TOXICOLOGICAL ASSESSMENT OF TOBACCO INGREDIENTS Richard R. Baker British American Tobacco...
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Transcript of TOXICOLOGICAL ASSESSMENT OF TOBACCO INGREDIENTS Richard R. Baker British American Tobacco...
TOXICOLOGICAL ASSESSMENT OF TOBACCO INGREDIENTS
Richard R. Baker
British American TobaccoSouthampton
UK
LSRO Meeting, Denver, CO, USA8/9 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 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
TYPICAL CIGARETTE TOBACCO BLENDS
COMPONENT USA (%) UK
(%)
Virginia lamina (flue-cured) 35 75
Burley lamina (air-cured) 26
Oriental lamina (sun-cured) 11
Stem 22
Reconstituted tobacco 25 3
Casings, humectants 2.5
Flavorings 0.5
TOTAL 100 100
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, low-nicotine 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 1950s
• 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 flavors1 flavor/solvent1 solvent7 preservatives 5 binders5 humectants1 filler2 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 (TA98 +S9)
0
50
100
150
200
250
300
350
0 50 100 150 200 250 300
NFDPM µg plate -1
TA
98 R
ever
tan
ts p
late
-1
Control
Vanilla
Citrus Fruit
Herbal
Spice
Caramel/Rum
Floral Fruit
Cocoa
Flavour Character
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
Volatile gases
Gases
Smoke
Air
Distillation-Pyrolysis Zone
Combustion Zone
TobaccoPyrolysis
DistillationResidual
Char
Char Oxidation Ash
Heat LossFeedback
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 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.
TOBACCO PYROLYSIS - DEVELOPMENT OF AUTHENTIC CONDITIONS - 1
• Mapped out cigarette combustion conditions
(Baker, 1970s/1980s)
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)
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)
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
Pyrolysis gas in
Heated interface
Septum purgeSplit vent
Injection port
GC column
Schematic of Pyroprobe interface with GC
To MS
Probe
Results of pyrolysis versus unchanged labelled transfer to mainstream smoke
Compound
Pyrolysis transfer
(%)
Unchanged transfer to mainstream (%)
Anisole 99 100
Benzaldehyde 95 100
Isoamyl isovalerate 100 100
Anisaldehyde 99 99
Methyl cinnamate 98 100
Vanillin 99.5 100
Menthol 99 99
Glycerol 94 83 – 85
Phenylacetic acid 52 80
n-Dotriacontane 54 95 – 99
Nicotine 98 76 - 100
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, 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
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
p-Tolyl acatate Cresol 0.09 11 - 37
FOR SINGLE-SUBSTANCE, SEMI-VOLATILE 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
Max. level predicted by
pyrolysis (µg/cig)
Smoke Analysis
% added
to cigarette
Test cig. yield
(µg/cig)
Control cig. yield (µg/cig)
Cellulose 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 non-volatile 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
FORMALDEHYDE
0
4
8
12
16
20
50 150 250 350 450 550 650 750 850
Temperature (oC)
Co
nce
ntr
atio
n (
a.u
.)
ACACIA GUMMOLASSESCELLULOSE
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
NNK variability over one year - 1R4F, ISO machine smoking
0
20
40
60
80
100
120
0 50 100 150
Measurement occasion
NN
K (
ng
)
Variability of HCHO over one year - 1R4F, ISO machine smoking
0.0
5.0
10.0
15.0
20.0
25.0
30.0
0 50 100 150 200
Measurement occasion
HC
HO
(mic
rog
)
Cigarette series A: First group of analytes
0
25
50
75
100
125TPM
NFDPM
Nicotine
CO
Ammonia
N oxides
HCN
1,3-ButadieneIsoprene
Acrylonitrile
Benzene
Toluene
Styrene
Pyridine
Quinoline
A1
A2
A3
A4
A5
A6
A7
A8
Cigarette series A: Second group of analytes
0
25
50
75
100
125Formaldehyde
Acetaldehyde
Acetone
Acrolein
Propionaldehyde
Crotonaldehyde
Methyl ethyl ketone
ButyraldehydePhenol
m+p-Cresol
o-Cresol
Catechol
Hydroquinone
Resorcinol
Benzo[a]pyrene
A1
A2
A3
A4
A5
A6
A7
A8
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
Cigarette series B: Second group of analytes
0
25
50
75
100
125
150
175
Formaldehyde
Acetaldehyde
Acetone
Acrolein
Propionaldehyde
Crotonaldehyde
Methyl ethyl ketone
ButyraldehydePhenol
m+p-Cresol
o-Cresol
Catechol
Hydroquinone
Resorcinol
Benzo[a]pyrene
B1
B2
B3
B4
B5
Cigarette series C: Second group of analytes
0
25
50
75
100
125
150
175Formaldehyde
Acetaldehyde
Acetone
Acrolein
Propionaldehyde
Crotonaldehyde
Methyl ethyl ketone
ButyraldehydePhenol
m+p-Cresol
o-Cresol
Catechol
Hydroquinone
Resorcinol
Benzo[a]pyrene
C1
C2
C3
C4
C5
C6
C7
C8
C9
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
Sugar - low Carmines et al. (2002)
10.7 65
Sugar - high Carmines et al. (2002)
9.9 60
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, 3-37.
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 16th 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 16th International Symposium on Analytical and Applied Pyrolysi, Alicante, Spain, May 2004.