 物质， 尽管 GC-MS被推荐用于甘油和三甘醇的全色谱分离。然而， RIVM的报告支持 GC-FID对电子烟液中溶剂的综合分析的适用性， 该报告使用该方法对丙二醇、甘油、聚乙二醇、二甘醇和烟碱进行 定量 [29]。 该方法被证明适用于电子烟液，并提供了包含烟碱的优点。建 议将其作为综合方法的起点，包括关注溶剂、化学相关的污染物， 如乙二醇和二甘醇以及烟碱。为了定量聚乙二醇，需要包括目的分 子量范围内的聚乙二醇分子的标准品。这样的标准品可以在市场上 购买（例如 Sigma Aldrich 81396）。 ENDS气溶胶中的溶剂可以采用标准的 44 mm CFP收集。R. J. Reynolds观察到 ENDS气溶胶中超过 98%的甘油和丙二醇被捕获在 ·78· 烟草制品管制科学基础报告： WHO研究组第六份报告 CFP上 [22]。对各种电子烟液（RIVM，个人通信）进行的实验表明， 过滤器上收集的 TPM的量与液体损失量密切相关。CDC烟草实验 室对这些结果进行了复制和证实（未发表的数据）。它们非常重要， 因为它们表明用于常规卷烟分析的玻璃纤维滤片可以高效地保留溶 剂，溶剂是从电子烟液产生的气溶胶 TPM中存在的含量最高的化学 物质。可以用甲醇从过滤器中提取溶剂，并且通过用于分析电子烟 液的相同方法将提取物直接注射到 GC上。建议将此方法作为 ENDS 气溶胶中溶剂定量详细方案的基础。 一个问题是电子烟液含有大量的香味成分 [53]，它们可能与溶剂 一起洗脱并干扰它们的定量。由于电子烟液中可能存在许多不同的 香味成分，因此优化色谱方法以确保溶剂完全分离将是非常耗时的。 因此宜使用更具选择性的 GC-MS方法代替 GC-FID。任何需要验 证的方法都应适用于各种产品类型，包括添加大量香味物质的产品 类型。 一些作者报道 GC-FID或 GC-MS方法可用于定量烟草中的保 润剂 [58]。用于测定烟草中植物甘油、丙二醇和三甘醇的 TobLabNet SOP-06已被验证 [59]，并提供了该方法的 GC-FID和 GC-MS变体。 预计 SOP将适用于 ENDS电子烟液和气溶胶中的溶剂分析。有人提 出， ENDS电子烟液中测量烟碱的方法可适用于同时测定溶剂（甘 油和丙二醇）和烟碱。 考虑到可能的干扰需要开发更多的方法来优化 ENDS电子烟液 和气溶胶中溶剂的测定。应该考虑调整现有的同时测定甘油、丙二 醇和电子烟液污染物的方法。 ·79· 3. WHO烟草实验室网络标准操作规程对电子烟碱传输系统评估的潜在应用 3.6.3  挥发性有机化合物 一些挥发性有机化合物（VOC）是强效致癌物质，因此也是降 低烟草制品毒性的政策和法规的潜在目标。例如，主流烟草烟气中 的苯和 1,3-丁二烯在WHO FCTC第 9条和第 10条中被列为重点关 注物质 [24]。关于 ENDS再填充电子烟液、烟弹和气溶胶中挥发性有 机化合物的分析报告已经发表。Laugesen[26]在 ENDS电子烟液烟弹 中发现了二甲苯和苯乙烯，国家烟草质量监督检验中心在 ENDS再填 充电子烟液中发现了几种VOC，包括苯、苯乙烯、乙苯和甲苯 [60]，其 中一些被 IARC归类为致癌物或可能致癌的物质 [23, 35, 61]。Goniewicz 等 [33]在 ENDS气溶胶中检测到甲苯、间二甲苯和对二甲苯，每个 ENDS的甲苯含量为 0.2~6.3 mg/ENDS（150次抽吸）。VOC可能来 源于烟草提取物、溶剂或其他来源。发现的水平差异可能是由于样 品（气溶胶或电子烟液）的不同性质或所用分析方法的灵敏度不同。 TobLabNet正在验证主流烟气中 VOC的 SOP。预计它适用于 ENDS电子烟液和气溶胶中 VOC的分析。 3.6.4  酚类化合物 酚类化合物是WHO首先列出的 18种优先有害物质，也被列入 39种烟草制品有害成分和释放物清单 [24]。大多数关于酚类化合物的 分析研究都集中在卷烟烟气上，关于它们在电子烟液或 ENDS气溶 胶中的研究很少。在再填充的电子烟液中检测到对二羟基苯和邻二 羟基苯、苯酚和间甲酚、对甲酚和邻甲酚的总量为 0.5~5 μg/g。烟碱 含量和酚类含量之间没有相关性，这意味着酚类化合物来源于烟碱 ·80· 烟草制品管制科学基础报告： WHO研究组第六份报告 以外的成分 [56]。 带荧光检测的 HPLC是测定卷烟烟气中酚类化合物最常用的方 法。加拿大卫生部 [62]和 CORESTA[63]都推荐用这种方法分析主流卷 烟烟气中选定的酚类化合物。我们期望用于测定烟草排放物中酚类 化合物的方法可以扩展到化学性质较差的电子烟液和 ENDS气溶胶 中。应该建立相应的 SOP。 3.6.5  金属 WHO FCTC第 9条和第 10条中，金属未被列入烟草和主流烟 草烟气的最初优先管制清单。但是，ENDS设备包含几个金属部件， 包括接线、加热元件、焊接连接和结构部件。ENDS设备中常见的 金属元素包括铬、镍、铝、铁、铅、锡和金。金属也可能在制造过 程中被引入电子烟液中，或在烟草植物中提取烟碱过程中被作为污 染物引入。几家实验室已经使用电感耦合等离子体质谱和扫描电子 显微镜鉴定了电子烟液和气溶胶中的金属 [29, 33, 64]。Williams等 [64]使 用这种方法来识别电子烟液中的无定形和纤维状颗粒。 电感耦合等离子体质谱技术是一种用于分析各种基质中金属的 高灵敏度、多功能的技术，已经证明其适用于分析电子烟液 [29, 33, 64]。金 属可能以小金属颗粒的形式存在于电子烟液中或以离子形式溶解 [64]。 由于不同形式的生物利用度和毒性差异很大，因此任何方法都应区 分不同形式。不同的物种可以通过 HPLC分离，建议开发 HPLC-电 感耦合等离子体质谱法来分析电子烟液中的金属。可能需要额外的 样品制备步骤来溶解金属颗粒。注意硝酸（用于溶解金属）和甘油（电 子烟液的常见组分）反应可能会产生硝酸甘油，硝酸甘油是一种摩 擦敏感型炸药。因此，安全有效的样品制备程序势在必行。 ·81· 3. WHO烟草实验室网络标准操作规程对电子烟碱传输系统评估的潜在应用 WHO尚未编制烟草或主流烟草烟气中的金属 SOP。一些研究 人员报告使用石英 CRF收集电子烟液中的金属 [29, 64]，然而，CFP已 经含有金属，这可能会导致基线水平较高 [65]。石英 CFP在使用前可 用稀盐酸和硝酸浸泡，以降低金属的背景水平 [65]。用于收集分析金 属气溶胶的 CFP的一种可能的替代方案是Whatman 47 mm QMA级 过滤器（货号：1851-047），其被发现含有低水平的金属 [22]。它们 直径较大因此需要制造适当尺寸的过滤器支架。建议采取预防措施 以确保准确测量电子烟液中金属以及收集和分析 ENDS气溶胶。 3.6.6  香精 WHO FCTC第 9条和第 10条中关于烟草和主流烟草烟气的优 先管制清单中未包括香精。电子烟液可提供超过 7500种独特口味， 每天都会引入新口味 [66]。食用大部分电子烟液香味剂“通常被认为 是安全的”（GRAS）。但 GRAS认证不适用于高温加热并吸入的化 学物质，因此，不保证 ENDS气溶胶吸入香精是安全的 [67]。尽管有 证据表明吸入 ENDS第一手气溶胶可对健康造成影响 [68, 69]，但香精 的作用在很大程度上是未知的。尽管如此，在电子烟液中报道的一 些类型的香味化合物会造成潜在的健康风险 [70]。 Farsalinos等 [71]在 69%的再填充电子烟液和甜味的气溶胶发现 了双乙酰和乙酰丙酰基化合物，这种化学物质具有黄油口味。电压 对 ENDS气溶胶中双乙酰水平没有明显影响，3.8 V下消耗 438 ng/mg ENDS烟液，而 4.8 V下消耗 433 ng/mg电子烟液 [1]。尽管测得的二 酮浓度明显低于传统卷烟，但一些测试产品含有浓度高于职业接触 限值的乙酰丙酰和双乙酰。双乙酰暴露与严重的呼吸系统疾病有关， 包括闭塞性细支气管炎或“爆米花肺”。最近报道了第一例由于使 ·82· 烟草制品管制科学基础报告： WHO研究组第六份报告 用有香味的电子烟液而导致的“爆米花肺”病例 [72]。 其他常见的香味添加剂也值得关注。例如，肉桂味电子烟液含 有对培养细胞有毒性的肉桂醛和 2-甲氧基肉桂醛 [73]，并且发现电子烟 液中肉桂香味化学物质的数量和浓度与有害性之间存在直接关系 [74]。 许多电子烟液使用吡嗪作为添加剂。这些化合物的添加使吸入更容 易，并且降低传统卷烟中与烟碱相关的刺激性 [75, 76]，减轻吸烟新手 使用 ENDS的反应 [77]。甜味或“糖果”味使 ENDS产品对儿童或新 手具有吸引力 [78]。 有关测量电子烟液和气溶胶中香味添加剂的文献是有限的。最 近对三种商业电子烟液进行 18种香味剂调查发现，一种名为“经典 烟草”的电子烟液中含有可检测到的香兰素，而另外两种（“泡沫” 和“莫吉托混合物”）检测到七种香味化合物 [1]。分析产品最常用 的技术是 HPLC、GC-MS和 GC-MS/MS。二酮类化合物如双乙酰和 乙酰丙酰基在 HPLC-MS平台上测定 [71]。通过 GC-MS和 GC-MS/MS 对电子烟液中的其他香精（包括薄荷醇、香草醛、邻氨基苯甲酸甲酯、 苯甲醛和胡椒醛）进行定量 [3, 79]。用于分析烟草中的成分和有害物 质的大多数方法可以扩展到电子烟液和气溶胶。对于含有大量不同风 味的电子烟液的分析，必须使用非特定检测器的方法保证色谱分离。 3.7  关于扩展方法的建议 2015年 9月，在菲律宾马尼拉的WHO烟草制品检测和研究合 作中心举行的一次会议上介绍了根据报告和观察到的 ENDS电子烟 液或气溶胶中有害物质的存在情况来考虑延伸WHO SOP的情况。 ·83· 3. WHO烟草实验室网络标准操作规程对电子烟碱传输系统评估的潜在应用 表 3.1列出了汇总的更新版本。 表 3.1 建议用于延伸当前和未决 WHO SOP 的决策矩阵 当前方法 当前 TobLabNet SOP对建议矩阵的适用性 电子烟液 气溶胶 烟草中的烟碱（填料） ？ 不适用 主流烟气中的烟草特有亚硝胺 ？ ？ 主流烟气中的苯并 [a]芘 不适用 不适用 主流烟气中的烟碱 ？ 适用 烟草中的保润剂 可能适用 可能适用 主流烟气中的挥发性有机化合物 不适用 可能适用 主流烟气中的羰基化合物 不适用 可能适用 许多因素，特别是电压和电子烟液的成分会影响一手烟的化学 成分。例如，对于给定的电池和雾化行为，线圈温度（形成烟气量 的主要因素）可能在不同设备之间差异很大 [1]。CORESTA推荐的 方法 No. 81[8]和WHO SOP-01具有固定数量的抽吸数（≥50以确保 CFP上足够的 TPM）足以用于 ENDS气溶胶抽吸生成的标准化公开 可用模式，评估WHO SOP在一次性和可再填充 ENDS样品中分析 烟碱、TSNA和苯并 [a]芘的应用情况。在方法扩展期间生成的报告 应该包含一条声明：CORESTA/WHO方法是为了方便而使用的，该 方法不是基于消费者如何使用 ENDS，其不适用于所有当前或未来 的 ENDS产品。 CORESTA方法第 81条的抽吸模式为 (55±0.3) mL的抽吸体积， (30±0.5) s一次的抽吸频率，18.5 mL/s的流速，矩形或方形的抽吸 分布形状，(3±0.1) s的持续时间，并记录口数。CORESTA推荐的 方法规定，它适用于各种一次性和可再填充的 ENDS（电子烟、电 子雪茄）。因此，它可以用于方法扩展研究中使用的“cigalike”产 ·84· 烟草制品管制科学基础报告： WHO研究组第六份报告 品。WHO关于深度抽吸的 SOP指定的吸烟量为 (55±0.1) mL，频率 为 30 s，持续时间为 2 s。由于WHO SOP是为烟草卷烟开发的，因 此没有规定流速。 在方法验证中使用的分析吸烟机应能够吸取固定体积的空气， 包含控制吸烟量、持续时间和频率的装置，可以通过机械和电力驱 动且两者能够充分互补，能够产生长方形的抽吸轮廓，并能够在吸 烟结束后清除。机器应在每个端口处计数抽吸次数。根据产品的不同， 启动时间应不迟于 0.1 s，或者在启动抽吸 0.1 s后流速上升到流量的 50%以上，并且不应在操作员或传感器完成并终止输出后 0.1 s内停 止。ENDS设备支架应该无泄漏，空气和气溶胶不能透过。仪器中 的压强降低不应超过 300 Pa，并且整个过程中，房间内的温度和相 对湿度应分别保持在±2℃和±5%的偏差范围内，例如吸烟机。气 雾捕集器支架应该是气密的，具有非吸湿性、化学惰性的端盖，过 滤器的保留效率应为 140 mm/s速度下邻苯二甲酸二辛酯气溶胶直径 ≥0.3 μm的所有颗粒保留 99.9%，按质量分数计，黏合剂的含量不得 超过 5%，气雾收集完成后压降不应超过 250 Pa。 对于任何新方法或交叉矩阵方法的扩展，应在低（例如 25%）、中等（例如 50%）和高（例如 75%）（对应于可报告的分析 范围，例如在 100%±10%回收率下可接受）加标水平下测量目标分 析物的回收率，以确定香味剂或液体制剂是否偏向目标分析物的结 果。只有新产品应该用于测试目的。 3.7.1  烟碱 由于 ENDS中的烟碱在密闭容器中为液体形式，因此应修改 ISO 3402中的 ENDS调节方法和 SOP-04中修订的 ISO标准程序。 ·85· 3. WHO烟草实验室网络标准操作规程对电子烟碱传输系统评估的潜在应用 在卷烟中，烟叶被纸包裹，容易受到介质及其储存条件的影响。 应该确定是否需要调节 ENDS或烟弹。样品中烟碱的含量取决于 ENDS的品牌和型号，因此，萃取和校准曲线的范围必须根据待 分析的烟液体积和烟碱浓度进行优化。电子烟液中烟碱的浓度通 常在 0~36 mg/mL范围内，上限范围远高于深度抽吸模式下吸烟 机（0.3 mg/mL）产生的卷烟烟气提取物的烟碱浓度（CDC，未发 表的数据）。加入萃取溶液中的分析物体积（例如约 0.25~0.5 mL）应 调整至现有校准范围内。由于丙二醇和甘油是烟碱的溶剂，因此应 评估烟碱回收率，因为这些化合物不溶于某些溶剂（如己烷）。电 子烟烟液中的烟碱可用异丙醇进行萃取后分析，如标准 ISO方法中 关于烟气中的焦油、烟碱和 CO分析或者对WHO SOP-04进行适当 修改。应重新计算含有丙二醇和 /或甘油的新基质的分析方法。 在 ENDS气溶胶中观察到的烟碱含量范围与烟草烟气中报道的 烟碱水平相当。吸烟者可能需要适应不同的 ENDS设计和配置。英 国一项有关各种 ENDS产品（罐装式，可充液式，一次性）的研究 发现，电子烟液中的烟碱浓度与气溶胶中的烟碱浓度之间的关联没 有统计学意义 [71]。但是该项研究中没有测量电压，而电压会对吸烟 机产生的气溶胶中的烟碱水平产生影响 [80, 81]。有关电压的设置目前 正在讨论中，具体应该考虑到向消费者递送最大化（类似于加拿大 传统烟草卷烟的“深度”抽吸模式）和使用制造商推荐的“预热” 选项。 3.7.2  烟草特有亚硝胺 研究发现电子烟液和气溶胶中的 TSNA含量很低，并且不同品 牌之间的差异很大 [33, 39, 82]。例如，Goniewicz和他的同事 [33]在波兰 ·86· 烟草制品管制科学基础报告： WHO研究组第六份报告 购买的 12个 ENDS中，有 10个 NNN含量范围为 0.8~4.3 ng，NNK 含量范围为 1.1~28.3 ng。由于在 Goniewicz研究中测试的 ENDS中不 含有烟草，因此气溶胶中测得的 TSNA可能是直接从电子烟液中转 移而来的。据推测，电子烟液中的 TSNA是烟碱提取时的污染物。 WHO SOP-03中一项有关烟草主流烟气中 TSNA的报道表明 ENDS 气溶胶的化学性质与WHO SOP规定的一致，可以进一步对 CFPS或 电子烟液中收集的气溶胶中的 TPM进行分析。 如上所述，ENDS气溶胶中 TSNA的报告水平低于WHO SOP规 定的烟草烟气中 TSNA的报告限值。如果纳入烟草的“混合型”ENDS 产品设计，气溶胶中的 TSNA水平可能更高。 3.7.3  苯并 [a] 芘 尽管预计气溶胶中的苯并 [a]芘浓度远低于卷烟烟气中的苯并 [a]芘浓度，但是WHO TobLabNet SOP-05仍适用于 ENDS气溶胶中 苯并 [a]芘的分析。因此，要分析一个烧瓶中 CFP的数量，提取溶 剂的体积以及校准曲线的范围需要进行相应的调整。 由于 ENDS气溶胶中丙二醇和甘油的浓度很高，所以当用环己 烷作为萃取溶剂时，应当评估丙二醇 -甘油基质中苯并 [a]芘的回收 率。如果环己烷中的回收率较低，则应评估其他萃取溶剂以确定丙 二醇和甘油的溶解度。对于新的检测条件，应重新计算分析校准。 由于在电子烟液或ENDS气溶胶中PAH含量很低甚至没有 [26,36-39]， 因此建议不对 ENDS或其气溶胶中的苯并 [a]芘进行分析，因为该结 果不会显著影响公众健康或监管决策。 ·87· 3. WHO烟草实验室网络标准操作规程对电子烟碱传输系统评估的潜在应用 3.7.4  挥发性有机化合物 在烟草主流烟气中挥发性有机化合物的 SOP在 TobLabNet中得 到了验证，它可以用于 ENDS气溶胶中 VOC的分析。除 1,3-丁二烯 和苯外，其他有害VOC也可能存在于电子烟液和气溶胶中，如甲苯、 苯乙烯和乙苯。然而，气溶胶中 VOC的浓度可能远低于烟草主流烟 气中的含量 [33]。因此，应对抽吸次数、碳分子筛的类型、萃取剂体 积和标准曲线的范围进行相应地调整。 3.7.5  羰基化合物 羰基化合物是在电子烟液蒸发过程中产生的，很多报道已表明 在 ENDS气溶胶中含有羰基化合物。在大多数研究中，ENDS气溶 胶中的羰基化合物含量是微量的或比烟草卷烟烟气中的含量低很多 [43]。 对其进行检测时应考虑溶剂、器件设计（如可填充式、一次性）和 电压的选择。 “干烧”，当烟芯不能充分地接触电子烟液时，由于烟弹是空 的或线圈过热，可能导致有害化学物质的形成 [83]；然而，这种现象 并不是一般吸烟者的使用模式 [84]。需要重新评估和修改WHO SOP 中有关烟草主流烟气中的羰基化合物的分析规范，以考虑 ENDS气 溶胶中潜在的 ENDS特定释放及浓度，包括乙二醛和甲基乙二醛， 据报道它们在 ENDS气溶胶中存在，但在卷烟烟气中不存在 [85]。 在气溶胶中检测到了有害和致癌的羰基化合物 [86]，因此是减轻 烟草制品有害性的政策和法规的潜在目标。因此，需要发展用于分 析烟草烟气及 ENDS气溶胶中羰基化合物的 SOP。 ·88· 烟草制品管制科学基础报告： WHO研究组第六份报告 3.8  为未来监管 ENDS提供数据所需的研究 • 识别或开发标准 ENDS研究产品。 • 识别或开发用于测试 ENDS电池的标准化研究材料。 • 审查和完善商业 ENDS气溶胶发生器的规格。 • 为各种 ENDS开发 ENDS支架和捕集系统。 • 确定当前分析方法是否适用于各种 ENDS，以及应该如何对 它们进行修改使检测结果准确率高、可重复性好。 • 定义 ENDS使用行为的关键方面，包括抽吸持续时间、频率、 容量和计数。 • 确定在气溶胶发生体系中应对哪些产品设计变量（例如可变 电压、电池功率、加热线圈温度设置）进行规定。 • 确定反映 ENDS使用行为的“标准”和“深度”气溶胶生成 方法，对于“强烈”的方法，调整产品设计变量，为监管决 策提供依据。 • 评估单独的监管限制是否适用于早期产品或新一代产品或不 同类型的 ENDS（例如电子雪茄、电子水烟）。 • 调查溶剂和烟碱提取物中杂质的含量，以确定是否需要对杂 质进行常规测试。 • 确定 ENDS气溶胶的 pH是否可以从电子烟液中推导出来， 过程类似于从无烟烟草中推导。 • 评估所有分析方法的干扰、回收率、基质比较和校准曲线的 适用范围。 ·89· 3. WHO烟草实验室网络标准操作规程对电子烟碱传输系统评估的潜在应用 3.9  结    论 在电子烟液和气溶胶中已经检测出了一系列化学物质。考虑 到 ENDS使用的普遍性和这些产品的演变性质，应用现有的和未决 WHO TobLabNet SOP来分析ENDS电子烟液和气溶胶的做法是合理的。 当电子烟液和香精暴露于高温时会生成羰基化合物，而产物中 的苯并 [a]芘和 TSNA则来源于烟碱提取物中的杂质；因此，在常规 检测中它们可能检测不到，即使它们存在，检测出来的水平也很低。 在对一家制造商为一个终端品牌出售的烟弹的研究中，随着烟碱水 平的增加，TSNA水平也增加。此外，有几个独立报告称吸烟机产生 的气溶胶中的 TSNA水平远低于卷烟烟气中的 TSNA水平，且低于 WHO SOP中规定的烟草主流烟气中的 TSNA限值。为了消除电子烟 液和气溶胶中的TSNA，应该要求制造商使用经认证未经污染的烟碱。 由于ENDS中不含有烟草，因此不需要对其中的TSNA和苯并 [a] 芘进行常规检测。但是，这些经过验证的 TSNA和苯并 [a]芘的检测 方法可以让监管机构和研究人员根据他们的意愿筛选电子烟液以及 新出现的“混合”产品。此外，将用于分析烟草主流烟气中 TSNA 和苯并 [a]芘的WHO SOP扩展到 ENDS，会为研究人员和监管者提 供适用于未来配置和设计变化的分析方法，其中包含烟草，这些方 法可能会导致有害物质的含量升高。一些主要跨国烟草公司推出了 名为 Heat-Not-Burn的“混合”产品。例如可以释放含烟碱气溶胶的 IQOS[91]，使含烟碱气溶胶通过烟草的 Vype[92]，以及释放穿过颗粒状 烟草胶囊气溶胶的 Ploom[93]。 ·90· 烟草制品管制科学基础报告： WHO研究组第六份报告 建议检测对公众健康及监管具有重要意义的烟碱和有害物质（如 金属），这些物质在 ENDS和其气溶胶中经常可以检测到（超过痕 量水平），且有利于描述潜在的暴露。ENDS装置设计的许多方面 都可以影响气溶胶的组成，包括加热线圈阻力、烟芯设计和材料、 储液器设计和气流开口。 例如，最初（前五口抽吸期间）、稳态条件（第 30口到第 40 口抽吸期间）以及两种电压设置的条件下，当电池设置为 3.8 V时， ENDS气溶胶中烟碱水平范围为 13.1~23.9 µg/mg；当电池设置为 4.8 V时，烟碱水平范围为 7.6~22.7 µg/mg[1]。因此，应明确规定用于验 证标准分析法的 ENDS装置中的这些特征。此外，预期可以影响以 化合（游离）形式存在的烟碱量的电子烟液 pH目前尚未完全规定。 在开发一种接近设备上限的“深度”气溶胶生成方法时，应对 产品设计变量进行研究。具有明确的关键设计参数的标准化 ENDS 设备的使用将有助于开发更多的 ENDS分析方法。相关研究应该解 决“设备设计中的哪些方面是最重要的”这一问题。诸如 Heat-Not- Burn之类的“混合”产品可能会导致不同的使用行为并且达到更 高的加热温度，进而定性和定量地影响烟气排放，包括可能产生的 CO。 完善烟草填充剂中保润剂的 SOP以检测和定量分析丙二醇、甘 油和聚乙烯（如乙二醇和二甘醇）中的杂质，有助于调查此类杂质 的分布情况，从而引起人们对单独使用乙二醇或甘油时不存在的毒 性的关注。烟碱分析可以与溶剂分析相结合，从而在一次 GC-FID 分析中同时测定这两种物质。在这种情况下，样品制备时应考虑选 用合适的溶剂如甲醇稀释液 [29]。 美国口味和提取物制造商协会（FEMA）发布了以下声明 [67]： ·91· 3. WHO烟草实验室网络标准操作规程对电子烟碱传输系统评估的潜在应用 “FEMA GRASTM中规定的在食品中可以使用的香味成分，不代表 它们可以在电子烟中使用”；且“电子烟和香精制造商和营销商不 应该表示或暗示电子烟中的香精成分是安全的，尽管它们在 FEMA GRASTM规定中可以用于食品。这些陈述不但是虚假的而且会误导 人”。应对新的和现有的电子烟和气溶胶中香精的分析方法进行单 独评估以确保数据质量。由于许多香精中含有酮或醛，当使用非特 异性检测方法分析羰基化合物时，电子烟中的香精会对结果产生干 扰。在对某些香精或短链羰基进行单独定量时，这可能是优势。但 如果结果证明常规分析存在问题，这时候应该使用特定检测器（MS） 进行检测。 鉴于不同装置的烟碱产量变化很大，且吸烟者可以通过调整吸 烟行为来改变烟碱产量，因此应开发不同的生成气溶胶的吸烟机抽吸 方法，来反映 ENDS使用行为的不断变化。不同吸烟者的 ENDS使 用行为差别很大。据报道，每分钟吸烟两至四次时，吸烟体积约为 50 mL，吸烟持续时间为 2~8 s，吸烟间隔为 18~30 s，流速约为 20 mL[1]。 关于吸烟状态变化的报告 [88, 89]表明为了更好地接近吸烟者的使用行 为，应该对吸烟持续时间、频率、容量和数量以及电池功率等几个 参数进行评估。对不同产品设计的程序或设备进行评估时，可能需 要根据 ENDS的研究结果、可能的标准和“深度”溶胶的产生体系 的讨论结果进行修改。此外，还可能需要对电压、加热线圈的功率 或温度进行设置以达到指定 ENDS电源的要求。不同的电池可以与 不同的设备组合使用，其中电池可能是未经调节的（直流电，电池 耗尽时电压较低）或受调节的。受调节的电池可以用于固定电压、 固定功率甚至固定加热元件的温度。最新的高端 ENDS电池模型被 用来测量和调节由某些金属（如钛）制成的加热线圈的温度。此外， ·92· 烟草制品管制科学基础报告： WHO研究组第六份报告 也可以使用实验室电源模拟出的电池。应进行研究以确定电源的 电气规格，包括电压、功率和温度的调节，最大输出的电压、功 率和温度，允许的纹波电流、电压以及频率。因此，需要对气溶胶 生成方案和仪器进行更多的研究，达成共识，以便今后对气溶胶进 行测试。 多位科学家 [78, 90]发现，ENDS的使用模式与传统卷烟的模式有 很大不同。吸烟者们常常通过调整吸烟行为，从而最大限度地提高 烟碱产量，实现血浆中烟碱和可替宁的含量与卷烟吸烟者中的含量 相当 [78]。据观察，ENDS的抽吸时间明显长于普通卷烟。目前尚不 清楚使用行为的程度是否会影响蒸气的化学组成。但是，据报道， 吸烟持续时间和吸烟频率会影响线圈的温度 [1]。仅仅依靠电池和线 圈特性不能预测 ENDS的工作温度 [1]，并且随着 ENDS设备的发展， 需要更多的研究来明确该区域。 总之，大多数国家通过零售和互联网销售，实现了 ENDS的营 销和推广以及它们在消费者中的普及，因此应对电子烟液和气溶胶 的化学成分进行检测，包括对烟碱、溶剂和羰基化合物的测定。其 中金属物质应该被测量以确定它们是否代表了一种健康风险，如果 发现风险，应制定测定金属成分的常规分析方法。如果政策制定者 和监管机构要求对烟碱提取物的质量进行认证，此时，常规的 TSNA 和苯并 [a]芘的测定方法并不适用。此外，诸如Heat-Not-Burn的“混 合”产品的出现，使得可能需要对烟草衍生有害物质进行额外的测试， 如 TSNA和多环芳烃。香精、酚类和 VOC以电子烟液和气溶胶的存 在可能会影响产品潜在的毒性，在将来的讨论中也应该考虑它们。 ·93· 3. WHO烟草实验室网络标准操作规程对电子烟碱传输系统评估的潜在应用 3.10  建    议 • 实验室已经有足够的数据证明 ENDS电子烟液和气溶胶中 存在烟碱、保润剂（溶剂）、羰基化合物、苯并 [a]芘和 TSNA。 • 如果政策制定者和监管机构要求对烟碱提取物的质量进行认 证，此时，常规的 TSNA和苯并 [a]芘的测定方法并不适用。 • 诸如 Heat-Not-Burn的“混合”产品的出现，使得可能需要 对烟草衍生有害物质进行额外的测试，如 TSNA和多环芳烃。 • 建议测量电子烟液的 pH，确定电子烟液的 pH范围，因为 这一信息可能有助于调查烟碱的成瘾潜力 [21]。 • 对金属物质的含量进行测定，确定它们是否代表了潜在的健 康风险；如果是的话，应该开发检测金属物质的常规分析方 法。 • 在将来的讨论中应考虑香精化合物、酚类和 VOC，因为它 们存在于电子烟液和气溶胶中，且可能会影响产品的毒性。 • 开发一种“深度”气溶胶生成方法以接近设备的上限，系统 地包括产品变量的相对重要性，并应建立一个可以用来比较 气溶胶的 ENDS标准装置。 • 适用于当前和未来的 ENDS产品设计在强烈吸烟条件下的 CORESTA方法或者 SOP（例如 SOP-01）仍有待确定。随着 数据的建立以及产品的不断发展，必须重新评估气溶胶的产 生机制。 ·94· 烟草制品管制科学基础报告： WHO研究组第六份报告 • 对于一种方法的交叉矩阵验证，应比较每个矩阵中每个分析 物的校准曲线的斜率，评估每种方法的等效性。 • 作为方法开发或新样本基质扩展的一部分，建议采用低中等 和高等水平的回收率研究，以确保适用性。 • 研究样品制备技术以确保其与电子烟液溶剂（丙二醇和甘油） 的相容性。在一些情况下，萃取溶剂和基质溶剂的混溶性可 能导致萃取不足。 • 测试程序应要求使用新的、未使用的产品，并遵循制造商提 供的预热的建议。 3.11  参 考 文 献 [1] Sleiman, M., et al., Emissions from Electronic Cigarettes: Key Pa- rameters Affecting the Release of Harmful Chemicals. Environ Sci Technol, 2016. 50(17): p. 9644-51. 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Japan Tobacco begins nationwide e-cigarette rollout next year. 2016 October 7, 2016; Available from: http://asia.nikkei.com/ Business/Companies/Japan-Tobacco-begins-nation wide-e-ciga- rette-rollout-next-year. ·107· 4. 水烟的有害内容物和释放物 Marielle Brinkman，美国 Battelle 烟草研究公共卫生中心 Alan Shihadeh，贝鲁特美国大学（黎巴嫩贝鲁特）烟草研究中心 目录 4.1 引言 4.2 抽吸方式和释放物测试方案 4.3 有害物质的含量及释放量 4.4 测试方法对水烟有害物质释放量的影响 4.4.1 抽吸模式 4.4.2 热源 4.4.3 烟草温度 4.4.4 水的影响 4.5 水烟设计对水烟烟草制品释放物的影响 4.5.1 组件和配件 4.5.2 实际水烟和研究级水烟 4.5.3 水烟软管 4.5.4 水烟托和铝箔 4.6 结论 4.7 对监管部门的建议 4.8 参考文献 ·108· 烟草制品管制科学基础报告： WHO研究组第六份报告 4.1  引    言 广义地说，“水烟”是一种常用于吸烟的器具，其特征是容器 中的烟气会通过水柱。在美洲、非洲和亚洲，甚至在引入烟草之前 就已经有使用水烟类似物的报道 [1]。近年来，在亚洲西南部和北非 地区，水烟类似物的使用更加流行——它们通常被称为“narghile”、 “shisha”或“hookah”， 吸引了全球各地的年轻人和新烟草使用者。 图 4.1展示了这种类型水烟的主要特征。它的组装元件有：水烟头（煅 烧的黏土）、瓶体（金属）、水碗（玻璃）和波纹软管（皮革或尼 龙缠绕在塑料管上），每个元件都有各种不同的尺寸。普通水烟的总 高度可以从大约40 cm到1 m以上，软管的长度从75 cm到150 cm不等。 图 4.1 Narghile 水烟 [2] ·109· 4. 水烟的有害内容物和释放物 水烟烟草的水分和保润剂含量高，不会自行维持燃烧，因此需 要将燃烧的炭块放在烟草顶部来维持它的燃烧。木炭需要定期补充 或进行调整以便维持吸烟者所需的烟气强度。通常，燃烧的木炭会 被放在附近的火箱中，特别是在提供水烟的餐馆和咖啡馆里。为了 避免每次吸烟时都要准备和维护火箱，水烟使用者也可以使用快速 点燃的木炭块。有趣的是，在一次会议中木炭的消耗量和maassel（一 种重度调味烟草混合物）的消耗量相当 [3]。 当吸烟者从软管吸吮时，空气也会被吸入并被头部的木炭加热。 热空气和木炭燃烧产物然后会通过烟草，产生烟气。因此，除了烟 草制品产生的烟气之外，水烟烟气中还含有木炭烟气。烟气从水烟 头通过瓶体内的中央管道，然后以气泡的方式过水后再进入软管。 最后，当烟气到达烟嘴时，它已经冷却至室温并被加湿。因此，在 吸水烟时，除了可以感受到凉爽、潮湿、甜味气溶胶外，在吸气时 还可以感受和听到烟气经过水的声音。 最常见的两种水烟头配置是 maassel配置和 ajami配置。其中 maassel配置的头部相对较深（约 3 cm），里面可以填充大约 10~20 g maassel（在阿拉伯语中被称为“honeyed”），它含有 65%的保润剂（主 要是甘油）[4]。另外，其中还含有烟草、水、香精和其他添加剂。市 场上有数百种口味的 maassel烟草，如水果、糖果、饮料、香料、鲜 花和香草等口味。maassel上覆盖有铝箔片，用于空气流通 [图 4.2(a)]， 燃烧的炭放置在铝箔的顶部。在第二种配置中，使用的是更传统的“未 经调味”的 ajami烟草（通常称为 tombac，或阿拉伯语中的“烟草”）。 吸烟者将少量的水与干的、切碎的烟草混合制成可模压的矩阵，它们 可以在浅黏土头上形成一个土墩 [图 4.2(b)]，炭直接放置在湿润的烟 草上。目前，世界上使用最多的是maassel配置。 ·110· 烟草制品管制科学基础报告： WHO研究组第六份报告 （a） （b） 图 4.2 水烟头 （a）maassel配置，烟草在铝箔下面；（b）ajami配置，烟草在头部上面且上面 没有铝箔将烟草与炭分开 近期，商店已经开始售卖无烟草的maassel，声称这是一个“健康” 的选择。然而，除了不含烟碱外，这些产品产生的烟气的有害物质 传递曲线和生物活性物质与含烟草的型号基本相同（见第 4.3节）。 4.2  抽吸方式和释放物测试方案 与卷烟不同的是，nurgices允许每口吸入的烟气量较多，主要 原因是它们的阻力较低，这与自由吸入非常类似。每口吸入体积为 1000 mL在水烟中很常见，但在卷烟中只有 30~50 mL。因此，吸一 口 narghile所吸入的烟气量可能与吸一支卷烟所吸入的烟气量相当。 一般吸烟时，会在一小时内吸数百口，累计吸入体积约 100 L[5]。此外， 与卷烟不同，抽水烟没有明确的终点，一般是到吸烟者认为水烟被 消耗为止。一般来说，当吸烟对吸烟者没有吸引力时，如味道改变、 饱腹感或社交圈改变（例如用餐的结束），吸烟者会停止吸烟。 由于吸烟方式对有害物质排放有很大影响，因此在对吸烟机的 有害物质排放进行实验室表征时需要确定抽吸方式，如口数、容量、 ·111· 4. 水烟的有害内容物和释放物 持续时间和间歇时间等 [3,6,7]。目前已经有了一些在不同人群、实验 室条件下或自然环境中有关吸烟参数的研究。在表 4.1中对这些研究 进行了总结，结果显示，平均吸烟体积为 500~1000 mL，持续时间 为 2~3 s，间歇时间为 10~35 s。表 4.1所示的研究结果的不同可能反 映了诸如吸烟年数、吸烟频率和环境等因素对抽吸参数的影响。 表 4.1 已报道的水烟抽吸方式 研究 水烟 卷烟 Shihadeh 等 [5] Maziak 等 [8] Katurji 等 [9] Cobb 等 [10] Alzoubi 等 [11] Pulcu和 McNeil[12] Brinkman 等 [13] Djordjevic 等 [6] 地点 黎巴嫩贝 鲁特 叙利亚 共和国 阿勒颇 黎巴嫩 贝鲁特 美国弗 吉尼亚 州里士 满 约旦伊 尔比德 土耳其伊 斯克斯坦 美国俄亥 俄州哥伦 布 美国纽约 州韦斯切 斯特 场所 咖啡馆 实验 室（30 min） 咖啡馆 实验 室（45 min） 实验室 实验室（30 min） 实验室 实验室 参与人数 52 61 61 54 59.2 20 35 77 吸每口烟之间 的间隔时间（s） 17.0 12.6 15.2 35.4 12.4/8.0 11.7 26.2 18.5 抽吸容量（mL） 530 511 590 834 520/480 1040 640 44.1 每口持续 时间（s） 2.6 3.2 2.8 未报道 2.3/2.7 3.5 4.5 1.5 总口数 171 169 169 75 157/199 120 71 12.1 总容量（L） 90.6 79.1 130 61.6 82.6/91.8 114 45.4 0.523 注：卷烟吸烟方式来源于 Djordjevic等 [6]，以供比较 一些实验数据表明，水烟的吸烟情况受烟碱含量的影响；在盲 法实验中，经验丰富的水烟使用者在吸无烟碱水烟时，吸烟强度更 大 [14]。实验数据还表明，吸烟情况与烟碱依赖程度有关 [11]。尽管如此， 值得注意的是，在吸水烟时所摄入的烟气量是吸卷烟的 10倍以上。 因此，很明显，卷烟抽吸参数不能直接用于水烟抽吸试验。 到目前为止，分析性研究中最常用的水烟抽吸参数是贝鲁特方法 [9]， ·112· 烟草制品管制科学基础报告： WHO研究组第六份报告 持续时间是 2.6 s，吸入体积是 530 mL，间歇时间是 17 s。这种方法是 在贝鲁特地区的咖啡馆进行的，在该咖啡馆中有提供水烟，实时测定 老顾客吸水烟时烟气中的焦油、烟碱和 CO含量以便进行验证 [5, 9]。 4.3  有害物质的含量及释放量 在十年前，实验室研究已经开始用现代分析方法、可靠的吸烟 机和采样方法来研究水烟烟气的化学性质。最近的一项系统综述表 明，水烟烟气中大约有 300种物质已经被确认，其中有 82种已经实 现了定量检测 [15]。除了成瘾物质烟碱外，可以定量检测的物质还包 括致癌物质如 TSNA、多环芳烃、苯、呋喃和重金属，以及其他重要 的有害物质，如挥发性醛类、一氧化氮和 CO。 与卷烟烟气一样，水烟烟气中包含的成分来源于原料（例如重 金属、烟碱、TSNA）、吸烟期间化学合成的成分（例如 CO、一氧 化氮）和原位转移合成的组分（例如多环芳烃）[16]。此外，由于燃 烧的木炭通常被用作水烟的热源，水烟烟气中除了含有来源于烟草 本身的有害物质外，还含有从木炭中产生的有害物质。因此，木炭 和烟草制剂的组成都可以影响烟气成分。水烟烟气中的多环芳烃和 重金属很大一部分取决于炭的 PAH含量 [16]和 maassel产品的重金属 含量 [17, 18]。研究发现，在不同的产品中，这些物质的含量差异很大， 提示颁布限制有害物质含量的法规对其进行调节可能是可行的。 由于已经出版的有关水烟有害物质生成量的报告是针对特定组 合的木炭和烟草产品、吸烟方式和水烟设计，因此所报告的有害物 质含量差别很大。 ·113· 4. 水烟的有害内容物和释放物 尽管如此，正如一项关于水烟烟气中的有害物质及生物活性的 综述所指出的 [15]，迄今为止的所有研究得出的结论都一致，即在吸 水烟时，吸烟者会吸入大剂量的有害物质，其卷烟当量范围从一到 几十不等（见图 4.3）。这些有害物质与吸烟者的成瘾、心脏和肺部 疾病以及癌症有关，在水烟的使用者中也有类似的结果。 T/N/CO 焦油（mg） 烟碱（μg/10） 一氧化碳（mg） 甲醛（μg） 乙醛（μg/10） 丙烯醛（μg） 多环芳烃 醛 砷（ng） 铬（ng/10） 铅（ng/10） NAB（ng） NNN（ng） NNK（ng） 烟草特有亚硝胺 重金属 苯并[a]芘（ng） 卷烟 水烟 浓度 二苯并[a, h]蒽（ng） 茚并[1, 2, 3-cd]蒽（ng） 0 100 200 300 400 500 600 700 800 900 1000 图 4.3 吸 1 h水烟和一根卷烟产生的主流烟气中有害物质的水平 [2] 卷烟数据来源于参考文献 [18,19]，水烟数据来源于参考文献 [3,20,21] ·114· 烟草制品管制科学基础报告： WHO研究组第六份报告 吸烟者的生物标志物检测结果与有关水烟烟气中有害物质排放 的报道一致，生物标志物检测结果显示吸烟者全身暴露于CO、烟碱、 多环芳烃和 TSNA[22-27]。此外，卷烟及水烟吸烟者的有害物质全身暴 露情况的差异与在吸卷烟及水烟时测得的烟气中有害物质释放量相 关。例如，在单位烟碱释放标准化的基础上，水烟吸烟者比卷烟吸 烟者的 CO和 PAH暴露高，TSNA的暴露低。暴露标记物与测得的 有害物质生成量之间的一致性验证了水烟烟气中含有并递送大量的 有害物质。 对这些发现的认识提高可能是无烟草 maassel出现的一个重要原 因，这类产品被称为“为了使用者的身体健康”。除烟碱外，使用 无烟草 maassel产品产生的烟气与常规烟草产品基本上具有相同的有 害物质分布和生物活性（表 4.2和参考文献 [17,28,29]）。 表 4.2 含烟草水烟和无烟草水烟主流烟气中有害物质含量的直接比较 有害物质 水烟（均值±95%置信区间） P 烟草 无烟草 焦油（mg） 464±159 51 ±115 NS 烟碱（mg） 1.04±0.30 <0.01 <0.001 CO（mg） 155±49 159±42 NS 一氧化氮（mg） 437±207 386±116 NS 多环芳烃（ng） 荧蒽 385±74 448±132 NS 芘 356±70 444±125 NS 苯并蒽 86.4±15.2 113±46 NS 䓛 106±16 124±36 NS 苯并 [b+k]荧蒽 64.7±11.3 72.9±12.6 NS 苯并 [a]芘 51.8±12.9 66.1±17.8 NS 苯并 [ghi]苝 33.6±10.2 39.6±10.7 NS ·115· 4. 水烟的有害内容物和释放物 续表 有害物质 水烟（均值±95%置信区间） P 烟草 无烟草 茚并 [1,2,3-cd] 芘 47.3±10.7 44.3±10.4 NS 羰基化合物（µg） 甲醛 58.7±21.6 117.6±78.7 NS 乙醛 383±121 566±370 NS 丙酮 118±36 163±68 NS 丙醛 51.7±15.3 98.4±65.0 NS 甲基丙烯醛 12.2±4.4 20.4±9.7 NS 注：改编自 Shihadeh等 [29]；NS表示无统计学差异；通过吸烟机模拟 31个水烟吸烟者在 62个随意吸烟期间的状态产生烟气，其中每个参与者在临床环境中完成了两次吸烟：一次是他 们喜欢的烟草产品，一次是与之味道匹配的无烟草产品 自从 20世纪 90年代初引入maselel——重口味的水烟烟草以来， 很少有研究对烟草制品中的香味物质进行鉴定和定量 [43]。随着这种 吸烟形式的普及 [44]，在过去的 20年中，制造商的数量和口味的数量 和种类都在稳步增加。在 maselel水烟主流烟气中有几种香精的含量 比卷烟主流烟气中高 1000倍，如香兰素、乙基香兰素和苄基醇 [45]。 Schubert等 [33]用非特异性检测方法初步鉴定了 79种挥发性香精， 并用顶空进样法对埃及、印度、约旦和阿拉伯联合酋长国等地的水 烟烟叶定量鉴定了其中的 11种香精。烟草制品中的香精可以直接通 过烟气被人体吸入，从而对人体健康产生危害。其中一个具体的例 子是肉桂口味的电子烟液。Behar等 [47]研究发现电子烟释放物的细 胞毒性与电子烟液中肉桂醛的浓度高度相关。另一个具体实例是甜 味添加剂如果糖、葡萄糖。Soussy等 [48]研究显示在电子烟雾化过程 中，糖可以热分解为 5-羟甲基糠醛和糠醛。尽管没详细的研究，但 ·116· 烟草制品管制科学基础报告： WHO研究组第六份报告 是一般认为这种分解途径在水烟中也存在，因为水烟中的糖含量高 达 70%，而且 Schubert等 [46]在水烟主流烟气中检测到了 5-羟甲基 糠醛和糠醛。也许在新烟民和烟草烟民中，口味对不良健康影响的 更大的潜在贡献为增加吸烟的吸引力。香精的加入会使原本粗糙的 烟气更加平滑和香甜，使初吸烟者更容易接受并吸入烟气，从而间 接地危害人体健康。美国一项有关有代表性的青少年人群（≤17岁） 的横断面研究表明第一次所吸的烟含有香精与现在吸烟之间具有正 相关 [49]。来自同一研究的最新纵向数据显示，第一次吸烟使用香精 烟草产品的年轻人比使用无香精烟草产品的年轻人更容易成为烟草 使用者④。 虽然二手烟不是本报告关注的重点，但应该注意的是，环境暴 露于水烟也会对健康造成重大危害。在实验室研究中，在吸烟期间 大量的挥发性醛类物质、CO、PAH和纳米粒子直接从烟头释放到 环境中 [50]。据估计，吸烟一个小时，一名水烟吸烟者释放的有害物 质量相当于 2~10名卷烟吸烟者释放的有害物质量。对使用水烟时的 周围环境进行研究，观测报告表明使用水烟会导致环境中细颗粒物 （PM2.5）浓度升高 [18, 51-53]。 4.4  测试方法对水烟有害物质释放量的影响 检测烟草产品排放的规程应包括取样，准备水烟烟草产品，从 ④ Villanti AC. Are youth and young adults who rst try a avored tobacco product more likely to continue using tobacco? 来源于 PATH研究，2017年 3月意大利佛罗伦萨烟碱和烟 草研究学会年会 ·117· 4. 水烟的有害内容物和释放物 产品中产生、收集和量化主流烟气中的有害物质整个过程。这些规 程中应该包括的内容和需要解决的问题见表 4.3。 表 4.3 需要烟草产品检测规程规范的程序、活性和变量 检测规程 活性 具体检测变量 准备烟草样品 和水烟 同质化 购买的数量？是否去除树枝和细枝？储存条件？储存 稳定性？ 条件 存储过的烟草还是新打开包装的烟草？如果有条件， 多长时间以及在什么温度和湿度下？ 烟草包装 用溶剂或水清洗烟头？烟草包装松散或紧密？铝箔穿 孔？烟草数量？ 管道和软管清洗 有机溶剂和 /或水清洗？每次使用新鲜软管？检查空 气渗入率？ 样品生成 抽吸模式 单级或多级抽吸？高度模仿人体抽吸？抽吸容量、持 续时间和频率？ 热源 木炭还是电？使用木炭的时间和数量？木炭的种类？ 吸烟机 抽吸机器？ 抽吸波形？ 样品收集 微粒和半挥发物质 过滤器的型号和尺寸？多少量过滤器足够？ 气相和挥发性物质 冲击器，吸收剂，罐或袋收集？ 有害物质定量 提取 溶剂？纯化方法？替代标准？ 定量 内部标准？仪器方法？ 由于没有测试水烟释放的标准方案，因此目前还没有有关检测 方案对水烟有害物质释放影响的研究。在这种没有标准规程的情况 下，研究小组已经使用了各种各样的设备和程序来研究水烟释放测 试方法。虽然无法对这些有关有害物质释放的研究进行直接比较， 但这些研究中收集的数据可以用于估计某些变量的影响，特别是抽 吸模式、热源、烟草温度和水碗中的水。目前已经有研究考察了这 些因素的影响，在下文中将对此进行简要讨论。其他因素如烟草包 装（松散与紧密包装）、样品生成（例如热源点火时间、抽吸机制）、 烟气收集条件（例如滤嘴类型、颗粒相的数量和直径、冲击器、吸 ·118· 烟草制品管制科学基础报告： WHO研究组第六份报告 附剂或实时收集）和有害物质定量方法的影响仍然需要调查。一些 用于定量卷烟烟叶中有害物质和释放物的方法经修改后已用于水烟 释放物的检测，但是这些方法都没有相关的参考文献支持或实验支 持，因此，还需要更多的研究来完善这方面内容。 人们已经用吸烟机和各种其他设备分析了水烟主流烟气中的成 分，研究的水烟类型见表 4.4，包括市售水烟 [3]、具有专门设计的实 验室水烟 [21]以及研究级水烟 [13]。市售水烟通常使用径向容积式真空 泵进行抽吸，如旋转叶片、隔膜、活塞或涡旋泵，流速恒定。用计 算机控制电磁阀或手动阀控制抽吸 [3, 17, 44]。在“shisha smoker”和研 究级水烟中，用缓和模式和单冲程活塞来进行抽吸，这种方法更比 真空泵更接近人体吸烟状况。“重放”吸烟机可以很好地模拟人体 吸烟，它也被用于研究水烟排放 [45]。但是不同的水烟设计和吸烟方 式如何影响主流烟气中的有害物质含量目前尚不清楚。 ·119· 4. 水烟的有害内容物和释放物 表 4. 4 水 烟 吸 烟 机 的 抽 吸 模 式 水 烟 泵 装 置 抽 吸 容 量 (L ) 持 续 时 间 (s ) 间 隔 时 间 (s ) 总 抽 吸 口 数 总 抽 吸 容 量 (L ) 抽 吸 时 间 (m in ) 炭 号 /种 类 / 直 径 (m m ) 烟 草 量 (g ) 托 盘 或 箔 (孔 的 数 量 ) 软 管 材 料 贝 鲁 特 方 法 ， Bl ac k Si ng le Pe ar l, K ha lil M am oo n [6 , 4 2] 数 字 电 磁 控 制 机 械 泵 0. 53 2. 6 17 17 1 90 .6 55 .6 1. 5C /3 K in gs /3 3 m m 10 箔 (1 8) 皮 革 和 塑 料 改 良 的 贝 鲁 特 方 法 [2 1] 气 动 单 冲 程 圆 筒 0. 53 2. 6 17 17 1 90 .6 55 .6 1C /3 K in gs / 4 0 m m 10 箔 (1 8) 塑 料 超 级 水 烟 [1 8] 真 空 泵 6 L/ m in 0. 3 3 15 10 0 30 30 1C /S w i -L ite /3 3 m m 10 箔 (1 9) 未 报 道 黏 土 碗 [4 3] a 机 械 泵 和 手 动 注 射 器 每 10 次 抽 吸 1. 0 5 25 10 0 10 0 50 1C /S w i -L ite /3 3 m m 8 托 盘 (未 报 道 ) 未 报 道 研 究 级 水 烟 b 单 冲 程 玻 璃 注 射 器 0. 72 0. 46 4. 6 3. 6 16 .4 28 .7 32 42 74 23 .0 19 .1 42 .1 11 .2 22 .6 33 .8 1C /3 K in gs / 4 0 m m 点 热 源 10 箔 (1 8) 托 盘 (3 0) 塑 料 a. 吸 水 烟 3 分 钟 后 再 采 集 主 流 烟 气 ； b. K ro eg er R R, B ri nk m an M C , B ue hl er S S, G or do n SM , K im H , C ro ss K M , e t a l. Th e im pa ct o f v ar ia tio n of h oo ka h co m po ne nt s on ch em ic al an d ph ys ic al em iss io ns . 2 01 4 年 2 月 6 日 美 国 华 盛 顿 西 雅 图 烟 碱 和 烟 草 研 究 学 会 年 会 ·120· 烟草制品管制科学基础报告： WHO研究组第六份报告 4.4.1  抽吸模式 大多数的水烟释放物的检测都是采用以下三种抽吸模式：水烟 咖啡馆中吸烟行为稳定的定期数据汇总模型 [46]；实验室环境中吸烟 行为的多阶段、稳定、周期性的汇总数据模型⑤；高分辨率、时间分 辨（10 Hz）的“playback”精确模拟每个人的行为 [29, 45, 47]。在第一 种情况下，利用贝鲁特方法 [5, 48]，设置吸烟机的程序为具有矩形波形， 中等吸入量，固定的吸入体积和抽吸时间、频率固定。在第二种情 况下，使用平滑的抛物线波形，具有两个波形和频率阶段——一个 用于吸烟过程的前第三分之一阶段（阶段 1），另一个用于其余阶段 （阶段 2）——说明吸烟者在开始抽水烟时，抽吸强度更大、更频繁、 更剧烈 [5]②。在第三种情况下，收集受试者的吸烟方式，将这种方式 “重现”或上传到吸烟机，使其精确地重复这种吸烟方式。为了比 较第一种和第三种抽吸方案中的释放量，Shihadeh和 Azar[45]对烟草 消耗量、焦油量、烟气温度和 CO生成量进行了比较。结果显示使 用周期性方案时主流烟气中 CO减少 20%，表明在这些方案期间得 到的 CO数据可能低于实际暴露。 Shihadeh[3]采用单级周期方案，探讨了喷水量和频率对水烟烟耗 和主流焦油和烟碱释放的影响。结果显示较大的抽吸容量会使烟草 的消耗增加，这可能是因为通过炭和头部的气流较大，进而使得烟 草温度较高；即使用烟草消耗量和总抽吸容量进行标准化的情况下， ⑤ Kroeger RR, Brinkman MC, Buehler SS, Gordon SM, Kim H, Cross KM, et al. The impact of variation of hookah components on chemical and physical emissions. 2014年 2月 6日 美国华盛顿西雅图烟碱和烟草研究学会年会 ·121· 4. 水烟的有害内容物和释放物 较大的抽吸容量也会引起主流烟气中的 TPM（潮湿和干燥）更多。 在用烟草消耗量标准化焦油量的情况下，将抽吸频率加倍（将抽吸 间隔从 30 s减少到 15 s），同时保持抽吸容量不变，结果显示抽吸 频率加倍并没有显著改变烟碱传送。 4.4.2  热源 一些研究人员已经研究了热源如何影响包括呋喃、VOC和 PAH 在内的有害物质的传送。为了更好地了解特定有害释放物的主要来源 是木炭还是maassel，研究人员用电和炭以及单独的炭（无maassel） 分别作为热源，进行机器吸烟。研究结果显示大部分呋喃在单独使 用炭作为热源的情况下未检出，表明maassel可能是其主要来源 [36]； 而对于像苯和甲苯这样的挥发性有机化合物，无论水烟头部是否有 maassel，烟气中检测出的含量都差不多 [49]。为了确定主流烟气中 CO和 PAH的主要来源，Monzer等 [20]设计了一种电热源来快速匹 配活性炭的空间和时间温度分布，单独收集每个热源的排放物，结 果显示木炭贡献了大部分的 CO（90%）和苯并 [a]芘（95%）。 Kroeger等⑤使用研究级水烟和两级抽吸模式，比较市售电热源 和熏香炭热源，结果发现与熏香炭热源相比，市售电热源组的主流 烟气中细颗粒 PAH（约 50倍）和烟碱（约 4倍）的产率降低，副流 烟气中 CO（约 2000倍）和苯（约 1200倍）的产率也降低了。 4.4.3  烟草温度 羰基化合物如乙醛、甲醛、丙酮和丙烯醛的浓度与烟草的峰值 温度有关，温度越高，这些物质的产率越高 [4]。反过来，烟草峰值 ·122· 烟草制品管制科学基础报告： WHO研究组第六份报告 温度受水烟烟草中的保润剂甘油和丙二醇的浓度的影响，保润剂的 含量越高，烟草峰值温度越低 [36]。 4.4.4  水的影响 一些研究直接或间接地描述了水烟烟气中的有害物质在水中的 溶解情况，通过这种方式这些有害物质可以从主流烟气中被“过滤” 出来。间接研究结果表明，水烟主流烟气中的有害物质浓度跟水的 存在有关。在水存在的情况下，烟碱含量降低了 4.4倍 [3]，羰基含量 降低了 3.7倍 [4]。Schubert等 [49]直接测定了水中酚的含量，发现其 中含有两种酚类物质：苯酚和愈创木酚（水中含量比烟气中分别高 了 7.9倍和 3.3倍）。但是，Shihadeh[3]报道显示无论水存在与否， 焦油的水平没有显著差异。 4.5  水烟设计对水烟烟草制品释放物的影响 4.5.1  组件和配件 在制定检测水烟释放物的规程时，对水烟的组件和配件进行区 分是非常有必要的。水烟的组件指的是水烟中的必要元件，配件则 指的是非必要的可选元件。表 4.5列出了水烟的组件和部分配件及可 能影响水烟排放的物理和化学属性。水烟的整体设计、组件及配件 如软管、托盘和箔片对排放物的影响将在下文作简要讨论。其他组 件和配件对水烟释放的影响目前尚不清楚。 ·123· 4. 水烟的有害内容物和释放物 表 4.5 可能影响烟气排放的水烟组件和配件 组件 用途 物理属性 水烟头 存放烟草 组成材料；几何结构；连接方式；位置，直径和孔 的数量；重量 瓶体 将烟气传送到碗口 组成材料；几何结构；连接方式；浸没深度 水烟杆 将烟气从碗口传送到水 组成材料；几何结构；连接方式；浸没深度 水烟碗 储存水 组成材料；形状（规模）；体积 水 形成气泡 体积；纯度；pH 软管 将烟气从碗传送给使用者 组成材料；长度；内外直径 木炭托盘 或箔 减慢烟草燃烧 制造材料；厚度；形状（直径）；放木炭的区域；位置， 直径和孔的数量；重量 水烟油 增加烟气或气溶胶的产生量 材料；使用量；使用方法（分层或堆放或与烟草混合） 气泡扩 散器 产生的气泡更小，更安静， 减少烟气粗糙感 材料；形状（直径）；位置，直径和孔的数量；安 装时覆盖杆的长度；密封接头的类型 水烟嘴 防止吸入细菌 材料；表面光滑度；长度；内外直径 防风罩 防止木炭熄灭 材料；厚度；形状（直径）；放木炭的区域；位置， 直径和孔的数量； 重量；密封接头的类型 4.5.2  实际水烟和研究级水烟 目前研究已经对商业用途的水烟 [3, 46]和实验室水烟 [13, 21]的释放 物进行了分析。商业用途的水烟的类型和组件在设计和耐用性方面 差异很大，具体包括制造杆、底座、碗和软管的材料；密封头的设计、 密封的程度以及流道的直径。这些所有的变量都会影响从头部传递到 烟草的净热能，进而影响主流烟气中颗粒相的性质和浓度 [4, 36]以及吸 烟者的吸烟行为。商业用途的水烟的制造材料和设计可能在不知不 觉中就已经改变，这可能会对排放测试有干扰。 为了解决上述问题，研究者们设计了研究级水烟，它的结构如 图 4.4所示。为了尽量减少表面化学吸附，并消除来自水烟本身的化 ·124· 烟草制品管制科学基础报告： WHO研究组第六份报告 学物质（如金属焊料和热降解产品）的影响，研究级水烟由惰性材 料制造而成。研究级水烟在精密度和准确度 [13]、批间和批内变异性 [50] 等方面都有基准指标，且临床试验显示吸烟者对它的接受度和满意 度都很高 [13]。 图 4.4 标准研究级水烟设备及吸烟数据的获取 4.5.3  水烟软管 大多数的吸烟机吸烟研究中使用的水烟软管都是由皮革或塑料 制成的。当使用这种软管时，主流烟气中的 TPM和 CO含量会升高 达到原来的两倍多，其原因是软管不仅导致空气透入 [43]，而且会导 致水分从塑料或皮革孔流失 [51]。但是烟碱水平没有显著改变。 ·125· 4. 水烟的有害内容物和释放物 4.5.4  水烟托和铝箔 在大多数吸烟机吸烟的研究中，都使用了箔片或金属托来隔绝 木炭和烟草。Kroeger等⑥比较了研究级水烟在使用金属托或箔片时 主流烟气和侧流烟气中的排放物，吸烟方式为两阶段抽吸模式。结 果发现，当使用金属托盘时，主流烟气细颗粒相中的一些有害物质 较低，包括 TSNA NNN和 NNK（低 2~3倍）和苯并 [a]芘和芘（低 2~3倍）。但是，测流烟气中的一些气相有害物质的含量明显较高， 包括乙醛、乙腈、丙烯腈、苯、1,3-丁二烯和异戊二烯（高 1~3倍）。 总之，水烟烟气排放的测试条件、水烟的组件和配件都可以影 响烟草消耗以及主流烟气和测流烟气的特征和浓度。表 4.6为基于目 前文献报道的测试条件及其对优先有害物质的影响的初步列表。总 的来说，热源对主流和侧流水管烟雾排放的影响最大。 表 4.6 水烟测试条件及其对有害物质释放检测结果的影响 条件 1 条件 2 MS、SS和 BW检测的有害物质 条件 1中的有害物质水平 按一定的周期 抽吸 [45] 相同抽吸模式 MS CO 高 1.2倍 MS TPM（干燥） 无显著差异 烟草消耗 高 1.2倍 抽吸容量 300 mL[3] 抽吸容量 150 mL[3] 烟草消耗 高 1.4倍 MS TPM（潮湿）a 高 3.8倍 MS TPM（干燥）a 高 3.2倍 MS 烟碱 无显著差异 ⑥ Kroeger RR, Brinkman MC, Buehler SS, Gordon SM, Kim H, Cross KM, et al. The impact of variation of hookah components on chemical and physical emissions. 2014年 2月 6日 美国华盛顿西雅图烟碱和烟草研究学会年会 ·126· 烟草制品管制科学基础报告： WHO研究组第六份报告 续表 条件 1 条件 2 MS、SS和 BW检测的有害物质 条件 1中的有害物质水平 每 30 s吸 一次 [3] 每15 s吸一次 [3] MS 焦油 高 1.5倍 MS 烟碱 无显著差异 含烟草 不含烟草 MS 呋喃 [48] 包含约 100%的呋喃 MS 苯 [36] 无显著差异 MS 甲苯 [36] 无显著差异 熏香炭 电热源 [20] MS CO 含有 90% CO MS 苯并 [a]芘 含有 95% 苯并 [a]芘 商用电煤 b MS 烟碱 高 4倍 SS CO 高 2000倍 SS 苯 高 1200倍 烟草达到的 最高温度， 277℃ 烟草达到的 最高温度， 203℃ [4] MS 乙醛 高 3.3倍 MS 丙烯醛 低 1.3倍 MS 甲醛 高 1.2倍 有水 无水 MS 烟碱 [3] 低 4.4倍 MS 乙醛 [4] 低 3.9倍 MS 丙烯醛 [4] 低 3.5倍 MS 甲醛 [4] 低 2.8倍 在水中 b 水烟主流烟气 BW苯酚 高 7.9倍 BW 愈创木酚 高 3.7倍 塑料软管 [43] 皮革软管 烟草消耗 高 1.4倍 MS TPM（湿） 高 2.4倍 MS CO 高 2.4倍 箔片 c 托盘 MS NNN 高 3.2倍 MS NNK 高 1.8倍 MS 芘 高 1.9倍 MS 苯并 [a]芘 高 2.6倍 SS 乙醛 低 1.5倍 ·127· 4. 水烟的有害内容物和释放物 续表 条件 1 条件 2 MS、SS和 BW检测的有害物质 条件 1中的有害物质水平 箔片 c 托盘 SS 乙腈 低 1.4倍 SS 氯乙烯 低 1.5倍 SS 苯 无显著差异 SS 1,3-丁二烯 低 1.9倍 SS 异丙二烯 低 1.6倍 注：MS，主流烟气（主动）；SS，测流烟气（被动）；BW，抽吸后水碗中的水 a. 用烟草消耗量对 TPM进行校正 b. 直接对水进行检测 c. Kroeger RR, Brinkman MC, Buehler SS, Gordon SM, Kim H, Cross KM, et al. e impact of variation of hookah components on chemical and physical emissions. 2014年 2月 6日美国华盛顿西 雅图烟碱和烟草研究学会年会 4.6  结    论 水烟的吸烟方式因人和环境而异，目前很少有研究对该变化的 程度进行定义。迄今为止的所有研究都表明水烟的抽吸量、流速和 口数都比卷烟大得多。所以必须对吸烟机测试方案进行相应的调整。 水烟烟草烟气中含有并可以向人体传送大量的有害物质，这些 物质与烟草相关疾病有关，如烟碱成瘾、肺部疾病、心脏病和癌症。 无烟草水烟的烟气中也含有许多有害物质，且也与烟草相关疾病有 关，如肺部疾病、心脏病和癌症。 水烟的有害物质释放不仅取决于烟草产品，而且还与水烟组件、 木炭类型、烟管设计、吸烟前的准备方法、吸烟方式以及它们之间 的相互作用有关。就目前来说，要保护公众健康，必须对烟草制品 ·128· 烟草制品管制科学基础报告： WHO研究组第六份报告 和木炭的特性和含量进行管理。 水烟吸烟者的数量在全球范围内不断增加，且吸水烟会产生高 浓度的有害物质暴露。因此，水烟应该被纳入烟草控制计划和政策中， 包括禁止香味添加剂和室内吸烟。 4.7  对监管部门的建议 • 要求制造商公布市售水烟（包括 maassel、herbal maassel、水 烟石和其他烟草与木炭混合的产品）中烟草和木炭包含的成 分和污染物（表 4.2） • 要求制造商提供水烟产品（含烟草和无烟草产品）中木炭、 组件（如软管）和配件（如铝箔）信息，并向监管者披露销 售此类产品的意图。 • 一旦水烟管制的法规通过，就要求水烟产品的销售点时刻记 录该类产品与管制措施的复合状况。 • 禁止在含烟草和无烟草的水烟产品中使用香精。 • 禁止在室内使用任何形式的水烟产品。 • 告知吸烟者吸水烟有害健康，因为水烟中含有有害化学物质 和细菌。 4.8  参  考 文 献 [1] Philips JE. African smoking and pipes. 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Tobacco control, pp.tobaccocontrol-2013. ·137· 5. 针对卷烟的 WHO 烟草实验室网络 标准操作规程对水烟的适用性 Marielle Brinkman，美国 Battelle 烟草研究公共卫生中心 Walther Klerx，荷兰国家公共卫生与环境研究所（RIVM）卫生防护中心 Alan Shihadeh，贝鲁特美国大学（黎巴嫩贝鲁特）烟草研究中心 Reinskje Talhout，荷兰国家公共卫生与环境研究所（RIVM）卫生防护 中心 Ghazi Zaatari，贝鲁特美国大学（黎巴嫩贝鲁特）病理学和实验医学系 目录 5.1 引言 5.2 抽吸方法 5.2.1 热源 5.2.2 水烟头 5.2.3 水烟头覆盖物 5.2.4 水 5.2.5 软管 5.2.6 滤嘴 5.3 吸烟机 5.4 水烟烟草取样 5.5 样品制备 5.6 内容物和释放物的测定 5.6.1 水烟烟草的内容物 5.6.1.1 保润剂 ·138· 烟草制品管制科学基础报告： WHO研究组第六份报告 5.6.1.2 烟碱 5.6.2 焦油、烟碱和一氧化碳的释放 5.7 讨论 5.8 结论和建议 5.8.1 对监管机构的建议 5.8.2 对研究人员的建议 5.9 参考文献 5.1  引    言 本部分包含现有和未决 ToBabNET SOP中对于吸水烟的建议，这 些建议是WHO FCTC COP工作组在 2016年 2月的第 9和 10号会议上 通过审议的。第 4章已经描述了全球所使用的水烟的特点。 通过吸烟机对烟草制品的有害物质释放进行实验室测试时，需 要规定抽吸条件，如抽吸容量、持续时间和间歇时间等。由于吸烟 机的抽吸参数对有害物质释放影响很大，因此有必要对抽吸条件进 行规范 [1-3]。到目前为止，某些科学研究已经报道了一些关于水烟吸 烟的研究，其中涵盖了各种人群在实验室和自然环境中的研究。在 第 4章表 4.1中对这些研究进行了总结，结果显示平均抽吸体积为 500~1000 mL，持续时间为 2~3 s，平均间歇时间为 10~35 s。表中各 个研究之间的结果差异可能表明吸烟年数、吸烟频率和环境等因素 会影响吸烟条件。一些研究表明水烟的吸烟形式会受烟气中的烟碱 水平影响。在一项双盲实验中，当吸烟者在使用无烟碱水烟时，吸 烟强度会增强 [4]。实验数据还表明，烟碱依赖的程度会影响吸烟者 ·139· 5. 针对卷烟的WHO烟草实验室网络标准操作规程对水烟的适用性 的吸烟形式 [5]。尽管如此，但值得注意的是水烟的吸烟量是一支卷 烟吸烟量的10倍以上，吸一口水烟的吸烟量与吸整支卷烟相当。因此， 卷烟的吸烟参数并不适用于水烟。 迄今为止，水烟研究中最常用的吸烟方案是贝鲁特方法 [6]，该方 法吸烟条件为：吸烟口数 171、持续时间 2.6 s、抽吸体积 530 mL、间 歇时间 17 s，此外，还需要有木炭的准备和添加程序。该方法是在贝 鲁特地区咖啡馆的两项实地活动的基础上提出的 [6, 7]，并通过测量咖 啡馆顾客吸烟时实时采集的烟气中“焦油”、烟碱和一氧化碳的含量 对该方法进行了验证 [6]。这是迄今为止唯一一种被验证了的方法。 5.2  抽  吸 方 法 如前一节所述，用于卷烟的测试方案并不适用于测试水烟的释 放物。在测定水烟释放物含量的时候，需要考虑到很多特殊因素， 在后文中将对此进行详细讨论。 5.2.1  热源 目前研究中应用最广的热源是熏香炭。用明火点燃木炭以后， 需要将其放在水烟头部 60[8]~100 s[1]后再开始吸烟机吸烟。 研究人员对两种电热源进行了研究，其中一个是实验室制造的 [9]， 另一种是在市场上购买的⑦。研究中，对热源下方和头部的烟草分 ⑦ Kroeger RR, Brinkman MC, Buehler SS, Gordon SM, Kim H, Cross KM, et al. The impact of variation of hookah components on chemical and physical emissions. 2014年 2月 6日 美国华盛顿西雅图烟碱和烟草研究学会年会 ·140· 烟草制品管制科学基础报告： WHO研究组第六份报告 别进行了温度测量，结果显示电热源可以用来模拟炭热源，而且烟气 中的大部分 CO和 PAHs来源于炭 [9]。Kroeger等②研究也表明烟气中的 绝大部分苯来源于炭燃烧。Schubertetal[10]使用其他研究方法也证实了 Kroeger的研究结果。因此，对水烟主流烟气进行研究时，应分别使用 电热源和炭热源，这样有利于确认有害物质的来源。我们建议，将炭 热源和电热源的规程纳入改编的 SOP或为炭排放制定的单独的 SOP。 5.2.2  水烟头 目前研究中常用的制造水烟头部的材料是陶瓷 [1]或金属 [8]，但 是也有少数研究使用的是玻璃 [11]①。尽管还没有被证实，但是普遍 认为由于材料的导热系数不同，可能会影响烟草的温度，进一步影 响主流烟气中的成分和浓度。我们建议在 SOP中对水烟头的材料类 型、厚度、尺寸（包括直径和孔的数量）进行规定。 释放量还取决于烟草的使用量。因此，需要标准尺寸的水烟头， 并计算每克烟草的释放量。为了保证加热装置与烟草之间的距离不 变，水烟头应完全填充，并在上面放上加热装置的特殊盖子。 5.2.3  水烟头覆盖物 在大多数吸烟机吸烟的研究中，都使用了铝箔或带有孔的金属 托盘来覆盖管道的头部，以使炭或其他热源不接触烟草。这两种材 料将热量传递给烟草的效率可能不同，进而可以影响主流烟气中的 有害物质含量。我们建议在相应的 SOP对箔片或托盘的厚度和尺寸 以及孔的数量和直径进行规定。根据所使用热源的不同，覆盖头部 可能会使热传递减少，因此，在进行检测不应该覆盖头部。 ·141· 5. 针对卷烟的WHO烟草实验室网络标准操作规程对水烟的适用性 5.2.4  水 应该对水烟中的水量进行规定和测量，因为它与压降或气流阻 力直接相关，而在吸烟过程中，吸烟者必须要克服这些阻力才能通 过软管吸入烟气。吸烟者通过软管吮吸使碗中的真空度大于另一边 时才能吸入烟气 [1]。这与孔的大小以及水位有很大关系。我们建议 在相应的 SOP中规定碗的尺寸、杆的长度和水覆盖杆的长度。 5.2.5  软管 大多数的吸烟机吸烟研究中使用的水烟软管都是由皮革或塑料 制成的。当使用这两种软管时，主流烟气中的 TPM和 CO含量会升 高达到原来的两倍多，其原因是软管不仅导致空气渗入 [12]而且会导 致水分从塑料或皮革孔流失 [13]。但是烟碱水平没有显著改变 [12]。我 们建议 SOP指定使用塑料软管以减少因皮革的孔隙率不同和湿度造 成的差异，此外，还应该指定软管的长度和直径，因为这些因素会 影响流动阻力和颗粒沉积。 5.2.6  滤嘴 卷烟主流烟气中的大部分烟碱（90%~99%）以质子化的形式存 在，它们会附着在烟气气溶胶上 [14, 15]。卷烟组分的标准分析方法为： 将 TPM收集到玻璃纤维滤纸上，用溶剂萃取，最后用 GC定量 [16]。 在抽水烟过程中产生的 TPM质量可能是卷烟的 10~100倍 [17]。因此， 在水烟机吸烟期间，要保证过滤器不能过载，因为这会造成压降增大， 进而导致样品滞留、损坏过滤器和 /或泵过载。 ·142· 烟草制品管制科学基础报告： WHO研究组第六份报告 在常规测试中，吸烟机吸烟过程中不应该更换过滤垫，因为这可 能会影响抽吸容量。在吸烟机吸烟方案保持不变的情况下，系统无法 在更换过滤垫后检查是否漏气，并且对于重复组分分析，密封系统是 至关重要的。有研究报道，在吸一口水烟时，主流烟气中的 TPM含 量为 1~2.7 g[1, 8]，60%的 TPM被水除去。烟碱可以在水中溶解，在有 水和没有水的情况下分别进行实验，结果表明，约 75%的烟碱被保留 在水中 [1]。水烟气溶胶含水量高，因此在采集烟雾样品时要避免使用 如聚四氟乙烯之类的疏水过滤介质，以避免堵塞；在诸如玻璃纤维芯 的亲水性介质中，水分会沿着过滤纤维传送。烟碱主要存在于粒相物 中，因此，在吸烟机吸烟过程中只要过滤器没有超载（或者变得饱和 以至于在过滤器的背面发现液滴），便可以有效地捕获到烟碱。对于 既可以分布在气相也可以分布于粒相的半挥发性分析物，它保留在过 滤器上的量受颗粒大小分布、吸湿性和抽吸持续时间等变量的影响 [18]。 在 SOP中，建议将主流烟气至少分流为两支，并且安装两个滤 片（直径为 92 mm），以确保粒相物负荷保持在过滤器的承载能力 范围内。对于由于颗粒大小不同和滤片的符合性而导致的半挥发性 化学品的穿透，则需要进行更多的测试。 5.3  吸  烟  机 由于水烟和卷烟的抽吸参数和机械设计不同，用于测试卷烟释 放的分析型吸烟机不能用于测试水烟。为了确定水烟烟气的释放量， 水烟吸烟机必须包含水烟的主要组件，如水烟头、体部、瓶子和抽 吸装置。尽管水烟吸烟机的吸烟参数仍有待确定，但它必须要满足 ·143· 5. 针对卷烟的WHO烟草实验室网络标准操作规程对水烟的适用性 以下要求： • 适用于各种类型和含量的水烟烟草或 molasses的检测； • 适用于不同类型的热源装置（如炭、电加热）； • 组件要耐化学腐蚀、惰性和无污染，包括所有的管道、软管 和连接器； • 对不同类型和尺寸的瓶子都适用； • 抽吸体积至少为 1000 mL； • 能够连接到不同的采集系统以获得粒相物和气相物； • 有管道、软管、收集装置和其他组分，每个组分的长度、直 径和位置都是确定的； • 包括用于设置参数、控制设备以及存储和打印数据的装置。 ISO/TC 126中的一个工作组正在为水烟吸烟机制定定义和标准 条件。而 Borgwaldt GmbH已经制造出了一种用于产生水烟烟气的吸 烟机（图 5.1）。水烟吸烟机中的加热装置和用于确定水烟烟气释放 量的设置都应根据未来的需求和法规进行相应的调整。第 5.4节中具 体描述了检测水烟排放的吸烟机的具体要求及其对释放的可能影响。 建议在检测水烟排放时使用标准的水烟设计和吸烟方案。 图 5.1 Borgwaldt GmbH 制造的分析式水烟吸烟机 ·144· 烟草制品管制科学基础报告： WHO研究组第六份报告 5.4  水烟烟草取样 目前，卷烟主要是根据 ISO 8243进行监管 [19]。该 ISO中描述了 对零售点、制造商及进口商的卷烟样品进行一次取样或一段时间取 样的方法。这个标准中还建立了在 ISO抽吸条件下的焦油、烟碱和 CO释放量的置信区间。自卷烟或自制烟草制品的取样在 ISO 15592 第 1部分 [20]进行了描述，与卷烟取样类似。 在对烟草制品（包括水管烟草）进行抽样时，必须在一次或一 段时间内获得特定产品的有代表性的样品。当消费者所使用的所有 样品都符合规定时，抽样和测试应该一次完成。当检查产品是否符 合规定时，建议隔一段时间采样，但每批次样品都要进行测试。 抽样地点（制造商或进口商的销售点或场所）要根据抽样目的 来选择：确定消费者使用的产品是否符合法规；制造商或进口商或 进口的水烟烟草是否符合规定。由于监管的目的是保护消费者，因 此最好在销售点进行取样。但这样做也存在不足，即制造商或进口 商可能声称在产品离开处所后不对其负责。为了避免制造商或进口 商的操作（预先选择符合要求的样品），建议由政府机构或独立机 构安排抽样。 这些取样建议也适用于相关的待测产品，如木炭。 ·145· 5. 针对卷烟的WHO烟草实验室网络标准操作规程对水烟的适用性 5.5  样  品 制 备 本节所述的样品制备是指从开始准备测试到测试程序开始时的 整个过程中对烟草样品所进行的处理。对于某个特定样品，对其进 行前处理时用到的特殊准备方法也包含在这部分。样品制备的主要 目标是从实验室样品中得到均匀、稳定、有代表性的部分进行测试。 其中比较重要的步骤是水烟烟叶的混合和纯化。 由于所有的水烟销售商都遵守法规限制，因此每个水烟产品的 包装都是一致的。但是水烟烟草却很不均匀。吸烟者在吸烟前可能 要自己剔除烟草中的某些成分，如茎等。对于这些不均一的成分， 需要进行进一步的调查来确定它们对水烟烟草含量及释放量的影响， 此外，也应该明确吸烟者会如何处理这些成分。根据研究结果来决 定在进行样品准备时，是否要剔除这些成分。 检测的次数取决于容许误差的范围和置信区间（CI）。对于卷烟， ISO 8243[19]规定需要对 20只卷烟的测试结果进行平均，其中每一支 卷烟都要在吸烟机上进行分析，以验证其是否符合法规。考虑到每 份水烟之间的差异性和可接受的 CI，对水烟进行测试时应选的样品 数仍然有待确定。测量的CI可以根据不同实验室的研究结果来确定。 ISO 8243[19]中焦油和烟碱的 CI为 20%，CO为 25%。CI主要取决于 每个产品之间的差异和特测定成分的分析变异性，可通过设定最大 可接受的 CI来限制待测物的数量。 测定水烟烟草组分和释放量的第一步是称量一定质量的样品， 在这个过程中，样品的水分含量是一个重要变量，在样品质量相等时， ·146· 烟草制品管制科学基础报告： WHO研究组第六份报告 水分含量越高，意味着烟草的量越少。对于其他烟草制品（卷烟和 自卷烟），相对湿度取决于所需的水分含量。按照 CORESTA推荐 的方法，卷烟约为 13%，自卷烟约为 20%[20]，对应于相对湿度分别 为 60%和 75%。由于所有的产品均储存在 22℃，因此不需要根据水 分含量来调节温度。对于水烟烟草来说，储存条件可能会影响某些 成分的测定结果。出于监管的目的，可以将水烟的检测结果告知消 费者。 水烟烟丝通常被装在密封容器中出售，由于水分含量不同，随 着时间的改变，同一实验室以及不同实验室间的检测结果可能会不 同。为了减少这种变化，可以对水烟烟草产品设定监管限制，如在 测定前先检测水分含量或者对其进行干燥。但是这两种额外的处理 势必会增加水烟烟草的检测成本。由于水和烟碱都可溶于异丙醇， 所以可以考虑同时检测烟碱含量和水分含量，但仍需要进一步的实 验来验证这种检测方法的可行性。 烟丝水分含量会影响吸烟过程中的烟气排放。为了尽量减少随 着时间的改变、水烟烟气释放量的变化，水烟产品应该在规定的条 件下进行保存，并且检测时的抽吸条件也要固定。用异丙醇萃取水 烟中的水分然后进行检测，结果显示水烟的水分含量为 10%~30%。 要调节烟草的相对湿度需要几个步骤，不适用于实际操作，因此 应该设置针对水烟烟草相对湿度的规范。将水烟与卷烟和自卷烟 进行比较，结果显示存储水烟时相对湿度应该调节为 75%，而不是 60%。但是由于极少数的实验室达到这个条件，这种情况下可以将水 烟储存在相对湿度为 60%，温度为 22℃的条件下，在 ISO 3402[22]中 对此进行了描述。此外，应该有进一步的研究以明确水烟的最短和 最长储存时间，并且应该在 SOP中明确表述。 ·147· 5. 针对卷烟的WHO烟草实验室网络标准操作规程对水烟的适用性 目前，环境温度和湿度对吸水烟的影响还不明确。当温度为 22℃、相对湿度为 60%时，尽管卷烟和自卷烟的含水量不同，它们 都可以使用。因此，建议水烟的机器吸烟条件为 22℃、相对湿度 60%。 5.6  内容物和释放物的测定 TobLabNet对 3种卷烟内容物含量的测定方法和 4种释放物含量 的测定方法进行了验证，下文对这些方法在水烟测定中的应用进行 讨论。 5.6.1  水烟烟草的内容物 在 TobLabNet SOP验证过的 3种卷烟烟草的测定方法中，讨论 了有关保湿剂和烟碱的测定方法在水烟中的应用。 5.6.1.1 保润剂 TobLabNet SOP-06中测定卷烟烟草填料中保润剂的方法适用于 甘油、丙二醇和三甘醇。卷烟中甘油和丙二醇的含量在 0.5%~4%， 而三甘醇作为生产过程中使用保润剂的可能污染物，在卷烟烟草中 很少存在。与此相反，在 44种水烟产品中，有 6种鉴定出含有三甘醇， 且几乎所有水烟产品中的甘油含量都比卷烟高 [23]。 Rainey等 [23]在水烟中对 TobLabNet SOP-06中的保润剂萃取方 法进行了验证，结果表明可以按照 TobLabNet SOP-06的方法对水烟 中的保润剂进行萃取，而无需对这个方法进行调整。 ·148· 烟草制品管制科学基础报告： WHO研究组第六份报告 由于水烟烟草中的甘油的含量很高，所以在设置 GC程序时应 注意避免甘油和三甘醇的共洗脱。并且由于水烟中甘油和丙二醇的 含量较高，因此应该对线性范围进行相应的调整。 5.6.1.2 烟碱 TobLabNet SOP-04中对卷烟中烟碱的测定方法进行了验证。在 该方法中，用水、氢氧化钠溶液和正己烷提取卷烟烟草中的烟碱。 在萃取过程中，烟碱将会被转移到正己烷中，最后用 GC-FID进行 检测。 水烟烟叶中高浓度的保润剂可能会导致烟碱提取不完全。应该 通过检测甘油、丙二醇或不同萃取液中烟碱的加标回收率来验证。 TobLabNet SOP-04中烟碱的测定方法是 GC-FID。目前，这项技 术已经广泛地被用于测定各种基质中的烟碱含量。但是，水烟烟草 中不仅含有高浓度的保润剂，还含有各种各样的香精，这些组分中 可能会含有干扰烟碱检测的化学物质（图 5.2）。 图 5.2 不同口味水烟中烟碱含量的色谱分析 ·149· 5. 针对卷烟的WHO烟草实验室网络标准操作规程对水烟的适用性 因为水烟烟草有很多种口味，因此要通过改变色谱参数来避免 香精的共洗脱是非常耗时且很难做到的。相比而言，使用气相色谱 - 质谱法来定量检测水烟中的烟碱可能会更实用且更准确。 5.6.2  焦油、烟碱和一氧化碳的释放 卷烟释放测定的结果取决于吸烟机的类型、抽吸方案、成分捕集、 样品制备和萃取以及测定方法。本部分主要讨论用于捕集目标组分、 样品制备和萃取方案的适用性，并讨论经过方案调整后该方案是否 可以用于水烟的检测。 在测定卷烟释放物的时候，TobLabNet SOP中规定的捕集系统为： • 针对焦油、烟碱和苯并 [a]芘和 TSNA的 CFP； • CO的气体采样袋； • 针对醛类和 VOC的 Carboxen 过滤管。 总的来说，是否可以将 TobLabNet SOP用于测定水烟释放物取 决于水烟中各组分的浓度、检测的灵敏度、是否存在干扰目标成分 捕获的物质以及所用的仪器。 如 ISO 3308[24]所述，用于收集卷烟烟气粒相物的 CFP可以收集 直径 ≥ 0.3 μm的微粒，捕获效率为 >99.9%。ISO 4387[16]中指出，对 于不同类型的卷烟吸烟机（直线或转盘），当滤片捕获超过 150 mg（直 线）或 600 mg（转盘）颗粒物时，可能会发生滤片破损。水烟的组 成会影响 CFP对颗粒相的收集。当水烟的总颗粒物与卷烟大致相同 时，可以使用与卷烟相同的 CFP。此外，需要有更多研究来明确用 于收集卷烟烟气中颗粒相的 CFP是否也适用于水烟烟气中颗粒相的 收集。 在水烟烟气组成成分和吸烟机抽吸方式一定的条件下，应该对 ·150· 烟草制品管制科学基础报告： WHO研究组第六份报告 捕获装置捕获水烟烟气中特定成分的效率进行研究。如果捕获装置 不能收集一轮抽吸中的所有成分，这时便需要对捕获装置进行更换， 其中包括调整吸烟机如引入多捕集系统或在吸烟期间引入压降监测 来确定何时需要更换捕集装置。在后一种情况下，要采取特殊防护 措施来防止更换疏水阀时发生泄漏。因为至少需要同时安装两个捕 集器，所以在这个过程中需要用到特殊的 CFP夹持器。 假设水烟中所有的烟碱都存在于粒相物中， CFP将能捕获到其 中所有的烟碱。这时，用异丙醇提取 CFP上的烟碱，水烟中的水分 含量不会对结果产生影响。需要进一步的研究来确定 CFP的数量和 萃取体积以达到定量烟碱的最优条件。 当水烟烟草含有大量的香精时，水烟烟气中也会有香精存在。 但是需要有进一步的研究来明确这些香精是否会被 CFP捕获从而影 响烟碱定量，以及香精物质是否都存在于气相中。 水烟烟气中 CO的含量显著高于卷烟烟气 [25, 26]。卷烟烟气中的 CO用吸烟机制造商提供的气体采样袋来收集，每个气体采样袋可容 纳 3 L（直线吸烟机）或 10 L（转盘吸烟机）气体。气体收集袋的尺 寸可以根据吸烟机抽吸方式来调整。另一种收集方式是保持吸烟的 口数不变，用气体采集袋收集一定口数的烟气。由于CO对人体有害， 因此在对其进行测定时必须采取一定的预防措施。为了保证实验室 人员不受 CO的影响，建议将水烟吸烟机放置在排气系统中，在 安全的环境中对气体收集袋进行放气且工作人员需要佩戴个人报 警系统。 实验室测试表明，水烟烟气中的 CO水平取决于加热烟丝的设 备 [9]。当使用电加热装置时，几乎没有 CO排放。因此可以推断 CO 是由炭加热装置产生的而并非来源于烟叶。目前还没有检测炭加热 ·151· 5. 针对卷烟的WHO烟草实验室网络标准操作规程对水烟的适用性 装置中 CO产生和释放的标准方法。 5.7  讨    论 WHO FCTC表示对烟草产品进行管制是非常有必要的，它对于 预防吸烟、戒烟以及保护公众不受二手烟的危害具有重要意义 [27]。 2003年，烟草制品管制科学咨询委员会 [28]对烟草制品的内容物和释 放量进行了讨论，建议对烟草制品中有害物质的含量和释放量设定 上限。通过 IARC与世界卫生组织无烟草行动组之间的合作，这项 建议已逐渐开始实施。与 IARC合作的目的是根据其毒性来限制排 放量。 虽 然 在 标 准 抽 吸 方 案（ISO/FTC[16]，Massachusetts Bench- mark[29]，Canadian Intense[30]）下，吸烟机抽吸卷烟产生的化学物质 释放量不能反映人体的暴露水平 [31]，但是它们可以提供建立和监管 卷烟化学物质产量阈值的框架。按照这种方法，当卷烟中化学物质 的含量超过阈值时，这种卷烟会被限制销售。随着时间的推移，允 许的排放水平可以不断降低，并且可以逐渐对其他有害物质进行监 管，从而逐渐降低市售卷烟的危害。 这种基于性能的模式特别适用于卷烟，因为卷烟与大多数烟草 制品不同，它呈现给消费者的形式是随时可用。正因为如此，卷烟 的排放量也相对固定，并可以通过给定的方法进行重复测定，一般 相对于标准偏差的变化范围在 15%以内 [32]。并且是否能满足监管要 求的主要责任在于生产制造商。但是这个特征在很多烟草制品中都 不适用，因为它们没有标准的产品规格，如比迪烟、自卷烟和水烟。 ·152· 烟草制品管制科学基础报告： WHO研究组第六份报告 对于水烟，虽然最终产品的每个组件都是按照统一的规格制造的， 但是这些组件怎么组合则是由吸烟者自己决定的或者是在无质量控 制的家庭工业中进行的。这种组合的非标准产品涉及了消耗品和硬 件的选择和准备。水烟吸烟者选择的组件和配件，如烟草制品、炭 和铝箔等，可能每种材料的来源都不同，且每种材料都可以通过烟 气中某些物质的直接来源或者与其他组分相互作用来影响烟气的排 放。例如，虽然大部分水烟烟气中的致癌 PAH来源于燃烧的炭，但 是 PAH只有与烟草混合物释放出的颗粒物结合后才能到达烟气。当 没有由烟草混合物产生的颗粒物质时，PAH会沉积在水烟的内表面 上，并且会大量存在于烟气中 [9]。另外一个交互作用影响有害物质 释放的例子是水烟软管的孔隙度和水烟的燃烧条件。软管孔隙度是 制造材料和施工质量的一个体现，它会影响经木炭到达水烟头的空 气量。软管的孔越多，则进入水烟头部的空气越少，这会影响炭燃 烧条件和水烟烟草制备的传热率，进而影响“焦油”和 CO排放 [12]。 因此，水烟的有害物质释放量取决于吸烟者选择的组件组合。并且 水烟释放量是否符合标准并非取决于市售的组件⑧。 此外，除烟碱释放外，不含烟草的水烟产品（宣传中常宣称： 关注健康者的选择）释放出的烟气中具有与常规含烟草的产品基本 相同的有害物质含量及生物活性 [33-35]。由于缺乏独立表征不同消耗 品（炭、烟草、铝箔）和硬件排放的方法，设置用于水烟制品监管 的排放标准是非常复杂的。 因此，一种更简单的方法——对产品的内容物进行监管——也 许是可行的。例如对已知的会导致释放物中有害物质含量升高或者 ⑧ 受 COP委托编写的本报道没有包含在发展 SOP时需要考虑的复杂的相互作用。 原因是现有的文献数量较少，无法基于这些文献发展考虑交互作用的 SOP ·153· 5. 针对卷烟的WHO烟草实验室网络标准操作规程对水烟的适用性 污染环境，且不会影响使用的水烟产品添加剂进行限制（例如烟叶 中的重金属）。根据 TobReg提出的排放标准，当在市售的产品中发 现污染物含量存在差异时，可以颁布法规以将其浓度限制为最小观 察值。这种方法可以立即应用到水烟木炭的管理上。不同类型水烟 木炭中的 PAH含量差异很大 [36]，并且烟气中很大一部分的多环芳烃 都来源于木炭。与此类似的是，烟草配方对烟气中重金属的含量（如 铅、铬、砷、镍）影响很大，因此需要限制烟草配方中的重金属浓 度不超过市售产品中已发现的最低浓度。有趣的是，呋喃和醛的释 放与烟草制剂中保润剂含量 [37, 38]成反比，这可能是因为当保润剂含 量高时，烟草燃烧时所达到的温度较低。 因此，在短期内，监管的重点可以放在市售水烟产品中已经发现 的有害污染物上——包括烟草和木炭——如无机金属和元素 [39, 40]、烟 碱 [41]、TNSA[26]和 PAH[9, 36]等。表 5.1对这些物质进行了总结。此外， 烟草 -保润剂混合物的 pH可能影响主流烟气中总烟碱含量，也就是 生物利用度更高的非质子化或“游离碱”形式的烟碱 [14, 42]。 表 5.1 可用于水烟监管的存在于烟草和炭中的化学物 水烟样品基质 监控的化学品 目标化学物和基质 烟草 碱度 pH 保润剂 二甘醇、乙二醇、甘油、丙二醇 无机金属和元素 砷、镉、铬、钴、铅、汞、镍、硒 烟碱 烟碱 TSNA NNN、NNK、NAT、NAB 炭 无机金属和元素 砷、镉、铬、钴、铅、汞、镍、硒 PAH 萘、苊烯、苊、芴、菲、蒽、荧蒽、芘、苯并 [a]蒽、䓛、 苯并 [b+k]荧蒽、苯并 [a]芘、苯并 [ghi]苝、二苯并 [a,h] 蒽、茚并 [1,2,3-cd] 从长远来看，随着证据支持和标准化测量方法的建立，水烟中 ·154· 烟草制品管制科学基础报告： WHO研究组第六份报告 需要进行管制的成分可能会逐渐增多，包括水烟中使有害物质排放 增加的成分，具体见表 5.2。 表 5.2 推荐对水烟烟草和炭中的以下化学物进行检测 监控的化学品 目标化学物质 醛 乙醛、丙烯醛、丁烯醛、甲醛 芳香胺 1-萘胺、2-萘胺、4-氨基联苯 香精 乙酰丙酰、丁二酮 呋喃 5-(羟甲基 )-2-糠醛、3-呋喃甲醇、呋喃甲醇、2-呋喃甲酸、2-呋喃甲醛、 3-呋喃甲醛、2-呋喃甲基酮、5-甲基 -2-呋喃甲醛、甲基 -2-糠酸盐 保润剂 二甘醇、乙二醇、甘油、丙二醇 无机金属和元素 砷、镉、铬、钴、铅、汞、镍、硒 烟碱 烟碱 PAH 萘、苊烯、苊、芴、菲、蒽、荧蒽、芘、苯并 [a]蒽、䓛、苯并 [b+k]荧蒽、 苯并 [a]芘、苯并 [ghi]苝、二苯并 [a,h]蒽、茚并 [1,2,3-cd] 酚类 邻苯二酚、间甲苯酚、邻甲苯酚、对甲苯酚、苯酚 TSNA NNN、NNK、NAT、NAB VOC 丙烯腈、苯、1,3-丁二烯 , CO、异戊二烯 5.8  结论和建议 有关水烟烟气毒性、成瘾性和吸引力的证据表明迫切需要相关 公共卫生干预来对烟草制品进行管制 [17]。然而，可以用于检测水烟 主流烟气中的有害物质含量的方法比较缺乏，且目前尚没有标准的 针对水烟烟气分析方法可以为水烟监管奠定基础。考虑到水烟中组 件、配件、烟草、热源和抽吸方式之间相互作用的复杂性，以及水 烟产品的类型众多，要对水烟产品进行管制，如对已知的可以增加 水烟有害物质释放量、成瘾性和吸引力的化学物质进行检测和报告， ·155· 5. 针对卷烟的WHO烟草实验室网络标准操作规程对水烟的适用性 也许会比从水烟设计、热源、烟草制剂、吸烟方式等方面对水烟排 放进行调节更有效。 回顾前文中的数据可以得出如下结论： • 与卷烟相比，水烟的特点为抽吸容量更大，流速更快，抽吸 口数更多。 • 吸烟机吸烟产生的水烟有害物质释放量受抽吸方式的影响。 • 需要有针对水烟的吸烟机来测定水烟释放物，这种吸烟机应 该是可以在市场上购买到。 • 有害物质释放量不仅仅是由水烟、炭或烟草配方单独决定的， 而是取决于这些因素的交互作用以及抽吸方式。 • 必须对测定水烟成分和释放量的标准 TobLabNet操作程序进 行修改，以便应用到水烟产品的测试中。 • 标准 TobLabNet操作程序不适用于测量炭的成分。 • 出于研究目的，可以用 Beirut方法来产生水烟烟气。 5.8.1  对监管机构的建议 （1）水烟管制的重点是在水烟烟草产品和炭的化学成分。 （2）应该对标准 TobLabNet操作程序进行调整，以用于测定水 烟中的烟碱、TSNA和保润剂的含量。 （3）应对现有的分析方法进行调整，并应用于测定水烟烟草（和 无烟草）产品的 pH和重金属含量。 （4）应对现有的分析方法进行调整，并应用于测定炭热源水烟 产品中的金属和 PAH释放量。 （5）按照 TobReg[43]推荐的方法，监管的重点应该放在降低水 烟产品中 TSNA、PAH和重金属含量上。受管制的成分清单应该随 ·156· 烟草制品管制科学基础报告： WHO研究组第六份报告 着有关有害物质释放和 /或健康效应的认识的更新而不断更新。 5.8.2  对研究人员的建议 应阐明水烟烟草产品成分、木炭组成、抽吸方式、水烟设计和 水烟使用环境对有害物质排放的影响，以促进水烟产品管制。 5.9  参  考 文 献 [1] Shihadeh, A. 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Tob Control 2008;17:132-41. ·163· 6. 无烟烟草制品的有害内容物和释放物 Stephen Stanll，美国疾病控制与预防中心 目录 6.1 引言 6.1.1 全球流行情况 6.1.2 无烟烟草制品在制造和物理特性上的多样性 6.2 产品构成 6.2.1 烟草 6.2.2 添加剂 6.3 无烟烟草制品的释放物 6.3.1 烟碱 6.3.2 有害物质和致癌物 6.3.2.1 烟草特有亚硝胺 6.3.2.2 挥发性亚硝胺 6.3.2.3 挥发性醛 6.3.2.4 多环芳烃 6.3.2.5 槟榔 6.3.2.6 金属 6.3.2.7 硝酸盐和亚硝酸盐 6.3.3 微生物及其组成 6.4 降低无烟烟草制品中的有害物质浓度 6.5 结论和建议 6.6 参考文献 ·164· 烟草制品管制科学基础报告： WHO研究组第六份报告 6.1  引    言 本报告是根据缔约方大会第六次会议（俄罗斯联邦莫斯科，2014 年 10月 13~18日）向公约秘书处提出的要求编写的，即邀请世界卫生 组织编写一份关于无烟烟草制品中的有害物质及其释放物的报告。 无烟烟草包含工业产品（湿鼻烟、干鼻烟、含化型、gutkha、 khaini、snus、咀嚼烟草、zarda）和手工制剂（槟榔嚼块、dohra、 tombol、toombak、iq’mik）（表 6.1）。大多数无烟烟草制品都是经 口服用的，但是也有部分干燥产品是经鼻使用的。口服的无烟烟草 制品和制剂可以通过咀嚼、吮吸的方式黏（“蘸”）在口腔黏膜或 牙齿和牙龈上。在产品的使用过程中，会释放具有成瘾性和有害的 化学物质，这些物质可以经过黏膜吸收 [1]，最后进入血液 [2, 3]。使用 无烟烟草制品会导致癌症发生 [4]。近期，有一项系统综述总结了使 用无烟烟草制品的不良健康影响 [5]。 表 6.1 全球无烟烟草制品的种类 制品 世界卫生组织地区 非洲 美国 地中海东部 欧洲 东南亚 西太平洋 Afzal（Oman） √ 槟榔嚼块（paan） √ √ √ 含咖啡因的湿鼻烟 √ Chimó √ 奶油鼻咽 √ 含化型 √ √ Dohra √ 干鼻烟 √ √ √ ·165· 6. 无烟烟草制品的有害内容物和释放物 续表 制品 世界卫生组织地区 非洲 美国 地中海东部 欧洲 东南亚 西太平洋 加纳传统鼻咽（tawa） √ Gudakhu或 gudakha √ Gul √ Gundi（kadapan） √ Gutka √ √ Hnat hsey √ Hogesoppu（烟叶） √ Iq’mik √ Kadapan √ Kaddipudi √ Khaini √ Kharra √ Kiwam（qiwam, kimam） √ √ Kuberi √ 松散烟叶 √ Mainpuri（kapoori） √ Mawa √ Mishri（masheri, misri） √ 湿鼻烟 √ √ Nass（naswar） √ √ √ Nasway（nasvay） √ √ √ Nea √ √ √ 烟碱咀嚼胶 √ 尼日利亚传统鼻烟（taaba） √ NuNu √ 无石灰 Pattiwalla √ Plug（咀嚼烟草） √ √ Rapé √ 红色牙粉（lal dant manjan） √ Sada pata √ ·166· 烟草制品管制科学基础报告： WHO研究组第六份报告 6.1.1  全球流行情况 据估计，在WHO的六个地区中，有 3亿多人使用无烟烟草制 品 [5]。在哈萨克斯坦和老挝人民民主共和国的成人中，无烟烟草制 品的使用非常普遍。在某些太平洋岛屿、挪威、瑞典和西欧其他地区、 非洲的几个国家、蒙古、南美和美国，无烟烟草制品也很流行 [6, 7]。 在全球范围内，所有使用无烟烟草制品的成年人中有 89%来自于东 南亚（主要是孟加拉国和印度），在东南亚约有 2.68亿成年人使用 无烟烟草制品 [5]。 无烟烟草制品的使用是一个全球性的公共卫生问题，据统计， 约有 170万伤残调整生命年（DALY）的损失是由无烟烟草相关癌症 引起的 [8]。印度无烟烟草的使用率很高，不吸烟者中约有 368000人 的死亡是由无烟烟草使用引起的 [9]。在全球范围内，652494人的死 亡是由无烟烟草使用引起的 [10]。 6.1.2  无烟烟草制品在制造和物理特性上的多样性 无烟烟草制品的外观、生产规模、成分和配方各不相同 [4, 5, 11, 12]。 具体来说，无烟烟草制品包括商业生产的产品，以及在传统环境下如 家庭、商店、市场摊位和街头贩卖场上生产的产品。这些产品包含了 从只含有烟草，到由烟草和非烟草植物材料及化学品混合而成的制剂。 无烟烟草制品有多种形式，包括全烟叶、细切烟叶、粉状烟草粉、压饼、 球团、粉糊、焦油、烟叶、化学药品和植物材料 [4, 5, 11, 12]。其中，可以 将松散的烟叶封装在小袋中，使其更便于使用（例如，snus和湿鼻 烟）。“含化型”包括片状的细碎烟草、薄的圆柱形棒（棍）、薄 ·167· 6. 无烟烟草制品的有害内容物和释放物 的细晶片和在使用时可溶解在嘴里的纸条 [13]。烟草棒是将干鼻烟 涂到一根细棒上，在吮吸时，可以释放出里面的干鼻烟 [4, 11, 12, 14]。 Verve®是一种含有烟草来源烟碱的口味纤维素聚合物盘的新产品，咀 嚼它可以使血液中的烟碱浓度升高，并且可以使生理功能更活跃（如 提高心率和血压）。据报道，Verve®还能满足吸烟者的烟碱成瘾 [15]。 6.2  产  品 构 成 6.2.1  烟草 大部分的无烟烟草制品是由一种或多种烟草（Nicotiana spp.） 制成的。还有一些特殊产品如 Verve®含有从烟草中提取的烟碱，但 是不含有烟末或松散的烟叶。尽管世界范围内 Nicotiana的品种有很 多，但在商业生产中最常用的一种是 N. tabacum。而在非洲、中东、 南美和南亚地区，最常用的是 N. rustica，它的烟碱、次要生物碱和 TSNA含量高于 N. tabacum[4, 16]。例如，据估计，在印度 35%~40%的 无烟烟草产品含有 N. rustica[17, 18]。红外分析结果表明，一些国家售 卖的无烟烟草制品中含有 N. rustica，如 gul、某些 toombak、zarda 和 rapé[19, 20]。其中在 toombak和 gul也可以包含另一种烟草，即 N. glauca[4, 21]，它不含有烟碱，但含有高浓度的 N-亚硝基假木贼碱 [22]。 尽管不含烟碱，但是普遍认为 N. glauca仍具有剧毒，在某些情况下 摄入它会致命 [17, 22]。因此，应该严格限制高烟碱烟草（N. rustica） 或剧毒品种烟草（N. glauca）的使用。 ·168· 烟草制品管制科学基础报告： WHO研究组第六份报告 6.2.2  添加剂 除烟草外，无烟烟草制品通常还含有甜味剂、保润剂、调味剂、 盐和碱剂。1994年，美国 10家无烟烟草制品制造商发布了一份清单， 列出了 560种用于制造无烟烟草制品的添加剂 [4]。 在手工或“家庭手工业”制作的产品中，常将烟草与其他植物 材料进行混合。在南亚，制造无烟烟草产品所需要的原料，如聚丙 烯酸钠（槟榔嚼块），含有烟草、槟榔、碱性药剂、儿茶和香料（如 生姜、丁香、樟脑、藏红花）的 dohra，可以用叶片对它们进行包裹 （蒌叶）。此外，在mainpuri、mawa、guthka、kharra和 zarda（南亚） 以及 tombol（中东）和 thinso（非洲）中也有使用槟榔 [5, 10]。也门的 一些 tombol是通过将烟草和神经活性植物阿拉伯茶（Catha edulis） 的混合物包裹进槟榔叶中制成的 [17]。南美的一种名称为 rapé的无烟 烟草产品中含有大量的薰草豆（Dipteryx odorata），它的香豆素含量 较高，是美国食品药品监督管理局颁布的禁止用于食品的“烟草制 品和烟草烟雾的有害及潜在有害成分”中的一种 [24]。其他非烟草植 物材料包括芫荽子、茴香、麝香、黑胡椒、香草、大蒜和人参等 [5]。 添加的甜味剂主要包括单糖、糖蜜、蜂蜜和木糖醇。商业 产品如松散的烟叶、gutkha和家庭手工业产品如 gul都添加了甜 味剂 [4]。一项关于美国市售无烟烟草的研究发现烟袋和栓形烟草 （13.5%~65.7%）中的糖含量远远高于鼻烟（1.9%），烟袋和栓形无 烟烟草中的糖含量高于烟斗、卷烟或雪茄烟 [25]。 保润剂，通常是指丙二醇和甘油，常被加入产品中以保持水分。 美国北卡罗来纳州州立大学对松叶咀嚼烟草产品的研究显示，其中 甘油浓度为 3.2% （CRP4）和 3.75%（STRP 1S1），丙二醇的含量为 3.0% ·169· 6. 无烟烟草制品的有害内容物和释放物 （CRP1，snus）[26]。在 snus制造的 GothiaTek®标准（在 6.4节中进 行描述）中提到，为减少微生物的生长，并防止形成 TSNA[28]，保润 剂的含量应为 1.5%~3.5%[27]。 调味料主要包括香味化合物、果汁、可可、朗姆酒、香料粉、 萃取物和 60多种精油 [11, 29, 30]。一项有关无烟烟草制品中化学物质的 研究显示，无烟烟草制品中最常发现的物质是水杨酸甲酯、水杨酸 乙酯、苯甲醛、香茅醇和薄荷醇 [31]。其他研究人员也在无烟烟草制 品中发现了水杨酸甲酯、水杨酸乙酯、薄荷醇和薄荷调味料 [32]。进 一步分析发现其中还可能包括一些生物活性物质如咖啡因、椰子、 甘草、草药、植物染料、色素、食用油、黄油、土壤、硝酸钾和斑 点的金属银等。可溶解的无烟烟草制品中还可能含有黏合剂、黏结 剂和增白剂 [5]。 无烟烟草制品中的碱剂包括碳酸盐、碳酸氢盐和石灰（氢氧化 钙）[5, 12, 29]。其中家庭手工业产品（toombak、shammah）和手工制 剂（iq’mik、nass、betel quid）中通常包括石灰、碳酸氢钠、某些植 物或真菌的灰烬 [4, 33, 34]。Iq’mik是北美北极地区土著居民所使用的一 种产品，它含有真菌或其灰烬与叶子的混合物 [35]。 6.3  无烟烟草制品的释放物 6.3.1  烟碱 烟草中主要的致瘾化学物质是烟碱，它在无烟烟草制品中使用 范围很广并且在重复使用中起着关键作用，会导致有害物质和致癌 ·170· 烟草制品管制科学基础报告： WHO研究组第六份报告 物的持续暴露。总烟碱是指一种产品中烟碱的总含量，不论它的存 在形式是什么。总烟碱很重要，但是 pH对烟碱的影响很大。未经 处理的烟草通常呈酸性（pH 5.0~6.5）[36]，其中非离子化烟碱的含量 很少（<5%）。非离子化烟碱也被称为“非质子化”或“游离”烟 碱，它容易被吸收。经口吸收烟碱时，通常需要添加碱性药剂以提 高 pH，从而将烟碱转化为游离烟碱 [5]。 总烟碱含量相近但 pH不同的产品中游离烟碱浓度相差很大 [5]。 随着 pH的升高，游离烟碱会从烟草中释放出来，穿过生物膜。因此， 碱性试剂在烟碱释放中起着关键作用，与总烟碱一起使血液烟碱浓 度升高，普遍认为这是无烟烟草致瘾的原因 [2-4,37]。烟碱本身是有毒的， 对健康会产生危害，如导致心血管疾病和糖尿病。因此，通过碱性 试剂增加其吸收不仅会使无烟烟草制品更容易上瘾，而且毒性可能 更强。 据报道，无烟烟草制品的 pH在 4.6~11.8之间不等，因此游离 烟碱含量范围为 0.02%~99.9%。Iq’mik和 nass中含有碱性灰分，pH 相对较高（11.0~11.8）[38, 39]。gul粉、naswar、khaini、南非干鼻烟 [19]和 afzal（阿曼）[19]的 pH也较高（9~10.5）[40]。调查显示 zarda产 品为碱性，pH为 8.1~9.0[41]。其他无烟烟草制品，如 toombak、chi- mo、rape和 snus，pH在酸性到强碱性之间 [5, 19]。咀嚼烟草（twist、 chew、plug和松散的烟叶）通常是酸性的（pH<7）[42]，湿鼻烟的 pH一般是 5.5~8.6[43, 44]。 在大约 700种产品中，烟碱的总浓度（湿重）在 0.39~95 mg/g 之间。其中最特殊的无烟烟草制品是美国生产的湿鼻烟（226种产 品）。在这些产品中，总烟碱浓度范围为 4.15~25.0 mg/g，而游离烟 碱的浓度为 0.01~15.2 mg/g[43, 44]。在不太常用的咀嚼烟草（扭花、咀嚼、 ·171· 6. 无烟烟草制品的有害内容物和释放物 栓形和松散的烟叶）中，总烟碱的浓度为 2.92~40.1 mg/g，游离烟碱 （0.01~0.47 mg/g）的含量较少。美国干鼻烟产品的 pH基本是在酸 性和中性之间，总烟碱浓度范围在 0.30~28.0 mg/g之间，游离烟碱浓 度范围在 0.05~3.12 mg/g之间 [42]。与此相反，南非制造的干鼻烟总 烟碱浓度较低（1.17~14.9 mg/g），但由于它的碱度较高，因此游离 烟碱浓度较高（1.16~13.8 mg/g）[19]。 有数据表明尼日利亚传统和药用鼻烟及南非传统鼻烟的 pH是 9.0~9.5，且总烟碱（2.49~7.41 mg/g）和游离烟碱（2.39~6.72 mg/g） 浓度接近。阿曼的一种强碱性产品 azfal的总烟碱（48.8 mg/g）和游 离烟碱（48.6 mg/g）含量均非常高 [40]。南非市售的 snus产品呈轻度 酸性，总烟碱含量居中（13.4~17.2 mg/g），游离烟碱浓度较低（0.47~1.19 mg/g）[18]。 瑞典 snus中总烟碱（6.83~20.6 mg/g）和游离烟碱（0.71~15.5 mg/g） 的含量变化范围很广 [45]，其中一些总烟碱含量高于美国湿鼻烟 [44]。 此外，研究表明，在 124个含化型产品的总烟碱含量（3.0~20.5 mg/g） 和游离烟碱（0.37~2.47 mg/g）含量较低。Verve®与含化型产品类似， 总烟碱（1.68 mg/g）和游离烟碱浓度（0.37 mg/g）含量较低 [15]。苏丹 的 toombak含有N. rustica，是已知总烟碱含量最高的产品（95 mg/g）[47]。 在南亚，无烟烟草制品包括红牙粉、以甘油为基础的奶油鼻烟 （两种都可以用作牙粉），gutkha和 zarda。在东南亚，人们常将烟 草和 supari包混合在一起，可以包括槟榔、香料、甜味剂和碱性试剂。 Gupta和 Sankar[48]发现，红牙粉呈轻度酸性，总烟碱浓度为 4.47~5.09 mg/g，游离烟碱含量为 0.03~0.23 mg/g。而碱性较强的奶油鼻烟的总 烟碱（5.62~10.0mg/g）和游离烟碱（0.71~3.39 mg/g）的含量均较高。 此外，他们还发现gutkha呈碱性（pH 8.6~9.2），总烟碱含量为0.71~3.39 ·172· 烟草制品管制科学基础报告： WHO研究组第六份报告 mg/g，游离烟碱含量为 0.03~0.25 mg/g。印度的 Zarda产品呈弱酸性， 总烟碱浓度为 2.61~9.5 mg/g，但几乎不含游离烟碱（0.01~0.02 mg/g）。 巴基斯坦的 Zarda产品碱性更强，总烟碱（7.35~26.7 mg/g）和游离 烟碱浓度也更高（5.52~21.4 mg/g）[41]。 在使用某些产品前，使用者会将加入碱剂，通过这种方式，可 以升高无烟烟草制品的 pH和游离烟碱的含量。Gupta和 Sankar[48]研 究发现五种 supari与烟草的混合物均为碱性（pH 8.6~10.1），总烟 碱浓度为 1.77~4.96 mg/g，游离烟碱浓度为 1.56~4.06 mg/g。此外，为 满足喜好，使用者也可以将碱剂添加到手工制品（如槟榔嚼块）中。 6.3.2  有害物质和致癌物 由于无烟烟草中存在致癌物质，因此它划分到了 IARC 1类（确 定人体致癌物）中 [4]。IARC研究工作组 [4, 11]在无烟烟草制剂中发现 了 40多种确定致癌物 [5]，包括活性无机离子（硝酸盐和亚硝酸盐）、 TSNA、N-硝基胺酸、挥发性 N-亚硝胺、霉菌毒素、多环芳烃、挥 发性醛、类金属、准金属和槟榔。其中无烟烟草致癌物中含量最高 的是 TSNA、N-亚硝基氨基酸、挥发性 N-亚硝胺和醛 [4]。此外，该 研究小组还得出了结论：有充分的证据表明，使用无烟烟草制品会 导致口腔癌前病变、口腔癌、食道癌和胰腺癌 [5]。 6.3.2.1 烟草特有亚硝胺 TSNA是在烟草的固化、加工、发酵和燃烧过程中形成的 [49, 50]。 在大多数烟草中，NNN的浓度超过 NNK的浓度，但在烤烟中， NNK的浓度超过 NNN[51]。因此，烟草的混合物决定了 NNN和 NNK的含量。在已知的 7种 TSNA中，通常在烟草制品中含有大量 ·173· 6. 无烟烟草制品的有害内容物和释放物 的 NNN和 NNK，它们具有致癌性 [52]。NNN和 NNK是 1类人类致 癌物 [4]，且是无烟烟草中最常见的“强”致癌物 [53]。NNN与无烟烟 草使用者口腔癌的发生密切相关，无烟烟草制品中 NNN的浓度水平 可以高达 79 µg/g[4, 53, 54]。表 6.2对世界各地商业和手工无烟烟草制品 中 TSNA浓度进行了总结。 表 6.2 商业和手工制造无烟烟草制品中的烟草特有亚硝胺浓度 [4] 产品 参考文献 浓度 (μg/g产品湿重 ) NNK NNN 所有 TSNA Toombak [47] 578~7300 395~2860 1500~12 630 Toombak [19] 147~516 115~368 295~992 鼻烟 湿鼻烟 [44] 0.38~9.95 2.20~42.6 5.11~90.0 干鼻烟 [42] 1.34~14.6 6.12~31.3 10.3~76.5 干鼻烟 (pouch) [42] 0.08~0.12 0.93~0.97 1.52~1.85 咀嚼烟草 栓型 [42] 0.34~0.94 2.92~4.64 4.09~7.75 松散的烟叶 [42] 0.24~0.31 0.94~2.83 1.55~4.10 扭曲型 [42] 0.31~0.56 0.83~2.46 2.59~4.95 Snus [19，42] 0.084~1.34 0.27~5.57 0.60~5.85 含化型 [42，46] 0.31 0.06~0.26 0.31~0.74 美国产品 Iq’mik [39] 0.19~0.54 1.99~4.00 5.64~8.84 Rapé [20] 0.04~3.30 0.013~14.5 0.04~24.2 Chimó [19] 0.31~2.60 0.32~4.62 0.95~9.39 南亚产品 Gul [19] 5.19~8.02 1.33~1.37 13.4~17.1 Khaini [19] 0.29~0.50 16.8~17.5 21.6~23.5 Zarda [19] 0.46~3.84 2.91~28.6 5.49~53.7 Gutha（手工） [19] 0.007~0.38 0.21~18.6 0.26~23.9 Gutkha [19] 0.057~0.46 0.17~1.28 0.37~2.25 ·174· 烟草制品管制科学基础报告： WHO研究组第六份报告 续表 产品 参考文献 浓度 (μg/g产品湿重 ) NNK NNN 所有 TSNA 中亚产品 Naswar [19] 0.029~0.31 0.36~0.54 0.48~1.38 非洲产品 尼日利亚传统鼻烟 [19] 0.28 0.71 1.52 药用干鼻烟 [19] 0.36 1.46 2.42 干鼻烟 [19] 0.13~0.35 0.89~3.40 1.71~4.67 传统鼻烟 [19] 1.61 5.57 20.5 TSNA浓度较高的无烟烟草制品往往是被微生物污染的产品。 可溶性无烟烟草制品中的 TSNA的浓度范围是 0.31~0.61 μg/g，它们 是固体且水分含量低 [46]。瑞典 snus经过了巴氏消毒 [19, 42, 45]，里面 TSNA浓度为 0.60~5.85 μg/g。这两种产品的 TSNA浓度均低于传统产 品。发酵过的无烟烟草制品中的 TSNA浓度较高，如印度 zarda（5.5~ 53.7 μg/g）[19]、湿鼻烟（5.11~90.0 μg/g）[44]和美国制造的干鼻烟（10.3~ 76.5 μg/g）[42]。尼日利亚和南非的传统鼻烟总 TSNA浓度分别为 1.52 μg/g 和 20.5 μg/g[19]。有趣的是，非洲生产的干鼻烟 TSNA浓度（1.71~4.67 μg/g）低于美国生产的干鼻烟。其他总 TSNA浓度较高的产品还有 khiani，naswar，iq’mik，rapé和 chimó。嚼烟烟草的 TSNA浓度非常 低（1.55~7.75 μg/g）[42]。 美国最畅销的湿鼻烟中 NNN（2.2~42.6 μg/g）的浓度高于 NNK （0.38~9.95 μg/g）[44]。瑞典 snus，TSNA浓度在 1983~2002年间下降 了大约 85%。在 2002年，27种瑞典 snus中的 NNN（0.49 μg/g）和 NNK（0.19 μg/g）平均浓度非常低 [56, 57]，在已报道的商业无烟烟草中 的NNN和NNK的最低含量。chaini khaini是印度 snus中的一种，它的 ·175· 6. 无烟烟草制品的有害内容物和释放物 NNN和NNK含量很高，分别为 (22.9±4.9) μg/g和 (2.6±1.0) μg/g[58]。 一项有关 117种“免吐出”和可溶解无烟烟草制品的研究发现， Camel Strips中总 TSNA（NNN，NNK，NAT和 NAB）的浓度为 0.53 μg/g，略低于 Camel Snus（1.19 μg/g）[46]。另一项有关九个国家 53种 产品的研究发现 [19]，苏丹的 toombak和孟加拉国的干 zarda中 NNK 浓度最高，而印度的 toombak、干 zarda和 khaini中的NNN浓度最高。 在这些产品中，巴基斯坦手工制作的 gutkha和 mawa 中 NNK浓度最 低。 已报道的 TSNA含量最高的无烟烟草制品为苏丹 toombak，为 12600 μg/g，它是一种高度发酵的产品，原因可能是苏丹 toombak中 的生物碱浓度极高。toombak中的 NNN浓度高达 2860 μg/g，NNK 浓度高达 7300 μg/g[47]。此外，在 toombak使用者的唾液中也发现了 很高浓度的 TSNA[47, 59, 60]。研究表明，苏丹男性中超过 50%的口腔癌 与 toombak或其他口服烟草制品相关，可能原因是其中的 TSNA很 高且其具有致癌性 [10, 60, 61]。 6.3.2.2 挥发性亚硝胺 与 TSNA相同，在微生物反应中也会产生亚硝酸盐积累 [5]。20 世纪 80年代早期一项研究表明，瑞典鼻烟和咀嚼烟草中的挥发性 N- 亚硝胺（N-亚硝基二甲基胺、N-亚硝基吡咯烷、N-亚硝基哌啶和 N-亚硝基吗啉）的湿重含量范围为 0.5~145.9 μg/kg[56]。农用化学品 马来酰肼二乙醇胺和生产化学品吗啉的用量减少，可以降低烟草制 品中的 N-亚硝基二乙醇胺和 N-亚硝基吗啉的含量 [62]。Nass（又称 nasswar）是阿富汗、印度、伊朗伊斯兰共和国、巴基斯坦、俄罗斯 联邦和中亚地区使用的一种混合物，它含有烟草、碱剂和棉花油 [63]。 研究发现 nass中的挥发性 N-亚硝胺的含量比咀嚼烟草或鼻烟低， ·176· 烟草制品管制科学基础报告： WHO研究组第六份报告 其原因是 nass生产中陈化时间较短 [64]。 6.3.2.3 挥发性醛 在包含鼻烟的无烟烟草制品中，致癌性醛（甲醛、丙烯醛、巴 豆醛和乙醛）的含量水平以百万分之一计。明火烤制烟草中的致癌 性醛含量往往高于空气调制烟草 [5, 55]。 6.3.2.4 多环芳烃 含有木头和锯末的无烟烟草在固化燃烧时会产生多环芳烃，且 多环芳烃的浓度比火烤烟和空气烤烟高 [5]。含火烤烟草的湿鼻烟中 的 PAH（包括 IARC 1类和 2类致癌物）的含量比 snus（不含火烤烟 草）高 [55, 65]。目前，在无烟烟草制品 [65]中已发现的 IARC 1类多环 芳烃为苯并 [a]芘，2A组的为二苯并 [a,h]蒽，2B组的有苯并 [b]荧蒽、 苯并 [j]荧蒽、苯并 [k]荧蒽、二苯并 [a,i ]芘、茚并 [1,2,3-cd]芘、5- 甲基、萘和苯并 [a]蒽）[66]。 对美国的 23种产品研究发现，湿鼻烟中的 PAH总浓度为 921~9070 ng/g，snus中PAH总浓度为660~1100 ng/g。湿鼻烟中苯并 [a] 芘的浓度（9.7~44.6 ng/g）高于 snus（3.0~12.3 ng/g），40%的 snus 中苯并 [a]芘浓度低于检测限（1.6 ng/g）。湿鼻烟中萘的浓度（409~1110 ng/g）与 snus类似（636~1065 ng/g）。但是当从总 PAH中排除萘时， 湿鼻烟中剩余 PAH（145~8120 ng/g）的含量高于 snus（21~213 ng/g）。 一个名为“starter”的产品中含有 145 ng/g PAH（排除萘），而萘的含 量为 776 ng/g。Marlboro snus中含有 7种 PAH，含量为 1.1~13.5 ng/g； 当排除萘时，PAH的总浓度为 20~70 ng/g。Camel snus品牌中含有 14种可检测的多环芳烃，其浓度范围为 3.1~79.4 ng/g，当排除萘时， PAH的总浓度为 110~320 ng/g[65]。当减少或消除无烟烟草制品中的 ·177· 6. 无烟烟草制品的有害内容物和释放物 火烤烟叶含量时，PAH的总浓度降低。 6.3.2.5 槟榔 未成熟的槟榔生物碱含量极高。在某些文化中它们“口碑很好”， 所以很受欢迎 [5]。目前，国际癌症研究机构工作组已将槟榔划分为 1 类致癌物 [66]。槟榔碱被认为是最重要的生物碱之一。在不与烟草混 合的情况下，槟榔提取物便对人口腔黏膜细胞和成纤维细胞具有强 细胞毒性和遗传毒性。研究证明，单独使用槟榔具有遗传毒性 [5]和 致癌性 [11]。 6.3.2.6 金属 金属和非金属物质可能在烟草植物或叶子表面积累，这与土壤 组成、pH和环境污染有关 [67]。在各种无烟烟草制品已发现的金属中， 含有 IARC 1类（人体致癌物）的有砷、铍、六价铬、镉和钋 -210， 2A类（可疑的致癌物）有镍化合物，2B类（可能致癌物）有铅和钴。 砷属于非金属，是 1类致癌物。此外，检测发现在无烟烟草制品中还 含有汞和铝。在加拿大、印度、巴基斯坦和美国的无烟烟草制品中发 现了砷（0.1~14.0 μg/g）、铍（0.01~0.038 μg/g）、铬（0.71~54.0 μg/g）、 镉（0.25~9.2 μg/g）、镍（0.84~64.8 μg/g）、铅（0.23~111 μg/g）和钴 （0.056~1.22 μg/g）[67]。一项有关印度无烟烟草制品（zarda，奶油鼻烟， khaini，gutkha）研究发现，四种 gutka产品中的铜浓度（237~656 μg/g） 高于其他产品（0.012~36.1 μg/g）[68]。此外，研究还在用于制作槟榔 嚼块的消石灰、槟榔叶和香味烟草（zarda）等组分中检测出了砷、 镉和铅 [69]。 6.3.2.7 硝酸盐和亚硝酸盐 植物可以从生长的土壤中摄取肥料衍生物硝酸盐。当烘烤干燥 ·178· 烟草制品管制科学基础报告： WHO研究组第六份报告 烤烟时，细胞会破裂释放出硝酸盐 [70-72]。在植物中，微生物常以内 生菌的形式存在 [73]。如果在植物中存在可降解硝酸盐的微生物，那 么在这个过程中就会产生并释放亚硝酸盐。目前研究已经在烟草 和烟草制品中鉴定出了几种可将硝酸盐转化为亚硝酸盐的细菌和 真菌 [71, 74, 75]。微生物释放的亚硝酸盐既可以与烟草生物碱反应生成 TSNA，也可以促进挥发性亚硝胺和亚硝基氨基酸的形成 [70, 76]。在烟 草发酵和烟草储存期间亚硝酸盐和 TSNA浓度也会增加 [71, 72]，尤其 是在高温和潮湿条件下 [77]。如果在加工过程中不消除硝酸盐还原性 微生物，将会影响烟草产品的化学性质 [70-72]。 6.3.3  微生物及其组成 烟草和烟草制品中经常含有细菌和真菌等微生物 [71, 75, 78, 79]。通 过对微生物 DNA进行测序 [75, 80]，在各种无烟烟草制品中共鉴定出了 33个细菌家族。其中通过对呼吸性硝酸还原酶的基因进行研究，预 计周质硝酸盐还原酶可能在亚硝酸盐的产生和细胞外释放过程中发 挥着重要作用。一些细菌家族（包括已知的厌氧菌）在缺氧（如发酵， 老化和储存的过程中）的条件下，会以硝酸盐而不是氧气为电子受 体 [81]，进而会导致细胞外亚硝酸盐积累 [71, 72, 77]。含有呼吸性硝酸盐 还原酶基因的细菌属有棒状杆菌属、乳杆菌属、葡萄球菌属以及肠 杆菌科中的某些细菌 [75]。 在烟草发酵的过程中，细菌和真菌会迅速增殖并形成有害的反 应性副产物 [70-72]。因此，在诸如 khaini[82]、干鼻烟 [82]、湿鼻烟 [65]和 苏丹 toombak[19, 47]等发酵产品中，亚硝酸盐和 TSNA的浓度都要高 于巴氏杀菌的 snus等 [19, 46]。 研究显示，烟草和烟草制品中还含有某些真菌（如镰孢菌、Al- ·179· 6. 无烟烟草制品的有害内容物和释放物 ternaria和 Candida）[71, 78, 79, 83]。黄曲霉毒素 B1是一种由黄曲霉真菌 产生的霉菌毒素，研究报道在美国生产的某 6种干鼻烟产品中含有 黄曲霉毒素 B1（0.01~0.27 g/g），但在 16种湿鼻烟和 3种 snus产品 中（84种）不含有。 6.4  降低无烟烟草制品中的有害物质浓度 要减少烟草制品中有害物质的浓度，需要了解烟草的农业生产 和制造过程以及其中的有害物质形成和积累。表 6.3中总结了已发现 的有害物质和致癌物质及其在烟草加工过程中的潜在来源。 表 6.3 无烟烟草制品中可能的致癌物质、有害物质和生物活性物质的来源 [85] 类型 IARC致癌物 (1、2A、2B)、有害物质或 生物活性物质 潜在来源 金属和非金属 1类：砷、铍、镉、镍化合物、钋 -210 2A类：无机铅化合物 2B类：钴致敏——铝、铬、钴、镍 皮肤刺激——钡、汞 可能导致口腔黏膜纤维化：铜（在槟榔中） 土壤吸收或存在于烟草中的土壤 颗粒 ;与烟草一起使用的其他成分 （槟榔叶、槟榔、熟石灰等）。 亚硝化剂 2B类：硝酸盐 2B类：亚硝酸盐 土壤吸收 微生物产生 真菌毒素 1类：黄曲霉毒素（混合物） 2B类：黄曲霉毒素M1、赭曲霉毒素 A 真菌（霉菌）产生 亚硝胺 TSNA 1类：NNN, NNK, NNAL 亚硝酸盐在固化、发酵和老化过程 中形成（亚硝酸盐与生物碱反应） 挥发性 N-亚 硝胺 2A类：N-亚硝基二甲胺 2B类：亚硝基吡咯烷标准品 N-亚硝基哌啶 N-亚硝基哌啶 N-亚硝基二乙醇胺 亚硝酸盐在腌制、发酵和老化过 程中形成（亚硝酸盐与二胺或三 胺反应） 亚硝酸 2B类：N-亚硝基肌氨酸 氨基甲酸酯 2A类：氨基甲酸乙酯 发酵过程产生（尿素和乙醇反应） ·180· 烟草制品管制科学基础报告： WHO研究组第六份报告 续表 类型 IARC致癌物 (1、2A、2B)、有害物质或 生物活性物质 潜在来源 多环芳烃 1类：苯并 [a]芘 2A类：二苯并 [a,h]蒽 2B类：苯并 [a]蒽、苯并 [b]荧蒽、苯并 [j] 荧蒽、苯并 [k]荧蒽、二苯并 [a,i]芘、茚 并 [1,2,3-cd]芘、5-甲基䓛和萘 在烤烟过程中沉积在烟草上 挥发性醛 1类：甲醛 2B类：乙醛 在烤烟过程中沉积在烟草上 非卷烟植物 材料 1类：槟榔子 肝脏有害物质：薰草豆 兴奋剂：阿拉伯茶 添加剂 在种植过程中，烟草等植物会从土壤中吸收金属、非金属、可 溶性离子（如硝酸盐和铵）[86]。因此，土壤颗粒（包括金属）、土 壤中的农药和微生物以及环境中的其他成分可以蓄积在烟叶中。其 中烟草中的金属含量受土壤 pH、土壤成分和环境污染物的影响 [67]。 烟草加工过程并不能除去其中所有的蓄积物质，但可以除去烟叶中 的土壤和微生物（包括产生亚硝酸盐的物质），这有助于减少 TSNA 和其他亚硝胺类物质的形成，且能降低蓄积在烟叶中的金属和农用 化学品的含量。 土壤和肥料中的硝酸盐会增加烟草中的硝酸盐含量 [5]。当烟 草中存在能够将硝酸盐转化为亚硝酸盐的微生物时，便会生成亚硝 酸盐。从微生物细胞排出的亚硝酸盐可以与烟草生物碱反应形成 TSNA。可以通过洗涤烟草 [88]、热处理封闭系统（巴氏消毒）[28]、清 洁发酵设备和在发酵过程中添加不产生亚硝酸盐的微生物 [75]等方法， 来降低 TSNA的产量。此外，冷藏储存也可以减缓微生物的生长， 减少亚硝胺化合物的形成。有多家制造商建议零售商对产品进行冷 藏，防止存储期间形成 TSNA[4]。可以通过减少或不使用含硝酸盐的 ·181· 6. 无烟烟草制品的有害内容物和释放物 肥料及其他策略（如在生长季节后期使用尿素或其他非硝酸盐肥料） 的方式，来减少烟草中的硝酸盐蓄积，从而减少亚硝胺的形成 [5]。 使用晾干的方式可以降低烟草中的多环芳烃和挥发性醛的含量。 瑞典的 GothiaTek®标准中明确规定了 snus中有害公众健康的物 质的最大含量，如亚硝酸盐、NNN、NNK、N-亚硝基二甲胺、苯并 [a] 芘、黄曲霉素、镉、铅、砷、镍、铬和农用化学品中。要减少 snus 产品中有害物质的含量，必须对相关原材料进行质量控制。并且在 产品使用过程中添加的香料香精必须符合瑞典食品法 [28]。遵守这些 标准，规范农业化肥的使用和生产加工工艺，可以针对性地降低无 烟烟草制品 snus（湿鼻烟）中有害物质浓度。瑞典这种严格限制产 品成分的方式也可能适用于降低其他类型烟草制品中有害物质含量。 世界卫生组织建议，在条件可行的情况下，无烟烟草中的 TSNA 含量上限应降低到 2 µg/g；如条件不可行，应尽可能地将其限制为 2 µg/g[88]。 6.5  结论和建议 无烟烟草制品种类繁多，包含了仅含烟草的产品、烟草和化学 物质及非烟草植物材料的混合制品等。这些产品在外观、生产方法、 内容物和添加剂及使用方式等方面各不相同。无烟烟草制品中的许 多有害物质来源于烟草中的有机、无机和微生物组分以及加工过程 中这些组分的交互作用。在无烟烟草制品使用过程中添加的植物添 加剂等会对制品的吸引力（味道或外观）、烟碱吸收、致瘾性、有 害性以及致癌和致病性产生影响 [4, 5, 11, 12]。 ·182· 烟草制品管制科学基础报告： WHO研究组第六份报告 由于 89%的无烟烟草使用者都在南亚，因此在评估与无烟烟草 制品相关的健康风险时，应优先考虑南亚制品中的特有成分，尤其 是槟榔。槟榔是一种 IARC 1类致癌物质 [66]，在全世界所有的人口中， 大约有 6亿人使用槟榔 [89]。槟榔的使用是一个全球性的公众健康问 题，因为它在全球范围内使用普遍 [89]，具有致癌性和成瘾性，并且 可以通过某种形式传播 [90]。 使用无烟烟草制品时，需要考虑以下方面： • 高烟碱（N. rustica）或有害（N. glauca）的烟草品种； • 烟草从污染的土壤中摄取的有害金属及其在烟叶中的蓄积； • 在收获季节施肥导致的烟草中硝酸盐含量升高； • 在收获季节施肥烟草中有害的农业化学残留物； • 微生物的污染促进了亚硝胺特别是 TSNA的形成； • 在厌氧环境条件下进行发酵或陈化，会促进亚硝酸盐和 TSNA的形成； • 火烤的加工方式会在制品中引入某些化学物质（如 PAH和 挥发性醛）； • 碱剂的使用会使制品 pH游离烟碱浓度增加； • 槟榔（IARC 1类人体致癌物）和其他公认的有害物质的添加。 在世界范围内，只有瑞典生产的 GothiaTek® snus产品测定了 其中某些农药、金属、亚硝胺以及亚硝酸盐和苯并 [a]芘（一种 PAH）的含量，以确保产品符合标准。虽然进行测定符合标准的产 品并不代表它没有健康风险，但有助于降低制品中某些有害物质的 含量 [28]。 无烟烟草制品的制造商可以选择产品中所用烟叶的类型和质量、 加工过程和添加剂的使用等。然而，虽然现有的技术可以降低产品 ·183· 6. 无烟烟草制品的有害内容物和释放物 中致癌物和其他有害物质的含量，但并没有强制要求制造商使用这 些技术。一般来说，新型产品通常 TSNA含量较低，而旧代和传统 产品中的 TSNA含量较高 [91]。监管机构可以监测和调节 pH、烟碱、 金属、PAH、TSNA和亚硝酸盐含量。通过控制原材料和加工过程可 以降低产品中有害物质的含量，尤其是在烟草固化和微生物反应中 产生的物质（这些反应主要会形成TSNA和挥发性N-亚硝胺）。目前， 检测 pH（pH纸、pH探针）、硝酸盐 /亚硝酸盐（指示剂、手持探针） 和微生物污染（培养板）的技术方法价格并不昂贵，适用于大多数 国家。手持式红外线扫描仪可用于识别有害烟草品种（N. rustica、N. glauca）、非烟草植物材料（槟榔、丁香豆、卡塔）和碱剂（碳酸镁、 熟石灰）。监管机构还应考虑储存条件，例如销售前冷藏，贴上生 产日期和规范包装材料等。此外，还应该要求制造商向零售商告知 储存条件对无烟烟草制品的影响。 本节概述的信息支持世界卫生组织 TobReg的建议，即无烟烟草 制品应由科学政府机构进行全面的监管控制 [92]。鉴于无烟烟草制品 的使用率高，有害物质成分和浓度多样性，对健康具有不良影响的 多样性，引起世界各地口腔癌发病率升高的程度不同等 [92]原因，也 许不能将所有的无烟烟草制品视为一类并采取统一的方法进行监管。 以瑞典湿鼻烟产品“snus”为例，它指的是一类产品，这类产品的 加工方式和特征各不相同 [93]，但是常常会混淆消费者和其他人。使 用含有的“鼻烟”一词是营销的一个例子，用来表示由不同流程和 不同特征生产的产品，它可能对无烟烟草制品的设计、成分和含量 以及加工过程进行仔细审查，有助于减少其在全球范围内使用而造 成的危害。 ·184· 烟草制品管制科学基础报告： WHO研究组第六份报告 6.6  参  考 文 献 [1] Boffetta P, Hecht S, Gray N, Gupta P, Straif K. 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Tob Control 2012;21:181-190 ·197· 7. 卷烟内容物和释放物中烟碱、烟草特 有亚硝胺和苯并 [a] 芘的标准操作 规程在无烟烟草制品中的应用 Nuan Ping Cheah，新加坡卫生科学局卷烟测试实验室 Patricia Richter，美国疾病控制与预防中心（佐治亚州亚特兰大） 侯宏卫 Hongwei Hou，中国国家烟草质量监督检验中心 胡清源 Qingyuan Hu，中国国家烟草质量监督检验中心 Cliord Watson，美国疾病控制与预防中心（佐治亚州亚特兰大） 目录 7.1 引言 7.2 无烟烟草制品中的烟碱、烟草特有亚硝胺和苯并 [a]芘 7.2.1 烟碱 7.2.2 烟草特有亚硝胺 7.2.3 苯并 [a]芘 7.3 WHO标准操作规程对无烟烟草制品分析的适用性评价 7.3.1 分析方法评价 7.3.2 烟碱的测定 7.3.3 烟草特有亚硝胺的测定 7.3.4 苯并 [a]芘的测定 7.4 讨论和建议 7.5 参考文献 烟草制品管制科学基础报告： WHO研究组第六份报告 ·198· 7.1  引    言 WHO烟草控制框架公约（FCTC）缔约方会议（COP）第五届 会议 [1]要求WHO确定管理无烟烟草制品中化合物的方案。COP第 六次会议请秘书处邀请WHO在两年内评估烟草内容物及释放物中 烟碱、烟草特有亚硝胺（TSNA）和苯并 [a]芘的标准操作规程（SOP） 是否适用于除卷烟以外的烟草制品，包括无烟烟草。中国国家烟草 质量监督检验中心、美国疾病控制与预防中心（CDC）和新加坡 卫生科学局同意承担此任务，以确定WHO关于烟草中烟碱含量的 SOP和主流烟草烟气中的 TSNA和苯并 [a]芘分析是否可用于无烟烟 草。选择代表商业和研究无烟烟草的鼻烟、潮湿鼻烟、干燥鼻烟和 散叶咀嚼烟草进行测试。为了符合WHO的最后期限，测试实验室 同意使用在某种程度上可代表常见形式无烟烟草化学特征并且在物 理和化学性质上存在差异的测试材料。本节介绍了WHO标准操作 规程对无烟烟草制品的适用性和适应性的评估以及推荐的方法。 7.2  无烟烟草制品中的烟碱、烟草特有 亚硝胺和苯并 [a] 芘 7.2.1  烟碱 如第 6节所述，烟碱被认为是无烟烟草中的主要致瘾物质。 7. 卷烟内容物和释放物中烟碱、烟草特有亚硝胺和苯并 [a]芘的 标准操作规程在无烟烟草制品中的应用 ·199· 它以离子化或非离子化（也称为未质子化或游离）状态存在。这种 非离子形式具有特殊的公共卫生和监管意义，因为它是烟碱快速通 过口腔黏膜吸收的形式 [2]。产品可能具有相似水平的总烟碱，但根 据其 pH提供不同量的非离子化烟碱。根据烟碱的 pKa和 Hender- son-Hasselbalch方程、测得 pH和总烟碱含量可计算出总非离子化烟 碱的量及其所占的百分比 [2]。因此，烟碱水平和 pH的测量对于通知 政策和监管至关重要。表 7.1列出了文献中报道的烟碱含量。 表 7.1 各种无烟烟草制品中烟碱、非离子化烟碱、pH、水分、TSNA 和苯并 [a] 芘的浓度 [3-12] 类型 总烟碱，湿 重（mg/g） 非离子化 烟碱（mg/ g） pH 水分 （%） 总TSNA（μg/g） 苯并 [a]芘 （μg/g） Gul powder tobacco leaf zarda 9.55~65.0 0.05~31.0 5.22~9.22 7.47~25.23（湿 重） 3~38.2 Khaini gutkha 0.16~21.3 0.12~4.68 7.43~9.65 0.14~127.93 （干重） Mawa Mainpuri Naswar toombak 0.16~40.6 0.11~13.2 7.38~11.0 6~60 0.10~7870 湿鼻烟（鼻烟） 7.76~26.92 （干重） <0.01~13.8 5.54~10.1 35~60 2.0~7870 ≤940 干鼻烟 <0.01~71.4 6~7 ≤1219 >0.1~90 7.2.2  烟草特有亚硝胺 TSNA是由烟草生物碱和亚硝化剂在固化、发酵、老化和高温 高相对湿度下储存过程中形成的强致癌物 [3, 13]。无烟烟草中的 TSNA 浓度是主流烟草烟气中的 500倍（表 7.2），尽管它们在国家和产品 间差异很大 [6, 14]。苏丹使用的无烟烟草 toombak报道的总 TSNA浓 度最高（992000 ng/g）[5]。 烟草制品管制科学基础报告： WHO研究组第六份报告 ·200· 表 7.2 无烟烟草和卷烟烟草及释放物中烟碱、TSNA 和苯并 [a] 芘的浓度 [3,15,16] 待测物 无烟烟草产品 烟草填充物 主流卷烟（ng/卷烟） 无烟烟草和卷烟浓度 差异倍数 烟碱 ≤71.4 mg/g 23.18 mg/g — >2.5 TSNA ≤992 000 ng/g — 1068.8 苯并 [a]芘 ≤940 ng/g — 29.93 7.2.3  苯并 [a] 芘 卷烟主流烟气中释放的苯并 [a]芘是烟草燃烧的结果，而无烟 烟草中的苯并 [a]芘则是由于使用了含有 PAH的火烤烟草 [17]。含有 火烤烟草的无烟烟草中含有苯并 [a]芘，其含量从低出检测限至 940 ng/g，明显高于主流卷烟烟草的产量（表 7.2）。苯并 [a]芘是 IARC 第一类人类致癌物。它经常作为 PAH暴露量的替代物来测量 [18]。 7.3   WHO标准操作规程对无烟烟草制品分析的 适用性评价 7.3.1  分析方法评价 目前已经发表了许多用于烟碱分析的方法，包括测定 pH和水 分含量。基于 GC-FID的技术是应用最广泛的技术 [2]，已经被美国马 萨诸塞联邦采用 [19]，并被 TobLabNet验证可用于分析卷烟烟丝。其 他已公布的程序包括使用MS进行检测 [4,5]。GC与热能分析仪或MS 结合常用于测定无烟烟草中的 TSNA[7,20]。用于分析卷烟烟气中 PAH 7. 卷烟内容物和释放物中烟碱、烟草特有亚硝胺和苯并 [a]芘的 标准操作规程在无烟烟草制品中的应用 ·201· 的气相色谱 -质谱法（GC-MS）的改进适用于无烟烟草的分析 [20,21]。 7.3.2  烟碱的测定 无烟烟草中烟碱的测定可以基于WHO SOP-04[22]。总烟碱和 非离子化烟碱的值对评估无烟烟草的成瘾性有重要的价值 [20,23]。在 SOP-04中，烟碱从卷烟烟丝中用氢氧化钠和正己烷的水溶液萃取出 来，在此过程中，所有烟碱都转移到正己烷中。提取物通过 GC-FID 分析，GC-FID通常用于分析主流卷烟烟气和电子液体中的烟碱。通 常分析实验室可提供该设备。 无烟烟草制品中烟碱的浓度与卷烟烟丝中报道的浓度相当或稍 高（表 7.2）。还应测量 pH和水分含量（在湿鼻烟中高达 50%）， 以便可以在干重和湿重基础上报告结果。TobLabNet SOP-04不包括 湿度和 pH的测量。重量测定法用于无烟烟草中的挥发性化合物的 测量，并描述了烟草与水混合物的 pH的测定 [2,5,20,24]。这些额外的测 量并不复杂，但需要处理烟草样品的设备（例如研磨含有大块烟叶 的样品，如散叶烟草）和能够保持 99~100℃几小时的干燥烘箱。一 种测量无烟烟草含水量的方法是对 AOAC 966.02方法的改进 [25]，被 称为“总水分测定”，用于测定在 99℃±1.0℃是挥发性的水和烟草 成分 [2]。 无烟烟草的 pH应使用标准 pH计测定。通常，将 2 g无烟烟草 与 20 mL分析级水混匀，在室温（20~25℃）下振荡或搅拌，60 min 内用校准的 pH计测量 pH。pH计用经过认证的标准缓冲液进行校准。 确认 pH没有系统误差很重要 [2]。根据样本的类型，再加入 10 mL水 稀释混合物以便于测量。 无烟烟草中总烟碱和非离子化烟碱含量由 Henderson-Hassel- 烟草制品管制科学基础报告： WHO研究组第六份报告 ·202· balch方程基于总测定的烟碱、pH和 pKa值（8.02）计算而来 [2,20]。 7.3.3  烟草特有亚硝胺的测定 目前 CDC用于确定烟气和烟草中 TSNA的方法与WHO SOP-03 类似 [26]，只有少数例外。 WHO SOP-03的样品制备和分析部分适用于无烟烟草，并且 To- bLabNet方法确定卷烟填充物中的 TSNA用于分析无烟烟草应该相对 简单。无烟烟草的 NNN和 NNK含量在 20~10000 ng/g之间，而主流 烟气中的 NNN和 NNK含量（表 7.2）处于标准工作曲线低浓度范 围内。因此，上限校准范围将不得不扩大到可以涵盖更高浓度 NNN 和 NNK的无烟烟草产品。线性度或探测器饱和度不应该成为问题。 SOP-03的适应性应基于与目前用于主流烟气释放物和烟草中 TSNA含量测定的 CDC方法的比较结果 [15]。具体而言，如上所述， 无烟烟草的校准曲线应该延长（并且保持线性），并且无烟烟草样 品可能需要研磨和过滤，使烟草“细粒”不会阻塞注射系统。样品 制备应与WHO TobLabNet方法和现行 CDC方法相同，包括提取 程序。提取的样本量必须进行优化，并且应考虑其他修正，如研磨 烟草以提高提取效率。因此，经过适当调整，用于测量卷烟排放中 TSNA的 TobLabNet方法也可测量无烟烟草中的 NNN和 NNK。 7.3.4  苯并 [a] 芘的测定 无烟烟草中苯并 [a]芘的测定可基于WHO SOP-05测定卷烟烟 气中苯并 [a]芘含量的方法 [27]。在WHO方法中，卷烟烟气捕集在 1 μm玻璃纤维制成的 CFP中。吸烟后，滤片用含有同位素标记的苯 7. 卷烟内容物和释放物中烟碱、烟草特有亚硝胺和苯并 [a]芘的 标准操作规程在无烟烟草制品中的应用 ·203· 并 [a]芘 -D12的环己烷溶液萃取。 环己烷萃取物通过二氧化硅固相萃取柱洗脱，收集洗脱液并通 过 GC-MS电子电离模式进行分析。0.2~1.0 g无烟烟草产品样品（验 证期间对量进行优化）应使用环己烷（室温下 10 mL）萃取，振荡 1 h并将提取物 200 r/min下离心 60~80 min。在 5 mL提取物中加入 苯并 [a]芘 -D12内标并充分混合。SOP-05中所示的样品净化包括在 硅胶柱上进行固相萃取，然后进行旋转蒸发。实验室应探讨是否需 要旋转蒸发。 将固相萃取的样品混合物装入预先清洗过的硅胶柱（Waters公 司的 Sep-pak Vac硅胶柱或同等产品）中进行样品清洗，并用环己烷 洗脱。将多次洗涤的洗脱液混合并干燥。然后用 1 mL环己烷重新溶 解并用 GC-MS进行分析。在方法验证过程中应探讨的可选步骤包括 采用固相萃取法净化样品和旋蒸，正如参考文献 [27]的规定那样。 复溶试样应通过 GC-MS进行分析。 CDC选择了七种无烟烟草产品（snus、湿鼻烟、干鼻烟和散叶） 进行本研究（表 7.3）。四种是从 CORESTA获得的参考产品，三种 是从供应商（Lab Depot, Atlanta, GA, USA）获得的商业产品。CDC 将这 7种无烟烟草产品邮寄到中国国家烟草质量监督检验中心和新 加坡卫生科学局进行方法验证。 表 7.3 选择无烟烟草测试材料进行方法验证 无烟烟草产品 类型 产品 总烟碱 pH 水分（%） TSNA 苯并 [a]芘 CRP1 鼻烟 参考 0.8%（湿重） 8.5 52 ~1.46 ppm 待定 CRP2 湿鼻烟 参考 1.2%（湿重） 7.7 54.6 ~4.40 ppm 待定 CRP3 干鼻烟 参考 1.2%（湿重） 7.7 54.6 18~19 ppm 待定 CRP4 散叶 参考 1.9%（湿重） 6.9 8.0 ~3.70 待定 烟草制品管制科学基础报告： WHO研究组第六份报告 ·204· 续表 无烟烟草产品 类型 产品 总烟碱 pH 水分（%） TSNA 苯并 [a]芘 Silvercreek Wintergreen[7] 湿鼻烟 商业 8.2-11.96mg/g （湿重） 6.29- 7.08 51.9-52.6 15.86 mg/g （湿重） 待定 Skoal Original [14,28] 湿鼻烟 商业 11.4 mg/g （湿重） 7.27 59 ？ 待定 Red Seal Wintergreen 湿鼻烟 商业 14.9 mg/g （湿重） 7.55 53.3 4.87~5.27mg/g（湿重） 待定 ppm：百万分之一 7.4  讨论和建议 本部分的目标是推荐量化分析程序，以适应现有的 TobLabNet 验证卷烟的方法并将其应用于无烟烟草的分析。已经发布了许多用 于分析烟碱和 TSNA的方法，还包括 pH测定和水分含量测定。GC- FID是首选方法，因为该设备在全球分析实验室中普遍可用。 知识渊博的专家对烟碱、苯并 [a]芘和 TSNA的 TobLabNet 标准 操作程序的审查结论是：这些方法适用于无烟烟草制品。必须对代 表性样品进行交叉基质研究以对方法进行确认。尽管为了涵盖一系 列物理和化学特性而选择了各种研究和商业无烟烟草测试样品（表 7.3），但样品没有涵盖不同类型的烟草产品的所有品种。样品制备 优化的方法有限，而对于 NNN和 NNK，校准范围将不得不扩大以 涵盖那些含量较高的无烟烟草制品（表 7.1）。另外，应该讨论测定 pH和水分的方法并达成共识。我们建议对无烟烟草产品中的烟碱、 pH、苯并 [a]芘、NNN和 NNK进行交叉基质验证实验，以适应 TobLabNet标准操作程序。 7. 卷烟内容物和释放物中烟碱、烟草特有亚硝胺和苯并 [a]芘的 标准操作规程在无烟烟草制品中的应用 ·205· 结论 • 用于测定 TSNA和烟碱含量的 TobLabNet方法可用于或适用 于测定无烟烟草制品； • 应该验证 TobLabNet方法测定苯并 [a]芘的适用性，因为基 质不同于标准操作规程中指定的方法； • 具体、有选择性的 TobLabNet方法和净化步骤应该能够提取 有害物质； • 标准曲线校准范围的延长或样品的稀释应涵盖无烟烟草产品 中的较高浓度值。 推荐 • 要求制造商通过独立实验室公布产品的 pH和有害物质 TSNA、苯并 [a]芘和烟碱的水平（用WHO验证的方法或国 家的官方方法测量）； • 合规性可以在政府机构指定的任何分析实验室中进行测试。 下一步工作 • 通过现有的实验室资源，利用烟草、食品、植物和环境基质 的现有方法，对无烟烟草制品中的金属、保润剂和醛类物质 进行分析。 烟草制品管制科学基础报告： WHO研究组第六份报告 ·206· 7.5  参  考 文 献 [1] Report of the sixth session of the Conference of the Parties to the WHO Framework Convention on Tobacco Control. 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J Natl Cancer Inst 1995;87:1862-9. ·210· 8. 总体建议 WHO烟草制品管制研究组（TobReg）出版了一系列报告，为 烟草制品管制提供了科学依据。根据世界卫生组织《烟草控制框架 公约》（WHO FCTC）第 9条和第 10条⑨，这些报告确定了以证据 为基础的烟草制品管理办法。 第八次会议侧重于推进烟草制品管理的关键问题，尤其是在 WHO FCTC第六次缔约方会议上概述的问题⑩。讨论的主题包括： ①卷烟特性和设计特点；②水烟烟草和无烟烟草中的有害物质；③世 界卫生组织烟草实验室网络（TobLabNet）将测定卷烟烟草制品特定 内容物和释放物中化学物质的标准操作程序（SOP）用于 ENDS、水 烟烟草和无烟烟草制品。 主要建议 本报告提供了有关指定卷烟设计特征的相关指导，以及测试和 披露各种无烟烟草制品、水烟烟草制品和其他产品（如 ENDS）的 内容物和释放物含量。 设计特点：成员国应该要求烟草制品制造商和进口商按照指定 ⑨ 更多信息见 http://apps.who.int/iris/bitstream/10665/42811/1/9241591013.pdf?ua=1 （2016年 9月 20日） ⑩ 欲了解更多关于世界卫生组织《烟草控制框架公约》缔约方大会的信息，见 http://apps.who.int/gb/fctc/E/E_cop6.htm 上的 FCTC/COP6(10) 号决定第 2(a) 段和 FCTC/ COP6(12)号决定第 2(b)段（2016年 9月 20日） ·211· 8. 总体建议 的时间间隔向世界卫生组织政府提供世界卫生组织《烟草控制框架 公约》部分指南附录 2中所列的设计特征信息，包括烟草行业进行 的测试结果。成员国还应考虑限制或禁止可能增加烟草产品吸引力 的其他设计特征，例如香味物质和胶囊。最后，如果特定品牌的烟 草产品的设计特征发生变化，成员国应该要求制造商通知政府这一 变化，并在变更时提供最新信息。 无烟烟草：要求制造商上报无烟烟草产品烟草特有亚硝胺 （TSNA），苯并 [a]芘（B[a]P）、烟碱含量及 pH，因为WHO To- bLabNet方法适用于测定这些特定的有害物质。此外，由于现有技术 可降低无烟烟草致癌物的含量，因此要求制造商使用这些技术以降 低产品的有害性。监管机构还应考虑要求制造商改善储存条件，例 如冷藏销售前的产品、标注生产日期和调整包装材料。最后，还应 要求制造商向零售商介绍无烟烟草产品的储存条件。 水烟烟草：水烟吸烟通常使用燃烧的木炭作为热源，因此，水 烟烟气除了来自烟草制品本身的炭之外，还包括由炭产生的有害物 质。由于这种复杂性，监管机构应该考虑一种方法，首先着重于测 量和报告水烟烟草制品中已知的增加有害性、成瘾性和吸引力的化 学成分，当评估和分析方法得到验证后，再将其扩大至释放物中的 化学物和有害物质。 ENDS：有足够的数据来支持现有和进行中的WHO SOP在 ENDS烟液和气溶胶中针对烟碱、保润剂（溶剂）、羰基化合物、（B[a]P） 和 TSNA的扩展。建议测量烟液的 pH以确定 ENDS烟液的 pH范围， 这将有助于调查提供给使用者的烟碱成瘾性。应对金属进行检测， 以确定是否存在潜在的相关健康风险。 ·212· 烟草制品管制科学基础报告： WHO研究组第六份报告 对公共卫生政策的意义 制定全面和有效的烟草控制政策面临的挑战之一是商业上可获 得的烟草产品具有广泛性和多样性。TobReg的报告为理解特定产品 （如卷烟、无烟烟草和水烟）的内容物、释放物和设计特点提供了 有益的指导。报告强调了它们的有害物质或特征对公众健康的影响。 此外，该报告阐述了WHO TobLabNet标准操作规程如何作为测试这 些产品的可靠方法。现有的知识表明，需要对多种烟草制品的使用 情况进行积极调查，并监测新兴的新型烟草制品。 对世界卫生组织计划的意义 该报告应 TobReg的要求向WHO总干事提供了成员国关于烟草 制品管制的科学合理、有据可依的建议。根据WHO FCTC第 9条和 第 10条的规定，TobReg已经确定了以证据为基础的方法来管理成员 国大量的烟草制品。TobReg的报告还列出了未来的研究领域，这将 扩大有关烟草制品监管的知识基础。