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.