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TOXICOLOGICAL ASSESSMENT OF TOBACCO INGREDIENTS Richard R. Baker British American Tobacco Southampton UK LSRO Meeting, D

TOXICOLOGICAL ASSESSMENT OF TOBACCO INGREDIENTS Richard R. Baker British American Tobacco Southampton UK LSRO Meeting, Denver, CO, USA 8/9 June 2004. PLAN OF PRESENTATION. General aspects, definitions etc. Briefly review past work Overview of BAT work: Bioassays Pyrolysis

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TOXICOLOGICAL ASSESSMENT OF TOBACCO INGREDIENTS Richard R. Baker British American Tobacco Southampton UK LSRO Meeting, D

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  1. TOXICOLOGICAL ASSESSMENT OF TOBACCO INGREDIENTS Richard R. Baker British American Tobacco Southampton UK LSRO Meeting, Denver, CO, USA 8/9 June 2004

  2. PLAN OF PRESENTATION • General aspects, definitions etc. • Briefly review past work • Overview of BAT work: Bioassays Pyrolysis Smoke chemistry

  3. SOME DEFINITIONS (a) Tobacco constituent: A substance naturally present in tobacco Tobacco ingredient: A substance, generally a flavor material, added to tobacco during the cigarette manufacturing process

  4. SOME DEFINITIONS (b) FLAVORS Impart a specific taste, flavor or aroma: • Casings-applied to pre-cut tobacco (few %) - often recognised foodstuffs • Flavorings (top flavors) - applied to cut and processed tobacco (ppm levels, several flavors in mixture)

  5. SOME DEFINITIONS (c) ADDITIVES Used for a specific technological purpose, e.g.: • Humectants – increase tobacco moisture-holding capacity • Preservatives – protect product deteriation from microorganisms • Binders and strengtheners – maintain physical state of product • Fillers – contribute to volume without contributing to odor, taste or flavor

  6. TYPICAL CIGARETTE TOBACCO BLENDS

  7. Potentially, ingredients can: • Distil into smoke • Decompose/oxidise and products enter smoke • Reaction products react with smoke constituents and affect their yields and generate other smoke products

  8. GENERAL ASSUMPTIONS BY HEALTH AUTHORITIES • Flavor ingredients increase the toxicity of smoke • Low ‘tar’ cigarettes have higher levels of flavor ingredients than higher yield cigarettes

  9. US Surgeon General’s Report, 1979 In a section discussing technical achievements to develop low ‘tar’ cigarettes, stated: “All of these developments have led to increased use of flavor additives, especially for low-tar, low-nicotine cigarettes. In fact, these new cigarettes require flavor corrections by additives in order to be acceptable to the consumer.”

  10. Wrong assumption Within British American Tobacco, flavor ingredients are not used any more on low ‘tar’ cigarettes than on higher yield cigarettes - menthol is an exception - its use increases as ‘tar’ yield decreases

  11. STUDIES ON INGREDIENTS SINCE 1950s • Pyrolysis • Effects on smoke chemistry • Mouse skin painting • Inhalation toxicity • In vitro bioassays - genotoxicity - cytotoxicity

  12. PUBLISHED REVIEWS ON TOBACCO INGREDIENTS • Paschke, Scherer and Heller, 2002 • Rodgman, 2002, two reviews, including much previously unpublished RJRT work • Dixon et al., 2000, effects of ammonia ingredients on nicotine transfer andbioavailability

  13. RECENT MAJOR STUDIES ON INGREDIENTS • Carmines et al., 2002, four papers – chemistry and biology • Gaworski et al., 1997-2002, four papers – biology • Baker et al., 2004, four papers – pyrolysis, chemistry and biology

  14. Paschke, Scherer and Heller 198 papers/patents from 1952-2002 on ingredients reviewed Over 300 ingredients Smoke chemistry – 150 single ingredients + 61 combinations Pyrolysis (161 papers) Smoke biological activity (37 papers)

  15. Paschke, Scherer and Heller - Conclusions Tobacco ingredients used commercially do not increase the biological activity of cigarette smoke Many gaps in knowledge on pyrolysis and transfer to smoke Standard analytical methods needed for influence of ingredients on smoke chemistry

  16. Rodgman Reviews- (1) Flavorings - (2) Casings Includes previously unpublished RJRT studies Includes work aimed at identifying precursors of smoke toxins -predicted that relatively volatile flavors would distil out of cigarette burning zone -studies on ingredients that could potentially generate smoke toxins

  17. Rodgman - Conclusions Neither flavorings nor casing and humectant ingredients added to tobacco during commercial cigarette manufacture in the USA increase the toxicity of cigarette smoke

  18. Carmines and co-workers, 2002 Study of 333 ingredients added to tobacco in 3 mixtures at normal and 1.5 – 3 x normal use Effects on 51 ‘Hoffmann analytes’ in smoke Effects on Ames and neutral red uptake bioassays Effects on sub-chronic inhalation toxicity (90-day rat inhalation)

  19. Carmines and co-workers - conclusions The addition of the 333 ingredients had not affected the toxicity of smoke, even in the exaggerated high level mixtures.

  20. Gaworski et al., 1997 - 2002 Effects on biological activity of 175 ingredients singly and in combinations: Sub-chronic smoke toxicity (90-day inhalation using rats) Mouse-skin painting

  21. Gaworski et al., conclusions Ingredients had no discernible effect on inhalation toxicity or tumor-promoting activity of smoke

  22. BAT STUDIES • Pyrolyse in isolation – look at products 2. Add to cigarette and see what happens to smoke chemistry – ‘Hoffmann analytes’ 3. In vitro bioassays 4. Inhalation toxicity

  23. ADD TO CIGARETTES • 482 ingredients: 460 flavors 1 flavor/solvent 1 solvent 7 preservatives 5 binders 5 humectants 1 filler 2 process aids (one is water) • Mixtures added to US blended tobaccos • 19 Test cigarettes in 3 series made • 44 ‘Hoffmann analytes’ determined • Bioassays and inhalation

  24. CIGARETTE SERIES Series AFlavorings Series BFlavorings and casings Sheet ingredients Series CCasings

  25. Inhalation toxicity 90-day inhalation with rats Series A, B and C cigarettes – no statistically-significant differences in the animals subjected to smoke from the test and control cigarettes

  26. Cigarette series A: Ames test (TA98 +S9)

  27. In vitro bioassays – on smoke particulate matter 1. Genotoxic endpoints - Ames - Micronucleus bioassay 2. Non-genotoxic endpoint - Neutral red uptake for cytotoxicity None of the test cigarette particulate matters produced changes different from their controls

  28. Three approaches to assess chemical effects of ingredients 1. Add to cigarette and see what happens to smoke chemistry 2. Pyrolyse in isolation 3. Add labelled substance and measure labelled products

  29. Approaches in present pyrolysis study • Develop pyrolysis to simulate conditions during smoking • Use pyrolysis to measure amount of decomposition during smoking

  30. SOME DEFINITIONS (1) Pyrolysis: Decomposition due to heat Pyrosynthesis: Thermal decomposition of substance followed by reaction of their decomposition products to form new, larger molecules

  31. SOME DEFINITIONS (2) Pyrolysis in inert atmosphere: Thermal decomposition, pyrosynthetic reactions can occur Pyrolysis in atmosphere containing oxygen: Combustion reactions can also occur Sometimes called ‘oxygen-sensitised’ or ‘combustion-sensitised’ pyrolysis

  32. Smoke Volatile gases Gases Pyrolysis Distillation Char Oxidation Tobacco Ash Residual Char Loss Feedback Heat Distillation-Pyrolysis Zone Combustion Zone Air

  33. PYROLYSIS • Pyrolysis techniques used in many studies over many years to establish component-smoke product relationships • Many false relationships published • Laboratory pyrolysis conditions must match combustion conditions inside cigarette

  34. Example (1) of a False Pyrolysis Relationship • Schmeltz & Schlotzhauer (1968) pyrolysed menthol at 600°C & 860°C • They found 22% & 84% pyrolysed respectively • The pyrolysis products included phenol & benzo[a]pyrene • BUT smoking of cigarettes containing radiolabelled menthol, shows that 99% of the menthol transfers to the mainstream intact. No phenol or benzo[a]pyrene is detected.

  35. Example (2) of a False Pyrolysis Relationship • Schmeltz et al. (1979) pyrolysed labelled nicotine added to tobacco in combustion tubes at 600 - 900°C • The nicotine underwent simple degradation to pyridines, and extensive degradation and re-arrangement to quinolines, arylnitriles, aromatic hydrocarbons…. • They also smoked the cigarettes. • They found much of the nicotine distilled unchanged to MS and SS smoke, small amount of simple degradation to pyridines, and no extensive degradation.

  36. TOBACCO PYROLYSIS - DEVELOPMENT OF AUTHENTIC CONDITIONS - 1 • Mapped out cigarette combustion conditions (Baker, 1970s/1980s)

  37. TOBACCO PYROLYSIS - DEVELOPMENT OF AUTHENTIC CONDITIONS - 2 • Effect of pyrolysis conditions: temperature, heating rate, atmosphere (Tiller & Gentry, 1977: Muramatsu et al., 1979; Baker, 1980s; Stotesbury, 1990s)

  38. TOBACCO PYROLYSIS - DEVELOPMENT OF AUTHENTIC CONDITIONS - 3 • Transfer of labelled substances from cigarette to smoke (Larson & Harlow, 1958; Jenkins et al., 1970s; Houseman,1973; Schmeltz el al., 1979; Best,1987; Eble, 1987; J. D. Green et al., 1989; Stevens and Borgerding, 1999, Stotesbury et al., 2000)

  39. TOBACCO PYROLYSIS CONDITIONS (BAT STUDIES) • Atmosphere of 9% O2 in N2 • Gas flow of 5 ml/s • Hold at 300oC for 5 s • Heat from 300°C to 900oC at 30 oC/s • Hold at 900oC for 5 s

  40. Schematic of Pyroprobe interface with GC Heated interface Pyrolysis gas in Probe Septum purge Injection port Split vent To MS GC column

  41. Results of pyrolysis versus unchanged labelled transfer to mainstream smoke

  42. USE OF PYROLYSIS IN ASSESSING INGREDIENTS • Pyrolysis system developed gives good predictions of smoke transfer/pyrolytic behaviour of relatively volatile tobacco ingredients added to cigarette in small amounts • For involatile substances, the pyrolysis system tends to overestimate the amount of decomposition that occurs during smoking • Useful screening tool to indicate which ingredients undergo significant decomposition during smoking

  43. PYROLYSIS OF SINGLE-SUBSTANCE, SEMI-VOLATILE INGREDIENTS (CUMULATIVE) • 291 flavour ingredients pyrolysed • 92 (32%) transfer to smoke with <1% decomposition • 184 (63%) transfer to smoke with <5% decomposition • 248 (85%) transfer to smoke with <20% decomposition

  44. FOR INGREDIENTS THAT DO UNDERGO PYROLYSIS, CAN CALCULATE MAXIMUM LEVEL OF EACH PYROLYSIS PRODUCT IN MAINSTREAM SMOKE FOR UNFILTERED CIGARETTE: Productmax (μg) = Weight of ingredient in cigarette (μg) [max. appication level] x Proportion of product in pyrolysate x Proportion of tobacco burnt in puffing [0.5] x Proportion of transfer of ingredient/product to MS smoke [100%]

  45. Examples of maximum pyrolysis yields from semi-volatile ingredients and cigarette smoke yields (μg/cigarette)

  46. FOR SINGLE-SUBSTANCE, SEMI-VOLATILE INGREDIENTS THAT DO UNDERGO PYROLYSIS: ‘Hoffmann analytes’ detected amongst pyrolysis products generally low/insignificant compared to smoke yields (<5%)

  47. Pyrolysis of non-volatile tobacco ingredients • 159 non-volatile and complex ingredients • Most ingredients decomposed in the pyrolyser • many products in small amounts • significant levels of some ‘Hoffmann’ analytes predicted • Pyrolysis products with toxicological concern - checked by adding ingredient to cigarette - smoked by machine - comparing smoke yields to control (no ingredient) cigarette

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