TOXICOLOGICAL ASSESSMENT OF TOBACCO INGREDIENTS Richard R Baker
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 Smoke chemistry
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
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)
SOME DEFINITIONS (c) ADDITIVES Used for a specific technological purpose, e. g. : • Humectants – increase tobacco moistureholding 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
TYPICAL CIGARETTE TOBACCO BLENDS COMPONENT USA (%) Virginia lamina (flue-cured) Burley lamina (air-cured) Oriental lamina (sun-cured) Stem Reconstituted tobacco 35 26 11 Casings, humectants 2. 5 Flavorings TOTAL 0. 5 100 25 UK (%) 75 22 3 100
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
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
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, lownicotine cigarettes. In fact, these new cigarettes require flavor corrections by additives in order to be acceptable to the consumer. ”
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
STUDIES ON INGREDIENTS SINCE 1950 s • • • Pyrolysis Effects on smoke chemistry Mouse skin painting Inhalation toxicity In vitro bioassays - genotoxicity - cytotoxicity
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 and bioavailability
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
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)
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
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
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
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)
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.
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
Gaworski et al. , conclusions Ingredients had no discernible effect on inhalation toxicity or tumor-promoting activity of smoke
BAT STUDIES 1. 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
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
CIGARETTE SERIES Series A Flavorings Series B Flavorings and casings Sheet ingredients Series C Casings
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
Cigarette series A: Ames test (TA 98 +S 9)
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
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
Approaches in present pyrolysis study • Develop pyrolysis to simulate conditions during smoking • Use pyrolysis to measure amount of decomposition during smoking
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
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
Distillation. Pyrolysis Zone Combustion Zone Smoke Volatile gases Gases Air Tobacco Pyrolysis Distillation Feedback Residual Char Oxidation Ash Heat Loss
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
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.
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 rearrangement 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.
TOBACCO PYROLYSIS - DEVELOPMENT OF AUTHENTIC CONDITIONS - 1 • Mapped out cigarette combustion conditions (Baker, 1970 s/1980 s)
TOBACCO PYROLYSIS - DEVELOPMENT OF AUTHENTIC CONDITIONS - 2 • Effect of pyrolysis conditions: temperature, heating rate, atmosphere (Tiller & Gentry, 1977: Muramatsu et al. , 1979; Baker, 1980 s; Stotesbury, 1990 s)
TOBACCO PYROLYSIS - DEVELOPMENT OF AUTHENTIC CONDITIONS - 3 • Transfer of labelled substances from cigarette to smoke (Larson & Harlow, 1958; Jenkins et al. , 1970 s; Houseman, 1973; Schmeltz el al. , 1979; Best, 1987; Eble, 1987; J. D. Green et al. , 1989; Stevens and Borgerding, 1999, Stotesbury et al. , 2000)
TOBACCO PYROLYSIS CONDITIONS (BAT STUDIES) • Atmosphere of 9% O 2 in N 2 • Gas flow of 5 ml/s • Hold at 300 o. C for 5 s • Heat from 300°C to 900 o. C at 30 o. C/s • Hold at 900 o. C for 5 s
Heated interface Schematic of Pyroprobe interface with GC Pyrolysis gas in Probe Injection port Septum purge Split vent To MS GC column
Results of pyrolysis versus unchanged labelled transfer to mainstream smoke
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
PYROLYSIS OF SINGLE-SUBSTANCE, SEMIVOLATILE 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
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%]
Examples of maximum pyrolysis yields from semi-volatile ingredients and cigarette smoke yields (μg/cigarette) Ingredient Product Max. level from ingredient Typical smoke level (non-filter cigarette) Anisyl phenylacetate Phenol 0. 03 80 - 160 Benzyl cinnamate Styrene 0. 2 10 - 20 Cinnamyl cinnamate Phenol 0. 2 80 - 160 α-Methylbenzyl acaetate Styrene 0. 1 10 - 20 Phenylacaetc acid Toluene 0. 07 100 - 200 Cresol 0. 09 11 - 37 p-Tolyl acatate
FOR SINGLE-SUBSTANCE, SEMIVOLATILE INGREDIENTS THAT DO UNDERGO PYROLYSIS: ‘Hoffmann analytes’ detected amongst pyrolysis products generally low/insignificant compared to smoke yields (<5%)
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
Comparison of 2 -furfural predicted by pyrolysis and measured by smoking Ingredient Cellulose Max. level predicted by pyrolysis (µg/cig) Smoke Analysis % added to cigarette Test cig. Control cig. yield (µg/cig) 410 2. 4 7. 7* 11. 0 Sorbitol 5, 500 3. 6 7. 8 8. 4 Sugar, brown 6, 900 6. 2 9. 4* 11. 0 Sugar, invert 11, 000 7. 0 6. 5* 4. 4 Sugar, white 10, 000 10. 5 12. 8 11. 0 Corn syrup 14, 000 6. 2 5. 2 4. 4 Honey 2, 100 4. 5 5. 4 4. 4
For 2 -furfural generated from nonvolatile saccharides, pyrolysis experiments have grossly overestimated the amount formed during smoking. Pyrolysis also predicts generation of formaldehyde from saccharide ingredients. (Formaldehyde not detected by MS system so used FTIR system. )
Generation of formaldehyde during pyrolysis
SMOKE CHEMISTRY Compare smoke yields of ‘Hoffmann analytes’ in test cigarette (with ingredients) with yields in control cigarette (without the ingredients)
CIGARETTE SERIES Series A Flavorings Series B Flavorings and casings Sheet ingredients Series C Casings
SMOKE CHEMISTRY RESULTS – FLAVORINGS - OVERALL SUMMARY • Flavorings have either no significant effect on mainstream yields of ‘Hoffmann analytes’ relative to control, or produce occasional changes in individual analyte levels (+ and -) • The significance of most of these occasional changes were not present when the long-term variability of the methodology was taken into account • Conclude that flavorings have no effect on smoke chemistry
SMOKE CHEMISTRY RESULTS – CASINGS, SHEET ADDITIVES - OVERALL SUMMARY • Usually no significant effect on TPM, nicotine and CO • Some ‘Hoffmann analyte’ levels affected, generally by up to +/- 15 %, not significant within long-term variability • Significant decreases in nitrosamines (up to 30%), phenols (up to 44%), and aromatic amines (up to 26%) with some mixtures • Carbonyls significantly increased with some mixtures - HCHO increased by up to 73% with mixtures containing high levels of sugars - HCHO increased by 68%, possibly due to cellulosic and polysccharide materials
HCHO YIELDS – DIFFERENT STUDIES INGREDIENT STUDY INCREASE (µg) INCREASE (%) Cellulose BAT 16. 1 68 Cellulose NCI (1980) 44 38 Sugar BAT 26. 0 73 10. 7 65 9. 9 60 Sugar - low Carmines et al. (2002) Sugar - high Carmines et al. (2002)
FORMALDEHYDE YIELDS FOR CIGARETTES WITH ‘TAR’ YIELD OF ca. 13 mg Experimental cigarette: 62 μg UK benchmark study: 30 - 56 μg World study: 30 - 90 μg
BAT STUDY - CONCLUSIONS - 1 • 2/3 of volatile flavorings transfer to smoke with <5% decomposition • Where decomposition does occur, ‘Hoffmann analytes’ detected amongst products generally low/insignificant compared to smoke yields (<5%) • Non-volatile ingredients generally decompose in pyrolyser and pyrolysis experiments overestimate amount of compounds formed during smoking
BAT STUDY - CONCLUSIONS - 2 • Flavorings have no significant effect on levels of ‘Hoffmann analytes’ in mainstream smoke • The vast majority of casings and sheet ingredients have little effect on level of ‘Hoffmann analytes’ in smoke. Several are decreased and one is increased.
BAT STUDY - CONCLUSIONS - 3 • The inhalation toxicity of the smoke from all the test cigarettes was the same as that from their respective control cigarettes • Within the sensitivity and specificity of three in vitro bioassays, the specific activity of smoke condensate was not changed by the addition of ingredients to the cigarette: -Ames test -Mammalian cell micronucleus assay -Neutral red uptake cytotoxicity assay
OVERALL CONCLUSIONS THERE IS BROAD AGREEMENT BETWEEN: • Chemical and biological studies published over 50 years (Paschke et al. , 2002, Rodgman, 2002) • Chemical and biological work undertaken by R. J. Reynolds (included in the Rodgman reviews) • Philip Morris chemical and biological studies (Carmines et al. , 2002) • Lorillard biological studies (Gaworski et al. , 1997 – 2002) • BAT pyrolysis, smoke chemistry and biological studies (Baker et al. , 2004)
BROAD CONCLUSIONS • Tobacco ingredients used commercially do not increase the biological activity of cigarette smoke • Most ingredients do not affect the smoke levels of ‘Hoffmann analytes’
BAT PAPERS ON INGREDIENTS 1. R. R. Baker and G. Smith, Toxicological aspects of tobacco flavour ingredients, Recent Advances in Tobacco Science, 2003, 29, 47 -76. 2. R. R. Baker and L. J. Bishop, The pyrolysis of tobacco ingredients, J. Anal. Appl. Pyrolysis, 2004, 71(1), 223 -311. 3. R. R. Baker, J. R. da Silva and G. Smith, The effect of tobacco ingredients on smoke chemistry. Part I: Flavourings and additives, Food Chem. Toxicol, 2004, 42 Supplement, 337. 4. R. R. Baker, J. R. da Silva and G. Smith, The effect of tobacco ingredients on smoke chemistry. Part II: Casing ingredients, Food Chem. Toxicol, 2004, 42 Supplement, 39 -52. 5. R. R. Baker, E. D. Massey and G. Smith, An overview of the effects of tobacco ingredients on smoke chemistry and toxicity, Food Chem. Toxicol, 2004, 42 Supplement, 53 -83. 6. R. R. Baker and L. J. Bishop, The pyrolysis of non-volatile tobacco ingredients using a system that simulates cigarette combustion conditions, Paper presented at 16 th International Symposium on Analytical and Applied Pyrolysi, Alicante, Spain, May 2004. 7. R. R. Baker, S. Coburn, C. Liu and J. Tetteh, Pyrolysis of eleven polysaccharide tobacco ingredients: a TGA-FTIR investigation, Paper presented at 16 th International Symposium on Analytical and Applied Pyrolysi, Alicante, Spain, May 2004.
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