An overview of the effects of tobacco ingredients on smoke chemistry and toxicity

https://doi.org/10.1016/j.fct.2004.01.001Get rights and content

Abstract

This paper presents an overview of a series of studies designed to assess the influence of 482 tobacco ingredients on cigarette smoke chemistry and toxicity. The studies are: pyrolysis of the ingredients; influence of the ingredients on smoke constituents believed by regulatory authorities to be relevant to smoking-related diseases (“Hoffmann analytes”); influence of the ingredients on in vitro genotoxicity and cytotoxicity of smoke partiulate matter; and influence of the ingredients on the inhalation toxicity of smoke. The present paper brings the salient features of these studies together. A pyrolysis technique has been developed which, as far as practicably possible, mimics the combustion conditions inside a burning cigarette. The results from 291 single-substance ingredients indicate that almost a third would transfer out of the cigarette burning zone at least 99% intact (i.e. less than 1% pyrolysis), and almost two thirds would transfer at least 95% intact. Of the ingredients that underwent some degree of pyrolysis, a few “Hoffmann analytes” were detected amongst the pyrolysis products of 19 ingredients. Taking into account maximum use levels, their maximum pyrolysis levels were generally small and often insignificant compared to the levels typically present in smoke. Possible exceptions were acetaldehyde and benzene from the pyrolysis of malic acid. However, subsequent smoke chemistry studies indicated that the maximum levels predicted from pyrolysis of this involatile substance were overestimated, suggesting that malic acid does not undergo complete pyrolysis in the burning cigarette and/or generates acetaldehyde and benzene at similar rates to that of tobacco on a per weight basis. When added to tobacco, many of the ingredient mixtures produced no significant effect on the levels of many of the “Hoffmann analytes” in smoke, while some produced increases or decreases relative to the relevant control cigarettes. The study has concentrated on the increases. Many of the differences were found to be not significant when the long-term variability of the analytical methodology was taken into account. However, even taking this into account, the smoke formaldehyde levels in two of the test cigarettes were significantly increased relative to their controls, by up to 26 μg (73%). These increases are likely to be due to the pyrolysis of sugars, cellulose and other polysaccharide materials. The activity of smoke particulate matter from cigarettes containing tobacco ingredients has been determined with three in vitro bioassays, two for genotoxicity and one for cytotoxicity. These were the Ames test, the mammalian cell micronucleus assay, and the neutral red uptake cytotoxicity assay. Within the sensitivity and specificity of these bioassays, the specific activity of the cigarette smoke particulate matter was not changed by the addition of ingredients to the cigarette. Three 90-day sub-chronic inhalation studies have been undertaken and histopathological and histomorphometric assessments made within the respiratory tracts of animals exposed to smoke from cigarettes containing the various ingredient mixtures and their control cigarettes. The response due to tobacco smoke exposure was not distinguishable between the test and control cigarettes, indicating that the presence of the ingredients had made no discernable differences to the type and severity of the treatment-related changes.

Introduction

In some parts of the world e.g. Germany and the U.S.A., tobacco companies add ingredients to tobacco products, either to impart a specific taste, flavour or aroma to the product, or for a specific technological purpose such as increasing the moisture-holding capacity of the tobacco. In other countries, e.g. the U.K., Australia and Canada, flavour ingredients are not generally used on cigarettes. Since the 1960s, it has been suggested that the use of such ingredients might increase the toxicity of tobacco smoke. In their classic 1967 book on tobacco smoke, Wynder and Hoffmann (1967) hypothesised that the use of tobacco ingredients might potentially either increase or decrease the toxicity of tobacco smoke. This has been repeated several times since, generally emphasising the possibility of increasing the toxicity of smoke (LaVoie et al., 1980, Hoffmann and Hoffmann, 1997, Hoffmann and Hoffmann, 2001, WHO, 2001). In the context of low ‘tar’ cigarettes, it has also been assumed in the 1979 and 1981 U.S. Surgeon General's reports, as well as by the World Health Organisation, that flavour ingredients are more likely to be used (United States Public Health Service, 1979, United States Public Health Service, 1981, WHO, 2001). It is emphasised that in these discussions of the potential changes in smoke toxicity through the use of tobacco ingredients, no evidence was presented to support the hypotheses. Furthermore, the increased use of flavour ingredients with low ‘tar’ cigarettes was an assumption. Low ‘tar’ cigarettes are developed with a variety of cigarette design options: filtration, filter ventilation, paper permeability, tobacco blend, reconstituted tobacco sheet and expanded tobacco. In general, within British American Tobacco, flavour ingredients are not used any more on low ‘tar’ cigarettes than on higher yield cigarettes; to do so would produce an ‘unbalanced’ taste. One exception to this generalisation is in mentholated cigarettes, where menthol usage tends to increase as ‘tar’ yields decrease.

The hypothesis of increased toxicity has been refuted by work recently published in several papers. Three major reviews were published in 2002 that examined studies on the chemical and biological effects of tobacco ingredients on smoke properties conducted over the last 50 years (Paschke et al., 2002, Rodgman, 2002a, Rodgman, 2002b). Paschke et al. gave an overview of 189 published papers that covered the effects of well over 300 ingredients, not all of which are used commercially. They concluded that the results from most studies clearly indicate that ingredients do not increase the biological activity of cigarette smoke (carcinogenicity, mutagenicity and cytotoxicity) but found many gaps in knowledge on the chemical effects of ingredients. Rodgman examined the extensive laboratory data on the effects of tobacco ingredients on smoke chemistry and various bioassays, in particular previously unpublished information from the R.J. Reynolds Tobacco Company undertaken from the 1950s to the 1970s (Rodgman, 2002a, Rodgman, 2002b). His conclusions were similar to those of Paschke et al., namely that ingredients added to tobacco during commercial cigarette manufacture in the U.S.A. do not increase the toxicity of cigarette smoke.

Also during 2002, Carmines and co-workers published a series of four papers that described a comprehensive study on the evaluation of 333 ingredients used in Philip Morris products. This study included smoke chemistry, in vitro genotoxicity and cytotoxicity, and animal sub-chronic inhalation toxicity (Carmines, E.L., 2002, Rustemeier et al., 2002, Roemer et al., 2002, Vanscheeuwijck et al., 2002). Taking into account all the smoke chemistry and biological data, they concluded that the addition of the ingredients to tobacco did not increase the toxicity of the smoke.

Gaworski et al. have also recently published details of studies in which the biological effects of single ingredients, or mixtures of ingredients, added to cigarettes were evaluated (Gaworski et al., 1997, Gaworski et al., 1998, Gaworski et al., 1999, Heck et al., 2002). They have assessed the effects of menthol (Gaworski et al., 1997), glycerol (glycerine) and propylene glycol (Heck et al., 2002) singly, and 172 flavour ingredients in six combinations (Gaworski et al., 1998) in various 13-week sub-chronic smoke inhalation toxicity studies with rats. They concluded that the addition of the flavour mixtures or menthol, glycerol and propylene glycol, as single ingredients at typical application levels to cigarettes, had no discernable effect on the character or extent of the biological responses normally associated with inhalation of cigarette mainstream smoke in rats. In 1999 Gaworski et al. also reported the results of a series of SENCAR mouse skin painting bioassays that were aimed at assessing the potential of six combinations of 150 flavour ingredients to affect the tumour-promoting activity of cigarette smoke particulate matter (Gaworski et al., 1999). No indications of any increase in tumour-promoting activity were found for any of the ingredient combinations evaluated.

In 2002 Seeman et al. published a comprehensive review of the formation of acetaldehyde in mainstream cigarette smoke and its bioavailability in the smoker (Seeman et al., 2002). They covered all known literature on the subject, including the effects of sugars as a tobacco ingredient on smoke acetaldehyde levels. They concluded that sugars do not produce greater yields of acetaldehyde in mainstream smoke than are produced from tobacco itself on a weight-for-weight basis. They also concluded that natural tobacco polysaccharides, including cellulose, were the primary precursors of acetaldehyde in mainstream smoke.

Thus, the results from numerous studies have indicated that the addition of either single ingredients, or various combinations of ingredients used commercially, to tobacco does not adversely change the smoke chemistry or toxicity as measured in specified assays. In spite of this extensive research, the present study was undertaken to assess the effect of cigarette tobacco ingredients at or above typical maximum levels used by British American Tobacco on smoke chemistry and toxicity.

Potentially, ingredients can undertake one or more of the following processes during tobacco combustion in the smoking process:

  • (a)

    Distil directly into smoke

  • (b)

    Pyrolyse or oxidise and their products enter smoke

  • (c)

    Pyrolyse or oxidise and the reaction products react with tobacco and smoke constituents and affect their yields or generate other smoke products.

Because of the possibilities of processes (b) and (c), justification for the use of tobacco ingredients based solely on their approved use in food may not always be appropriate. There are no regulatory requirements for testing tobacco ingredients in the U.S.A. although a few individual states have specific requirements of disclosure. In the European Union there is a requirement for all available information on ingredients relevant to smoke toxicity to be submitted to the individual member states.

In the present paper, an overview is given on studies undertaken to investigate the potential chemical changes and biological activity of smoke from cigarettes with added ingredients used by British American Tobacco. The detailed results of the individual chemical studies have been published elsewhere, as indicated below. This present paper brings the salient features together and includes the biological studies. This covers the following aspects:

  • 1.

    Determination of the relative amounts of distillation and pyrolysis/oxidation of the ingredients during the combustion of tobacco (Baker and Bishop, 2004).

  • 2.

    Evaluation of the effect of the ingredients on the generation of known toxic chemical constituents of cigarette smoke (Baker et al., 2004a, Baker et al., 2004b).

  • 3.

    Evaluation of the effect of ingredients on the potential of cigarette smoke particulate matter to damage DNA (genotoxicity) as measured by:

    • (a)

      the Ames test, an in vitro bacterial mutation assay (Massey et al., 2002)

    • (b)

      the in vitro micronucleus test, a mammalian cell assay for chromosome damage.

  • 4.

    Evaluation of the effect of ingredients on the in vitro cytotoxicity of cigarette smoke particulate matter, as measured by the neutral red uptake assay, a mammalian cell viability test. Cytotoxicity is regarded as having a potential role in several chronic disease processes associated with smoking, including carcinogenesis and emphysema (Bombick, 2002).

  • 5.

    Evaluation of the effect of ingredients on the potential sub-chronic inhalation toxicity of cigarette smoke.

Individually, each of these assessments may have some limitations. However, collectively they provide comprehensive information on the potential effects of tobacco ingredients on the chemical and toxicological profile of cigarette smoke. Most of the assessments are recommended in the U.K. Guidelines for the assessment of new tobacco ingredients (Report of the Scientific Committee on Tobacco and Health, 1998). Two of the three in vitro bioassay studies and the sub-chronic inhalation toxicity studies were done at external laboratories, as indicated in 2.4 In vitro bioassay studies, 2.5 In vivo sub-chronic inhalation studies below. These laboratories operated in compliance with the Good Laboratory Practice Regulations as set forth by the U.S. Environmental Protection Agency, Code of Federal Regulations, 40, Part 792 (Code of Federal Regulations, 2001).

Section snippets

Pyrolysis studies

For the pyrolysis experiments, 200 μg of the flavour ingredient was pyrolysed, usually in solution in ethanol, in the pyroprobe of a CDS Pyrolyser 2000. The products of pyrolysis were separated by passage through a Hewlett Packard 6890 gas chromatograph and identified with a Hewlett Packard 5973 mass spectrometer. The pyrolysis conditions were carefully chosen to simulate the average conditions inside the burning zone of the cigarette during smoking. Much is known about these conditions:

Pyrolysis studies

Pyrolysis of individual tobacco constituents has been used in many studies since the 1950s to establish relationships between smoke constituents and their precursors in tobacco (e.g. Chortyk and Schlotzhauer, 1973, Schlotzhauer and Chortyk, 1987, Baker, 1987, Baker and Bishop, 2004). If the pyrolysis conditions are the same as those that occur in the cigarette burning zone during smoking, a series of reaction pathways can be established for the generation of specific smoke constituents from

Overall summary

  • 1.

    An overview is given of the effects of 482 tobacco ingredients on various aspects of the chemistry and biological properties of cigarette mainstream smoke. The ingredients comprise: 462 flavours, 1 flavour/solvent, 1 solvent, 7 preservatives, 3 humectants, 5 binders, 1 filler and 2 process aids (one of which is water). The ingredients were added to 19 test cigarettes at or above their typical maximum use levels, in various combinations in three experimental cigarette series. The levels of smoke

General conclusions

This work has contributed to the enormous amount of work that has been conducted over the last fifty years on the effects of tobacco ingredients on the chemical and biological properties of cigarette smoke. This work was comprehensively reviewed in three major reviews published in 2002. Two of these reviews contained details of many previously unpublished studies from the R.J. Reynolds Tobacco Company. In addition, large studies from Philip Morris and Lorillard Tobacco Company have recently

Acknowledgements

It is a pleasure to acknowledge Alan Rodgman, who has made significant contributions to this paper, both by reviewing it and also sharing his considerable knowledge on the subject with us.

References (61)

  • K Rustemeier et al.

    Evaluation of the potential effects of ingredients added to cigarettes. Part 2: Chemical composition of mainstream smoke

    Food and Chem. Toxicol.

    (2002)
  • W.S Schlotzhauer et al.

    Recent advances in studies on the pyrosynthesis of cigarette smoke constituents

    J. Anal. Appl. Pyrol

    (1987)
  • Y.S Stein et al.

    A study of the gas-phase pyrolysis of glycerol

    J. Anal. Appl. Pyrol.

    (1983)
  • P.M Vanscheeuwijck et al.

    Evaluation of the potential effects of ingredients added to cigarettes. Part 4: Subchronic inhalation toxicity

    Food Chem. Toxicol.

    (2002)
  • R.R Baker

    Temperature variation within a cigarette combustion coal during the smoking cycle

    High Temp. Sci.

    (1975)
  • R.R Baker

    Variation of the gas formation regions within a cigarette combustion coal during the smoking cycle

    Beitr. Tabakforsch. Int

    (1981)
  • R.R Baker

    Smoke chemistry

  • Baker, R.R., Bishop, L.J., 2004. The pyrolysis of tobacco ingredients. J. Anal. Appl. Pyrol....
  • Baker, R.R., Pereira da Silva, J.R., Smith, G., 2004a. The effect of tobacco ingredients on smoke chemistry. Part 1:...
  • Baker, R.R., Pereira da Silva, J.R., Smith, G., 2004b. The effect of ingredients on smoke chemistry. Part II: Casing...
  • D.W Bombick

    Assessment of tobacco smoke by in vitro cell toxicity assays

    Recent Adv. Tob. Sci.

    (2002)
  • O.T Chortyk et al.

    Studies on the pyrogenesis of tobacco smoke constituents (a review)

    Beitr. Tabak

    (1973)
  • Code of Federal Regulations (U.S.A.), revised 2001. Title 40, Protection of the environment, Part 792, Good laboratory...
  • Department of Health, Canada, 2000. Proposed Tobacco Reporting Regulations, Schedule 2, Official methods for the...
  • C.W Dunnett

    A multiple comparison procedure for comparing treatments with a control

    J. Am. Stat. Assoc.

    (1955)
  • EEC (European Economic Commission) Directive 67/548, 1967. Toxicology and chemical substances. Testing methods of Annex...
  • D.G Gatehouse et al.

    Bacterial mutation assays

  • C.L Gaworski et al.

    Toxicological evaluation of flavor ingredients added to cigarette tobacco13-week inhalation exposure in rats

    Inhal. Toxicol.

    (1998)
  • C.R Green et al.

    The Tobacco Chemists’ Research Conferencea half century forum for advances in analytical methodology of tobacco and its products

    Recent Adv. Tob. Sci.

    (1996)
  • J.D Heck et al.

    Toxicological evaluation of humectants added to cigarette tobacco13-week smoke inhalation study of glycerine and propylene glycol in Fischer 344 rats

    Inhal. Toxicol

    (2002)
  • Cited by (216)

    • A comparative study on the physicochemical properties of tobacco ash from lit bright cigarettes with different cut tobacco fillers

      2022, Journal of Analytical and Applied Pyrolysis
      Citation Excerpt :

      The research on cigarette combustion process mainly focuses on the measurement, influencing factors and changing rules of process parameters such as temperature, pressure, velocity field, combustion rate and so on [1–9]. The release rules, influencing factors and regulation technology of smoke aerosol and chemical components are the key points of smoke research [10–13], while the research on cigarette combustion ash is mainly emphasized on the physical state of cigarette ash, i.e. ash appearance quality [14–18]. Until now, lots works have been focused on the characterization analysis of cigarette combustion process and the research on smoke release and influencing factors [1–13], which have describe us a clear heat and mass transfer process during cigarette combustion, deepening our understanding of the basic law of cigarette combustion.

    View all citing articles on Scopus
    View full text