REVIEW OF INGREDIENTS ADDED TO … internal use of Philip Morris personnel only REVIEW OF INGREDIENTS ADDED TO CIGARETTES PHASE TWO: ... D. Case Study Summaries for …

For internal use of Philip Morris personnel only

REVIEW OF INGREDIENTS ADDED TO CIGARETTES

PHASE TWO: SCIENTIFIC CRITERIAFOR THE EVALUATION OF INGREDIENTS ADDED TO CIGARETTES

LSRO

Life Sciences Research Office9650 Rockville PikeBethesda, Maryland

EditorsDaniel M. Byrd III, Ph.D., D.A.B.T.

Kara D. Lewis, Ph.D.Paula M. Nixon, Ph.D.

Robin S. Feldman, B.S., M.B.A.


Copyright © 2004 Life Sciences Research Office

No part of this document may be reproduced by any mechanical, photographic, orelectronic process, or in form of a phonographic recording, nor may it be stored in aretrieval system, transmitted, or otherwise copied for public or private use, withoutwritten permission from the publisher, except for the purposes of official use by theU.S. Government.

Copies of the publication may be obtained from the Life Sciences Research Office.Orders and inquiries may be directed to: LSRO, 9650 Rockville Pike, Bethesda,MD 20814-3998. Tel: 301-634-7030; Fax 301-634-7876; web site: www.LSRO.org

ISBN: 0-9753167-3-7Library of Congress Catalog Number: 2004111771


FOREWORD

The Life Sciences Research Office, Inc. (LSRO) provides scientific assessmentsof topics in the biomedical sciences. Reports are based on comprehensive literaturereviews and the scientific opinions of knowledgeable investigators engaged in workin relevant areas of science and medicine. This LSRO report was developed for andsupported by Philip Morris, USA, Inc., P.O. Box 26583, Richmond, VA 23261 (PhilipMorris) in accordance with a contract between Philip Morris and LSRO.

An Expert Panel provided scientific oversight and direction for all aspects of thisproject. LSRO independently appointed members of the Expert Panel based ontheir qualifications, experience, judgment, and freedom from conflict of interest,with due considerations for balance and breadth in the appropriate professionaldisciplines. The Expert Panel was selected with the concurrence of LSRO’s Boardof Directors. The Expert Panel convened six times to assess the available data.LSRO invited submission of data, information, and views bearing on the topic understudy, held a widely advertised Open Meeting on November 5, 2003, and acceptedwritten submissions. Information about the process, including the critical literatureand presentations upon which the Expert Panel based their deliberations, was madepublicly available by posting on the LSRO web site.

LSRO’s staff and special consultants considered available information, and thedeliberations of the Expert Panel in drafting the report. The LSRO report wasedited by LSRO’s staff, who submitted the report for review by independentreviewers, incorporated the reviewers’comments, and provided additionaldocumentation and viewpoints for incorporation into the final report. The ExpertPanel and the LSRO Board of Directors reviewed and approved the final report.On completion of these reviews, the report was transmitted to the Sponsor fortechnical comments by the Executive Director, LSRO.

The listing of members of the Expert Panel, others who assisted in preparation ofthis report, and the LSRO Board of Directors, does not imply their endorsement ofall statements in this report. The report was developed independently of PhilipMorris and conclusions drawn therein do not necessarily represent the views ofPhilip Morris or any of its employees. LSRO accepts full responsibility for thestudy’s conclusions and accuracy.

Michael Falk, Ph.D.Executive Director

Life Sciences Research Office, Inc.

August 2004


Table of Contents � v


TABLE OF CONTENTS

Foreword ............................................................................................................. iii

1 Executive Summary ...................................................................................... 1

2 Introduction ................................................................................................... 72.1 Background Information ........................................................................... 82.2 Basis of Knowledge .................................................................................. 92.3 LSRO’s Approach ...................................................................................112.4 The “Toxins-in-Smoke” Hypothesis ........................................................ 12

3 Strategy ......................................................................................................... 143.1 Introduction ............................................................................................. 153.2 LSRO’s Scientific Criteria ...................................................................... 193.3 Testing Substances Not Meeting LSRO’s Three Scientific Criteria ...... 223.4 Summary ................................................................................................. 22

4 Relevance of Testing Methods of Other Organizations ....................... 244.1 Introduction ............................................................................................. 254.2 Guidelines and Published Methods ......................................................... 274.3 Some Product Safety Testing Guidelines ................................................ 284.4 Summary ................................................................................................. 29

5 Definition, Specification, and Characterization of the Test Substance .. 305.1 Introduction ............................................................................................. 315.2 Definition of the Test Substance ............................................................. 325.3 Classification of the Test Substance ....................................................... 325.4 Consideration of “What to Test” ............................................................ 335.5 Analytical Testing ................................................................................... 345.6 Special Considerations for Complex Mixtures

and Biological Products .......................................................................... 35

6 Chemical Fate of Added Ingredients in Cigarette Smoke ................... 366.1 Introduction ............................................................................................. 376.2 Detecting Fate of Ingredients in a Complex System .............................. 416.3 Some Relevant Tests .............................................................................. 426.4 Studies of Individual Chemical Ingredients ............................................. 476.5 Additional Chemical Analyses ................................................................ 526.6 Summary ................................................................................................. 52

7 Exposure ...................................................................................................... 547.1 Introduction ............................................................................................. 557.2 Initial Studies ........................................................................................... 597.3 Other Considerations .............................................................................. 677.4 Relative Risk and Additional Testing ...................................................... 687.5 Summary ................................................................................................. 68

vi � Evaluation of Cigarette Ingredients: Feasibility


8 Kinetics and Dosimetry of Chemical Substances .................................. 708.1 Introduction ............................................................................................. 718.2 Disposition of Nicotine ............................................................................ 728.3 Pharmacokinetics of Selected Other Ingredients ................................... 778.4 Experimental Considerations .................................................................. 798.5 Summary ................................................................................................. 84

9 Biological Activity of Cigarette Smoke ................................................... 869.1 Introduction ............................................................................................. 879.2 Tests of Biological Activity ..................................................................... 899.3 Limitations and Issues ............................................................................. 989.4 Summary ............................................................................................... 101

10 Human Cigarette Smoking Behavior .................................................... 10310.1 Introduction ........................................................................................... 10410.2 Ways in Which Added Ingredients May Affect Smoking Behavior ..... 10510.3 Clinical Studies of Human Smoking Behavior ...................................... 10910.4 Additional Measures of Smoking Behavior and Exposure .................... 11010.5 Summary ................................................................................................114

11 The Evaluation of Ingredients Added To Cigarettes ...........................11611.1 Introduction ............................................................................................ 11711.2 Assembling Existing Data ...................................................................... 11911.3 Selection of Test Methods .................................................................... 12011.4 Expert Evaluation .................................................................................. 12511.5 Summary ............................................................................................... 127

12 Research Opportunities ........................................................................... 12912.1 Introduction ........................................................................................... 13112.2 Kinds of Information ............................................................................. 13112.3 Summary ............................................................................................... 142

13 Conclusions ................................................................................................ 14313.1 Scientific Criteria .................................................................................. 14413.2 Significance of Change ......................................................................... 14513.3 Scientific Judgment ............................................................................... 14713.4 Relevance of Scientific Data to Adverse Human Health Effects ........ 14713.5 Limitations ............................................................................................. 14913.6 Summary ............................................................................................... 150

14 Literature Citations .................................................................................. 152

15 Study Participants ...................................................................................... 182Ad Hoc Expert Panel ................................................................................... 182Life Sciences Research Office Staff ........................................................... 183

Table of Contents � vii


Tables

Table 4-1 Guidance Documents Relating to Inhalation Toxicity .......................... 29

Figures

Figure 3.1 Testing Smoke From Cigarettes With and Without an Added Ingredient . 17Figure 3.2 Information Flow Diagram ................................................................. 18Figure 4.1 Testing Guidelines Flow Diagram ....................................................... 26Figure 5.1 Definition of an Added Ingredient Flow Diagram .............................. 31Figure 7.1 Illustration of Exposure ....................................................................... 57Figure 11.1 Information Flow Diagram ...............................................................118Figure D.1 Substance A ..................................................................................... 247Figure D.2 Substance B ..................................................................................... 250Figure D.3 Substance C ..................................................................................... 253Figure D.4 Substance D .................................................................................... 255Figure D.5 Substance E ..................................................................................... 257Figure D.6 Substance F ..................................................................................... 259Figure D.7 Substance G ..................................................................................... 262

Appendices

A. Life Sciences Research Office (LSRO) ...................................................... 184Added Ingredient Review Ad Hoc Expert Panel ........................................ 184Added Ingredient Review Meeting Speakers .............................................. 189Added Ingredient Review Open Meeting Speakers .................................... 192LSRO Staff .................................................................................................. 193LSRO Consultants ....................................................................................... 196LSRO Board of Directors ............................................................................ 199

B. Glossary ....................................................................................................... 200C. Public and Invited Comments ...................................................................... 215D. Case Study Summaries for Some Hypothetical Ingredients ........................ 245E. Closure Letter .............................................................................................. 263F. Reproductive and Developmental Effects of Cigarette Smoke Exposure ... 264G. Some Perspectives on Limits ....................................................................... 268

Index ................................................................................................................. 271


1EXECUTIVE SUMMARY

A U.S. National Academy of Sciences (NAS) report, Clearing the Smoke,recommended that manufacturers test non-tobacco ingredients added to cigarettes(Institute of Medicine, 2001). Philip Morris, USA, tasked the Life Sciences ResearchOffice (LSRO) with development of an approach to testing. LSRO divided theproject into three phases: (1) feasibility, (2) scientific criteria, and (3) reviews ofindividual ingredients.

LSRO previously published a report about Phase One (Feasibility) of the project.The report concluded that testing ingredients added to cigarettes for potential adversehealth effects is both feasible and worthwhile (Life Sciences Research Office, 2004).This report, Scientific Criteria, covers the second phase of the project. It explainsLSRO’s scientific criteria for testing and how a data submitter might want to presentdata to LSRO for review.

Current public health initiatives aim to prevent initiation of smoking and to encouragecurrent smokers to quit, eliminating any risk of smoking. LSRO also sees the optimalpublic health goal as prevention of smoking initiation and encouragement of smokingcessation. This project does not attempt to lower the risk of adverse health effectsfrom smoking cigarettes for the smoker through the addition of ingredients. Aninherent consequence of the approach described within this report is that successfulimplementation will leave the consumers’ adverse health risk of smoking cigarettesunchanged.

Toxicologists have developed standardized methods to evaluate the potential ofsubstances to induce adverse human health effects. These methods apply when ause of a chemical substance is contemplated before human experience with thesubstance accumulates. Thus, both prevention and protection motivate this approachto testing. This report recognizes that traditional toxicology methods have limitations,since most conventional toxicological methods have been developed to test puresubstances at relatively high concentrations. These conditions are the inverse ofthose involving substances added to cigarette tobacco. Non-tobacco substancesadded to cigarettes are present in complex mixtures and at low concentrations. Thesubstances of interest may not exist in smoke. Instead, their pyrolysis products may

EXECUTIVE SUMMARY

2 � Evaluation of Cigarette Ingredients: Scientific Criteria


merit testing. Standard regulatory approaches, like U.S. Food and DrugAdministration’s (FDA’s) Redbook 2000: Toxicological Principles for the SafetyAssessment of Food Ingredients (2001), do not address the background toxicity ofsmoke or the inhalation route of administration. At present no appropriate experimentalanimal model is known for human exposure to cigarette smoke.

LSRO has sought to learn whether an ingredient added to a cigarette might changethe risk of adverse human health effects from smoking cigarettes with the ingredient,relative to cigarettes lacking the ingredient. Comparing otherwise identicallymanufactured cigarettes, with and without a non-tobacco ingredient, should producethe necessary data to understand the relative risks of adverse human health effects.

LSRO’s approach applies to the inhalation route of exposure and includes analysisof pyrolysis products of an ingredient that may be produced in a burning cigarette.Initially, LSRO’s approach will apply mostly to substances already in use. LSRO’sapproach calls for testing within the matrix of cigarette smoke, not the isolatedsubstance.

The approach recommended by LSRO also differs from the “toxins-in-smoke”hypothesis, which suggests that bioassays of added ingredients (1) in isolation, (2) athigh concentrations, and (3) in non-human subjects would predict the adverse humanhealth effects of smoking observed in epidemiology studies. LSRO considerssubstances in smoke to be responsible for the morbidity and mortality associatedwith cigarette smoking. However, LSRO does not know which individual substanceswithin the smoke are responsible for the diseases associated with cigarette smokingand is not certain how this issue would be resolved in any practical way.

The approach in this report seeks to eliminate changes in premature mortality andmorbidity from the addition of an ingredient to cigarettes. This is, intrinsically, arelative risk approach. Eliminating any change in relative risk will eliminate anychange in adverse health effects. To this end, LSRO seeks information to showwhether:

(a) either the added ingredient or a pyrolysis product of the addedingredient, detectably transfers into smoke in such a way that smokersare subject to a change in adverse health effects,

(b) addition of the ingredient changes the physics, chemistry, orbiological activity of smoke significantly, and

(c) addition of the ingredient changes exposure to cigarette smokethrough altered human smoking behavior.

The approach outlined in Scientific Criteria has limitations. It depends critically oncomparisons of otherwise identically manufactured cigarettes, containing or lacking

Executive Summary � 3


an ingredient. If these test cigarettes cannot be prepared, LSRO’s approach willnot work. Until LSRO better understands the effects of variation in smoke compositionand smoke effects, LSRO will limit its conclusions to the cigarettes tested. LSROcannot conclude that an upper limit on the amount of an ingredient would conferabsolutely no change in relative risk, because of negative test results with indicatorsubstances. Like all areas of science, LSRO’s inference will relate to the informationknown at some point in time.

In response to a request for an evaluation of an added ingredient, LSRO willreview all submitted data and respond with (1) an explicit request for additionaldata, (2) advice to use no more than a specified maximum amount of an ingredientper cigarette, or (3) advice not to use the ingredient in cigarettes. LSRO also willprovide a scientific rationale for its advice. Thus, given data showing that transferoccurs, or that smoke composition or exposure changes, a data submitter coulddecrease the ingredient added to a cigarette and retest. Alternatively, a datasubmitter could accumulate data to show that relative risk does not change.Chapters 5 through 10 explain how LSRO might go about such a demonstration(ingredient definition, smoke chemistry analysis, exposure, dosimetry, biologicaleffects, and behavioral effects).

Existing test guidelines and published methods for other uses, such as testing ofsubstances added to foods intended for ingestion, usually do not provide the kinds ofinformation necessary for LSRO to assess the potential health effects of adding aningredient to cigarettes. (See Chapter 3.) LSRO will initially seek to understandwhether the tested ingredient is the same ingredient used in cigarettes. For everyingredient submitted to LSRO for review, the substance that is added to cigarettesand a rationale for the selection of that substance should be described. Regardlessof the naming conventions, the test substance should be representative of the chemicalcompound, mixture, or natural substance added to cigarettes during the manufacturingprocess and should be thoroughly characterized to ensure its identity, purity,composition, and concentration. For complex or natural substances, the origin andmethod of preparation are also important. (See Chapter 5.)

Determining whether the addition of an ingredient changes the chemical compositionof cigarette smoke is integral to LSRO’s approach to evaluate ingredients added tocigarettes. The chemical fingerprint of a test cigarette and that of the test cigaretteminus the added ingredient can be compared. The chemical fingerprint is determinedby the measurement of analytes present in cigarette smoke by specified analyticalmethods. Looking for change due to the presence of an added ingredient in morethan 4,800 components currently identified in smoke is not recommended in thisreport. LSRO has not set requirements on the selection of specific components insmoke. However the effects of an ingredient on indicator substances includingnicotine, water, carbon monoxide, ‘tar’, and nitrogen oxides would provide importantinformation. (See Chapter 6.)



An ingredient could change the composition of cigarette smoke either by contributinga substance derived from the pyrolyzed ingredient, or by influencing the combustionof tobacco. Understanding whether that ingredient, or a novel pyrolysis-relatedproduct of the ingredient, transfers in smoke into the lungs of smokers, and whetherthe smoker absorbs the transferred substance, is essential to an evaluation of potentialadverse human health effects of smoking cigarettes containing that ingredient.(See Chapter 7.)

LSRO deliberated and decided to define the transfer into the smoker’s lung as the“exposure.” The dividing line between exposure and dose is not sharp, and theappropriate nomenclature varies between groups of scientists. (See Chapter 7.)However, in this report, the amount of absorbed material, that crosses the outermembrane of the body, is designated the “dose.” Thus, no difference should exist inthe biological effects of an intravenous and an inhaled dose. Assessing the potentialexposure of smokers to smoke from cigarettes containing added ingredients haslimitations. However, reasonable estimates of potential pulmonary exposures ofsmokers can be obtained. Further, hypothetical estimates of exposure can beconfirmed and refined experimentally.

If transfer occurs, or if smoke composition changes, a sponsor may choose to reducethe amount of the ingredient and retest, until reduction in amount eliminates detectionof the changes. Alternatively, the sponsor may choose to retain the same amount ofingredient and show that its presence does not change the relative risk of adversehuman health effects of smoking cigarettes. In this last instance, an exposure estimatelikely will be a step in a process, extending from studies of smoke composition todosimetry and biological testing.

Even if no change occurs in a single test, a new method of increased sensitivitymight detect change later. Multiple tests and tests of different smoke characteristicswill reduce the probability of a false negative result, which might influence an upperlimit. Ultimately, a “no detectable change” approach assumes a nonlinear relationshipbetween exposure and the amount of ingredient added. LSRO recommends the useof several qualitatively different kinds of assays of smoke, including physical, chemical,and biological tests. If the addition of an ingredient does not change smokecomposition, the possibility of a change in the relative risk of adverse human healtheffects associated with inhaling cigarette smoke seems unlikely. If the addition ofthe ingredient changes smoke composition, the data may prove useful in estimatingthe likelihood of a change in the relative risk of adverse health effects.

To assess whether an added ingredient has the potential to change the relative riskof adverse health effects of smoking cigarettes, a sponsor could compare the biologicalactivity of smoke from cigarettes with and without the added ingredient. Chapter 9provides examples of some of the biological tests a data submitter might conduct for



each ingredient, including comparisons of cigarette smoke cytotoxicity andmutagenicity. Also described are additional tests that the sponsor might choose toconduct, if evaluation of the ingredients warrants additional data.

An ingredient that changes smokers’ exposure to smoke has the potential to changethe risk of adverse health effects of smoking cigarettes. LSRO will regard anychange in smoke either through transfer of a novel substance or through a significantchange in smoke composition as potentially adverse, until shown otherwise throughfurther testing. An added ingredient could generate no detectable change in smokecomposition yet elicit a change in smoking behavior and smoke exposure. A changein exposure could result from modified smoking behaviors, such as smoking morecigarettes or smoking cigarettes differently, that is, puffing more frequently or inhalingcigarette smoke more deeply and for a longer period of time. Chapters 7 and 10describe other measures of smoking behavior that data submitters could apply toaddress smoke exposure through measuring the number of cigarettes smoked perday and concentrations of surrogate biomarkers of smoke exposure, e.g., nicotineand cotinine.

LSRO suggests pharmacokinetic studies of substances that transfer into cigarettesmoke in biologically significant amounts. The ingredient itself may have toxicityand may warrant kinetic study. In addition, the ingredient may alter exposure totoxic cigarette smoke, and therefore change the kinetics of other smoke components.This integration of pharmacokinetics into an evaluation of the relative risk of adversehuman health effects involves the measurement of specific parameters that allowthe dose of an ingredient or smoke component received by a smoker to be determined.In addition to the distribution of an added ingredient within the body, identification ofany metabolites, as well as the methods of conversion, may provide an indication ofthe sites and mechanisms causing potential adverse effects. Chapter 8 providesinformation about testing which may produce useful pharmacokinetic data for anevaluation of ingredients added to cigarettes.

LSRO has not followed standard regulatory approaches in this report. LSROencourages sponsors to take a flexible approach to testing and to utilize existingtoxicological data about the added ingredient to guide decision-making about testingadded ingredients. The application of a set of diverse and relevant tests will providesome assurance of the potential effects of adding ingredients to cigarettes, althoughno single test predicts the range of health effects associated with cigarette smoking.Standard approaches would not address the background toxicity of smoke, theinhalation route of exposure, or testing within the matrix of intended use.

LSRO can only reach conclusions from available data, which in the current approachwill lead to one of three potential outcomes detailed in Chapter 11. Submission ofdata which proves insufficient or inconclusive, will lead LSRO to return the



submission and explain what additional testing is necessary to complete the evaluation.Evaluation of all of the data initially submitted and later requested could lead to aconclusion that the data submitter might prefer not to use the ingredient or that theingredient has a scientific rationale permitting the use in cigarettes up to some statedamount without a change in adverse health effects. LSRO will publish a reportstating the conclusion of its evaluation. To improve the process, LSRO has listedsome research needs in Chapter 12. Similarly, LSRO has included some appendicesto improve practical applications of the approach, including some hypothetical casestudies. (See Appendix D.)


2INTRODUCTION

2.1 BACKGROUND INFORMATION

2.2 BASIS OF KNOWLEDGE

2.3 LSRO’S APPROACH

2.4 THE “TOXINS-IN-SMOKE” HYPOTHESIS



22.1 BACKGROUND INFORMATION

The U.S. government does not assert regulatory jurisdiction over non-tobaccoingredients added to cigarettes or for testing ingredients added to cigarettes. Federallaw requires manufacturers to submit, annually, a list of ingredients added to tobaccoand used in the manufacture of cigarettes, to the U.S. Department of Health andHuman Services (DHHS) but without disclosure of the manufacturer, brand, orquantities (U.S. Congress, 2004). However, DHHS has no legal authority tocontrol these ingredients. A U.S. National Academy of Sciences (NAS) report,Clearing the Smoke, recommended development of a surveillance systemconsisting of mandatory, industry-supplied data, including test data about ingredientsadded to cigarettes, as a public health and scientific research objective (Instituteof Medicine, 2001). Philip Morris USA, a manufacturer of cigarettes, engagedthe Life Sciences Research Office (LSRO) to develop a more detailed approachto testing ingredients added to cigarettes.

Throughout this report, the term “LSRO” refers to the staff of the Life SciencesResearch Office, a nonprofit corporation in Bethesda, MD, its advisors for thepurpose of this project, and the members of the Added Ingredients Review AdHoc Expert Panel. By added ingredients, LSRO means chemical substancesdirectly added during manufacturing, not indirect additives derived from agronomicpractices, such as pesticides applied to tobacco plants and thus present in cigarettesindirectly.

The overall project and this Phase Two report (Scientific Criteria) do not attemptto change the risk of adverse human health effects of smoking cigarettes throughthe addition of an ingredient or ingredients. Instead, LSRO seeks to eliminate thepossibility of a significant change in the risk of adverse human health effects fromthe addition of ingredients, that is, prevent any change in relative risk. Thus, theapproach taken in this project aims to leave the risk of adverse human healtheffects of smoking cigarettes unchanged. To meet the objectives of this project infull, LSRO reviewed the potential adverse human health effects of smokingcigarettes containing additional, non-tobacco ingredients, in three phases: (1)feasibility of testing, (2) scientific criteria, and (3) subsequent reviews of individualingredients.

INTRODUCTION

Introduction � 9


The Phase One report concluded that testing ingredients added to cigarettes, as totheir potential adverse health effects, is both feasible and worthwhile (Life SciencesResearch Office, 2004). LSRO has pursued the objective specified in the PhaseOne report (Life Sciences Research Office, 2004), that the purpose of testing theseingredients should be to assure, to the extent feasible, that the ingredients do notincrease the premature mortality and morbidity associated with cigarette smoking.

The purpose of this report is to explain the rationale for LSRO’s conclusions aboutscientific criteria for testing ingredients added to cigarettes. This report advancesthree measurement-based criteria for determining relative risk, describes LSRO’scurrent thinking about testing of cigarettes, and explains how an outside party mightwant to obtain and present data to LSRO for review. The report provides specificexamples of test methods to illustrate that the scientific criteria, as presented, can beachieved.

2.2 BASIS OF KNOWLEDGE

Manufacturers make cigarettes from tobacco, a natural product. Burning a cigaretteproduces smoke. Smoke is a cloud of fine particles suspended in a gas, possessinglittle mass and usually produced by combustion or incomplete combustion. An aerosolis a colloidal dispersion of a liquid or solid in a gas. Normally a combustion aerosol,like cigarette smoke, consists of both particles and gases. Cigarette smoke containsa mixture of many thousands of chemical substances, some short-lived and reactive.The smoke is dynamic in nature, changing rapidly with time, puff number, and type.Aerosol particles exchange substances with the gas phase, and their chemicalcomposition varies over time.

When humans inhale cigarette smoke, their mouths and respiratory tracts are exposedto particles and gases. The surfaces react with and absorb many chemical substances,sometimes producing effects at the site of deposition. Once deposited, thesechemicals and/or their reactive products may be transported to systemic tissues bycirculation. Biological effects may depend on complex interactions between thesubstances and/or their components and the target tissues.

Exposure sometimes is defined as the crossing of a chemical substance through anopening into the body. However, LSRO is not using this definition. LSRO definesexposure as a state of vulnerability. Thus, deposition or absorption of substances inthe lung is the exposure of interest. The framework put forward by Zartarian andcoworkers (1997) best applies to this task of evaluating the exposure to potentialreaction products of added ingredients. As described in Chapter 7, exposure to achemical substance involves contact with a body membrane, such as the skin or theinternal, epithelial surface of the lung. Exposure and dose are closely linked concepts.



In this report, LSRO defines chemical dose as the amount of substance that entersthe circulation after crossing an outer membrane.

In 1948, the National Heart Institute, now known as the National Heart, Lung andBlood Institute (NHLBI), investigated risk factors contributing to increased rates ofcardiovascular disease-associated mortality in Framingham, Massachusetts.Investigators noted a significant association between cigarette smoking and theaverage annual death rate (Freund et al., 1993). By now, many epidemiologicalstudies support an association between smoking and premature death from chronicobstructive pulmonary disease (COPD), cardiovascular disease, cancer, and otherdisorders (National Institutes of Health, 1997; Peto et al., 2000).

Currently, the optimal public health goal remains prevention of smoking initiationand, failing this objective, accomplishment of smoking cessation. However, preventionand cessation measures are not the only things that public health and science can dofor smokers. The public health and scientific communities can also take measuresto protect against exposure of current smokers to increased levels of cigarette smokeor to novel substances in smoke that have the potential to increase the risk of adversehealth effects for smokers.

LSRO believes that the primary, if not the only, objective of testing ingredients addedto cigarettes should be to assure, to the extent practical, that ingredients do notincrease the premature mortality and morbidity associated with cigarette smoking.Epidemiological studies have revealed that certain major causes of death: cancer,cardiovascular diseases, COPD, and stroke contribute more than half of thepremature mortality associated with cigarette smoking. These diseases increase inincidence as non-smokers age, as smokers age, as smokers consume more cigarettes,and as smokers consume cigarettes for longer periods of time. Among smokers,latency and reversibility of disease vary. After cessation of smoking, lung cancerincidence eventually decreases to nearly that observed in lifetime non-smokers.However, COPD incidences do not revert to background levels after smokingcessation.

Thus, the descriptive standard for adverse health effects of cigarettes remains thelong-term observation of human smokers. The optimal scientific comparison wouldbe between smokers of cigarettes containing an added ingredient and smokers ofnearly identical cigarettes lacking the ingredient. However, such observational studiespresent many challenges.

Predicting the risk of adverse health effects from smoking a cigarette with an addedingredient, relative to the risk of adverse health effects from smoking a nearly identicalcigarette lacking the ingredient, is a difficult task. The attribution of adverse healtheffects, if any, to any one or to a combination of these ingredients added to cigarettes

Introduction � 11


to facilitate processing, modify burns, and/or achieve unique flavor or brandcharacteristics, will challenge the science of toxicology. No predictive animal modelof the range of health effects of smoking currently exists.

Except for menthol (Brooks et al., 2003; Carpenter et al., 1999; Kabat & Hebert,1991; Sidney et al., 1995), no epidemiological studies have examined the adversehealth effects of added ingredients in cigarettes. Previous epidemiological studiesseldom provided adequate information to attribute incidence to an ingredient in acigarette, because: (1) smokers changed brands, (2) manufacturers changed brandingredients, (3) smoking may have been intermittent, and (4) almost none of thesechanges tracked to ingredients in ways that smokers could explain to interviewers.An appropriate epidemiological study, particularly for ingredients added to cigarettesat undetectably low levels, is not feasible. Cigarette smoking-associated humandiseases have long latencies. Diverse human populations, different living conditionsand environments, and variable background rates of diseases, all limit quantitativeepidemiological observations. Susceptibility factors, such as predisposing genetictraits, age, ethnicity, gender, or nutritional status, also vary in human populations(Perera, 1997).

2.3 LSRO’S APPROACH

LSRO believes that it has made an initial step in resolving the issue of ingredientsadded to cigarettes. This report presents an integrative approach. In developingscientific criteria that are intellectual in nature, this report emphasizes the informationnecessary to show, to the extent practical, that the relative risk of adverse healtheffects from smoking cigarettes does not change due to the addition of an addedingredient.

LSRO seeks data which document the following scientific criteria:

(a) Either the added ingredient or a pyrolysis product of the addedingredient transfers into smoke in such a way that smokers are subjectto a change in risk of adverse health effects.

(b) Addition of the ingredient does not change the physics, chemistry, orbiological activity of smoke significantly.

(c) Addition of the ingredient does not change exposure to cigarette smokethrough altered human smoking behavior.

These criteria apply only to the relative risk of adverse health effects of smoking forsmokers, not to potential adverse human health effects for bystanders. LSRO hasnot addressed subjects such as environmental tobacco smoke (exposure of non-smokers to cigarette smoke). The focus of this report does not imply that LSROconsiders these risks negligible or unimportant. LSRO has included an appendix on



the developmental effects of exposure to smoke. (See Appendix F.) The risk ofadverse human health effects for cigarette smokers is high and of immediate concern.Of necessity, LSRO has focused on the problem of understanding the relative riskof adverse health effects of ingredients added to cigarettes for the smokers ofcigarettes.

2.4 THE “TOXINS-IN-SMOKE” HYPOTHESIS

Substances in smoke are responsible for the morbidity and mortality associated withcigarette smoking. However, the scientific basis of LSRO’s approach to testingingredients added to cigarettes contrasts with the currently popular “toxins-in-smoke”hypothesis. This hypothesis singles out specific chemical substances in smoke asthe causes of the adverse human health effects observed in smokers, implying thatmost of the other substances in smoke have no effect. The “toxins-in-smoke”hypothesis also holds that bioassays of these substances: (1) in isolation, (2) at highexposures, and (3) in non-human subjects, characterize these substances adequatelyto predict the adverse human health effects of smoking observed in epidemiologicalstudies.

Chemical substances typically cited as demonstrating the “toxins-in-smoke” hypothesisare benzo(a)pyrene and 4-(N ′-Nitrosomethylamino)-1-(3-pyridyl)-1-butanone(NNK), which are representatives of two, chemical classes of substances, polycyclicaromatic hydrocarbons and nitrosamines, respectively. These substances arecharacterized by isolation from smoke and by administration to laboratory animals athigh exposures (Goldstein et al., 1998; Hecht et al., 1986; Hecht, 1999; Hoffmannet al., 1991). In such studies, substances which induce a higher incidence of tumorsat exposures lower than the exposures which cause overt toxicity (mortality orinhibition of body weight gain) are classified as “carcinogens.”

The “toxins-in-smoke” hypothesis is plausible. In some instances, one or a few verypotent substances do explain the toxicological properties of naturally-arising mixtures.In some instances, animal models of tumor induction mimic the etiology and pathologyof cancer observed in humans. However, in application to cigarette smoking, theevidence supporting the “toxins-in-smoke” hypothesis is problematic. Knowledgeabout the adverse human health effects of cigarette smoking is limited almost entirelyto information arising from human epidemiological studies. Animal bioassays do notpredict the range of human health endpoints associated with cigarette smoking.Mouse cancer bioassays do not predict the organ specificity of rat cancer bioassays,and vice-versa (Freedman et al., 1996). To date, bioassays of smoke substancesmay not have tested the substances generating adverse human health effects. Smokeemerging from the end of a cigarette is chemically active, in a state of dynamicchange. Bioassays of purified chemical substances at high exposure levels cannotreplicate the chemical properties of fresh smoke. Almost all of the existing data

Introduction � 13


relate to substances isolated from the particulate fraction of mainstream smoke.However, Witschi and coworkers (1997a; 1998) have shown that the gas, not theparticulate, fraction of a mixture of sidestream and mainstream cigarette smokeincreased lung cancer in strain A mice.

An alternative hypothesis is that the entire smoke obtained from cigarettes generatesmost of the observed effects. Another alternative is that some substance in smoke,which generates most of the observed human health effects, remains undiscovered.Given more research, the “toxins-in-smoke” hypothesis could acquire better supportfrom data. While it may prove correct eventually, LSRO declines to base a currentapproach to testing ingredients in cigarettes on an unproven hypothesis.

LSRO seeks to avoid an approach to testing that would reduce one adverse healthendpoint at the expense of increasing another. Lung cancer, even increased cancerincidence in general, is not the only observed effect of cigarette smoking. LSROdoes not want to facilitate the use of ingredients which decrease the incidence ofhuman lung cancer, but increase the incidence of cardiovascular effects, particularlyif doing so might lead to an increase in overall premature death. A testing approachconsistent with LSRO’s view of the data will, of necessity, be more restrictive.

These restrictions lead LSRO to aim for an approach involving “no change,” insteadof “no increase.” “No change” means that LSRO seeks an approach whicheliminates potential increases in any of the experimental parameters tested withoutnecessarily characterizing the biological meaning of a potential change. (“Nodetectable change” emphasizes the empirical nature of the experimental data.) Anincrease in risk might occur when a change in a measured parameter is observed.However, if a measured parameter related to smoking cigarettes does not change inresponse to an ingredient, relative to smoking cigarettes lacking the ingredient, thenincreased relative risk, related to that parameter, would seem possible.

An approach based on “no increase” in specific experimental parameters wouldimply that LSRO understands the basis of adverse human health effects of smoking.At present, LSRO lacks such insight. The “no change” approach adopted in thisreport does not imply that LSRO does not care about potential decreases in diseaseincidence or that LSRO would not accept decreases while eliminating increases inadverse human health effects. Given the current state of knowledge, LSRO believesthat the “no change” approach is the most rational.


3.1 INTRODUCTION

3.2 LSRO’S SCIENTIFIC CRITERIA3.2.1 No detectable transfer3.2.2 No detectable change in smoke physics, chemistry,

or biological activity3.2.3 No detectable change in human exposure

3.3 TESTING SUBSTANCES NOT MEETING LSRO’S THREESCIENTIFIC CRITERIA

3.4 SUMMARY

3STRATEGY

Strategy � 15


3STRATEGY

3.1 INTRODUCTION

This report proposes an approach to identify risk and decrease the probability of anypremature mortality and morbidity from ingredients added to cigarettes. LSROoutlines a program to provide sufficient information to determine whether an addedingredient changes the risk of adverse human health effects, when applied to cigarettesat some maximum amount. LSRO has developed three scientific criteria to evaluateeach added ingredient, which are discussed in more detail later in this chapter. Thesescientific criteria specifically address whether the added ingredient:

(a) transfers significantly to smokers through smoke,

(b) changes the physics, chemistry, or biological activity of the smoke, and

(c) alters exposure of smokers by modifying their behavior.

The mechanisms responsible for cigarette smoking-associated premature deathsand morbidity continue to elude scientific investigation. For some scientists, thediscovery of substances in smoke with activities in animal bioassays is sufficient toexplain cigarette-associated premature mortality or some cause of cigarette-associated mortality (Hecht, 1999). LSRO’s Phase One report (2004) of this project,Feasibility, documented that cigarette smoke is a complex mixture resulting in manyadverse human health effects. The report concluded that it is feasible and worthwhileto initiate research studies to assess the risk of smoking cigarettes with addedingredients when compared to smoking cigarettes without such ingredients.

The pursuit of a cause and effect relationship is a worthwhile scientific objective, onevalidated by the history of natural products pharmacology. However, scientists haveneither discovered one-to-one relationships between individual substances in cigarettesmoke and the human diseases associated with cigarette smoking nor found anoverarching explanation of the phenomenon of adverse human health effects of cigarettesmoking (Shields, 2002; Thun et al., 2002; Wynder & Muscat, 1995). Specification ofthe substances in smoke which lead to adverse human health effects remains elusive.Cigarette smoke consists of thousands of substances, and it causes numerous humandiseases at many organ sites (National Institutes of Health, 1997; Vineis et al., 2004).



Any hypothesis proposing that a reduction of certain substances or increases inother substances would change the overall incidence of adverse health effectsassociated with cigarette smoking requires the development of a database necessaryfor monitoring the impact of such products on public health. Future research mightdemonstrate the irrelevance, or even the benefits, of excluding substances that arecurrently thought to lead to adverse health effects. If new scientific informationdoes link specific substances in cigarette smoke to specific adverse human healtheffects, manufacturers could test to see whether the inclusion of an added ingredientchanges the levels of those specific substances found in smoke in ways predicted toincrease adverse human health effects. However, LSRO cannot find substantialevidence to support such a hypothesis at this time.

While cigarette smoke induces lung cancer in some smokers, leading to death, themammalian species commonly used in experimental studies as animal models havelimited usefulness in predicting lung cancer. Developing animal models of tobacco-related diseases, including cancer, is difficult in part because animals breathe smokedifferently than humans (Institute of Medicine, 2001). Replicating all of theparameters of human smoking in animals is not possible.

The relationship between the results of bioassays of cigarette smoke in experimentalstudies and the observed health effects of human smokers remains elusive.Quantitatively, the results from bioassays of substances found in cigarettes do notexplain the overall incidence in smokers of diseases linked to these substances.Overall, experiments to demonstrate one of the effects of smoke inhalation, such aslung cancer, cannot be used as evidence that the same mechanism is responsible forthe entire concert of diseases associated with human smoking. Much of theexperimental animal evidence only involves the ‘tar’ portion of cigarette smoke.Even for experiments with intact smoke, processing of the smoke prior to delivery toexperimental animal lungs may change the chemical properties and dosimetry of thesmoke as compared to mainstream smoke experienced with human smoking.

Prevention of initiation and encouragement of cessation of smoking remain the primary,if not the only, means of avoiding the adverse human health effects of cigarettesmoking. This report does not attempt to lower the risk of adverse health effects ofsmoking through the addition of an ingredient. Instead, LSRO seeks to eliminatesignificant changes in the relative risk of potential adverse human health effects ofsmoking cigarettes from the addition of the ingredient, leaving the effects of smokingunchanged.

The approach described in this report is not the only possible way to test and evaluateingredients for potential adverse health effects. LSRO is not a regulatory agencyand therefore does not set or enforce testing requirements for ingredients added tocigarettes. With this report, LSRO outlines a program anticipated to provide sufficient

Strategy � 17


information to determine that an ingredient added to a cigarette will not changeadverse human health effects from smoking, when added to some maximum uselevel (MUL). Within this approach, LSRO encourages flexibility and innovation.

LSRO also recommends testing substances added to cigarettes within the matrix ofsmoke. This approach differs from traditional safety assessments, such as the U.S.Food and Drug Administration’s (FDA’s) food additive review, where testing isconducted on the additive isolated from the foods proposed to contain the additive(U.S. Food and Drug Administration, 1982; 1993).

LSRO has no preconceived test regime, checklist, or format for data to demonstratean absence of change in relative risk. However, LSRO’s approach is predicated onthe availability of otherwise identically manufactured test cigarettes, with and withoutan ingredient (Figure 3.1). LSRO seeks to encourage scientific innovation and willreceive proposals and reports from researchers who go about this task with multipleapproaches.

Figure 3.1 illustrates paired test cigarettes. LSRO’s approach is predicated on the availability ofotherwise identically manufactured test cigarettes, with and without an ingredient. In this way, anyeffects of an added ingredient can be identified.



Each chapter in this report illustrates various possibilities of how such data could beobtained. Chapters 6, 7, 8, 9, and 10 have bifurcated structures and explains:

(a) the acquisition and interpretation of data aimed at the three scientificcriteria that LSRO poses, and

(b) how to proceed with relevant testing if one of more of the criteria isunattainable.

Figure 3.2 proposes a simplified flow chart that will be useful for documenting atesting approach for an added ingredient. The figure integrates the flow of information,as explained further in Chapter 11. Appendix D applies the approach to somehypothetical substances.

The intent of the approach in this report is to provide examples of the data necessaryfor a scientific reviewing panel to document non-tobacco ingredients and relevantconditions for their addition that would provide adequate evidence that the additionof the ingredient under those conditions would not be expected to change the adverse

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Figure 3.2 A packet of information submitted to LSRO for review will consist of both existingpublic information and data from ingredient-specific testing. Evaluation of all relevant informationwill lead to one of three outcomes: a) setting a maximum use level for an ingredient to add to acigarette where no change in the relative risk of adverse health effects is likely, b) the ingredient wouldnot be recommended for addition to cigarettes where data shows the potential for associated healtheffects, and c) the absence of sufficient data will lead LSRO to detail what further information isrequired.

Strategy � 19


human health effects of cigarette smoking. The approach is challenging. It will onlyprove possible if adequate data are collected.

LSRO expects that a data submitter will assemble a package of relevant informationorganized to demonstrate that inclusion of the added ingredient (up to some maximumamount per cigarette) will not change the relative risk of adverse human healtheffects of smoking cigarettes. The assembled data package likely will include areview of relevant scientific literature, data from ingredient-specific testing, andother information about the ingredient (i.e., function, MUL, chemical, and physicalcharacteristics). LSRO does not expect a point-to-point correspondence betweenexperimental tests and interpretational objectives. LSRO also will interpret the datato make a decision about one of three potential outcomes:

(1) not to add the substance to cigarettes,

(2) to add the substance contingent on specified limitations, usually amaximum amount per cigarette, or

(3) to request further information due to the absence of sufficient data.(See Figure 3.2.)

The approach to testing in this report does not address the adverse human healtheffects of cigarette smoking by requesting human epidemiological studies. [See thePhase One report for related information (Life Sciences Research Office, 2004)].LSRO will not attempt to explain the adverse human health effects of cigarettes inthis report. However, LSRO will review cogent epidemiological data whenappropriate. (See Appendix C, Peter Lee presentation.) While LSRO does notexpect initiation of such studies, where available a well-conducted epidemiologystudy that compares the adverse human health effects of smoking cigarettes withand without an added ingredient in LSRO’s view would, provide definitive evidenceof the health consequences of inclusion of the added ingredient.

LSRO proposes meeting the overall objective through a demonstration of the threescientific criteria stated at the beginning of this chapter. The following sections furtherexplain the three scientific criteria, and subsequent topics in the report develop theoverall approach in greater detail. Ideally, scientific data are measurements that arereproducible between different observers. LSRO’s Phase One report (2004) addressesthe need for direct measurement. However, LSRO understands that the scientificprocess requires explicit methods to resolve problems in measurement replication.

3.2 LSRO’S SCIENTIFIC CRITERIA

Data submitted for evaluation should focus on demonstrating that all three criteriacould be met. A substance not meeting one of the criteria need not immediatelyundergo testing for the other criteria.



3.2.1 No detectable transfer

(a) Neither the added ingredient, nor a pyrolysis product of the addedingredient (which is not a substance already found in smoke), detectablytransfers into smoke, in such a way that smokers could inhale a novelsubstance.

One example of an approach to identify ingredient transfer is to isotopically label aningredient and quantify the amount, if any, of mass originating from this ingredient inthe substances within smoke. Use of either stable or radioactive labels couldaccomplish this goal, and different chromatographic methods are available to separatesmoke substances sufficiently to obtain unique chemical identification. This methodcan be viewed as the “gold standard”; other methods exist which can also be appliedto address this criterion. (See Chapter 6.)

Test data that are scientifically achievable demonstrate that the addition of aningredient to cigarettes meets this criterion. In LSRO’s view, the alternatives todemonstrating a lack of transfer are (a) to reduce the amount of the added ingredientin a cigarette and retest or (b) to demonstrate the absence of change in relative riskof the amount of an ingredient already tested.

If a demonstration of the absence of a change in relative risk, given the addition ofan ingredient, is the objective, then knowledge of the structure of the substances(s)which transfer(s) will be desirable.

3.2.2 No detectable change in smoke physics, chemistry, orbiological activity

(b) Addition of the ingredient does not detectably change the physics,chemistry, or biological activity of smoke.

For the composition of smoke, LSRO recommends testing paired cigarettes, withand without the ingredient, for changes in indicator substances. If the smoke fromcigarettes with the ingredient is indistinguishable from the smoke from cigaretteswithout the ingredient, no change can be expected in adverse human health effects.Epidemiological studies define these known adverse health effects of smoking.

For purposes of discussion, in the U.S. the American Cancer Society’s (ACS’s)epidemiology studies (CPS-I and CPS-II) are good benchmarks (National Institutesof Health, 1997). Smokers in those studies consumed cigarettes that generated arange of nicotine, water, carbon monoxide, and nitrogen dioxide, when smoked. Ifthe cigarettes without an ingredient do not generate smoke distinguishable from thecigarettes used in the CPS-I and CPS-II, LSRO has no reason to expect that the

Strategy � 21


adverse human health effects of the test cigarettes would differ from those seen inthe ACS’s studies. However, it is worth pointing out that the cigarettes used duringthe conduct of CPS-I and CPS-II are not necessarily representative of what issmoked today. The CPS-I participant enrollment ended in 1960 when the sales-weighted average tar was around 27 mg. CPS-II enrollment ended in 1982 whenthe sales-weighted average was about 12 mg. A definition of the variance of indicatorsubstances in typical cigarettes smoked today is a research need.

Ingredients added to cigarettes can change the levels of smoke constituents (Gager,Jr. et al., 1971a; 1971b; Sato et al., 1979; Schlotzhauer et al., 1986). Testing canestablish whether ingredient-induced changes in indicator substances in smoke, ifany, lie within the range generated from cigarettes smoked today. A change whichlies outside this range could indicate the potential for a change in adverse humanhealth effects.

Because animals breathe smoke differently than humans, animal models of adversehuman health effects associated with cigarette smoking are limited (Martonen &Schroeter, 2003). A predictive inhalation test using laboratory animals to modelmany cigarette smoking-related health effects is currently unavailable to investigatorsof ingredients added to cigarettes. Thus, LSRO relies on previous epidemiologicalstudies to define the adverse human health effects of smoking.

In the Phase One report (2004), LSRO described a “no detectable change” approach,which interprets test data and other health effects information to set an upper limiton an ingredient added to a cigarette. The upper limit relates to signals from specifictests. If investigators cannot reasonably detect differences between cigarettes withand without an ingredient, no reason would exist to expect a change in adversehuman health effects. Here, the anticipated effects are those previously observedin epidemiological studies.

LSRO will regard any detected changes in smoke constituents as potentially adverse.The first question to address, given a measured difference, is whether the changearose by chance. Circumstances may arise where investigators cannot reduce aningredient added to cigarettes below a point of no detectable change and achieve auseful purpose with the ingredient. The investigation of relative risk, given a detectablechange, differs from achieving compliance with the three criteria outlined here.LSRO recommends that investigators begin by estimating the chemical fate(Chapter 6), exposure (Chapter 7), biological effects (Chapter 9), and influence onsmoking behavior (Chapter 10) of the substance which changes detectably. Thissubstance could be a pyrolysis product which transfers or a substance usually foundin smoke.



3.2.3 No detectable change in human exposure

(c) Addition of the ingredient does not change exposure to cigarettesmoke through altered human smoking behavior.

LSRO does not want to restrict ingredients which induce behavioral changes that donot alter the exposure of smokers. However, in response to an ingredient in cigarettes,smokers might change their behavior such that they smoke significantly differentnumbers of cigarettes per day or they smoke their usual number of cigarettes with adifferent intensity such that their biomarkers of exposure change. Reliable tests toobtain such data are presently available. If data indicate that consumers changetheir exposure to cigarette smoke in response to an ingredient, the manufacturer canreduce the ingredient and retest, or demonstrate that the change in exposure doesnot change the relative health risk of smoking.

3.3 TESTING SUBSTANCES NOT MEETING LSRO’STHREE SCIENTIFIC CRITERIA

If an ingredient meets all three criteria, LSRO cannot foresee how the relative riskof smoking cigarettes containing the ingredient would change the adverse humanhealth effects.

The most reliable and direct demonstration of an unchanged relative risk ultimatelywould involve epidemiological studies of people smoking cigarettes containing aningredient compared to smokers of identically manufactured cigarettes that lack theingredient. However, such studies take a long time. For ingredients which cannotmeet the above LSRO scientific criteria, LSRO proposes that the data submitterreduce the amount of ingredient added and retest or that investigators try todemonstrate that relative risk will not change using more extensive chemical andbiological testing methods (i.e., standard regulatory toxicology bioassays, adjustedfor inhalation). These data would embed within an overall framework which includesinterpretations of exposure, dosimetry, and biological effects, including behavioralmodification of exposure.

3.4 SUMMARY

The perspective that cigarette smoking induces premature mortality and morbidityof humans strongly influenced LSRO’s development of this approach. Even if somescientists believe that a particular test (e.g., a chemical assay for a specific chemicalor a bioassay for a specific endpoint) yields information relevant to the adversehuman health effects of cigarette smoking, LSRO might not rely on the test. UntilLSRO better understands the effects of variation in smoke composition and smokeeffects, LSRO will limit its conclusions to the cigarettes tested.



LSRO cannot conclude that an upper limit on the amount of an ingredient wouldconfer absolutely no change in relative risk, because of negative test results withindicator substances. Even if LSRO obtained data from every known test, a newtest might arise, which would detect a difference. Like all areas of science, LSRO’sinference will relate to the information known at some point in time. A test also cangenerate a false negative result, a finding of no effect, when one exists. Employinga battery of tests, instead of a single test, reduces the probability that a false negativeresult will influence estimation of the upper limit. Each test has characteristic statisticallimitations, such that retesting with more samples might detect a difference, wherepreviously none was noted. Testing with reliable tests that have good signal-to-noise characteristics will minimize the possibility of underestimating the upper limitfor an ingredient.


44.1 INTRODUCTION

4.2 GUIDELINES AND PUBLISHED METHODS

4.3 SOME PRODUCT SAFETY TESTING GUIDELINES

4.4 SUMMARY

RELEVANCE OF TESTING METHODS OFOTHER ORGANIZATIONS

Relevance of Testing Methods of Other Organizations � 25


4.1 INTRODUCTION

Because LSRO is not aligned with a regulatory agency, no legal or policy contextsupports LSRO’s decisions. LSRO’s science-based analysis probably would notsupport a construct such as a “safe level” or a “de minimis risk.” LSRO neitherobjects to outside parties having such policies and submitting their documents forreview, nor can LSRO constrain the use of its reviews, once published. LSRO willcomment on the scientific support for policy goals. However, when potential sponsorsbase their decisions on such goals, LSRO will appreciate a clear articulation of thepolicy.

In response to a request, LSRO will review submitted data and respond with anexplicit inquiry for additional data or with final advice regarding the use of an addedingredient. LSRO will provide a scientific rationale for its advice and will not ask foradditional data beyond the initial request. As described in Chapter 5, sponsors willsubmit data about the ingredient in cigarettes for LSRO to accomplish a review.Existing test guidelines and published methods for other uses, such as testing ofsubstances added to foods intended for ingestion, usually do not provide the kinds ofinformation necessary for LSRO to assess the potential health effects of adding aningredient to cigarettes. However, modifications of guidelines might help a sponsorobtain the data of interest: when a substance transfers, changes the physics, chemistry,or biological activity of smoke, or alters human exposure through a change in smokingbehavior. Sponsors may want to modify existing guidelines to apply to differentcircumstances, such as inhalation. Figure 4.1 illustrates the potential influence ofexisting test guidelines on experimental design for ingredient specific testing.Traditional toxicological methods do not translate well to an assessment of the healtheffects of non-tobacco ingredients in cigarettes.

The main purpose of this chapter is to define the term “guideline.” However, aguideline without accompanying study data cannot contribute to the review of aningredient. LSRO has provided this information for the convenience of potentialdata submitters, not to indicate prior “approval” of studies conducted within theframework of any guidelines or to suggest that some kind of requirement exists forstudies conducted in particular ways.

4RELEVANCE OF TESTING METHODS OFOTHER ORGANIZATIONS



This chapter also serves to clarify LSRO’s purpose in referencing test guidelines.LSRO has considered the possibility of publishing guidelines and presently has declinedto do so for several reasons:

(1) LSRO is under no obligation to interpret data in any particular wayand will not necessarily endorse a sponsor’s interpretation because astudy has been conducted in accordance with a guideline.

(2) LSRO seeks not to freeze the particular methods of research in usenow; this might serve as a barrier to progress. Instead, LSRO willrely primarily on case-by-case scientific judgment.

(3) LSRO does not want to indicate a relationship between test resultsand any change in “safety,” as this term is commonly understood.

Figure 4.1 shows how existing test guidelines could add to an evaluation of the potential healtheffects of added ingredients. Sponsors may need to modify existing guidelines to apply tocircumstances of inhalation of cigarette smoke.

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4.2 GUIDELINES AND PUBLISHED METHODS

In LSRO’s view, a test guideline is a codification of experience about how to proceedwith a particular test under ideal circumstances. Regulatory agencies, particularlyagencies that want to instruct the public about the best ways to conduct tests toobtain data for submission, will add additional details to their guidelines. For example,a regulatory guideline may specify a minimum number of animals per dose group,but LSRO already understands group size as a convention and has no rationale toachieve a particular statistical result.

Guidelines are statements about how to proceed under ideal circumstances, basedon past experience. Domestic regulatory agencies issue relevant guidelines, includingthe U.S. Environmental Protection Agency (EPA), and the U.S. Food and DrugAdministration (FDA). Many countries have harmonized their test guidelines,including member countries of the Organization for Economic Cooperation andDevelopment (OECD) and the International Conference on Harmonisation (ICH).In addition to regulatory agencies and governmental bodies, voluntary organizationspromulgate test guidelines, including the International Standards Organization (ISO),Centre de Coopération pour les Recherches Scientifiques Relatives au Tabac(CORESTA), the Association of Official Agricultural Chemists (AOAC), or AOACInternational, the Flavors and Extracts Manufacturers Association (FEMA), theAmerican Society for Testing Materials (ASTM), and the International Society forPharmacoepidemiology (ISPE).

Guidelines differ from published methods in several ways. The existence of aguideline implies that scientists have reviewed one or more published methods, thatthe test itself has sufficient currency to merit attention, that use of the guideline canlead to reliable results, and that the data from the test meet at least one specific use.Testing guidelines cover a wide range of scientific domains, such as chemistry,epidemiology, and toxicology. Some are specific to the kind materials tested, someto the use pattern, still others to the endpoint of interest. For example, CORESTArestricts itself to tobacco products and substances added to tobacco products.However, the distinctions between method, validated test, and guideline are notsharp.

Data developed in accord with a test guideline and submitted to LSRO should citethe guideline and organization responsible for the guideline. This report and otherLSRO reports refer to the guidelines of other organizations for convenience, not asa form of endorsement. Thus, this topic only gives a brief outline of the subject andsome examples of testing guidelines; it does not attempt a comprehensive review.

Tests are specific to substance, use pattern, endpoint, and objective. Few organizationshave examined the testing of cigarette smoke for purposes of reaching conclusionsabout potential adverse human health effects. Even ICH’s inhalation guidance has



different materials and different objectives - looking at exposures intended to achievea biological effect, whereas optimally an ingredient added to cigarettes would lackany biological effect. Broadly, LSRO found that guidelines of other organizationsconcerned with product safety lacked relevance to its objectives.

4.3 SOME PRODUCT SAFETY TESTING GUIDELINES

The FDA’s Center for Food Safety and Nutrition (CFSAN) issued and updated foodadditive test guidelines, which aim to cover comprehensive needs for this use (U.S.Food and Drug Administration 2001). Manufacturers use the test results to satisfyregulatory requirements. Similarly, the EPA’s Office of Pesticides, Prevention, andToxic Substances (OPPTS) developed harmonized test guidelines for regulatorysubmissions to cover both pesticides and industrial chemicals (U.S. EnvironmentalProtection Agency, 2004). EPA issued these guidelines over multiple years.

The OECD consists of delegations from partner countries interested in trade witheach other. To this end, OECD is interested in the elimination of non-tariff tradebarriers, such as a lack of acceptance of one partner’s safety data by another.OECD’s purpose in issuing chemical testing guidance was to achieve common waysof conducting regulatory tests that trading partners would all accept (Organizationfor Economic Cooperation and Development, 2004). OECD’s testing guidelinespredate EPA’s more recent harmonized guidelines, and EPA officials participated inthe development of the OECD guidance. OECD countries must accept data generatedin other countries if the tests are performed according to these guidelines. The mostrecent OECD guidance covers five areas: physical-chemical properties, effects onbiotic systems, degradation and accumulation, health effects, and special activities.

The ICH Technical Requirements for Registration of Pharmaceuticals for HumanUse developed guidelines to meet the technical requirements of registration ofpharmaceuticals for human use in the U.S., Europe, and Japan. ICH recently updatednonclinical safety testing guidance in 2003 (International Conference onHarmonisation, 2004). Of interest here, ICH has issued inhalation toxicologyguidelines.

Guidelines may be specific to a kind of material or to a particular endpoint. Thus,AOAC guidelines usually relate to analytical chemistry methods, whereas Table 4-1illustrates that several organizations have issued guidelines relating to inhalation toxicity.



Table 4-1 Guidance Documents Relating to Inhalation Toxicity

Date Source Number/ Pages/ ApplicabilityVolume Section

1981 Inhalation n/a 279-298 S.B. Gross, Regulatory guidelinesToxicology for inhalation toxicity testing.

(Ed.) B.K. Leong, Ann Arbor, MI,Ann Arbor Science (Gross, 1981)

1981 OECD 413 Subchronic inhalation toxicity:90-day study

1981 OECD 413 Acute inhalation toxicity

1996 EPA OPPTS 875 2500 Occupational and ResidentialExposure Test GuidelinesInhalation Exposure—Indoor

1998 EPA OPPTS 870 3465 90-Day Inhalation Toxicity

2000 EPA 40 CFR799 9130 Acute Inhalation Toxicity

2002 NAS (NRC) 2 Acute Exposure Guideline Levelsfor Selected Airborne Chemicals

EPA = U.S. Environmental Protection Agency; NAS (NRC) = National Academy of Sciences (NationalResearch Council); OECD = Organization for Economic Cooperation and Development; n/a = notapplicable

4.4 SUMMARY

Since LSRO is neither a regulatory agency nor aligned with a regulatory agency, nolegal or policy contexts support decisions as to the relevance of testing. Existing testguidelines and published methods for other uses usually do not provide the kinds ofinformation necessary for LSRO to assess the potential health effects of adding aningredient to cigarettes. In this context, data submitters may want to modify existingguidelines to apply to inhalation studies. LSRO does not provide a complete listingof specific guidelines, but indicates a few broad guidelines, so as not to suggest priorapproval of studies conducted within the framework of any guideline, or that somekind of requirement for studies conducted in any particular way, or to endorse anygiven guideline. Thus, LSRO emphasizes the needs for (a) scientific judgment inevaluating data and (b) not setting specific criteria that might inhibit innovation.


55.1 INTRODUCTION

5.2 DEFINITION OF THE TEST SUBSTANCE

5.3 CLASSIFICATION OF THE TEST SUBSTANCE5.3.1 Well-defined chemical compounds5.3.2 Less well-defined chemical compounds5.3.3 Chemical mixtures

5.4 CONSIDERATION OF “WHAT TO TEST”5.4.1 Factors that alter the chemistry and physics of tobacco smoke

5.5 ANALYTICAL TESTING5.5.1 Identity5.5.2 Physical constants5.5.3 Purity and composition5.5.4 Concentration5.5.5 Description of analytical procedures

5.6 SPECIAL CONSIDERATIONS FOR COMPLEX MIXTURESAND BIOLOGICAL PRODUCTS

DEFINITION, SPECIFICATION, ANDCHARACTERIZATION OF THE TESTSUBSTANCE

Definition, Specification, and Characterization of the Test Substance � 31


5.1 INTRODUCTION

For every ingredient submitted to the Life Sciences Research Office (LSRO) forreview, a detailed description of the substance added to cigarettes and a rationalefor the selection of that substance should be included as relevant data. (See Figure5.1.) Because shorthand naming conventions exist, LSRO defines the test substanceas a sample of the ingredient that is in use, including its origin and method of

5DEFINITION, SPECIFICATION, ANDCHARACTERIZATION OF THE TESTSUBSTANCE

Figure 5.1 shows how a definition of an added ingredient fits into the overall flow of information.An accurate definition of an added ingredient provides necessary information and should be providedby a data submitter as part of an information packet detailing added ingredient data.

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preparation, which are often important for complex or natural substances. Theobjective is to prevent testing of one form of the ingredient, while cigarettes containa different form. The following guidelines are intended to serve as a basis fordecisions about what should be tested and what information a data submitter shouldprovide. LSRO requires this information for the accurate identification andcharacterization of the substance undergoing review.

5.2 DEFINITION OF THE TEST SUBSTANCE

The test substance is a representative compound specifically prepared for use inanalytical or biological assays. As defined by LSRO, the test substance should berepresentative of the chemical compound or mixture added to cigarettes during themanufacturing process and should be thoroughly characterized to ensure its identity,purity, composition, and concentration.

5.3 CLASSIFICATION OF THE TEST SUBSTANCE

Regulatory agencies have had experience in defining test substances (U.S.Environmental Protection Agency, 1998a; 1999; U.S. Food and Drug Administration,2000). Using this information, LSRO has adopted the following chemical classificationfor test substances:

5.3.1 Well-defined chemical compounds

Well-defined chemical compounds are single compounds of known molecular formulaand structure (e.g., acetone and benzene).

5.3.2 Less well-defined chemical compounds

Less well-defined chemical compounds are compounds that can be represented bya definite molecular formula, but have either a variable structural diagram in whichthe position of substituent groups may change or an unknown structural diagram(e.g., aluminum cerium nickel sulfide, AlCe3NiS7).

5.3.3 Chemical mixtures

Chemical mixtures are complex mixtures composed of two or more compounds thatdo not react with one another or are reactive mixtures where one might expect achemical reaction. The category of mixtures may include, but is not limited to,natural products (e.g., honey, apple juice), complex reaction products (e.g., propyleneglycol, benzaldehyde, 2-ethylpyridine, methylpyrazine and other hydrocarbonconstituents), and chemical substances mixed together for the production of product(e.g., chocolate).



5.4 CONSIDERATION OF “WHAT TO TEST”

The LSRO approach to “what to test” utilizes the Screening Information Data Set(SIDS) Manual (1998a) that offers the following general guidance:

…it is recommended that tests for physical-chemical properties beconducted on the purified substance because they are basic informationon a specific chemical. The other tests, however, should generally becarried out on the substance with any essential additives and impuritiesit normally contains in order to know the effects of the marketedproduct. Ideally, the same batch of substance should be used.

LSRO suggests that the American Society for Testing and Materials (ASTM) standard(2004) or some other generally recognized industry standard be used to define thetest substance. LSRO recognizes that many chemical compounds and mixtureslack rigorous standards and that chemical compounds may have multiple ChemicalAbstracts Service (CAS) registry numbers, grades, or manufacturers. The burdenof proof, therefore, is on the data submitter to identify accurately all of the chemicalcompounds added to cigarettes during the production process. A data submittershould consider the various differences that exist for a chemical compound that isused industry-wide when selecting the test substance, although it is ultimately withina submitter’s best judgment to choose the test substance and justify its selection.

5.4.1 Factors that alter the chemistry and physics of tobaccosmoke

LSRO recognizes that many factors alter the chemistry and physics of tobaccosmoke and thus, the chemical and toxicological aspects of inhaled smoke.

An important factor in determining the character of tobacco smoke is the specifictobacco blend used in the manufacture of a cigarette. Tobacco leaves are complexbiological substances that differ significantly depending on the type of tobacco plant,curing method used, geographic region, season, and the position of the leaf on thestalk. Blending can be defined as the combining of raw materials that alterscharacteristics of the cigarette. Blending is dictated by each manufacturer’sproprietary knowledge, chemical and physical characterization of the tobacco quality,availability, and cost. A typical blend may consist of flue-cured, Burley, Oriental,cut-rolled stems, and reconstituted tobacco. Since this character can be modifiedby the use of added ingredients, the degree of inherent variability of these blends,and the added ingredients being used, it is important that chemical and biologicaltesting be conducted within the matrix of the marketed product.

Many other factors can influence smoke characteristics including the type of cigarettepaper used, presence or absence of filters, and filter ventilation. In addition, anyevaluation of the chemical and toxicological aspects of cigarette smoke are dependent



on the accurate reporting of the mixing of side-stream and mainstream smoke, thetime between smoke generation and exposure, and the dilution factors used.

5.5 ANALYTICAL TESTING

LSRO suggests that for every chemical compound, the data submitter should includethe following analytical information, where applicable:

5.5.1 Identity

Information identifying the test substance should be as complete as possible andinclude the name of the compound, CAS-number/descriptor, structural formula,empirical formula, molecular weight, the amount of the compound added to cigarettesas a percentage of tobacco weight, and a physical description of the material, includingits color and physical form. Information to substantiate proof of structure shouldinclude appropriate analytical tests, such as elemental analysis, infraredspectrophotometry (IR), ultraviolet spectrophotometry (UV), nuclear magneticresonance (NMR), and mass spectrometry (MS), as well as applicable functionalgroup analysis. Interpretation of the test data in support of the claimed structureshould be provided.

5.5.2 Physical constants

Any measurable parameter which delineates the physical composition and/orproperties of the test substance should be reported. The appropriate physicalconstants such as melting point, boiling range, density, refractive index, partitioncoefficient, dissociation constants (pK values), and optical rotation should be included.

5.5.3 Purity and composition

Purity data should be obtained using appropriate tests, such as thin-layerchromatography (TLC), gas chromatography (GC), high-pressure liquidchromatography (HPLC), phase solubility analysis, appropriate thermometricanalytical procedures, and others as necessary. The purity of the test substanceshould be reported as percent of weight. The identities of all impurities and degradationproducts that represent 0.1 % or more of the total composition should be included(U.S. Food and Drug Administration, 2004). If the test substance is a racemicmixture, the percentage of each optical isomer should be reported.

5.5.4 Concentration

An accurate concentration or the maximum use level (MUL) of the test substanceshould be reported.



5.5.5 Description of analytical procedures

A detailed description of the analytical procedures used to characterize the referencestandard should be included.

5.6 SPECIAL CONSIDERATIONS FOR COMPLEXMIXTURES AND BIOLOGICAL PRODUCTS

The complexity of most chemical mixtures and biological products precludes extensiveanalytical testing. Therefore, chemical mixtures are often defined by their processhistory and product use specifications, not by detailed compositional information thatidentifies each molecular component. Therefore, for chemical mixtures, especiallynatural products, the country of origin, processing methods, manufacturer, testingprocedures, and ratios of components in the final product become critically important.This information must be submitted to LSRO for the evaluation of chemical mixtures.


6.1 INTRODUCTION6.1.1 Ingredients added to cigarettes6.1.2 Standards in chemical smoke analysis

6.2 DETECTING FATE OF INGREDIENTS IN A COMPLEX SYSTEM6.2.1 Detection of new chemicals, not present in the test cigarette

smoke6.2.2 Change in the amounts of existing smoke components

6.3 SOME RELEVANT TESTS6.3.1 Pyrolysis6.3.2 Isotopic tracer methods6.3.3 Smoke composition

6.4 STUDIES OF INDIVIDUAL CHEMICAL INGREDIENTS6.4.1 Menthol6.4.2 Sugars (e.g., sucrose and glucose)6.4.3 Ingredients that partially survive the burning process6.4.4 Complex Mixtures

6.5 ADDITIONAL CHEMICAL ANALYSES

6.6 SUMMARY

6CHEMICAL FATE OF ADDEDINGREDIENTS IN CIGARETTE SMOKE

Chemical Fate of Added Ingredients in Cigarette Smoke � 37


6.1 INTRODUCTION

Chapter 3 of this report outlined LSRO’s scientific criteria for the evaluation ofingredients added to cigarettes. An ingredient that meets these three criteria wouldnot be expected to add to the adverse health effects of cigarette smoking. The firsttwo scientific criteria involve an investigation of the effects of added ingredients onsmoke chemistry with a demonstration that:

(a) neither the added ingredient, nor a pyrolysis product of the addedingredient, detectably transfers into smoke, in such a way that smokersare subject to a change in adverse health effects, and

(b) addition of the ingredient does not significantly change the physics,chemistry, or biological activity of smoke.

An investigation of the contribution of an added ingredient to smoke makes up anintegral part of LSRO’s approach. In order to address the chemical aspects ofthese two criteria and to demonstrate that obtaining such data is feasible, this chapteraddresses and provides examples of test methods which are available and can beapplied. The focus of this chapter is therefore to look objectively at the chemicalfate of ingredients used in cigarettes and methods of analysis of cigarette smoke todetermine their individual contributions.

6.1.1 Ingredients added to cigarettes

An expert panel (Doull et al., 1994; 1998) identified 599 individual chemicals andcomplex mixtures, primarily plant extracts, that may be added to cigarette tobaccoand/or paper to condition the tobacco, modify the burn, or to enhance flavor, aroma,and general smoking quality. These added ingredients in cigarettes fall into severalclasses humectants, sugars, and ingredients that lend desirable flavors and/or aromasthat otherwise enhance the smoking experience.

Individual flavor ingredients may be used at very low concentrations, less than0.0001 % (10-6) of the weight of the cigarette (Doull et al., 1994). The fate of thesechemical ingredients in cigarettes is a product of their individual volatility, pyrolysis,and/or reaction with other chemicals in the burning mixture (pyrosynthesis) andvaries with the smoking habits of the individual smoker (Baker & Bishop, 2004;

6CHEMICAL FATE OF ADDEDINGREDIENTS IN CIGARETTE SMOKE



Bridges et al., 1990). Thus, design of analytical methods to determine the contributionof individual ingredients to cigarette smoke presents a complex problem that is notgoing to be addressed by a single solution.

Combustion of cigarette tobacco and the paper in which it is wrapped involves aninteracting series of complex chemical reactions that occur at the end of a rod ofreactants (Baker, 1999; Pankow, 2001). The result of these reactions is the productionof a complex mixture of chemicals. The complexity of these mixtures is exemplifiedby the fact that, to date, more than 4,800 different chemicals have been identified incigarette smoke (Green & Rodgman, 1996). By far, the bulk of this material isderived from tobacco and less than 5 % from the paper. Paper is a plant derived,mostly carbohydrate material, and therefore combusts mostly to carbon dioxide andwater. Those substances added to enhance flavor and smoking characteristicsaccount for another significant, but less obvious fraction of the material in a cigarette.These added ingredients, do however, contribute to the contents of cigarette smoke(Baker et al., 2004a; 2004b; Rustemeier et al., 2002).

Assessing whether these ingredients change the risks of adverse health effectsfrom smoking cigarettes requires a determination of chemical fate. Fate in thiscontext takes the form of chemicals that result from the overall process of burningthe cigarette: vaporization without loss of chemical identity; creation of chemicalproducts due to the processes that occur during combustion in the cigarette; and acombination of the two. The complexity of smoke and low levels of certain ingredientsadded to cigarettes present a challenge to determining which of the paths an ingredientmay take.

The chemical complexity of smoke, is in essence, a large noise background.Determining the fate of an added ingredient becomes a signal-to-noise problem inidentifying small changes in a large chemical signal. The use of stable and radioactiveisotopes to label the ingredients provides a method for filtering the chemical noisebackground so that the products of the different processes can be identified by theobservation of the isotope with various analytical methods such as mass spectrometrycoupled with chromatography techniques (Gager, Jr. et al., 1971b; Green et al.,1989; Jenkins, Jr. et al., 1970). Total mass balance of the isotopic label can also beevaluated. This approach has been used in determining the mechanisms for theburning of a cigarette (Green et al., 1989; Jenkins, Jr. et al., 1970). Complex mixturesof added ingredients from natural sources will present special problems for isotopiclabeling. These approaches and others will be discussed later in this chapter. (SeeSection 6.3.)

6.1.2 Standards in chemical smoke analysis

Smoke arising from a burning cigarette is comprised of at least two parts (Baker,1999). That portion of the smoke drawn through the cigarette by the smoker (whether



inhaled or not) is referred to as mainstream smoke (MS). Smoke released from thetip of the burning cigarette into the atmosphere is referred to as sidestream smoke(SS). Environmental tobacco smoke (ETS), a mixture of aged SS and exhaled MSdisseminated in air, can be thought of as a third type of smoke.

Cigarette smoke contains submicron particles, perhaps as many as one billion permilliliter. Particle formation is due to the condensation of materials released bycombustion, which then adhere or condense onto nuclei formed in the burning (fire)cone of the cigarette (Kiefer & Touey, 1967). The temperature of the fire cone willaffect the nature of the smoke. Nicotine and other semi-volatile compounds thatdistill from the tobacco rod immediately behind the burning zone of the cigarettesubsequently condense, along with the smoke particles that form as they cool ontheir passage through the cigarette. Non-volatile materials such as sugars are largelypyrolyzed to carbon monoxide (CO) and carbon dioxide (CO2) and other productsthat are found in SS. Chemicals added to cigarette tobacco or paper to enhancecigarette flavor, smoking, or burning characteristics may, depending on their volatility,be found primarily intact in smoke (transfer) or they may be pyrolyzed to variousproducts that are found in both SS and MS.

Materials in smoke are the products of the cigarette from which they are derived.Individual cigarette brands are many and varied, and cigarette content andconstruction can modify the contents of smoke (Hoffmann et al., 1995). Variationsin cigarettes include the tobacco used, the presence or absence of a filter, as well asthe paper in which they are wrapped. Many less obvious variations are the result ofingredients used in the tobacco, filter, or paper as well as the construction of each ofthese components. Thus, no exact standard cigarette exists among those productsthat are sold commercially.

The tobacco industry, in association with the Tobacco and Health Research Instituteat the University of Kentucky, the National Cancer Institute (NCI) of the NationalInstitutes of Health (NIH), and the Agriculture and Health Research department atU.S. Department of Agriculture (USDA), developed a series of “reference” cigarettesto be used in smoking studies (Davies & Vaught, 1990). More recent cigarettesmoke studies have included the Kentucky 1R1, 1R4F, 2R4F, and others as referencecigarettes for unfiltered and filtered brands. In the case of the 1R4F, the blendspecifications are based on fabricating a cigarette that delivers approximately 11 mgof ‘tar’ and 0.8 mg of nicotine when smoked by a machine under standard FederalTrade Commission (FTC) conditions (Federal Trade Commission, 1967). Theselevels are accomplished through filtration, air dilution, and tobacco blend selectionand are near the sales-weighted average of cigarettes currently being smoked in theUS (Davies & Vaught, 1990). The latest reference cigarette, Kentucky 2R4F, wasmade available in 2002 (Tobacco and Health Research Institute, 2002). The 2R4Fis the second manufacturing run of the 1R4F cigarette blend and has a similar deliveryof nicotine and ‘tar’ to the 1R4F. A further reference cigarette, 1R5F, is representative



of ultra low yield cigarettes and consistently delivers approximately 1.67 mg ‘tar’and 0.16 mg nicotine when machine smoked under standard FTC conditions (Davies& Vaught, 1990). The use of reference cigarettes, such as the Kentucky referencecigarettes, is important to demonstrate consistency between laboratories in theirapplication of analytical methodology as well as allowing for comparison of databetween laboratories.

Smoke varies with the smoking habits of individual smokers (Bridges et al., 1990) aswell as with individual cigarette brands (Swauger et al., 2002). At present, inhaledsmoke cannot be accurately collected and analyzed. Further, if smoke wereaccurately collected from an individual, any analysis would be representative only ofthe individual smoker from whom it was collected. Therefore, standard methodshave been developed by which tobacco smoke is collected and analyzed in order toobtain an approximation of those materials in smoke.

The FTC and International Organization for Standardization (ISO) methods arecommonly cited as being the basis for methods used in smoking studies. Bothinvolve a 35 mL puff volume, 2 second puff duration, and a 60 second inter-puffinterval with no ventilation holes blocked (Federal Trade Commission, 1967;International Organization for Standardization, 2000c). In order to develop smokingmethods which better reflect current human smoking, some regulatory authoritieshave proposed and implemented methods involving more intense smoking conditions(Massachusetts Department of Public Health, 2001; Province of British Columbia,2001).

The Canadian province of British Columbia (BC) formulated a tobacco testing(machine smoking) regimen and subsequent testing and disclosure regulation thatrequires cigarette companies to report smoke constituent amounts based on filter/gas collections from a machine smoking method that uses a 55 mL puff volume, 2second puff duration, and a 30 second inter-puff interval (Province of BritishColumbia, 2001). In addition, the BC method involves blocking 100 % of the ventilationholes present in the cigarette filter. These conditions were based on an effort toachieve a more representative, maximum smoke yield that might be expected to beobtained by a current smoker. Another “intense” smoking method was developedby the state of Massachusetts Department of Public Health (MDPH). This methodconsists of a 45 mL puff volume, 2 second puff duration, and a 30 second inter-puffinterval with 50 % of the ventilation holes blocked (Massachusetts Department ofPublic Health, 2001).

A major distinction between the FTC methods and those developed by BC andMDPH is the inclusion of vent blocking. At the time the FTC developed its originalmethod, vent holes did not exist in commercially available cigarettes. The smokingmethods formulated by BC and MDPH have also altered both the puff volume and



the inter-puff interval compared to the standard FTC/ISO methods. The combinationof puff volume and duration defines the average puff flow rate and therefore, theburning temperature of the puff. An increased puff volume with increased frequencyincreases smoke yield. Changes in the puff flow rate also alter the smoke composition.

The UK Department of Health (2000) carried out a study of the effects of the ISO,BC, and MDPH methods on the yields of ‘tar’, nicotine, and CO. In addition to theincreased yields of nicotine, ‘tar’, and CO, an approximate increase of up to 10 % inthe ‘tar’/nicotine ratio was observed under “intense” smoking conditions. A furthercomparison of cigarettes smoked under FTC/ISO conditions to MDPH conditionsalso identified an approximate 2.5 fold increase in the yields of 49 smoke constituentsunder the more intense MDPH conditions (Roemer et al., 2004). The concentrations[constituent yield relative to total particulate matter (TPM) yield] of nearly all theconstituents measured (75 %), decreased with increasing TPM yields. This maysuggest an increase in the concentrations of other, unmeasured smoke constituentsthat make up ‘tar’.

The smoking method chosen impacts the yields of smoke components. A validatedmethod should be consistently used in cigarette smoking studies. Machine smokinghas its limitations, but a satisfactory alternative is not yet available. The Phase Onereport (2004) suggests use of the FTC smoking method.

6.2 DETECTING FATE OF INGREDIENTS IN ACOMPLEX SYSTEM

Change and its detection limits must be clearly defined when trying to identify thedifference the presence of an added ingredient confers to the overall chemistry ofcigarette smoke. A change in smoke chemistry due to the ingredient in question canbe defined in two ways:

6.2.1 Detection of new chemicals, not present in the testcigarette smoke

The use of radiolabeled chemicals allows the determination of the total contributionof the added ingredient to the smoke chemistry. This can either be by the intacttransfer of the added ingredient itself, or through the formation of pyrolysis orpyrosynthesis products from the ingredient, which transfer into smoke. Change canbe assessed by the use of current analytical methods such as gas chromatography-mass spectrometry (GC-MS or GC-MS/MS) to identify the novel chemicals.However, the latter requires some knowledge of the products expected from burningthe ingredient. Potential products of an ingredient can be identified by pyrolysis ofthe ingredient in isolation without the presence of the cigarette matrix (Baker et al.,2004a; 2004b; Stotesbury et al., 2000). (See Section 6.1.) Detection of biologically



relevant levels of an added ingredient or a pyrolysis product, in cigarette smoke,corresponds to an ingredient “failing” the first of the scientific criteria, as the ingredienthas caused a change in smoke chemistry. Further experimentation should concentrateon a demonstration of no change in the relative risk of adverse health effects.

6.2.2 Change in the amounts of existing smoke components

The chemical fingerprints of a test cigarette and that of the test cigarette minus theadded ingredient can be compared (Baker et al., 2004a; 2004b; Rustemeier et al.,2002). The chemical fingerprint is determined by the measurement of analytespresent in cigarette smoke by specified analytical methods. No one method candetect all 4,800 known products at once. Since no single component or combinationsof substances have a proven direct correlation with the range of adverse humanhealth effects of cigarette smoking, no single substance or combination of substancescan be singled out. Singling out individual components that may have a high interestsuch as benzo(a)pyrene, increases the ability to detect change, if the focus is on thatsingle component.

Detecting a change in the amounts of existing smoke components relates to thesecond of LSRO’s scientific criteria. Looking for change from the presence of anadded ingredient among more than 4,800 smoke components is not recommended.LSRO has not set requirements on the selection of specific components in smoke.Instead, LSRO will review all data submitted. However the effects of an ingredienton indicator substances including nicotine, water, CO, and nitrogen oxides wouldprovide important information. Standardized methodologies are published for theanalysis of these smoke components (excluding nitrogen oxides) (InternationalOrganization for Standardization, 1995; 1999; 2000a; 2000b). Recently, theCoopération Pour Les Recherches Scientifiques Relative au Tobac (CORESTA)published a recommended method for the determination of benzo(a)pyrene in cigarettemainstream smoke (CORESTA, 2004). No other analytes have standard methods.Instead, individual laboratories develop and apply their own protocols. Therefore, awide variability in measurements can be found even from smoke from the samecigarette (e.g., Kentucky 1R4F) (Baker et al., 2004a; Purkis et al., 2003).

6.3 SOME RELEVANT TESTS

Determination of the contribution of an added ingredient and its pyrolysis productsto cigarette smoke presents a multitude of technical problems. Primary amongthese is the background chemical noise relative to the signal of interest. That is,whereas some ingredients (e.g., humectants and sugars) are used in appreciablequantities most ingredients are used at very low concentrations relative to thoseoccurring naturally in tobacco (Doull et al., 1994). Thus, the contributions of tobaccoand paper to cigarette smoke are overwhelming compared to those of individualadded ingredients.



Determination of the contribution of ingredients is also complicated by the fact thatsome ingredients (e.g., sugars) are naturally present in tobacco, and others areformed naturally in the pyrolysis of tobacco (Gager, Jr. et al., 1971b; Thornton &Massey, 1975). In these cases, a determination of the portion of the productscontributed by the added ingredient, versus those naturally present or formed in theburning cigarette is recommended. The cigarette minus an added ingredient in acomparison defines the noise above which the contribution of the added ingredient(signal) needs to be seen. Some methods exist to identify the signal. In mentioningsome approaches from the literature, LSRO is demonstrating that effective methodsexist. Development of novel techniques and improvement of current methods isencouraged.

6.3.1 Pyrolysis

Model pyrolysis experiments on isolated ingredients have been used to define thecontribution of added ingredients to smoke chemistry. Some attempts tended tooversimplify the experiment, pyrolyzing ingredients under conditions that do not relateto the burning cone of a cigarette (Schmeltz & Schlotzhauer, 1968). A pyrolysisscan allows for rapid test of the potential products from burning added ingredients;however the limitations of the technique must be recognized.

Stotesbury and coworkers (1999) applied a pyrolysis GC-MS method that allowedthe effects of pyrolyzing a single material to be studied in isolation. The authorsperformed pyrolysis under a range of different conditions in an attempt to mimic theconditions of the burning zone of a cigarette. Important variables determine theoutcome of pyrolysis including temperature, rate of temperature change, oxygenconcentration, and chemical environment. The dynamic changes in chemicalenvironment and temperature, which occur during smoking of a cigarette, are difficultto reproduce under laboratory conditions and were not modeled in these experiments.However, this technique was useful in the prediction of temperature of transfer aswell as the identification of potential decomposition products. The authors concededthat it is not entirely valid to base an assessment of an added ingredient on a pyrolysisexperiment alone, unless it demonstrably relates to smoke chemistry (Stotesbury etal., 1999).

A more recent study by Baker and Bishop (2004) pyrolyzed 291 single compoundingredients to estimate their behavior in a burning cigarette in terms of intact transferand/or pyrolytic decomposition. Taking into account temperature, heating rate, oxygenlevels, and gas flow conditions in a burning cigarette, the conditions of pyrolysiswere set to heat the sample at 30°C per second, from 300°C to 900°C, under a flowof 9 % oxygen in nitrogen. A comparison of the results of this study to previousstudies, using selected isotopically labeled ingredients burned as part of a smokedcigarette, better predicted results for volatile ingredients than non-volatile ingredients.The authors reported an overestimation of the level of pyrolysis for non-volatile



ingredients. The authors of this study also mention that these data should be regardedas a first screen of the effect of added ingredients on smoke chemistry.

Results of pyrolysis experiments alone do not adequately determine the chemicalfate of an added ingredient in a burning cigarette. However, results from this kind ofanalysis can provide relevant information which can be used in the design of morein-depth chemical analysis.

6.3.2 Isotopic tracer methods

The most representative analysis for both naturally occurring and added chemicalsis obtained with an ingredient incorporated into test cigarettes (Carmines, 2002;Schmeltz et al., 1979). Radiochemical tracer methods facilitate the study of individualcomponents at very low levels within the complex smoke environment (Gager, Jr. etal., 1971b; Green et al., 1989; Jenkins, Jr. et al., 1970). Studies that use 14Cradiolabeled chemicals, permit the detection of the very low quantities of addedingredients and their pyrolysis products in smoke by effectively “isolating the signal”from the chemical background. These studies allow differentiation from chemicalsarising from tobacco. Radiolabeling of the added ingredient in question using 14Cprovides a means of monitoring the behavior of the ingredient as well as allowingidentification of pyrolysis products.

Stotesbury and coworkers (2000) used the stable isotopes 18O and 13C, to determinethe extent of degradation of p-anisaldehyde and vanillin. Initial pyrolysis experimentsdetermined that added ingredients labeled with stable isotopes produced identicalchromatograms to their unlabeled counterparts. This experiment allowed foridentification of degradation products of the ingredients within smoke extracts.Radiolabeled isotopes can be detected very easily and selectively at lowconcentrations. However, specificity may not be possible since the species that islabeled may not be directly detected. The amount of an added ingredient labeledwith a stable isotope added to a test cigarette may need to be much higher than withradiolabeling, but the use of stable isotopes together with a mass spectrometer as adetector, allows for straightforward detection and identification of pyrolysis andpyrosynthesis products.

Currently the highly sensitive methods of accelerator mass spectrometry are combinedwith chromatography techniques (high pressure liquid chromatography and gaschromatography) to allow for the determination and detection of 14C labeledcompounds (Buchholz & Mueller, 2002; Vogel & Turteltaub, 1992; Vogel et al.,1995). The combination of chromatographic methods with a low resolution massspectrometry and then accelerator mass spectrometry, allows for the identificationof combustion products that contain a 14C label. This procedure removes one of thelimitations of using 14C labeling mentioned previously, the specificity of detection.



However, the procedure is not generally available, which limits its application. Manyof the necessary instruments are found only in specially equipped national or academiclaboratories.

Isotopic labeling of single ingredients allows tracing of added ingredients and theirpyrolysis within the smoke matrix. Complex mixtures such as cocoa present a morechallenging problem. Cocoa powder contains more than 300 volatile compoundsidentified during the burning process, most of these compounds are also producedduring the burning of tobacco (Harllee & Leffingwell, 1979). The complexity ofthese mixtures means that not all components will behave the same way, some willpyrolyze and others will transfer intact.

Comparative analysis of the smoke produced from a test cigarette and a test cigaretteminus the added ingredient through measurement of a set of smoke analytes mayindicate a change in the chemical footprint of the cigarette smoke caused by thepresence of an added ingredient. (See section 6.3.3.) However, detectable changesin cigarette smoke caused by these complex mixtures are likely to be due to chemicalsunique to the mixture, that do not arise from burning tobacco. These chemicalsprovide a unique signal, which if known, may be measurable in cigarette smoke.

6.3.3 Smoke composition

Data submitters can generate comparative data, using test cigarettes smoked on asmoking machine, to compare analysis of smoke from otherwise identical testcigarettes smoked in an identical manner containing or lacking the substance ofinterest, for a defined battery of “indicator substances” (Baker et al., 2004a; 2004b;Rustemeier et al., 2002).

Rustemeier and coworkers (2002) compared smoke by looking at 51 smokeconstituents to identify the effects of 333 added ingredients separated into threedifferent cigarettes. Any change observed in the level of a smoke constituent wasexpressed both on a per ‘tar’ and a per cigarette basis. An increase (13 - 28 %) inTPM yield compared to the control cigarette was observed. The authors suggestedthat this was caused by the higher transfer rates of added ingredients to the smokecompared to tobacco. Changes in individual biologically active smoke constituentswere also seen depending on the group of ingredients used. Further research isnecessary to identify the source and mechanisms underlying these changes(Rustemeier et al., 2002) as well as the biological relevance.

More recently Baker and coworkers (2004a; 2004b) performed a similar study on482 ingredients separated into several different test cigarettes, depending on theidentity and function of the ingredient. The effects of these groups of ingredients on44 smoke components were investigated.



This study attempted to take into account the variability of smoke. Comparison ofthe yields of smoke constituents from the Kentucky 1R4F reference cigarette,measured on three occasions over the period of a year, allowed an assessment ofthe variability in the measurements of selected components measured in the samelaboratory over time. The standard deviations in yields of nicotine, CO, and TPMwere similar to those obtained from measurements taken on a single occasion.However most of the analytes measured did show greater variation over timecompared to that obtained from a single occasion. In addition, the inter-laboratoryvariation was also examined through comparing yields of smoke constituents fromKentucky 1R4F under the same smoking conditions determined in eight differentstudies. Again the variation in the values for nicotine free dry particulate matter(NFDPM, referred to as ‘tar’ in this report), nicotine, and CO were relatively small(9.5 %, 13.5 %, and 16.4 % respectively). Other smoke components showed muchwider variation, greater than 300 % for some metals. This increase in variation mayrelate to the existence of standard ISO methodologies for ‘tar’, nicotine, and COlevels in smoke but not for metals (International Organization for Standardization,1995; 2000a; 2000b).

The Canadian government has stipulated analytical methods for the measurementof smoke constituents for cigarettes sold in Canada (Health Canada Tobacco ControlProgramme, 2004). This is currently the only known requirement, within a regulation,for a specific methodology to be used for the measurement of smoke constituents.

CORESTA formed a task force in 1999 to develop methods for the determination ofcigarette smoke constituents. In 2004, a recommended method for the determinationof benzo(a)pyrene in MS smoke was published (CORESTA, 2004). Other smokeconstituents under consideration are the tobacco specific nitrosamines (TSNAs):N′-Nitrosonornicotine (NNN), 4-(N′-Nitrosomethylamino)-1-(3-pyridyl)-1-butanone(NNK), N′-Nitrosoanabasine (NAB), and N′-Nitrosoanatabine (NAT).

These studies allow for identification of the effects of single ingredients or mixtureson the smoke within the complex smoke environment. However, the identificationof any ingredient related changes is limited to selected smoke components. Thestudies mentioned above do not address the first objective of LSRO’s scientificcriteria. The ingredient and specific products of the ingredient(s) were not measuredin the smoke, therefore the degree of transfer can not be determined.

No individual method is best for all added ingredients, but studies using one or moreof these various assays have determined that the more volatile compounds may berecovered nearly quantitatively intact, whereas less volatile added ingredients maybe partially or almost completely pyrolyzed. Pyrolysis products may in turn reactwith one another or with chemicals that form during the pyrolysis of tobacco and/orpaper to form still more chemicals. In addition, some ingredients might be sufficiently



pyrolyzed that neither parent substance nor its pyrolysis products are detectable incigarette smoke using currently available techniques.

6.4 STUDIES OF INDIVIDUAL CHEMICALINGREDIENTS

The above approaches identify the fates of individual added ingredients as well asgroups of ingredients added together. A large amount of information exists for someingredients (e.g., menthol, sugars) where the effects of the ingredient on smokechemistry have been addressed in a number of ways.

6.4.1 Menthol

Volatile top flavorings are applied to cut tobacco after final drying to minimizeevaporation of the ingredient (Carmines, 2002). Chemicals added as top flavoringswould be expected to be retained in an unlit cigarette while in the cigarette packaging,but completely volatized at the elevated temperature in the rod of a burning cigarette.Such chemical ingredients are largely unchanged in the burning cigarette asexemplified by menthol. Menthol has been used in cigarettes since the 1920s and,of the chemicals added to flavor cigarette tobacco, it is probably the most obviousbecause of the unique flavor and aroma and a “cooling” effect it imparts tomainstream smoke (Gaworski et al., 1997). This fact probably accounts, in part, forthe fact that menthol has been the most studied of tobacco flavor ingredients. Mentholis also commonly used in other applications such as cold remedies, cosmetics,toothpaste, and as a flavoring in food and drink products (Eccles, 1994). (SeeAppendix C, Peter Lee presentation.)

Menthol has a boiling point of 212°C and is a naturally occurring monocyclic terpenefound in many essential oils (Eccles, 1994). The Flavoring Extract Manufacturer’sAssociation (FEMA) classified menthol as “Generally Regarded as Safe” or GRAS,and the Food and Drug Administration (FDA) approved its use in non-prescriptiondrug products (U.S. Food and Drug Administration, 1994). As a result of its volatilityat elevated temperatures encountered in the rod of a burning cigarette, menthol isnot significantly pyrolyzed in the burning cone of a cigarette and serves as a modelfor the fate of chemicals of similar volatility. Virtually all the menthol added tocigarette tobacco remains intact in MS or SS smoke. Studies of menthol containinga 14C label determined that 99 % of the menthol in MS remains intact and onlyapproximately 1 % is pyrolyzed to menthane and menthone or oxidized to carbonoxides (Jenkins, Jr. et al., 1970). Approximately 30 % of the menthol added tocigarette tobacco is delivered in MS and approximately 70 % of that is absorbed bythe smoker (Haggard & Greenberg, 1941). Menthol absorbed into the body israpidly metabolized and excreted primarily as the glucuronide conjugate in urine(Eccles, 1994; Gelal et al., 1999).



Chemicals of an equal or greater volatility than that of menthol can be assumed tobe largely volatized intact from the burning tobacco. This assumption is supportedby a study of six volatile chemicals used as flavor ingredients in cigarettes;anisaldehyde, anisole, benzaldehyde, methyl cinnamate, isoamyl isovalerate, andvanillin (Green et al., 1989). These chemicals were added to the tobacco of testcigarettes at concentrations representative of their actual use levels ranging from8.7 - 223 µg/cigarette. Thus, 14C labeled chemicals were used to permit detection ofthe very low concentrations of these chemicals and their pyrolysis products in smoke.

Four of these six chemicals have boiling points similar to that of menthol and wererecovered quantitatively in smoke or the residual tobacco and filter (Green et al.,1989). Two of the chemicals studied have higher boiling points than that of menthol.Anisaldehyde (248°C) showed some decomposition (< 10 %). The radiolabeledchemical vanillin, with a boiling point of 285oC, showed a lower recovery of 14Clabel in MS smoke (63.2 %). No labeled decomposition products were detected inMS smoke (Green et al., 1989). The authors suggested that the low recovery ofradiolabel was due to unidentified decomposition products released in the SS.Stotesbury and coworkers (1999) identified over 100 different potential decompositionproducts of vanillin. Stotesbury and coworkers (2000) also measured the pyrolysisof vanillin and anisaldehyde labeled with the stable isotope 13C in experimentalcigarettes. Five-thousand micrograms of each ingredient was added per cigarette.In this experiment, 99.1 % of anisaldehyde and 99.9 % of vanillin was found totransfer intact.

Volatile chemicals such as menthol which transfer intact into cigarette smoke changethe composition of smoke. Such an ingredient would not meet LSRO’s first twoscientific criteria. Further experimentation would be necessary to demonstrate theabsence of change in relative risk of adverse health effects associated with anupper limit of the amount of the ingredient.

6.4.2 Sugars (e.g., sucrose and glucose)

Added ingredients that are unlikely to survive the burning process are exemplifiedby sugars that are added to increase the smoking quality of tobacco and to serve ascasing for cigarette tobaccos (Baker et al., 2004b; Rodgman, 2002; Thornton &Massey, 1975). Casings tend to be hygroscopic agents applied to tobacco to conditionit for processing (Carmines, 2002). Sugars occur naturally in tobacco. Sugars,usually glucose or sucrose, are also added to cigarette tobacco in the form of casing,at up to 9 % of the total weight to increase overall smoking quality (Doull et al.,1994; Gager, Jr. et al., 1971b). As opposed to menthol, sugars are not volatile andare pyrolyzed in the burning cone of the cigarette so that little of the added ornaturally occurring sugar in cigarette tobacco, approximately 0.5 %, is transferredto MS unchanged (Gager, Jr. et al., 1971b).



In a study of 14C labeled glucose and sucrose added to test cigarettes, Gager andcoworkers (1971b) reported that of the total radioactivity added, 6.4 and 8.9 % ofthat added as glucose or sucrose, respectively, were recovered in MS smoke primarilyas pyrolysis products. Distribution of the radioactivity in MS smoke was similarwith each sugar. Approximately two-thirds of the radioactivity was in the gas phase.In each case, a little less than half of the radioactivity in the gas phase was in theform of CO2, with CO and other gases accounting for the remainder. The remainingmaterial was recovered as TPM. Approximately 50 % of the radioactivity added tothe cigarettes as each sugar was in SS, primarily in the form of CO2. CO and othergases accounted for the remainder of the radioactivity in SS. The unburned butt ofthe cigarette accounted for approximately 40 % of the total cigarette tobacco andcontained a similar amount of the total radioactivity (Gager, Jr. et al., 1971b).

In a follow-up study utilizing gas chromatography combined with mass spectrometricanalysis Gager and coworkers (1971a) isolated, identified, and quantified chemicalsin mainstream smoke arising from 14C labeled sugars. Chemicals arising from glucoseand sucrose included acetaldehyde, furan, propionaldehyde, acetone, 2-methylfuran,2-butanone, benzene, 2-buten-2-one, 2,5-dimethylfuran, acetonitrile, 2,3-butanedione,and crotonaldehyde. The amount of radiolabel recovered as each of these chemicalsamounted to less than 0.1 % (10-3 by weight) of that added as each of the respectivesugars. Thus, these compounds would not be expected to exert significant biologicaleffects. The International Agency for Research on Cancer (IARC) classifiedacetaldehyde and furan as “Reasonably Anticipated to be Carcinogens,” based onanimal studies (International Agency for Research on Cancer, 1985; 1987; 1995).Benzene and formaldehyde are similarly classified as known “Human Carcinogens”(International Agency for Research on Cancer, 1982; 2004). Risks posed by smallamounts of these pyrolysis products are unknown. Gager and coworkers (1971a)also noted that acetone and acetaldehyde in smoke that did not contain a 14C labelexceeded the amount arising from the added sugars. Thus, the bulk of thesechemicals in smoke were assumed to have arisen as pyrolysis products of sugarsand/or other compounds naturally present in tobacco. Gager and coworkers (1971a)also pointed out that the presence of acetonitrile containing 14C in mainstream smokewas evidence of pyrosynthesis resulting from pyrolysis products of the sugars reactingwith nitrogenous products in tobacco.

Baker and coworkers (2004b) studied the effects of casings on cigarette smoke bymeasuring the levels of smoke components. Twenty-nine casing ingredients indifferent combinations were tested as experimental mixtures. A large increase informaldehyde levels (73 %) occurred with one casing mixture containing sugar.Smaller changes in formaldehyde levels were observed with other mixtures ofingredients (Baker et al., 2004a; 2004b). A common element of these mixtures wasthe inclusion of sugar in various forms (e.g., white sugar, honey, corn syrup, sugarcane molasses, invert sugar, and brown sugar). In a similar study, Rustemeier andcoworkers (2002) found increased formaldehyde in smoke from cigarettes containing



corn syrup sugar. They postulated that the generation of formaldehyde comes fromthe pyrolysis of sugars.

Few examples are available, but it can be assumed that the fate of non-volatileorganic chemicals will be similar to that of sugars. That is, they will be nearly orcompletely pyrolyzed and found primarily in smoke as CO2, CO, and various pyrolysisproducts. Detection, identification, and attribution of the respective products to theappropriate chemicals will require the use of labeled compounds and/or very sensitiveanalytical instrumentation such as gas or liquid chromatography in conjunction withmass spectrometry.

6.4.3 Ingredients that partially survive the burning process

Approximately 75 % of the added ingredients that may be used in cigarettes arepure chemicals (Doull et al., 1994). However, the fate of very few of these chemicalshas been studied in cigarettes. Most of these chemicals have some volatility at thetemperature of a burning cigarette, thus it may be assumed that those chemicalshaving boiling points in the range of that of vanillin will have a similar fate. That is,like vanillin, they will be recovered at least partially intact and as pyrolyzed orpyrosynthesis products in smoke and the remaining butt and filter (Green et al.,1989). The relative amounts found in each fraction will be determined by the volatilityof the respective starting material and the reactivity of its pyrolysis products.

The most effective method to study individual compounds would be to add therespective chemical containing a radiolabel to test cigarettes smoked by machineand follow the distribution of the label into SS, MS, ash, butt, and filter. Chemicalsstudied in this manner should be added at concentrations at which they are normallyused in cigarettes in order to avoid artifacts arising from abnormal pyrosynthesisand/or pyrolysis. Whenever available, model pyrolysis studies of individual compoundsin isolation can be reviewed in order to determine likely pyrolysis products, but suchstudies are not a substitute for studies using test cigarettes.

6.4.4 Complex mixtures

Of the 599 different compounds in use as added ingredients, many are used invarious combinations as proprietary mixtures in cigarette tobacco (Doull et al., 1994).By far the greatest number of compounds is used as flavorings. Flavorings areadded to cigarette tobacco to enhance its natural flavor, to impart distinctive brandspecific flavors, or to mask the aroma of environmental tobacco smoke (Connolly etal., 2000; Doull et al., 1994). Approximately 75 % of the list of chemicals used asflavor ingredients consists of individual compounds and their analysis is discussedabove. The remaining substances, approximately 25 % of the list, include manyplant extracts commonly used in food. Of these, licorice and cocoa are primaryflavor ingredients added to tobacco. Cocoa powder may account for as much as



5 % of the weight of the tobacco while the total of other flavorings account for onlyabout 0.2 % or less of the weight of most cigarettes sold in the United States. Mostindividual flavor ingredients account for less than 0.05 % (5 x 10-4 by weight) of thetobacco and a third account for less than 0.0001 % (10-6 by weight) (Doull et al.,1994).

Literature describing the components of most of the complex mixtures used astobacco flavors is limited. Since they are botanical substances (oils or extracts), itmight be assumed that they consist of varying amounts of proteins, carbohydrates,and fats (e.g., cocoa, see below). That is, they are composed primarily of the samematerials as the tobacco in cigarettes. Since these mixtures are generally used atlow concentrations relative to tobacco, these classes of components are unlikely tocontribute significantly to the products formed by combustion of the tobacco portionof the cigarette.

This could be shown by a simple calculation indicating the current sensitivity ofavailable analytical methods for the measurement of these classes of componentsand the potential total increase in signal if all of an ingredient were added to thesignal. In theory should the fat from cocoa convert totally to CO and water, adetermination of whether the associated increase in CO would be detectable for theanalytical method being used would support a conclusion that an ingredient would beunlikely to significantly add to cigarette smoke.

The component(s) of the respective mixtures that are most likely to contribute totobacco smoke at detectable levels are those that are unique to the substance inquestion, as they will create a unique signal in the smoke matrix. In most cases,these chemicals will be those that impart a distinctive flavor. For some ingredients,these flavor components have been identified, though their pyrolysis has probablynot been studied (Müller et al., 2000). Studies of complex mixtures can focus on thefate of the respective flavor component.

As mentioned above, cocoa powder and licorice are two complex mixtures used inthe greatest quantities in cigarettes (Doull et al., 1994). Cocoa powder is cited asan example of a complex mixture because it has long been used as a casing forcigarette tobacco as well as to enhance the tobacco flavor (Müller et al., 2000).Cocoa powder is composed primarily of carbohydrates and triglyerides of steric,oleic, and palmitic acids (Schlotzhauer, 1978). Pyrolysis studies of cocoa powderhave demonstrated its degradation to phenolic products and fatty acids anticipatedfrom the respective triglycerides.

Cocoa powder also contains a pharmacologically active compound, theobromine, asa minor constituent, at approximately 2.6 % of added cocoa (Müller et al., 2000).At higher concentrations theobromine acts as a bronchodilator, diuretic, and



cardiostimulant (Simons et al., 1985). However, Müller and coworkers (2000)estimate that theobromine is present at a concentration of approximately 1 mg/cigarette, and of that 1 mg only approximately 13 % is transferred to MS. Thus,they speculate that the resulting 0.13 mg of theobromine delivered per cigarette istoo low to result in plasma levels consistent with detectable pharmacological effects.Even for a complex mixture such as cocoa powder that is used in relatively highconcentrations, the fats and carbohydrates derived from it are likely to contributeminimal amounts when compared to those naturally present in the tobacco. Thiscontention could be supported using a calculation similar to the one mentioned above.

6.5 ADDITIONAL CHEMICAL ANALYSES

Once a substance has been shown to transfer into smoke, change smoke physics,chemistry, or biological activity, or change exposure to whole cigarette smoke,additional chemical studies could provide further relevant information for an ingredientevaluation. In addition to the methods already outlined in this chapter, it may interesta manufacturer to investigate further the pyrolysis pathway of an ingredient to identifythe source and mechanisms underlying these changes. It may be useful to speculateor obtain experimental data about the mechanism of formation and the structure ofpyrolysis breakdown products after combustion.

Pyrolysis and pyrosynthesis of added ingredients in the burning cigarette involvesspecific patterns of bond breaking and formation which depend on thermodynamicconsiderations. Some of these reactions and the associated, overall considerationsare theoretically predictable, for example, using the Rice-Ramsperger-Kassel-Marcus(RRKM) theory. This was discussed in the LSRO Phase One report (2004) andelsewhere (Davis et al., 1999; Kiefer et al., 1997; Schranz & Sewell, 1996). Suchtheoretical prediction of the mechanisms involved in the pyrolysis of added ingredientsmay provide additional relevant information which allow for better understanding ofthe changes observed. In addition, they suggest predictable pathways of pyrolysisand pyrosynthesis.

6.6 SUMMARY

An integral part of LSRO’s scientific criteria is a demonstration of whether: (a) aningredient or pyrolysis product(s) transfers into smoke, and/or (b) an ingredient changesthe chemical composition of cigarette smoke. This chapter presented some of themethods available to address these criteria. These methods are meant as examplesto demonstrate that LSRO’s approach is feasible. Other techniques not mentionedhere, that address LSRO’s scientific criteria, may have equal validity.

An added ingredient that transfers into smoke, will change the composition of smoke.Such an ingredient would not satisfy the first two scientific criteria of this strategy.



LSRO has proposed that the data submitter either: (a) reduce the level of ingredientadded to a cigarette to meet the criteria, or (b) carry out further experimentation todemonstrate the absence of change in relative risk of adverse human health effects.Further analysis to demonstrate no change in the relative risk of adverse healtheffects associated with transfer is outlined here and in the subsequent chapters.

In the absence of transfer, the second criterion of no change in smoke compositionshould also be confirmed. Section 6.3.3 of this chapter identified a few examples ofindicator substances including nicotine and CO, which identify change. Publishedstudies have looked at the variation of measurements obtained for these substancesand others (Baker et al., 2004a; 2004b; Purkis et al., 2003). The range ofmeasurements for these components defines smoke composition. Only ‘tar’, TPM,nicotine, water, and CO have internationally recognized ISO standard methodology(International Organization for Standardization, 1995; 1999; 2000a; 2000b). Consistentuse of these methods between different laboratories results in a relatively narrowrange of values for these substances within and between labs as well as over time(Baker et al., 2004a).

LSRO is not setting restrictions on what components to measure in cigarette smoke.Studies of smoke composition often examine between 40 and 50 different componentsof smoke (Baker et al., 2004a; 2004b; Rustemeier et al., 2002). However, nostandard methods exist for measuring the levels of any other components of smokeapart from those mentioned. These differences in analytical methodology cause awide variation in measurements taken in different laboratories, even with the samecigarette smoked under the same conditions. Few inter-laboratory studies examinevariation of these smoke components (Baker et al., 2004a; Purkis et al., 2003).

LSRO regards any change in smoke either through transfer of a novel substanceinto smoke or through significant change in the level of an indicator compound(s) aspotentially adverse. As described in Chapter 3, potential options for a data submitterare to reduce the level of an ingredient added to a cigarette for compliance withLSRO’s scientific criteria or to shift the emphasis to a demonstration of no changein relative risk of adverse human health effects. Subsequent chapters will providefurther examples of how to demonstrate no change in relative risk once an ingredienthas failed these chemical aspects of the scientific criteria.


77.1 INTRODUCTION

7.1.1 Relationship to other topics7.1.2 Overall approach to exposure7.1.3 Exposure limits

7.2 INITIAL STUDIES7.2.1 Transfer7.2.2 Smoke composition7.2.3 Smoking behavior7.2.4 Biomarkers of exposure

7.3 OTHER CONSIDERATIONS7.3.1 Prior information7.3.2 Dilution effects

7.4 RELATIVE RISK AND ADDITIONAL TESTING

7.5 SUMMARY

EXPOSURE

Exposure � 55


77.1 INTRODUCTION

7.1.1 Relationship to other topics

This report advances three scientific criteria to evaluate ingredients added tocigarettes. (See Chapter 3.) LSRO does not expect that an ingredient meeting allthree of these criteria would change the relative risk of adverse health effects fromsmoking cigarettes. All three of LSRO’s scientific criteria involve exposure.Specifically:

(a) neither the added ingredient nor a pyrolysis product of the addedingredient detectably transfers into smoke in such a way that smokersmight be subject to a different health risk through inhalation of a novelsubstance,

(b) addition of the ingredient does not change the physics, chemistry, orbiological activity of smoke in such a way that smokers are exposed tosignificantly different amounts of substances usually present in smokefrom cigarettes, and

(c) addition of the ingredient does not change exposure to cigarette smokethrough altered human smoking behavior.

An investigation of exposure would begin with the acquisition of data necessary tomeet one or more of LSRO’s scientific criteria. A more detailed exploration ofexposure could follow an inability to meet one or more of these criteria where areduction in the maximum amount of the ingredient added per cigarette would precludeits use in cigarettes. A more in-depth analysis of exposure would be part of an effortto demonstrate that the ingredient did not change the relative risk of adverse healtheffects. This more detailed exploration of exposure probably would follow a moredetailed analysis of smoke chemistry. (See Chapter 6.)

7.1.2 Overall approach to exposure

LSRO reviewed the general subject of exposure to cigarette smoke (Life SciencesResearch Office, 2004). Ott and coworkers (1997; 1998) advanced a frameworkfor exposure analysis, which helps in considering exposure to cigarette smoke. Themechanics of smoking cigarettes dictate a focus on pulmonary exposure. Smokers

EXPOSURE



draw cigarette smoke into their oral cavities and from that site into their lungs.These actions bypass the nasal cavity and concentrate exposure within the pulmonarytree. Thus, the lung and pulmonary passageways are the most relevant exposuretargets (Byrd & Cothern, 2000).

Ott and coworkers (1997; 1998) defined exposure as an event-related quantity of asubstance, such as smoke, or a smoke component, potentially accessible to (ordeposited at) the air/lung interface. Investigators could define an event as theinhalation of smoke over a lifetime, during one day, from one cigarette, or even fromone puff. Smoking, by its very nature, produces intermittent exposures of therespiratory tract to cigarette smoke. However, at longer time scales this jagged orsaw-toothed exposure pattern appears nearly constant. Consequently, investigatorsgenerally express the intensity of exposure as an average rate term, (i.e., the averagenumber of cigarettes or packs of cigarettes smoked per day). This traditional measureof smoking intensity (packs/day) averages a series of inhalation events during a day.To express exposure over longer durations, epidemiologists usually multiply smokingintensity by the duration of exposure in years to yield a quantity called “pack-years.”

For purposes of estimating quantitative inhalation exposure, the relevant contactboundary is the surface of the lung external to the plasma membrane of cells at theair/lung interface. The relevant contact zone is the volume of smoke in the lung.The concentration of smoke at the contact boundary is a measure of exposure. Theconcentration of smoke in the contact zone is a measure of potential exposure. Oneway to define pulmonary exposure is to understand it as the same as a “potentialdose” (Ott & Roberts, 1998; Zartarian et al., 1997). An inhalation dose usually isthe quantity of deposited smoke, or the quantity of a smoke component, absorbed bylung cells, as measured in circulating body fluids, such as the blood.

Figure 7.1 illustrates this approach to exposure. Previously, the amounts of substances(in either gas or particulate form) passing into a space in body constituted “doses.”Investigators might define an amount passing through the mouth into the body as an“oral dose.” This approach requires multiple definitions of “dose” because not all ofthis “oral dose” would be absorbed and not all of the “absorbed dose” would bedelivered to target organs. In this report, LSRO defines the amounts of substancesoutside of the outer membranes as “exposures.” The amounts of substances thatcross the outer membranes of the body are redefined as “doses.” The spirit of Ott’sapproach is that authors should clearly state what they mean by these terms (Ott &Roberts, 1998; Zartarian et al., 1997). LSRO will follow the conventions illustratedin Figure 7.1 and define the contact boundary as the outer membranes of the body.Dose is the mass of the substance that has crossed the outer membranes.

These definitions permit subsequent definition of the “exposure” as the specificconcentration of the substance of interest in contact with the outer membrane andthe “dose” as amount of substance which crosses this boundary. Both exposure

Exposure � 57


Figure 7.1 illustrates an approach to exposure. Exposure to a chemical substance involves contactwith an outer membrane of the human body, such as the skin (dermal), or the internal, epithelialsurface of the lung (inhalation). Exposure and dose are closely linked. In this report, LSRO defineschemical dose as the amount of substance that enters the circulation after crossing an outer membrane.E = exposure, D = dose.

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and dose implicitly require consideration of kinetics, whether time is instantaneous,averaged, or integrated. Thus, dose rate and dose are separate concepts. Whilescientists working with inhalation dosimetry understood each other when they referredconventionally to a dose as the amount inhaled into the lung, problems arose whenthe same material was absorbed in the gastrointestinal tract or given intravenously.The units of exposure could differ, depending on route.

LSRO realizes that many investigators use a different nomenclature, in which the“dose” is the amount of smoke passing the smoker’s lips into the smoker’s mouth.Similar problems exist with pharmaceutical substances, with which “dose” is theweight of an ingested tablet. Theoretically, smokers could exhale all of a smokesubstance they inhaled. Calling the dose, the “delivered dose” or the “absorbeddose,” to circumvent this problem, changes the terminology; it does not change thephysical, chemical, and biological processes.

Because the quantity of a smoke component deposited in the lungs directly relatesto, but is less than, the quantity accessible at the air/lung interface, the calculatedconcentration of a substance in the lung represents an upper bound of actual exposure.The same definitions apply to components of cigarette smoke generated fromcigarettes with or without an added ingredient. Investigators can estimate componentyields from the compositional analyses of smoke generated with smoking machines.While smoking behavior will vary between humans and machines, among machines,and among smokers in a population, point estimates usually will suffice for purposesof exposure estimation.

These definitions are in the spirit of observations by Brodie, Axelrod, and theircollaborators (1955) that blood levels of drugs and their metabolites better predictbiological responses than do exposures. Thus, no difference should exist betweenan intravenous dose and an inhaled dose. Dose also should predict biological response,whereas exposure may require the intervention of pharmacokinetic information topredict response.

7.1.3 Exposure limits

Investigators can calculate component yields from the number of cigarettes smokedper day. Yields of ingredient-related smoke components generated by cigaretteswith and without the added ingredient can be directly compared as measures ofrelative potential exposure. While such yields are precisely valid only for the smokingparameters (e.g., type of tobacco, puffs/cigarette, puff duration, puff volume, andbutt length) used in the mechanical generation of the cigarette smoke, comparativeanalyses under conditions of human smoking are unlikely to yield very differentresults. Both calculations based on the population consumption of cigarettes andcalculations based on human smoking patterns will yield probability distributions.

Exposure � 59


Because human smoking is of interest, behavior is an important variable in exposure.LSRO wants to know that an ingredient is not added simply to increase cigaretteconsumption.

In Appendix D, LSRO presents some hypothetical examples of an ingredienttransferring to smoke or changing the physics, chemistry, or biological activity ofsmoke. Another concept in chemistry is the “detection limit.” (See Appendix G.)Current technology can identify a chemical substance in cigarette smoke in theparts per trillion (ppt) range. Therefore, a transfer or compositional change that ismeasurable and analytically significant may occur, but the concentration of thesubstance may be too low to cause a significant lifetime exposure or to have anadverse human health effect. Assuming a change in the chemical composition ortransfer of a new substance, an important question is whether the level of a substancein smoke is likely to pose a lifetime exposure sufficient to cause an adverse healtheffect. For ingredients that transfer, calculation of potential lifetime exposure maybe the most informative.

In essence, a perspective on a detection limit is a statement about the significance ofthe maximum measured amount of a substance in smoke and its potential for adversehuman health effects. Different ways exist to estimate maximum levels of substanceswhich merit toxicological interest. LSRO describes two potential approaches inAppendix G. One approach is analogous to the U.S. Food and Drug Administration’sthreshold of regulation policy; the other approach links exposure levels to existingregulatory standards for chronic exposure.

7.2 INITIAL STUDIES

7.2.1 Transfer

LSRO recommends that potential data submitters search for new chemical substancesin smoke related to the addition of an ingredient to a cigarette. One way to acquiresuch data is to survey smoke for new substances, present when test cigarettescontain the ingredient, but not when test cigarettes lack the ingredient. (See Chapter6.) This experimental design is capable of repetition, and thus, it intrinsically canundergo statistical analysis and validation. For example, the use of isotopically labeledingredients will facilitate an understanding of the total chemical contribution of theadded ingredient to the smoke.

Validated detection of either an unpyrolyzed ingredient in smoke or a newpyrolysis product, not already occurring in smoke but derived from the added ingredient,will constitute sufficient evidence of potential exposure. Baker and Bishop(2004) suggest that most added ingredients transfer in smoke. Initially, LSRO expectsinvestigators to estimate an upper bound on exposure for these ingredients.



Investigators can assess the transfer of novel substances from the formation ofpyrolysis-related products of an ingredient that transfers into smoke. Current analyticalmethods, such as gas chromatography-mass spectrometry/mass spectrometry (GC-MS/MS), can identify novel chemical substances in smoke and often provide cluesabout the structures of these novel substances. These investigations require a workingknowledge of the products expected from burning the ingredient. Chapter 6, Section6.3.1, explains how investigators can identify potential products of an ingredient bypyrolysis of the ingredient in isolation from a cigarette matrix (Baker et al., 2004a;2004b; Baker & Bishop, 2004; Stotesbury et al., 2000).

Detection of a transferring chemical substance not usually found in cigarette smoke,indicates an ingredient which does not meet LSRO’s first criterion. If the datasubmitter does not want to reduce the amount of added ingredient and retest, theremaining work would require an explanation of the biological significance of thetransferred substance. Investigators might accomplish this task for a DNA adduct-forming, mutagenic, or carcinogenic substance by demonstrating that the maximumplausible risk of lifetime exposure to the substance is trivial. Given a maximumplausible potency of a substance, multiplying this potency by the upper bound ofexposure will yield a maximum plausible risk. A data submitter also could obtainpotencies from the scientific literature, by modeling in comparison to similar chemicalstructures, or by testing the substance in isolation.

No evidence supports the idea that substances in cigarette smoke exert independentactions, such that a mixture of chemical substances produces a predictable group ofadverse human health effects. Instead, chemical substances may interact witheach other, such that the adverse health effects observed are a function of the entiresmoke. Thus, estimation of a maximum plausible risk could overestimate orunderestimate the effect of a chemical substance in the smoke matrix. Estimates ofmaximum plausible risk are rough guides to relative contributions, when viewedfrom this perspective.

For substances thought to elicit only reversible toxicities, an alternative approachused by some regulatory agencies, is to calculate the margin of safety for a substance.This calculation involves dividing the maximum exposure of a lifetime smoker to thesubstance by the lifetime no-effect level of the same substance. Margins of safetyfor a group of chemical substances have the same utility as potencies for a group ofchemical substances. Margins of safety provide rough guides to relative importance,when viewed from the perspective that health effects of cigarette smoking may notdepend on independent action of isolated substances.

If transfer is detected, or if a novel degradation product appears in smoke when theingredient is added to a cigarette, a decision will arise. Smokers of cigarettescontaining the substance probably will inhale the novel substance, which by definition

Exposure � 61


is one not usually detected in smoke. A new, adverse health effect could occur.One option is to reduce the maximum amount of the ingredient per cigarette andretest for transfer, which may not occur at the new, lower level, particularly if thecombustion-related production of the new substance is a nonlinear function ofconditions.


The addition of an ingredient to a test cigarette could potentially change the amounts,or concentrations, of chemical substances already present in smoke. Even if pyrolysisof the ingredient produces substances usually found in cigarette smoke, an addedingredient has the potential to change composition or yield. Changed concentrationsof components could arise as either pyrolysis products of the added ingredient or aschanges induced by the ingredient in the usual combustion products of tobacco.Right now, the adverse human health effects observed in cigarette smokers lackdetailed scientific and mechanistic explanations. Thus, testing must account for thepossibility that a substance in smoke lacks effects itself but modifies the effects ofother substances (Matsuka et al., 1997; Miller, 1990). (See Chapter 6.)

One way to demonstrate that an ingredient does not change smoke composition is tocompare measurements of smoke components between otherwise identicallymanufactured cigarettes, possessing or lacking the ingredient. The application ofexisting analytical methods leads to a “fingerprint” of the smoke, in essence a tableor graph of the concentrations of chemical substances present in the smoke (Bakeret al., 2004a; 2004b; Rustemeier et al., 2002). Unfortunately, comparison of a largenumber of smoke components likely will lead to apparent differences, based onchance alone.

Comparison inherently involves measurement variation, resulting from both thevariation in the experimentally generated smoke and the variation in the measurementtechnology. No single analytical method detects all known smoke componentssimultaneously. In addition, an inherent problem arises from the measurement ofmultiple substances. As a data submitter measures more substances in a singlecomparison, a statistical difference is more likely to arise from random fluctuationsin smoke generation and measurement variation. Since no single component, orcombinations of smoke components, has a proven direct correlation with the directhealth risks of cigarette smoking, LSRO cannot single out a substance, or combinationof substances, for testing. Thus, LSRO does not recommend efforts to detect anychange in the more than 4,800 smoke components. Therefore, LSRO recommendsthe chemical measurement of key smoke substances that are characteristic ofcigarette smoke, easily measured, measured with proven methods, and measuredwith methods having good signal-to-noise characteristics (Life Sciences ResearchOffice, 2004). LSRO will prefer more reliable measurements of fewer substances(Calafat et al., 2004).



Some examples of substances characteristic of smoke are: water, which is alwayspresent; carbon monoxide, which has known toxic properties in isolation; somenitrogen-containing smoke components, such as nitrogen oxides and nicotine, thesubstance most characteristic of tobacco combustion. The analysis of these smokecomponents is standardized (International Organization for Standardization, 1995;1999; 2000a; 2000b). These substances are present in high concentrations (relativeto other known smoke substances) the assays for them are well known, the assayshave good signal-to-noise ratios, and multiple laboratories have conducted replicatetesting.

Unless a prior hypothesis suggests change in one or a few specific substances,measurement of key substances should adequately characterize the smoke as arisingfrom cigarettes. Currently, most manufacturers contemplate measuring a largenumber of analytes. Manufacturers often select analytes for measurement on thebasis of the analyte’s assumed biological activity. The crucial question is whetheraddition of the ingredient changes the overall smoke profile. Statistical inferencewill be relevant, as the question is whether an observed difference arose by chance.Testing multiple cigarettes (and retesting multiple times) will assist with interpretation,when a difference appears to arise by chance. However, LSRO’s suggestionsinclude easily measured, characteristic substances found in greater amounts incigarette smoke.

Instead of measuring a larger number of substances in smoke with chemical testswhen investigating the effects of an added ingredient, LSRO recommends that adata submitter rely on a larger number of kinds of different tests (e.g., physical,chemical, biological, and clinical). Thus, data submitters can compare smokeproperties (e.g., particle size through light scattering or change in concentrationover time with infrared), concentration of chemical substances (e.g., water, carbonmonoxide, nicotine, and nitrogen dioxide), biological potencies (e.g., mutagenicreversion and cell killing), and smoking behaviors (e.g., cigarettes consumed andbiomarker levels).

Two kinds of data lead to inferences about smoke composition: the fate of isotopicatoms in the ingredient and the concentrations of characteristic, unlabeled substancesin smoke. Such data might lead to two fundamentally different circumstances, thatmay occur when smoke composition changes: either all substances in the smoke willchange in parallel or only one (perhaps a few) substance will change concentrationin the smoke.

Investigators also may be able to focus on detection of changes in a few smokesubstances, based on knowledge of the pyrolysis of the ingredient. If the addedingredient induces a change in smoke pyrolysis and pyrosynthesis of many substances,detection will prove more difficult. A “complete” analysis, which compares all of

Exposure � 63


the thousands of substances in smoke, when the ingredient is present or absent, isnot feasible. As data about more substances accumulates, correction for multiplecomparisons becomes more problematic (Pocock et al., 1987). Traditionalapproaches, such as the Bonferroni correction, may not be appropriate. Their useleads to situations where large numbers of uncorrected differences “dilute” thestatistical significance of one change. Instead, investigators may want to comparethe concentrations of individual smoke components generated in the presence andabsence of the ingredient. This approach will generate more opportunities for changesgenerated by the possibility of random chance, but repeated testing can overcomethis deficiency.

If only one substance (or one chemical class of substances) changes in the smokeas a consequence of addition of the ingredient, the data submitter may find existingliterature about the human health consequences of this substance or may developnew information about it. If a change in smoke composition is detected, the datasubmitter ultimately will have to make a decision. One option will be to reduce theconcentration of the ingredient in cigarettes and retest for changed composition. Achange in smoke composition might not occur when less of the ingredient is addedper cigarette, particularly if the relationship between ingredient and compositionchange is nonlinear.

The alternative decision, not to reduce and retest, will arise when a reduction in theamount of the ingredient would eliminate its utility in cigarette manufacturing. Inthis event, the data submitter should include data about the possibility that the changedsmoke might create different risks of adverse human health effects. In this event,an exposure assessment would be a step in producing evidence to test whether ornot the ingredient changes the relative risk of smoking. Pursuit of this hypothesismight end with an exposure assessment. Evidence of changed smoke compositionmight show that maximum exposure to the new substance, which was not found insmoke from cigarettes lacking the ingredient, would only occur in trivial amounts,relative to its known biological effects. An exposure assessment also might be aninitial step in a series of analyses (dosimetry and biological testing, possibly evenpostmarketing evaluation) aimed at demonstrating that the relative risk of adversehealth effects does not change.

If all of the substances in the experimental smoke increase or decrease together, theeffect will be similar to smoking more or fewer cigarettes. (See Section 7.2.3).Investigators could expect compensation in these circumstances. The incidenceand magnitude of the adverse health effects from smoking cigarettes containingingredients, which change the overall amount of smoke, should be proportional tothe change in smoke concentration. Thus, direct testing of human exposure seemsadvisable.



7.2.3 Smoking behavior

LSRO recommends a hypothesis testing study design to test whether human smokershave higher exposures to cigarettes, induced by addition of an ingredient. A minimaldesign would compare the number of cigarettes spontaneously consumed and abiomarker of smoke exposure in smokers consuming otherwise identicallymanufactured cigarettes with and without the ingredient. Such a study will inherentlyinvolve random variation, and thus, the design will need to incorporate an adequatenumber of smokers. [See Appendix H of the Phase One report (2004).] In addition,one smoker is not likely to resemble another with respect to these two study outcomes.For this reason, an adequate study design likely will involve comparison of a smokerto the same smoker, which is a crossover design. (See Chapter 10.) However, sucha design does not allow the subject to choose freely between kinds of cigarettes.Thus, it is not a consumer preference study.

Observing cigarette consumption seems straightforward (Höfer et al., 1992).Counting the number of cigarettes consumed by a smoker seems straightforward,as does correcting for different butt lengths (Hughes et al., 2004). Similarly,metabolites like cotinine are convenient, validated biomarkers of cigarette exposure,and investigators can monitor cotinine levels in urine non-invasively (Benowitz, 1999).LSRO does not contemplate that these two measures of cigarette consumption willnecessarily move in parallel. For example, comparison between a commercial brandof a cigarette and a similarly manufactured cigarette containing less of an ingredientthan usually present, could reveal that a smoker consumes approximately the samenumber of cigarettes but smokes them more intensely, as estimated from a highersmoke biomarker level per cigarette consumed.

If human exposure does not detectably change in response to an ingredient, LSROwill want to examine the data. In most cases, a changed exposure to smokesubstances implies a change in relative risk. However, some alternative explanationsof changed smoking behavior will not necessarily imply a change in relative risk(e.g., removal of an ingredient might create a tobacco which cannot be made into areasonable cigarette). In a double-blinded, crossover study design, investigatorswould instruct subjects to smoke their usual cigarettes. However, during one phaseof the study, these smokers would consume experimental cigarettes which lack ausual ingredient. Investigators would compare the numbers of cigarettesspontaneously consumed by each smoker with and without the ingredient. Duringthe same study, investigators could measure a biomarker of cigarette exposure inthe same smokers. Investigators would compare changes in cigarette consumptionand biomarkers of exposure in each smoker.

Exposure � 65


7.2.4 Biomarkers of exposure

Added ingredients in cigarettes could alter the adverse health effects of cigarettesmoking by introducing an effect directly related to the ingredient or by changing theeffects of cigarette smoke by changing exposure to cigarette smoke. Both of theseeffects can be investigated through studies of general biomarkers of smoke exposure,particularly when comparing the effect of an ingredient on smoke from highly similarcigarettes. These biomarkers contrast with biomarkers of ingredient exposure. Theamount of an added ingredient, or its metabolite, detected in smokers would measureexposure to the ingredient. The amount of a biomarker of exposure, such as cotinine,would indicate whether exposure to smoke changes.

Measurements of biological markers in physiological fluids can link internal doses toexternal exposures. An ingredient, its metabolite, or macromolecule adduct of aningredient detected in an exposed subject (e.g., in blood, urine, or exhaled breath)will indicate dose and may permit a calculation of exposure (Benowitz, 1999). Torelate the level or amount of a biomarker to exposure quantitatively, rates of formationand removal (pharmacokinetic parameters or clearance) must be determined throughindependent measurements (Henderson, 1995).

A British American Tobacco (BAT) report described the excretion rates of mentholfrom humans smoking mentholated cigarettes (Lugton et al., 1978). No mentholwas detected in blood or urine. However, the conjugate, l-menthol-D-glucuronidewas measurable in urine during and four hours after the smoking period. Lugtonand coworkers (1978) calculated that the conjugate, l-menthol-D-glucuronide inurine accounted for 52.6 % of the l-menthol inhaled from mainstream smoke. Thus,dividing the amount of l-menthol-D-glucuronide in urine by 0.526 should show theamount of menthol inhaled. The concentration of menthol in smoke can be estimatedfrom this calculation. However, Lugton and coworkers (1978) also observed alarge variation in menthol metabolism and excretion between smokers in the study.

Benowitz and coworkers (1990) published a similar approach for estimating thedaily intake of nicotine from cotinine measures. The calculation assumes that smokerswith relatively stable smoking habits are at steady state, that is, elimination of cotinineis the same as the daily conversion of nicotine to cotinine. Daily nicotine intake(Dnic) was calculated as the product of average cotinine concentration (Ccot ) andtotal body cotinine clearance (CLcot) divided by the fractional conversion (F) ofnicotine to cotinine.

F

CLCDnic

cotcot ×=



Added ingredients could also affect the delivery of smoke components to the smoker.Vanscheeuwijck and coworkers (2002) looked at the inhalation toxicity of smoke inrats. They compared otherwise identical cigarettes to cigarettes containing threegroups of ingredients with respect to carboxyhemoglobin (COHb) and several nicotinemetabolites (5'-hydroxycotinine, cis-3-hydroxycotinine, trans-3'-hydroxycotinine,norcotinine, cotinine, nicotine, nicotine-N ′-oxide, and nornicotine). Exposure to smokewas held constant. The authors found no significant differences in either COHb ornicotine metabolite levels in male or female rats between test and control cigarettes.

Gaworski and coworkers (1997) conducted a similar inhalation toxicity study of rats,which compared equivalent exposures to smoke from otherwise identical mentholatedand non-mentholated cigarettes. These authors found no significant differences inserum nicotine or cotinine levels after exposure to mentholated cigarette smokecompared to non-mentholated smoke. They did observe a significant reduction inthe COHb levels of rats exposed to mentholated cigarette smoke compared to controls.This decrease was consistent with the observation of reduced chamber CO levelsresulting from mentholated cigarette smoke. When comparing humans smokingmentholated cigarettes to those smoking non-mentholated cigarettes, Ahijevych andcoworkers (1996) observed similar reductions in exhaled CO boost. CO boost isdefined as the change in CO levels before and after smoking.

Some studies of the effects of cigarette mentholation on CO exposure reach differentconclusions. Miller and coworkers (1994) reported an increased CO boost measuredfrom exhaled breath of mentholated cigarette smokers as did Clark and coworkers(1996), once they adjusted the expired CO levels for the amount of cigarettes smoked.The increase could not be attributed to a change in smoking topography. Jarvik andcoworkers (1994) measured both expired CO levels and blood COHb of subjectswho smoked either a mentholated or non-mentholated cigarette. Again, once thelevels were corrected for the cumulative puff volume, a significantly greater boostin both expired CO levels and blood COHb was recorded from the mentholatedcigarette. These reported increases in CO boost and COHb levels contrast with thedecrease observed by Gaworski and coworkers (1997) and Ahijevych and coworkers(1996).

In contrast, Pickworth and coworkers (2002) did not observe a different CO boostamong smokers of mentholated and non-mentholated cigarettes. Changes in COand COHb were also not observed among rats in the study by Vanscheeuwijck andcoworkers (2002) where menthol was included as one of the ingredients. Thedirection of effect of menthol on CO levels, if any, is not clear from these studies.

Exposure � 67


7.3 OTHER CONSIDERATIONS

7.3.1 Prior information

As a data submitter contemplates testing, prior information will strongly influenceboth the interpretation of, and desire for, additional data. In addition to an exposureassessment being an integral part of the scientific criteria, exposure assessment willalso become relevant when the data submitter detects transfer, or a change in oneor a few smoke constituents, but does not want to reduce the maximum amount ofthe ingredient and retest. For review purposes, additional data should persuadeLSRO that the relative risk of adverse health effects does not change after additionof the ingredient to some maximum amount.

At present, LSRO envisions the process of data development beginning with anassessment of relative smoke composition (in the presence and absence of theingredient). Calculations should reveal the maximum human exposure and whetherthis exposure is trivial in amount. If the ingredient, or a unique pyrolysis product ofthe ingredient, transfers in smoke, or if the ingredient induces a change in the chemicalcomposition of smoke, the studies outlined in Section 7.2.1 and Chapter 6 will specifythe chemical substance(s) of interest. Exposure analysis need not focus on allsubstances in the smoke. If the ingredient induces a change in smoke compositionwhich affects only one or a few substances, the studies outlined in Section 7.2.2 willspecify the chemical substance of interest. Exposure analysis can concentrate onexposure to these substances.

Initially, investigators usually will calculate an exposure, using assumed maximumextent of several variables (e.g., transfer and retention). However, additional studiescan refine the initial estimate. Since deposition of particulate matter and gas extractionoperate through different principles in the human lung, and since few substancespartition equally between the particulate and gas phases, studies on machine generatedsmoke will help to define the location of the substance in the aerosol. Humans willbreathe out some portion of the substance of interest, and breath analysis studiescould show the extent of retention. Thus, breath analysis studies may becomerelevant. Studies of the substance, or metabolites of the substance, in blood or urineof human smokers will permit back-calculation of exposure from biomarker levels.Investigators can compare calculated biomarker exposures to calculations of exposurebased on the chemical composition of inhaled smoke through the application ofminimally invasive techniques on existing smokers.

7.3.2 Dilution effects

Adding an ingredient to a cigarette implies that an equal weight of tobacco is removed.Thus, the pyrolysis products of any ingredients intrinsically dilute the pyrolysis productsof tobacco in cigarette smoke. If an ingredient is present in small amounts, relative



to the amount of tobacco, this concern will be trivial. However, larger proportions ofan ingredient will displace significant amounts of tobacco. If the ingredient burns tothe same smoke substances as tobacco, the smoke fingerprint should not change.Asking for no detectable change after introduction of a large proportion of a non-tobacco ingredient is equivalent to expecting that the ingredient will yield the samesmoke substances as tobacco. Interpretation of such data calls for scientific judgment.

Thus, a “thought” experiment is relevant to the interpretation of smoke compositionstudies. Investigators should consider the effect of removing tobacco, as well as theeffect of the ingredient, on smoke generation and the chemical composition of smoke.When a non-tobacco ingredient produces different pyrolysis-related products theproblem of interpretation increases. Similarly, investigators should consider the effectof introducing a large amount of inert gas to the pyrolysis-related processes ofburned tobacco.

7.4 RELATIVE RISK AND ADDITIONAL TESTING

Standard toxicology tests do not predict the human health effects seen in observationalstudies of smokers, and epidemiological studies of smokers of cigarettes with andwithout added ingredients do not appear feasible. As part of a testing strategy, thekinds of tests employed by federal regulatory agencies for safety assessments canbe appropriate. These tests include standard genetic toxicology and intact animalbioassays, as recommended by FDA and as adapted to the inhalation route ofadministration. If the pyrolysis of an ingredient is understood, it may prove meaningfulto test the ingredient or its pyrolysis products in isolation from smoke to compare theexposure-response relationship for the isolated substance to the concentration typicallypresent in smoke.

7.5 SUMMARY

The convention that LSRO adopted for this report is that smoke exposure is theconcentration of a substance inside of the lung (Zartarian et al., 1997). This definitionof pulmonary exposure is not exactly the same as the definition of mainstreamsmoke. An exposure estimate based on an analysis of mainstream smoke, eitherfrom machine-generated smoke or from another source, usually represents anapproximate estimate of maximum potential exposure. As such, these upper boundestimates also represent research opportunities to improve the accuracy of pulmonaryexposure estimates. The influence of humidification of airway linings and otherfactors may make the deposition of cigarette smoke difficult to predict.

Pulmonary dose directly relates to pulmonary exposure, after adjustment forabsorption, distribution, metabolism, and elimination. It is the amount of a substance

Exposure � 69


that passes through the plasma membrane of the epithelial cells lining the air/lunginterface into the general circulation. So, exposures also can be back-calculatedfrom biomarker levels in biological fluids. Intravenous dose closely correlates with,but is not exactly the same as, the pulmonary dose. LSRO essentially has adjustedthe definition of exposure so that little difference exists between an intravenousdose and an inhaled dose.

Exposure estimates are important in evaluating the potential adverse human healtheffects of smoking cigarettes containing an ingredient. LSRO will want to understandwhether the ingredient, or a novel pyrolysis-related product of the ingredient, transfersin smoke into the lungs of smokers. Similarly, understanding whether the ingredientchanges the composition of smoke, either incrementally in the amount of a substanceusually found in cigarette smoke, but derived from the pyrolyzed ingredient, or bythe influence of the ingredient on the combustion of tobacco, is an essential step inevaluating the potential adverse human health effects of smoking cigarettes containingthis ingredient. While assessing the potential exposures of smokers from cigarettescontaining added ingredients has limitations, reasonable estimates of potentialpulmonary exposures of smokers can be obtained. Further, hypothetical estimatesof exposure can be confirmed and refined experimentally.

If transfer occurs, or if smoke composition changes, a data submitter may choose toreduce the amount of the ingredient and retest, until reduction in amount eliminatesany biologically significant change. Alternatively, the data submitter may choose toretain the same amount of ingredient and produce data to show that the ingredient isnot likely to change the relative risk of adverse human health effects from smokingcigarettes containing the ingredient. In this last instance, an exposure estimate likelywill be an initial step in a process, which may include dosimetry and biological testingand which aims to demonstrate unchanged relative risk.


8.1 INTRODUCTION

8.2 DISPOSITION OF NICOTINE8.2.1 Absorption8.2.2 Distribution8.2.3 Metabolism8.2.4 Elimination8.2.5 Detection of chemical substances in biological fluids

8.3 PHARMACOKINETICS OF SELECTED OTHER INGREDIENTS8.3.1 Menthol8.3.2 Rosemary oil

8.4 EXPERIMENTAL CONSIDERATIONS8.4.1 Rodent models8.4.2 Human studies8.4.3 Selection of dose range

8.4.3.1 Repeated dose toxicity studies8.4.3.2 Repeated dose tissue distribution studies

8.4.4 Pharmacokinetic models

8.5 SUMMARY

8KINETICS AND DOSIMETRY OFCHEMICAL SUBSTANCES

Kinetics and Dosimetry of Chemical Substances � 71


8.1 INTRODUCTION

The scientific criteria, described in Chapter 3, involve a determination that: (a) theingredient does not detectably transfer into smoke, (b) the ingredient does not changethe physics, chemistry, or biological activity of smoke, and (c) there is no change inhuman exposure to smoke. For ingredients which fail to meet these criteria, LSROhas proposed that a data submitter reduce the level of an ingredient added to acigarette to result in compliance with the criteria. Alternatively, furtherexperimentation can be carried out to demonstrate that although the scientific criteriawere not met, no change in the relative risk of adverse health effects due to additionof an ingredient would be expected.

This chapter focuses on those ingredients failing to meet the scientific criteria.Published literature and data from the previous studies of the effect of the ingredienton smoke chemistry, biological activity, and human exposure to cigarette smoke willprovide important information and allow for a priority-based investigation of therelative risk of adverse human health effects. In some cases, this prior informationmay highlight the need for a determination of the kinetics and dosimetry of an addedingredient.

Transfer of an added ingredient or a pyrolysis/pyrosynthesis product into themainstream cigarette smoke matrix introduces the possibility of increasing the incidenceof a current adverse health effect associated with cigarette smoke, or causing anew one. (See Chapter 6.) Further testing to evaluate potential toxicity relating tothe inhalation of an added ingredient is important. Added ingredients that do nottransfer and are instead pyrolyzed or undergo complete combustion resulting in nodetectable change in smoke chemistry are not recommended for pharmacokinetictesting as the sensitivity of such tests are unlikely to provide a measurable effect ina human or animal model.

Exposure as it relates to added ingredients has two implications. The ingredientitself may have related toxicity and warrant kinetic study. In addition, the ingredientmay alter exposure to cigarette smoke and therefore change the kinetics of othersmoke components. The implications of a change in exposure, discussed inChapter 7, may indicate a need for kinetic studies. The subsequent integration of

8KINETICS AND DOSIMETRY OFCHEMICAL SUBSTANCES



pharmacokinetics into an evaluation of the relative risk of adverse health effects,involves the measurement of specific parameters that allow the dose of an ingredientor smoke component received by a smoker to be determined. The distribution of anadded ingredient within the human body, the identification of its metabolites, and itsmechanism of biotransformation may provide an indication of the sites andmechanisms causing potential adverse effects.

This chapter outlines how LSRO would approach obtaining pharmacokinetic data todemonstrate that such studies are possible. Reference is made to the publishedInternational Conference on Harmonisation (ICH) guidelines (1994a; 1994b).Application of these pharmacokinetic guidelines to the inhalation of added ingredientsprovides the basis for this chapter. The purpose of this chapter is therefore toprovide a reference for testing which produces useful pharmacokinetic data for anevaluation of ingredients added to cigarettes.

8.2 DISPOSITION OF NICOTINE

Nicotine is an illustrative example of relevant pharmacokinetics. Its selection is notmeant to imply that nicotine is or is not added to cigarettes. Instead, nicotine isrepresentative of substances which transfer in smoke, and data about nicotinepharmacokinetics are available in the scientific literature, reviewed in Benowitzand coworkers (1990). Nicotine is a major component of cigarette smoke, and itprovides a good illustration of the kind of data obtained with currently developedtechniques.

The absorption, distribution, metabolism, and elimination (ADME) of chemicalsubstances over time define their pharmacokinetics (Klaassen, 2001). Thepharmacokinetic behavior of inhaled aerosols may differ from that of ingestedmaterials in important ways. The particulate material contained within an aerosol(for cigarette smoke, the ‘tar’) mostly coats surface lung tissue before absorptioninto the body (Pankow, 2001). With aerosols, transport time to the brain may occurquickly, in a matter of seconds, distribution differs, and “first pass metabolism” throughthe liver does not occur (Klaassen, 2001).

After absorption through the gastrointestinal tract, blood flow takes a chemicalsubstance directly to the liver, but after absorption through the pulmonary tract,blood containing a chemical substance mixes in the heart. After inhalation, thematerial is not passed through the liver before the cycle of distribution to othertissues in contrast with ingested agents (Klaassen, 2001).

Nicotine is a natural liquid alkaloid. It is colorless, alkaline, and is volatilized in thecone of burning tobacco. The free base is present in smoke suspended on droplets(0.3 - 1.0 µm) of ‘tar’ (Russell & Feyerabend, 1978). The effects of inhaled nicotine



are mediated through nicotinic cholinergic receptors (nAChRs) which have beenidentified in the brain, neuromuscular junctions, autonomic ganglia, and adrenal medulla(Gundisch, 2000). Nicotinic cholinergic receptors are pentamers made up fromdifferent combinations of four subunits (α, β, γ, and δ). Specific combinations ofsubunits result in the diverse pharmacodynamic effects of nicotine (McGehee &Role, 1995). Binding to the receptor by nicotine can cause secondary release ofdifferent neurotransmitters including serotonin (appetite suppression and moodmodulation), epinephrine, norepinephrine (arousal and appetite suppression), anddopamine. All have effects which relate to nicotine addiction (Corrigall et al., 1994).

8.2.1 Absorption

Absorption is the process by which substances cross body membranes and enterthe blood (Klaassen, 2001). Both timing and extent of absorption have importantbiological consequences. The concentration of the added ingredient or its metabolitesshould be measured in an appropriate biological fluid (e.g., blood) over a relevantperiod of time and values for the Cmax (maximum concentration reached), Ctime(time at which the maximum concentration is reached), and AUC (area under theconcentration time curve) determined. The values of these pharmacokineticparameters describe the rate and extent of absorption. Calculation of AUC fromblood is important as it describes the dose of a chemical received by a smoker aswell as the residence time of the chemical in the body. Blood levels can be afunction of the amount of chemical distributed in the body.

Inhaled nicotine in smoke is rapidly absorbed (Benowitz et al., 1989). Nicotineabsorbed through the lungs reaches the brain in seven seconds (Russell & Feyerabend,1978). The large surface area, thin endothelial layers, and extensive capillary bed ofthe respiratory system facilitate rapid absorption. The arterial concentration ofnicotine consistently exceeds the venous concentration after smoking (Gourlay &Benowitz, 1997; Henningfield et al., 1990). Significant inter-individual differencesexist in the absorption rate of nicotine (Cholerton et al., 1993). Smoker-controlledfactors exert a great influence on the amount of nicotine absorbed by smokers(Byrd et al., 1998).

The absorption rate of nicotine in buccal and nasal cavities is pH dependent (Cholertonet al., 1993; Pankow et al., 2003). Absorption rate increases as the pH increases(Russell & Feyerabend, 1978). The oral cavity absorbs nicotine more slowly thanthe respiratory tract (Schneider et al., 2001). Nicotine is also absorbed from thegastrointestinal tract, nasal mucosa, and through the skin (Svensson, 1987). In addition,some nicotine is reabsorbed from the urinary bladder (Schievelbein, 1982).

The rate of nicotine absorption reportedly influences self-regulation of nicotine dose(Russell & Feyerabend, 1978). Rapid absorption leads to transiently high levels ofnicotine in the brain which activates, then desensitizes nAChRs (Benowitz et al.,



1989; Lena & Changeux, 1993). A subsequent drop in brain nicotine levels allowstime for re-sensitization of brain nAChRs. This rapid change in brain levels allowsthe smoker to titrate nicotine dose to achieve the desired effect (Benowitz et al.,1989; Benowitz, 1998). The rate of binding to nicotine receptors is, therefore, animportant determinant of physiological effect.

8.2.2 Distribution

After absorption, substances distribute to organs and tissues. Blood flow and diffusionrates control the initial rate of distribution. The apparent volume of distribution isdescribed as the volume of fluid through which a chemical or substance appears tobe distributed, judging from its concentration in the blood or plasma (Klaassen, 2001).It provides a reference for the plasma concentration expected for a given dose andfor the dose required to produce a given concentration. Volume of distribution can,therefore, provide estimates of exposure to an added ingredient, inhaled in cigarettesmoke, for a known dose. This measurement, however, does not provide specificinformation on the pattern of distribution. The pattern reflects physiological factorsand physiochemical properties of the substance or metabolite in question. Eventualdistribution is determined by the affinity of a substance for a particular organ ortissue.

Nicotine is a weak base and is ionized at physiologic pH. It seems to be bound atacidic tissue sites (Waddell & Marlowe, 1976). Nicotine is extensively distributed totarget tissues with a mean steady state volume of distribution reported as 183 Lafter an intravenous infusion into healthy male cigarette smokers (Benowitz et al.,1982a).

Measurements of nicotine concentration in the blood over time (AUC) reflectdistribution to body tissues. A perfusion model simulation of nicotine distributionfrom infusion of 1.5 mg of nicotine into the lung compartment was used to simulatetissue levels of nicotine (Benowitz et al., 1990). Uptake of nicotine into the brainwas rapid, occurring within 1 or 2 minutes of peak arterial concentrations. Bloodlevels fell due to peripheral tissue uptake for 20 - 30 minutes after administration.After this time, blood concentration declined at a slower rate due to distribution tostorage tissues and elimination.

The distribution of added ingredients and any metabolites to specific organs can bedetermined through the use of isotopically labeled compounds. Radioisotope studieshave been conducted. 14C labeled nicotine, and its metabolite, cotinine, wereadministered to mice by many routes including inhalation (Tjalve et al., 1968; Waddell& Marlowe, 1976). By sacrificing the mice at specific time points followingadministration both the pattern and time course of distribution could be determined.Whole body sectioning of exposed mice revealed the distribution of radioactivity ateach time point within organs and tissues. However, unless metabolism is minimal,



the presence of radiolabel does not necessarily indicate the presence of the originalsubstance.

Radioactivity from inhaled 14C labeled nicotine was detected in the bronchi, olfactoryand septal epithelium, as well as the contents of the stomach. In addition,concentrations of nicotine in the salivary gland, Harder’s gland, adrenal medulla,kidney, thymus, brain, bone, spleen, liver, and intestine exceeded the bloodconcentration (Waddell & Marlowe, 1976). Postmortem determination of nicotineand cotinine distribution from a person using an excess of nicotine patches revealedhigh concentrations in the liver and brain. Urine had the highest concentration ofnicotine. The concentration in heart blood exceeded femoral blood (Kemp et al.,1997).

Nicotine readily traverses the placenta. Amniotic fluid concentrations exceed thematernal serum concentration in smoking mothers. The milk/serum concentrationratio averaged 2.92 in a group of smoking mothers (Svensson, 1987).

8.2.3 Metabolism

In-depth analysis of exposure to the metabolites of added ingredients will not benecessary for most ingredients under investigation. ICH guidelines on toxicokineticsprovide general guidance for circumstances where testing added ingredientmetabolites would provide useful data (International Conference on HarmonisationSteering Committee, 1994b). Measurement of metabolite concentrations in blood orurine will provide important information if:

(a) an added ingredient is metabolized to a toxicologically active metabolitethat could contribute to adverse health effects, or

(b) an added ingredient is significantly metabolized so that measurementof the major metabolite is the only practical means of establishingexposure.

As an example, the liver metabolizes most of a dose of nicotine. The primarymetabolite of nicotine, cotinine, forms through a C-oxidation reaction catalyzed bythe enzyme P450A6 (CYP2A6) (Nakajima et al., 1996). Cotinine is biologicallyactive (Benowitz et al., 1983). An estimated 70 - 80 % of nicotine is converted tocotinine in this way before conversion to other metabolites (Benowitz & Jacob, III,1994). So, cotinine meets both of the above ICH criteria. Genetic polymorphismswithin the CYP gene could influence nicotine metabolism reactions. Smoking behaviorsalso may reflect postulated ethnic differences in allele frequencies of the polymorphicCYP genes (Nakajima et al., 2002). Such differences in metabolism of nicotine tocotinine limit the usefulness of cotinine as a biomarker for population exposure tonicotine (Benowitz & Jacob, III, 1994).



Nicotine also undergoes N-oxidation to the polar metabolite nicotine-N ′-oxide. Hepaticflavin-containing monooxygenase form three (FMO3) catalyzes this reaction(Cashman et al., 1992). Smaller amounts of other polar metabolites are also formedfrom nicotine and cotinine. Both nicotine and cotinine undergo glucuronidation (Tricker,2003). Smoking induces O-glucuronidation of 3'-hydroxycotinine, but does not affectthe N-glucuronidation of nicotine or cotinine (Benowitz & Jacob, III, 2000).

8.2.4 Elimination

Elimination is not synonymous with excretion. Elimination of an added ingredientfrom the body follows its removal from the blood, possibly its metabolism, and finallyexcretion from the body. The measurements of pharmacokinetic parameters thatdescribe the time course of exposure are the half-life of elimination (time taken forthe concentration or amount of an added ingredient in the body to decrease by half)and total clearance.

Clearance describes the efficiency of removal of a chemical substance or itsmetabolite from the blood. The half-life of a substance depends on its clearance.Clearance relates to the period of time the body is exposed to the substance (Klaassen,2001). The plasma concentration of a substance at steady state depends on boththe rate and extent of absorption and elimination (half-life and clearance). Multipleexposures within the half-life of elimination will result in accumulation of the substancewithin cells and tissues.

Nicotine has a relatively short plasma elimination half-life, with a mean value of 2.3hours (range 1.6 - 2.8 hrs) (Benowitz & Jacob, III, 1994). About 80 % of nicotineis cleared metabolically. The liver is the main metabolic organ for nicotine. Oxidationto cotinine is the major pathway (Tricker, 2003). The urinary excretion of nicotine,like its absorption, is pH dependent (Benowitz et al., 1990). Normally, the urinaryexcretion of unchanged nicotine is low. Approximately 10 %, as a molar percentageof total recovered nicotine and metabolites, is found unchanged in smokers’ urine(Andersson et al., 1997; Benowitz & Jacob, III, 1994).

In smokers, the plasma elimination half-life of cotinine is approximately 17.5 hours(range 8.1 - 29.3 hrs), and the urinary half-life of cotinine is approximately 15 hours(Benowitz & Jacob, III, 1994). Cotinine is preferred to nicotine as a biologicalmarker of exposure, because of its longer half-life. As a percent of the averagesystemic dose of nicotine in smokers, 37.2 % is excreted as trans-3'-hydroxycotinine,12 % as conjugated cotinine, 11.8 % as cotinine, and 8.0 % as trans-3'-hydroxycotinineconjugate (Jacob, III & Benowitz, 1993).



8.2.5 Detection of chemical substances in biological fluids

Chemical methods have been developed for the detection and quantitation ofsubstances, including nicotine and its metabolites, from small biological samples.Gas-liquid chromatography is often used for the detection of nicotine in plasmasamples (Feyerabend & Russell, 1979; Hengen & Hengen, 1978) as is highperformance liquid chromatography (HPLC) (Kyerematen et al., 1987) andradioimmunoassay which has been modified for high throughput applications (Langoneet al., 1973). Gas chromatography-based methods when combined with massspectrometry (GC-MS and GC-MS/MS) can detect minute quantities of nicotine inblood or urine. However, the high sensitivity of the technique can often cause aproblem with background contamination from nicotine in the atmosphere(Schievelbein, 1982).

Stable isotopic methods for the quantitative analysis of nicotine metabolism anddisposition were developed by Benowitz and coworkers (1991). Capillary gaschromatography allowed separation of compounds within the complex biologicalmatrix. Subsequently, mass spectrometry distinguished labeled and unlabeledsubstances. A method was developed for the quantization of nicotine and cotinine inbiological fluids using deuterium labeled analogues (Benowitz & Jacob, III, 1993).GC-MS with selected ion monitoring (suitable for blood or urine) was applied toquantify concentrations of less than 1 ng/mL in a 1 mL sample.

The analysis of a chemical substance in biological fluids requires accurate andsensitive methods sufficient to measure appropriate concentrations in small samples.Given an isotopic labeled ingredient and knowledge of its mass-spectrometric behavior,it and/or its metabolites can be identified in biological fluids. These techniques havebeen used in the identification of nicotine and its metabolites. Identification ofbiologically active metabolites as well as the site and process of metabolism mayindicate a potential adverse effect linked to an added ingredient.

8.3 PHARMACOKINETICS OF SELECTED OTHERINGREDIENTS

Little work has been published about the pharmacokinetics of added ingredients incigarettes, using inhalation as the route of administration. Several studies assay thetoxicity of smoke from cigarettes with groups of ingredients added to cigarettes.These studies do not contain pharmacokinetic information, but suggest that the additionof the ingredients did not produce adverse biological effects in the test systems(Heck et al., 2002; Vanscheeuwijck et al., 2002). Because of the lack of toxiceffects, absorption was not a concern. Thus, these studies did not determinepharmacokinetic parameters of individual added ingredients that transfer intact intorespiratory tracts of smokers. Pharmacokinetic data available for some of the



ingredients currently used in cigarettes, even by non-inhalation routes of exposure,illustrate the relevance.

8.3.1 Menthol

Menthol is added to flavor cigarettes, to distinguish brands, and to impart aromas(Fisher, 1999; Gutcho, 1972). LSRO’s Phase One report (2004) briefly reviews thementhol literature. Menthol is the most studied of ingredients added to cigarettes(Eccles, 1994; Gaworski et al., 1997; Gelal et al., 1999; Jenkins, Jr. et al., 1970;Lugton et al., 1978). (See Appendix C, Peter Lee presentation.)

Menthol vapor is rapidly absorbed when exposed by the oral or inhalation route(Gaworski et al., 1997; Gelal et al., 1999; Lugton et al., 1978). Menthol ismetabolized through hydroxylation in the liver by microsomal enzymes to form p-menthane-3,8 diol. The enzyme, UDP-glucuronyl transferase, glucuronidateshydroxylated menthol (Eccles, 1994), which is excreted in urine (Haggard &Greenberg, 1941). Conjugated forms in the urine account for 100 % of orallyadministered, radiolabeled l-menthol (Lugton et al., 1978). Menthol induces theliver microsomal enzymes, cytochrome P450 and NADPH-cytochrome C (P450)reductase (Eccles, 1994).

Lugton and coworkers (1978) studied the pharmacokinetics of menthol obtained byvolunteers smoking filtered cigarettes. The cigarettes were manufactured with2.99 mg/l-menthol/cigarette. Subsequent analysis indicated that the l-menthol contentwas 2.83 mg per cigarette at the time of study. Of this amount, 15.6 % (0.442 mg)of menthol was found in mainstream smoke, and 43.0 % (1.217 mg) remained in thecigarette butt. Blood and urine samples were collected from three men and threewomen. A second group of five men had only their urine analyzed. Samples werecollected during an 8-hour smoking period and for 24 hours after smoking cessation.Group one smoked an average of 19 cigarettes, and group two smoked an averageof 21 cigarettes.

Neither l-menthol, nor the conjugate, l-menthyl-D-glucuronic acid, was detected inthe blood of the first group of smokers. Free l-menthol was not detected in the urinewhereas the levels of conjugated menthol in the urine increased rapidly during thesmoking period. Of the total conjugated menthol excreted over the 24-hour testperiod, 80 - 90 % was excreted during the smoking period and for four hours aftercessation (Lugton et al., 1978). Inhaled menthol is therefore rapidly conjugated andexcreted in the urine of smokers. This conclusion is supported by the dispositionkinetics of orally ingested l-menthol (Gelal et al., 1999). Menthol rapidly and totallymetabolizes to the conjugate, menthol glucuronide, as measured in plasma or urine.Menthol glucuronide had a plasma half-life of 56.2 minutes when ingested in capsuleform. The ratio of menthol dose to plasma AUC of menthol glucuronide was calculated(0.23 ± 0.08 L/min) to estimate oral clearance by way of the glucuronide pathway.



8.3.2 Rosemary oil

The 599 list indicates that rosemary oil is an ingredient in some cigarettes (Doull etal., 1994; Life Sciences Research Office, 2004). One of the biologically activecomponents of rosemary oil is 1,8-cineol. Jager and coworkers (1996) exposed foursubjects (two men and two women) to 4 mL of vaporized 1,8-cineol for 20 minuteswithin a closed breathing circuit. They took blood samples during a 60-minute studyperiod after inhalation started. They measured 1,8-cineol in serum by gaschromatography. Like menthol, 1,8-cineol was quickly absorbed into the blood afterinhalation (Jager et al., 1996). The time to reach the peak plasma level averaged15.57 ± 2.34 minutes. However, peak concentrations varied from 459 to 1135 ng/mL. The distributional half-life ranged from 1.98 to 13.1 minutes and the eliminationhalf-life ranged from 31.1 to 281 minutes. Blood concentrations remained elevatedat the end of the one hour study period.

A previous study on a single subject (Stimpfl et al., 1995) suggested a half-life ofelimination of 10 minutes. In this subject 1,8-cineol was still detectable in the bloodafter 120 minutes. Jager and coworkers (1996) suggested that the variation in half-life might reflect variation in body fat, which would also account for prolongedretention. Distribution half-lives also differed with gender. Women eliminated 1,8-cineol more slowly, and women generally have more body fat.

Nasel and coworkers (1994) found that prolonged inhalation of 1,8-cineol increasedcerebral blood flow. If so, 1,8-cineol inhaled in cigarette smoke might increase theeffect of other smoke components acting on the central nervous system (CNS).However, the plasma concentrations reported in these studies seem unlikely to bereached by smoking a cigarette containing rosemary oil. Compared to an exposureof 4 mL of the active component 1,8-cineol used in these experiments, rosemary oilis added to cigarettes at levels below 0.05 % by weight of tobacco (Doull et al.,1998).

8.4 EXPERIMENTAL CONSIDERATIONS

The ICH guidelines on the Assessment of Systemic Exposure in Toxicity Studies(1994a; 1994b) can be used as a basis for experimentation to determine the level ofexposure to an added ingredient in a smoked cigarette through the measurement ofpharmacokinetic parameters.

8.4.1 Rodent models

The disposition of an added ingredient as described by pharmacokinetic parametersmeasured in animal models provides a means to predict pharmacokinetic events inhumans (Gabrielsson & Gumbleton, 1993). To describe systemic exposure in animalsand the relationship to dose, measurements of a substance or a relevant metabolite



in a biological fluid should be taken at appropriate time points. Exposure can berepresented through measurements of plasma (serum or blood) concentration and acalculation of the AUC. Justification should be given for the biological matrix(blood, urine) and the time course in which the ingredient concentration was measured.

Rodent studies can also be used to determine if the added ingredient alters thekinetics of other inhaled smoke constituents (e.g., nicotine or carbon monoxide)(Gaworski et al., 1997; Vanscheeuwijck et al., 2002). A comparison of kineticmeasurements after exposure to cigarette smoke with the ingredient or to cigarettesmoke without the ingredient may indicate an effect of the ingredient on the dispositionof specific smoke components.

The vast majority of these studies are carried out in the rat or mouse. Investigatorsshould give attention to the most relevant species for the study and reasons forspecies selection stated. The number of animals included in each study should allowfor generation of adequate pharmacokinetic data.

8.4.2 Human studies

Human studies provide valuable information. The ability to measure and quantitatechemical substances in a biological fluid allows for a kinetic description of exposurein smokers (Benowitz & Jacob, III, 1984; Benowitz et al., 1990; Jacob, III &Benowitz, 1993). Initial experimentation using rodents might indicate the need forhuman data. For example, a rodent study showing a large variation in the values ofkinetic parameters could suggest genotypic or physiological influences. Theimplication is therefore that humans would also show significantly different kineticvalues on exposure to the same chemical substance. Specific differences in factorssuch as protein binding, tissue uptake, receptor properties, and metabolic profile willaffect the kinetic parameters measured. A study by Jager and coworkers (1996)illustrates how different human physiological conditions, in this case fat stores, affectthe disposition of a substance.

Measurement of the concentration of an added ingredient or a metabolite in a biologicalfluid over time provides a good description of the disposition (Lugton et al., 1978).Urine or blood can be used for the analysis of a substance, although for the purposesof human experimentation, urine measurements require a non-invasive procedurecompared to blood sampling and is, therefore, often preferred.

8.4.3 Selection of dose range

Toxicological findings and pharmacodynamic responses will influence the desirabilityof measurements during initial experimentation with the test species. For example,investigators would want to avoid a species which does not produce a metabolite



found prominently in humans. However, the doses and dosages for kinetic studiesalso merit consideration. The initial experiments probably will begin with singleexposures which relate to the levels of an ingredient added to cigarettes. If aneffect is produced at that exposure, most investigators will want to repeat theexperiment to confirm the findings. If no effect is observed, the exposure can beincreased. Increasing the exposure until an effect is observed will define the activedose range.

Rodent experimental design should consider the exposure and dose in humans relatingto the levels of an added ingredient currently used in cigarette brands, in order toachieve a relevant test exposure in the animal model. Recent studies evaluating thesubchronic inhalation toxicity of certain groups of added ingredients tested the effectsof the added ingredients at levels thought to be representative of current use levels(Vanscheeuwijck et al., 2002). In addition, they also tested higher levels of ingredientsthat were 1.5 or 3.0 times the lower level.

8.4.3.1 Repeated dose toxicity studies

For the study of the pharmacokinetic effect for some added ingredients in cigarettes,a repeat dose regimen would produce more relevant data. Human smokers, thesubjects of interest, are subject to many repeated doses. Added ingredients withlong half-lives would be subject to bioaccumulation, where the interval betweencigarettes is shorter than the half-life of the ingredient. A repeated dose toxicitystudy explores the possibility of a toxic effect associated with a higher dose causedby bioaccumulation of an added ingredient. Employing single dose studies to formulatean appropriate dose interval for an added ingredient may help, as would adetermination of the pharmacokinetic parameters of the AUC and the half-life.

The frequency of administration usually is frequent as necessary, but not so frequentto interfere with the conduct of the study or to cause undue physiological stress tothe subjects. The number of time points needed is usually justified in terms ofadequacy to estimate exposure based on kinetic data.

8.4.3.2 Repeated dose tissue distribution studies

Tissue distribution studies provide information about the distribution and accumulationof a compound, and/or its metabolites, in relation to potential sites of action(International Conference on Harmonisation Steering Committee, 1994a). LSROdoes not envision tissue distribution studies for many ingredients. Initial studieswould highlight situations where distribution data would provide important additionalinformation (e.g., where a species exhibits prolonged retention or biological studiessuggest a toxic effect in a specific organ). Tissue distribution studies can be appliedto study the distribution of an added ingredient repeatedly inhaled in cigarette smoke.



ICH guideline S3B (1994a) also states circumstances to consider performing repeateddose distribution studies:

(a) Single dose studies suggest that the apparent half-life of an addedingredient in organs and tissues significantly exceeds the apparent half-life of the elimination phase in plasma and is more than twice thedosing interval in the toxicity studies.

(b) Steady state levels of the ingredient/metabolite in the circulation,determined from repeated dose PK studies are markedly higher thanthose predicted from single dose kinetic studies.

(c) Histopathological changes occurred that were not predicted from singledose studies. The focus of such studies should be the tissues or organsthat were the site of the lesions.

Repeated dose distribution studies can be accomplished with isotopically labeledcompounds or alternative methods of sufficient sensitivity (Tjalve et al., 1968; Waddell& Marlowe, 1976). As with all pharmacokinetic studies, investigators will want toemploy appropriate doses and durations as gauged from prior experiments.

8.4.4 Pharmacokinetic models

Pharmacokinetic models explain the relationships between blood concentration(sometimes described as bioavailable dose) and exposure (concentrations of aninhaled chemical, such as an added ingredient transferred in cigarette smoke).Investigators can use pharmacokinetic models to estimate the clearance or the organdistribution of an added ingredient or its metabolites. Ultimately, pharmacokineticmodels involve the simultaneous solution of differential equations. However, manysimpler investigations assume a one compartment model for distribution of a substanceand fit data to a first order elimination curve.

Physiological-based pharmacokinetic (PBPK) models differ from standardpharmacokinetic models in their use of anatomically and physiologically correct,independently acquired parameters to describe the kinetics of dosimetry of asubstance. These parameters include compartments (e.g., blood volume) and rates(e.g., velocity of circulation). To these species-specific parameters, PBPK modelsadd chemical-specific data, such as partition coefficients and metabolic rates. Thus,PBPK models allow ready extrapolation between species, by adjusting the species-specific parameters, often by using allometric relationships. In addition to theindependently obtained parameters, most PBPK models go through several roundsof validation by fitting the model to experimental data obtained in different speciesor under different dosing circumstances. When model predictions do not fit thedata, the model is rejected and usually modified, by both adjusting parameters andintroducing new compartments and pathways (i.e., the addition of new equations).



After several rounds of such validation, the models achieve powerful predictivecapabilities.

Four PBPK models exist for nicotine and its major metabolite, cotinine:

(a) Gabrielsson and Bondesson (1987) based one model on a 6-day constantinfusion of nicotine and cotinine into rats to simulate the disposition ofcotinine.

(b) Benowitz and coworkers (1990) based another model on subcutaneousnicotine infusion into rats and intravenous infusion into rabbits.Apparently, this model was not tested against additional experimentaldata.

(c) Robinson and coworkers (1992) based a PBPK model on intravenousinfusion into rats. Although intended to represent the disposition kineticsof nicotine and cotinine in humans, the authors did not include a braincompartment in this model, and the brain is a target organ for nicotineexposure. Also, this model apparently was not tested against additionalexperimental data.

(d) Plowchalk and coworkers (1992) developed a model based on anarterial bolus dose of tritiated nicotine in the Sprague-Dawley rat.Parameters for the model were either taken from relevant literatureor determined experimentally. This model included nicotine metabolismto cotinine. The ability of the model to simulate nicotine plasma kineticswas tested by comparison to experimental data. After an arterialbolus of labeled nicotine into rats, the highest concentrations indescending order were in the kidney, liver, brain, lung, and heart. Theorder of labeled concentrations was consistent with the tissue/plasmapartition coefficients predicted by the model. The decline in nicotineover time was slower in the brain, heart, and lung, indicating saturablebinding in these tissues. The volume of distribution of 3.7 L/kg alsosuggests such binding. This model indicated that 91.5 % of nicotinewas cleared via metabolism, whereas 8.5 % was cleared through thekidney without undergoing metabolism (Plowchalk et al., 1992). Theauthors made no attempt to extrapolate their rat model to humans.

An approach to the modeling of an added ingredient would assume that thepharmacokinetics were similar to that in a PBPK model for nicotine. The model byPlowchalk and coworkers (1992) would appear preferable as the arterial infusionmost closely mimics smoking. However, this model should be converted to a humanmodel. This could be achieved by substituting human blood flow and tissue volumesfor those of the rat. These data have been compiled by Brown and coworkers(1997). Brain and fat would have the greatest variation with both flow and volumebeing larger in humans. Most often the compound specific tissue partition coefficients



for humans are assumed to be similar to that of the rat. Carrying this forward thehuman blood/air coefficient could easily be determined and the others corrected tothe tissue/blood partitioning. The Michaelis-Menton constants and renal eliminationconstant could be optimized or scaled up as needed on the basis of body weight andthe results compared to human data for nicotine.

If this comparison confirms that the model is valid for humans, the model could beconverted to apply to an added ingredient. The partition coefficients could bedetermined and the constants for the added ingredient optimized for the rat. Theadded ingredient’s inhalation dose could be used in the simulation. The conversionfor estimating the human blood or tissue levels would be as previously described.The dose regimen could be modified so as to simulate repeated exposure as occurswith smokers. This would permit the estimation of accumulation and maximumtissue concentrations.

8.5 SUMMARY

Chapter 8 describes a component of a testing framework to provide data relevant tothe potential adverse health effects of an ingredient added to cigarettes. Currentmethods permit the measurement of extremely small amounts of chemical substancesfrom very small volumes of biological materials, including blood and urine. Thereforekinetic measurements, such as metabolites, AUC, and half-lives of elimination, canbe made easily with either human or experimental animal subjects. Dispositionstudies may also indicate biologically relevant doses, potential adverse health effects,and target organs of an added ingredient. Sites of absorption may identify sites ofaction, identification of metabolites may identify a potential risk, and sites ofdistribution may provide sites of action of adverse effects associated with the addedingredient.

Even for substances known to transfer, when pharmacokinetic data are not available,the kinetics of disposition should follow the principles laid out in this chapter andelsewhere (Klaassen, 2001). Thus, investigators might want to apply apharmacokinetic model of a similar substance to the study of an ingredient added tocigarettes or its pyrolysis products.

Potential lifetime exposure to inhaled chemicals can be calculated. Assume a onepack per day smoker, who smokes for 45 years. This hypothetical individual smokesin excess of 3 x 105 cigarettes, each cigarette generating approximately 500 mL ofsmoke in ten, 50 mL puffs. Most of each cigarette burns to smoke and ash (Baker,1999). Each cigarette contains approximately 850 mg of tobacco, which combustionconverts into a dense aerosol of between 109 and 1010 nonaqueous particles per cm3

in a surrounding volume of 350 mg of vapor phase. Most of this vapor is atmosphericgas (90 – 96 % by weight) (Baker, 1999; Pankow, 2001). The smoker’s lifetime



exposure to foreign substances from tobacco combustion exceeds 5 x 104 g or 3 x1016 particles. The proportion of a chemical substance partitioned between the gasand particulate phases of cigarette smoke varies from substance to substance.

Adsorption of the particles onto the surface of the lung generates a high localconcentration of chemical substances in direct contact with lung cells (Martonen,1992; Oberdörster, 1996). Some portion of a substance is absorbed into the generalcirculation. In addition, the smoker absorbs gases through general pulmonarymechanisms, and some of these gases are not common atmospheric gases. Theseprocesses are also substance-specific and will apply to ingredients added to cigarettesand their pyrolysis products, which transfer in cigarette smoke in biologically significantamounts.

LSRO recommends pharmacokinetic studies of substances which transfer in cigarettesmoke in biologically significant amounts. Pharmacokinetic studies would provide adescription of the disposition of a substance and the relevant dose and enable backcalculation of exposure from the biologically-relevant dose. (See Chapter 7.)Pharmacokinetics reflects the transfer of these chemical substances into lung cellsand the general circulation. Fortunately, pharmacokinetic processes are wellunderstood, because of the study of drugs (Abdel-Rahman & Kauffman, 2004;Anderson et al.,1993; Cashman et al., 1996). Inhalation pharmacokinetics are morecomplex than ingestion pharmacokinetics, but this subject also has been reviewed(Schneider et al., 2001; Witek, Jr., 2000), both for gases and particles.

This chapter presents some considerations for designing kinetic studies. Rodentmodels play an important role in kinetic testing. However, relating the findings ofthese models to human situations is important. An interaction between rodent andhuman studies is likely to provide the most useful data. Modeling techniques such asPBPK modeling can also yield useful information although these must be testedagainst actual experimental data.

The need for kinetic data and the extent of exposure assessment inindividual toxicity studies should be based on a flexible step-by-stepapproach and a case-by-case decision-making process to providesufficient information for a risk and safety assessment (InternationalConference on Harmonisation Steering Committee, 1994b).


99.1 INTRODUCTION

9.2 TESTS OF BIOLOGICAL ACTIVITY9.2.1 Cytotoxicity9.2.2 Sensory irritation9.2.3 Genomic effects: mutations and gene expression

9.2.3.1 Bacterial reverse mutation (Salmonella mutagenicity test)9.2.3.1.1 Cigarette smoke mutagenicity9.2.3.1.2 Urine mutagenicity of cigarette smokers

9.2.3.2 Sister chromatid exchange assay9.2.3.3 Mouse lymphoma TK+/- gene mutation assay9.2.3.4 Mammalian erythrocyte micronucleus test

9.2.4 DNA adducts9.2.5 Skin tumorigenicity bioassay9.2.6 Inhalation studies of non-human species9.2.7 Human studies

9.2.7.1 Clinical studies9.2.7.1.1 Human smoking behavior9.2.7.1.2 Biomarkers of exposure

9.2.7.2 Epidemiological studies

9.3 LIMITATIONS AND ISSUES9.3.1 Smoke exposure9.3.2 Interpretation of data9.3.3 Range of endpoints tested9.3.4 Application of new technologies

9.4 SUMMARY

BIOLOGICAL ACTIVITY OF CIGARETTESMOKE

Biological Activity of Cigarette Smoke � 87


9.1 INTRODUCTION

This report describes an integrative approach to assessing the potential for ingredientsadded to cigarettes to change the risk of adverse human health effects from smokingcigarettes. Data submitted to LSRO for evaluation of an added ingredient shouldaim to demonstrate that the added ingredient and its pyrolysis products do notdetectably: (a) transfer into smoke, (b) change the physics, chemistry, or biologicalactivity of cigarette smoke, or (c) alter smokers’ exposures to cigarette smoke bychanging their smoking behavior. If the added ingredient does not meet the threecriteria, stated above, LSRO will interpret this as a signal of possible change in riskof cigarette smoking-related diseases for individuals who consume cigarettescontaining the ingredient. LSRO will suggest a maximum limit for inclusion of theadded ingredient in cigarettes or recommend omission of the ingredient fromcigarettes.

An added ingredient that satisfies all three criteria is considered unlikely to modifythe risk of adverse health effects from smoking cigarettes. This chapter describesapproaches to generating adequate information about the biological effects of smoke,generated from cigarettes with and without specific added ingredients, for reviewby LSRO. The intent of this chapter is to outline the kinds of tests that LSROconsiders instructive for evaluating the effect of the added ingredient(s) on thebiological activity of cigarette smoke.

LSRO’s recommendation of an inclusion level for an added ingredient will derivefrom relevant, published literature, and as made available to LSRO, unpublished,scientific data about the added ingredient. Some information about the biologicalactivity of smoke from cigarettes with and without the added ingredient is desirablefor evaluation of every added ingredient. Prior information about the ingredient willinform the scope of additional testing necessary to generate sufficient data foringredient evaluation. LSRO considers comparisons of the mutagenicity andcytotoxicity of smoke from cigarettes with and without added ingredients to beadvisable for all added ingredients. However, if the added ingredient, or its pyrolysisproducts, transfer into cigarette smoke or confer a significant change in themutagenicity or cytotoxicity of cigarette smoke, further biological testing (e.g., sub-chronic inhalation toxicity studies, dermal tumor promotion assays) and submission

9BIOLOGICAL ACTIVITY OF CIGARETTESMOKE



of additional data to LSRO would be warranted. In such cases, the review ofscientific literature and data about the added ingredient could provide a rationale fortargeted research efforts about the biological activity of cigarette smoke. Alternatively,the data submitter could lower the concentration of the added ingredient to a level atwhich testing detects no significant differences between cigarettes with and withoutadded ingredients.

Epidemiological studies have identified lung cancer, cardiovascular disease, andchronic obstructive pulmonary disease (COPD) as the major diseases associatedwith cigarette smoking. Latencies in the development of these diseases, ethics, andother issues surrounding human testing preclude timely, direct measurements of theadverse human health effects of smoking cigarettes with and without the addedingredient(s). Historically, investigators have conducted biological tests on multiple,diverse, surrogate in vivo and in vitro systems to gain insight about the potentialadverse human health effects of smoking cigarettes. Ideally, tests most useful forevaluating an added ingredient would investigate the influence of the added ingredienton endpoints related to known cigarette smoking associated diseases. LSROapproached the development of scientific criteria for biological testing of addedingredients in cigarettes by identifying previously applied tests that are responsive towhole cigarette smoke or fractions derived from cigarette smoke, and reviewingpublished testing guidelines from organizations for various toxic endpoints of a varietyof substances. (See Chapter 4.)

LSRO’s intent is not to prescribe generic, experimental protocols or preciseexperimental designs for testing the biological effects of added ingredients. Instead,the objective is to describe a spectrum of tests that might prove useful for theevaluation of the effects of an added ingredient on the existing biological activity ofcigarette smoke. A complete or even partial description of all useful tests of biologicalactivity of cigarette smoke falls beyond the scope of this chapter. Therefore, LSROencourages the data submitter to exercise a flexible approach to testing. Testsdescribed in this chapter, considered by the data submitter to be irrelevant forevaluation of the ingredient may very well be omitted. Tests considered by the datasubmitter to be relevant for the evaluation of a specific added ingredient, but are notdescribed in this chapter should be conducted. LSRO will review all available dataabout the biological effects of an added ingredient in cigarettes to evaluate whetherthe ingredient contributes an additional risk of adverse health effects to smokers.



9.2 TESTS OF BIOLOGICAL ACTIVITY

Any test of the biological activity of smoke from cigarettes with and without addedingredients should be conducted with adherence to principles of scientific testing.Tests should exhibit characteristics such as:

• Good signal-to-noise ratio• External validity• Internal validity• Sensitivity• Specificity• Adequate sample size and power to discriminate• Minimization of bias and differential error• Adequate and appropriate controls

9.2.1 Cytotoxicity

The cytotoxicity of smoke from cigarettes with and without an added ingredientmay be compared using an assay such as the neutral red cytotoxicity assay. Otherassays could be used. Cell damage and growth inhibition decrease the uptake andretention of neutral red dye by lysosomes (Bombick et al., 1997a; Borenfreund &Puerner, 1985). Following exposure to whole smoke , Chinese hamster ovary (CHO)or mouse embryo BALB/c 3T3 cells are incubated with neutral red dye for a definedperiod of time and the uptake of dye in viable cells is determined (Bombick et al.,1997b; 1997c; 1998a). Tissue culture fluid is collected and measured spectrophoto-metrically at 540 nm. The cytotoxicity of cigarette smoke condensate can also beendetermined. After cells in wells are exposed to cigarette smoke condensate dissolvedin dimethyl sulfoxide (DMSO) (Roemer et al., 2004), neutral red dye is applied tothe cells. The dye is then removed, a wash/fix solution is applied, and absorbance ismeasured at 540 nm. A negative control for this assay is humidified, HEPA (high-efficiency particulate accumulation) charcoal filtered air.

Bombick and coworkers (1997c) analyzed data by calculating the mean and standarddeviations of absorbances for each concentration of cigarette smoke tested. Theeffective concentration that decreased the neutral red effect by 50 %, relative to thesolvent control, the EC50 value, was ascertained by probit regression analysis.Comparisons of EC50 values of smoke from different cigarettes were then made(Bombick et al., 1997c). As one example, Bombick and coworkers (2001) reportedno significant differences in cytotoxicity of cigarette smoke condensate preparedfrom cigarettes with and without menthol.



9.2.2 Sensory irritation

Sensory irritants decrease the respiratory rates of mice by stimulating trigeminalnerve endings in the respiratory tract mucosa. The Alarie test for sensory irritationprovides a measure of the potency of the respiratory tract irritation that is caused bysmoke from cigarettes (Alarie, 1981). Respiratory rates are measured prior to, during,and following cigarette smoke exposure. From a plot of percentage decrease inrespiratory rate vs. the logarithm of the exposure concentration, the concentrationof smoke that elicits a 50 % decrease in respiratory frequency (RD50) is identified.When concentration response plots are sufficiently parallel, sufficient theoreticalevidence exists to allow RD50 values to be used to compare potencies of a variety ofsubstances to which mice are exposed.

9.2.3 Genomic effects: mutations and gene expression

According to the International Conference of Harmonisation (ICH) Guidelines forGenotoxicity Testing (1997), tests that assess the ability of a chemical to cause genemutations, more extensive chromosomal damage, chromosome recombinations, andalterations in chromosome number may identify potential human carcinogens.Examples of some tests that can be applied to assess the genomic effects of cigarettesmoke are described below.

9.2.3.1 Bacterial reverse mutation (Salmonella mutagenicity test)

The Salmonella mutagenicity assay or Ames test is typically used as a screeningstudy for carcinogenicity. The test determines whether a substance is mutagenic in anumber of Salmonella typhimurium strains. Approximately 54 % of substances thatare carcinogenic in a rat or mouse model induce reversions in Salmonella (Zeiger,1987).

The strains of Salmonella typhimurium used in this assay have a defective genethat prevents them from growing in the absence of histidine. The mutation of asingle gene can revert these “defective” strains to organisms that will grow in theabsence of histidine. Several strains of Salmonella typhimurium are used in thisassay to permit the detection of both point mutations (e.g., strains TA100 and TA1535)and frame-shift mutations (e.g., strains TA98, TA1537, TA1538) (McCann et al.,1975).

A number of chemicals are not mutagenic, but are metabolized to other chemicalswhich are mutagenic. Human cells and Salmonella typhimurium can metabolizechemicals in significantly different ways. To allow for possible differences inmetabolism, activated rat or hamster liver enzymes (S9), which activate mutagensand carcinogens, are added to approximately half of the test plates. In brief, severalstrains of Salmonella typhimurium are exposed to different levels of the testsubstance in the absence of histidine, in the presence or absence of S9. If the



Salmonella grow, this is generally taken as an indication that the test substance ismutagenic in the test system (National Toxicology Program, 2001b).

9.2.3.1.1 Cigarette smoke mutagenicity

The Salmonella mutagenicity assay has been applied to study whole cigarette smoke(Bombick et al., 1997c; 1998a), which includes the particulate and vapor phases, aswell as to cigarette smoke condensate, which includes only the particulate fractionof smoke (Bombick et al., 1997c; 1998b; Chepiga et al., 2000; Roemer et al., 2002;2004; Steele et al., 1995; Theophilus et al., 2003; 2004). Strains of Salmonellaexhibit varying sensitivities to cigarette smoke condensate (De Flora et al., 1996).Avalos and others (2001) report that strains TA98 and TA100, in combination withAroclor 1254-induced male Sprague-Dawley rat-liver S9 fraction of mouse liverhomogenate (Mol-Tox, Annapolis, MD), produce the most sensitive, reproducibleresponses to mainstream cigarette smoke. In addition to Salmonella strains TA98and TA100, strains TA1537, TA97a, TA1538, YG1021, YG1024 (frame-shift mutationstrains) and YG1029 (base substitution strain) are responsive to cigarette smokecondensate (CSC) prepared from Kentucky reference 1R4F cigarettes (Sinclair etal., 1998). The Organisation for Economic Co-operation and Development (OECD)and the International Conference on Harmonisation (ICH) recommend testing witha minimum of 5 Salmonella strains: TA98, TA100, TA102, TA1535, and TA1537 orTA97 or TA97a (International Conference on Harmonisation Steering Committee,1995; Organization for Economic Cooperation and Development, 1997).

DMSO, the solvent for cigarette smoke condensate, is used as a solvent control forassays involving condensate. Positive controls for mutagenicity for Salmonellastrains TA98 and YG1024 with and without S9 activation are 2-aminoanthracene(0.5 µg/plate) and 2-nitrofluorene (1.0 µg/plate), respectively (Smith et al., 2000)and condensate from reference cigarettes. For exposures to whole smoke, smokemay be drawn over humidified, HEPA filtered air and smoke mixed with filtered airmay then be passed over a culture of cells. A negative control for smoke mutagenicityis humidified, HEPA filtered, diluted air (Bombick et al., 1997c). Bombick andothers (2001) reported no significant difference between the number of revertantsobtained from exposure of Salmonella to smoke condensate from cigarettes withand without menthol.

Statistical analyses of data have included calculations of the means and standarddeviations of the number of revertant colonies per plate. Slopes were calculatedfrom linear regression lines constructed from the log of the concentration versusrevertant number. Comparisons of the Ames test responses may be made using a t-test with Bonferroni’s adjustment for multiple comparisons at p = 0.05 (Bombick etal., 1997c).



9.2.3.1.2 Urine mutagenicity of cigarette smokers

Ames test bacteria have also been applied to study the reversion potency of urinefrom smokers and non-smokers (Doolittle et al., 1990; World Health Organization,1986). Smokers’ urine is more mutagenic than non-smokers’ urine (Bartsch et al.,1990; Granella et al., 1996; Yamasaki & Ames, 1977). Strains TA98 and TA1538are described as being most sensitive to smokers’ urine. Strain YG1024 is alsosensitive to urine mutagenicity (Smith et al., 2000). In the method described byYamasaki and Ames (1977), urine samples are activated with S9 liver fraction fromrats and are concentrated with XAD-resin prior to testing. Abstinence of studyparticipants from broiled, charcoal cooked, or pan-fried meats can reduce potentialconfounding from dietary mutagens generated by these cooking processes (Pavanelloet al., 2002; Smith et al., 2000).

9.2.3.2 Sister chromatid exchange assay

The sister chromatid exchange (SCE) assay assesses the potential for chemicalexposure to cause exchanges of arms of sister chromatids undergoing replication.This assay may be conducted in vitro using cultured mammalian cells or in vivo inmammals or non-mammals (U.S. Environmental Protection Agency, 1998b).According to EPA, bone marrow and lymphocytes from mice, rats, and hamstersare used most frequently in the SCE assay. Human lymphocytes are also used.The sister chromatid exchange assay has previously been applied to cigarette smokeand cigarette smoke condensate. The assay can be conducted with or without S9metabolic activation. Tests without metabolic activation are considered to be moresensitive for cigarette smoke substances (Bombick et al., 1995; Doolittle et al.,1990).

For studies without metabolic activation, the thymidine analog, 5-bromo-2'-deoxyuridine is added to cells immediately prior to exposure to whole smoke (Bombicket al., 1997c; 1998a) or after exposure to cigarette smoke condensate (Bombick etal., 1997c; Theophilus et al., 2003). Positive controls for metabolic activation andnon-metabolic activation assays are cyclophosphamide and mitomycin C, respectively(Theophilus et al., 2003). The negative control for smoke exposure is humidifiedHEPA filtered air drawn through the tissue culture at the same rate at which thesmoke is drawn (Bombick et al., 1998a). Colcemid or colchicine, a mitotic spindleinhibitor, is added to halt the cells in metaphase toward the end of the incubationperiod with smoke, cigarette smoke condensate, positive controls, or negative controls.Cells are collected and fixed on slides and stained with fluorescence plus Giemsa(FPG) technique (Goto et al., 1978; Perry & Wolff, 1974).

According to EPA, the number of cells scored per animal is determined by thenumber of animals in the experiment, the frequency of negative control, test sensitivity,and power of the test (U.S. Environmental Protection Agency, 1998b). The number



of SCE for each metaphase and the number of SCE per chromosome should bedetermined. In the approach to data analysis described by Bombick and coworkers(1997c), following a test for transformation for variance stabilization (Healy, 1968),data may be transformed, (e.g., log transformation and square root transformation)and regression lines fitted to data. Slope values can be determined for the varioussmoke exposures. Slopes of regression lines may then be compared with regressionanalyses (p < 0.05) to determine whether cigarettes with and without the ingredienthave significantly different SCE frequencies (Bombick et al., 1997c). Student’st-tests have also been used to analyze data (Theophilus et al., 2003). Considerationsof both biological and statistical significance of data are encouraged (U.S.Environmental Protection Agency, 1998b).

9.2.3.3 Mouse lymphoma TK+/- gene mutation assay

The in vitro mouse lymphoma thymidine kinase gene mutation assay, using theL5178Y+/--3.7.2C mouse lymphoma cell line, is capable of detecting various typesof gene mutations and chromosome aberrations (Auletta et al., 1993; Clive et al.,1979; 1983; Combes et al., 1995; Dearfield et al., 1991; Mitchell et al., 1997; U.S.Food and Drug Administration, 1993). According to FDA (2001), an advantage ofthis assay is its ability to detect multiple types of mutations (e.g., point mutations)and chromosomal events (deletions, translocations, mitotic recombination/geneconversion, and aneuploidy) (Applegate et al., 1990; Hozier et al., 1981; Moore etal., 1985; Sawyer et al., 1985; 1989). While cells with mutations in the thymidinekinase gene proliferate in the presence of the cellular metabolism inhibitor,trifluorothymidine, wild-type cells are unable to proliferate in the presence of thissubstance (U.S. Food and Drug Administration, 2001).

Two approaches may be applied to conducting the assay (Clive et al., 1979; Cole etal., 1986; Turner et al., 1984). Some ICH recommendations for this assay includethat the test be conducted with and without metabolic activation, positive controlsand solvent controls, and a minimum of a single positive test compound dose, if oneexists (Diamond, 1986). Positive controls yielding high mutation frequencies (500 -1000 x 10-6) include hycanthone methanesulfonate (10 µg/mL) and methylmethanesulfonate (MMS 10 - 20 µg/mL). A positive control that will generatemoderate mutagenicity (300 x 10-6) is 2-acetylaminofluorene (2-AAF 50 µg/mL)(Turner et al., 1984). Cytotoxicity and mutation frequency are determined.

ICH accepts other tests of mammalian gene mutation. These include tests examiningthe tk locus of human lymphoblastoid TK6 cells, the hprt locus in CHO, V79, orL5178Y cells and the gpt locus with AS52 cells (Diamond, 1986).



9.2.3.4 Mammalian erythrocyte micronucleus test

The National Toxicology Program (NTP) employs the in vivo mouse micronucleustest as a component of its battery of toxicology tests. This test is used to evaluatethe ability of chemicals to induce changes in the structure and number of chromosomes(National Toxicology Program, 2001a). Inclusion of the micronucleus test is thoughtto improve the characterization of chemical carcinogenicity. Micronuclei form duringcell division when a chromosome or fragment of a chromosome is not incorporatedinto daughter cells and instead forms a separate nucleus. Formation of micronucleicould represent an increased risk of carcinogenesis (National Toxicology Program,2002a). The NTP reports that the majority of human carcinogens are positive inmammalian micronucleus tests.

Micronucleus tests are performed on bone marrow tissue or peripheral blood of ratsor mice. Mammalian erythrocytes are particularly useful as they: 1) do not containa nucleus, 2) are constantly produced to make up for the loss of “worn out” cells and3) may readily be distinguished from erythrocytes which contain a micronucleus.This test may be conducted as a component of the sub-chronic inhalation exposuretests. (See Section 9.2.5.) After exposure to the chemical of interest, blood is takenfrom mice for each dose. Slides are prepared and micronucleated cells are countedunder a microscope (National Toxicology Program, 2002a).

Statistical analysis of data may include a trend test. Data are typically expressed asthe mean number of micronuclei per 1,000 cells for each exposure type. Masseyand others (1998) describe an exposure approach wherein V79 cells derived fromChinese hamster lung cells are exposed to whole cigarette smoke. Using thisapproach, they observed a significant level of formation of micronuclei.

9.2.4 DNA adducts

DNA adducts are not direct evidence of mutation. However, DNA adducts oftencorrelate with biological mutations (Pottenger et al., 2004). DNA adducts aregenerated from covalent reactions between electrophilic molecules and DNA. DNAadduct formation may serve as a precursor to cancer. DNA adducts are “intermediatebiomarkers for evaluating the molecular dose: i.e., the biologically effective dose atthe molecular level” (De Flora et al., 1996).

DNA adducts have been measured in mice which have undergone dermal tumorpromotion studies with cigarette smoke condensate. DNA adduct formation hasalso been measured in Sprague-Dawley rats and B6C3/F1 mice following inhalationexposure to whole cigarette smoke (ECLIPSE Expert Panel, 2000). The Kentucky1R4F reference cigarette has been shown to induce development of DNA adductsin the lungs and heart of mice in a dose and time dependent fashion (Brown et al.,1998). A 32P post-labeling technique may be used to identify and quantify DNAadducts.



There is evidence for considerable variation in the levels of DNA adducts measured incurrent smokers with similar cigarette consumptions (Wiencke et al., 1999). Althoughsome of this difference may be due to differences in cigarette smoking profiles, it islikely that variations in susceptibility to DNA adduct formation are due to differentialmetabolism of the myriad of carcinogenic substances present in cigarette smoke(Caporaso et al., 1991; Degawa et al., 1994; Kato et al., 1995; Shields et al., 1993)and/or individual differences in DNA repair capacities (Wei & Spitz, 1997). Thisshould be factored into any interpretation of studies in which DNA adducts are measured.

9.2.5. Skin tumorigenicity bioassay

The mouse skin painting/dermal tumor promotion assay has been used to studytumor promotion of cigarette smoke condensate applied to mouse skin (Coggins etal., 1982a; Davies et al., 1974; Gori, 1977; 1980; Hoffmann et al., 1983; Roemer &Hackenberg, 1990; Van Duuren & Goldschmidt, 1976). This assay has been usedfor the evaluation of groups of added ingredients (Gaworski et al., 1999) and individualadded ingredients such as cocoa (Gori, 1974; Roemer & Hackenberg, 1990) andhoney (Stavanja et al., 2003). The SENCAR mouse strain, bred for sensitivity toskin tumor development (Slaga & Nesnow, 1985) is often used in this assay. Theability of the assay to predict cancer arising from human cigarette smoking remainscontroversial. However, in the absence of a standard animal model for cigarettesmoke-induced carcinogenesis, the mouse dermal tumor model represents anacceptable approach to evaluating the tumorigenicity of the particulate phase ofcigarette smoke (ECLIPSE Expert Panel, 2000; Gaworski et al., 1999).

In the 30-week dermal tumor promotion assay, tumor formation is initiatedby injecting mice with one dose of a known carcinogen, for example, 7, 12-dimethylbenz[a]anthracene (DMBA) in acetone, or mice are injected withacetone alone (control for tumor initiation) (Meckley et al., 2004; Stavanja et al.,2003; Theophilus et al., 2003). One week later, acetone (negative control for tumorpromotion) or cigarette smoke condensate dissolved in acetone is applied once orseveral times per week to a shaved area of the mouse back for 29 weeks. Apositive control for tumor promotion is 12-O-tetradecanoyl-phorbol-13-acetate (TPA)(Gaworski et al., 1999).

In addition to the general assessment of mouse health (e.g., excretion, respiration,eyes and ear condition, behavior, and mortality, and other parameters), mice arechecked once or several times weekly for development of tumors or “other types oflesions” (Gaworski et al., 1999; ECLIPSE Expert Panel, 2000; Roemer &Hackenberg, 1990). Specific aspects of tumor development monitored include tumorincidence (percentage of animals that developed at least one lung tumor), tumormultiplicity (the mean number of tumors in each lung for tumor-bearing and non-tumor-bearing animals), tumor latency, and malignancy (Gaworski et al., 1999).Body and organ weights and other biological endpoints can be scored.



Means and standard deviation calculations can be performed for various parameters.Chi-squared statistics may be used to analyze mortality and non-tumorous lesionformation. Statistical analyses of the data can include tumor probability (Kaplan &Meier, 1958). The Mantel-Haenszel test (log-rank test) (Peto et al., 1977) may beused to compare tumor incidences. A probability of 0.05 or less for test statistics isinterpreted as indicating a significant difference between tumor promotion ofcondensates.

9.2.6 Inhalation studies of non-human species

A 13-week toxicity study (90-day study) has been conducted to determine the effectsof inhalation exposure of rodents to smoke from cigarettes with different addedingredients (Gaworski et al., 1998; Stavanja et al., 2003; Vanscheeuwijck et al.,2002). Ingredients were added in groups at typical inclusion levels in cigarettes or amultiple of the inclusion level, unless addition at the higher level changed the staticburn rate of the cigarettes (Gaworski et al., 1998; Vanscheeuwijck et al., 2002).Various clinical signs, body and organ weights, clinical chemistry, hematology, generalbehavior, eye condition, food intake, biomarkers of smoke exposure, respiratoryparameters, gross pathology, and histopathology were assessed.

Studies of the effects of inhalation exposure to smoke from cigarettes with andwithout added ingredients have focused on assessing the histopathologic changes inthe rodent respiratory tract, the main locus of effects due to cigarette smoking(Coggins et al., 1982b; Dalbey et al., 1980; Wehner et al., 1981). The OECD(1981) has published guidelines for conducting these studies. Male and female ratswere exposed (nose only) to diluted mainstream cigarette smoke or air (sham-control)for a defined number of hours per day, and days per week for 13 weeks after whichthey were sacrificed. In order to assess whether any effects of added ingredients,beyond smoke exposure effects, were reversible, a subset of rats from differentexposure groups can be sacrificed 42 days after the end of the cigarette smokeexposure period (2002).

Vanscheeuwijck and coworkers (2002) analyzed data by calculating means andstandard deviations of the mean. Comparisons were made between data obtainedfrom reference cigarettes, cigarettes without added ingredients, and cigarettes withdifferent concentrations of the added ingredients. Continuous data were analyzedwith the one-way Analysis of Variance (ANOVA) and a modified t-test (Welch) orpair-wise comparisons (Duncan). Results were considered to be statisticallysignificant at p ≤ 0.05 and there was no adjustment for multiple testing.



9.2.7 Human studies

9.2.7.1 Clinical studies

9.2.7.1.1 Human smoking behavior

Substances in cigarette smoke can elicit various kinds of biological effects on smokers,including changes in smoking behavior. Changes in smoking behavior can altersmokers’ exposures to cigarette smoke constituents (Scherer, 1999). Since a changein exposure to cigarette smoke could alter the risk of adverse health effects ofsmoking, whether an added ingredient modifies the exposure of smokers to cigarettesmoke substances is an important component of the evaluation of an ingredient.

Added ingredients could alter exposure through various mechanisms, such as changingthe number of cigarettes smoked by a smoker per day or the way in which cigarettesare smoked (e.g., increased volume of inhalation or longer puff duration). Since thevolume of inhalation of cigarette smoke and duration of the inspiratory breath-holdaffect cigarette smoke delivery and deposition, these factors presumably wouldhave an effect on overall cigarette smoke toxicity. Approaches to assessing theeffects of added ingredients on human smoking behavior and human exposure tocigarette smoke are discussed in detail in Chapter 10.

9.2.7.1.2 Biomarkers of exposure

The overall magnitude of exposure to cigarette smoke constituents can be evaluatedby measuring biomarkers such as nicotine, cotinine, and cotinine metabolites, suchas trans-3'-hydroxycotinine, cotinine glucuronide, cotinine-N-oxide, norcotinine, andtrans-3'-hydroxycotinine glucuronide. Carbon monoxide and thiocyanate have alsobeen used as biomarkers of cigarette smoke exposure. Chapter 10 describesadvantages and disadvantages of using nicotine, cotinine, carbon monoxide, andthiocyanate as surrogate measures of cigarette smoke exposure.


LSRO does not recommend that the data submitter initiate epidemiological studiesin order to determine the effect of inclusion of added ingredients on risk of theadverse health effects of smoking cigarettes. Epidemiological studies, in whichsubjects would smoke cigarettes with or without added ingredients, for the decadesrequired for cigarette smoking-related diseases to develop, are not feasible. It ispossible that specific populations exist that exclusively smoke cigarettes withoutadditives. A comparison of the adverse health effects from cigarette smoking thatexist in such a population, to the adverse health effects from cigarette smokingexperienced by populations that smoke cigarettes with added ingredients, could provideinsight about the change in risk of adverse health effects that may be conferred byadded ingredients. As new, relevant, epidemiological studies are published by othergroups, they should be incorporated into an added ingredient review. Data arising



from epidemiological investigations of the role of smoking cigarettes with specificadded ingredients on the development on cigarette smoking related adverse healtheffects can be an important component of ingredient evaluation.

Some epidemiological data exist about the change in risk of developing some cigarettesmoking-related diseases conferred by smoking cigarettes with an added ingredient.Multiple epidemiological studies examining the effects of smoking menthol cigaretteson the development of cancer have been published (Brooks et al., 2003; Carpenteret al., 1999; Hebert & Kabat, 1989; Kabat & Hebert 1991; 1994; Muscat et al.,2002; Sidney et al., 1995; Stellman et al., 2003; Tsujimoto et al., 1975). Based onanalysis of four epidemiological studies on mentholation of cigarettes and lung cancer(Brooks et al., 2003; Carpenter et al., 1999; Kabat & Hebert, 1991; Sidney et al.,1995), Lee reports no evidence for an elevated risk of lung cancer due to smokingmentholated cigarettes (Appendix C). However, some caveats exist. These includequestions about the reliability of data about cigarette mentholation, whether aspectsof smoking behavior that may be influenced by menthol (and may differ for regularsmokers of mentholated cigarettes and regular smokers of non-mentholatedcigarettes) were appropriately controlled for, and the effect of cigarette mentholationon smoking cessation (Appendix C). According to Clark and coworkers (2004), andLee (Appendix C) another concern about these data is the short duration of time forwhich smokers smoked menthol cigarettes. Clark and coworkers (2004) describedmenthol cigarette epidemiological studies as yielding “mixed results”.

9.3 LIMITATIONS AND ISSUES

9.3.1 Smoke exposure

LSRO’s goal is to assess the potential for an added ingredient to change the risk ofadverse health effects of human smoking. To this end, cells, tissues, or organismsused in biological tests should be exposed to smoke that mimics, to as great anextent as possible, the smoke that human smokers inhale. Model smoke used inexperiments is generated by automated smoking machines under conditions specifiedby organizations such as the Federal Trade Commission (1967), the InternationalOrganization for Standardization (2000c), and the Massachusetts Department ofPublic Health (2001). Since human smokers change the way that they smoke, evenwhile smoking a single cigarette, standard ISO or FTC protocol machine-generatedsmoke cannot mirror, exactly, the smoke to which smokers are exposed.

One caveat of some guidelines for testing is that they do not address testing of thesubstance of interest within the context of its use. The burning of a cigarette mayresult in the transfer of an added ingredient and/or the pyrolysis and pyrosynthesisproducts of the added ingredient into smoke. LSRO recommends, when possible,testing added ingredients in the smoke matrix in which they are inhaled.



Studies have described exposure to varied forms of cigarette smoke (e.g., wholesmoke, cooled diluted smoke, the vapor (gas) phase of smoke, and cigarette smokecondensate). The World Health Organization (1986) reports that whole cigarettesmoke includes some volatile and semi-volatile compounds not present in cigarettesmoke condensate. Testing with condensate may not reflect the effects of wholesmoke on cellular structure and function.

Human smokers are exposed to a mixture of mainstream (MS), sidestream (SS),and environmental tobacco smoke (ETS). Therefore, exposure to whole mainstreamsmoke, alone, may not reflect the effects of exposure to the mixture of smoke towhich smokers are exposed. LSRO recognizes the challenges inherent in achievingrelevant cigarette smoke exposure.

There are limits as to what standard toxicology tests can reveal about the potentialfor ingredients added to cigarettes to alter the risk of adverse health effects fromsmoking cigarettes. Standard toxicology tests generally involve exposures to highconcentrations of pure substances including the maximum tolerated dose (MTD) ofthe substance. The MTD is the highest dose of a substance that an animal canreceive without showing evidence of acute toxicity (Lave & Ennever, 1990). Highdoses of substances are used in toxicology tests to increase the likelihood of detectingan effect of the substance when tested on a small group of animals (Lave & Ennever,1990). In contrast, ingredients are added to cigarettes at relatively low levels, someat less than 0.0001 % of the weight of the cigarette (Doull et al., 1994). Since theburning of tobacco produces it own toxicity, any further toxicity contributed by lowlevels of added ingredients may prove difficult to detect in standard toxicologicalassays.

According to the World Health Organization, carboxyhemoglobin levels in smokersrange from 3 to 8 % and average 4 % (World Health Organization, 2003). Theaffinity of carbon monoxide (CO) for hemoglobin is 240 times greater than theaffinity of oxygen for hemoglobin (Douglas et al., 1912). CO reduces the oxygencarrying ability of hemoglobin, leading to tissue hypoxia. As described in Clearingthe Smoke (Institute of Medicine, 2001), rats tend to accumulate carboxyhemoglobinmore rapidly than humans at the same level of carbon monoxide exposure (Guerinet al., 1974; Silbaugh & Horvath, 1982) and therefore are less able to withstandexposure to carbon monoxide levels to which humans are exposed (Institute ofMedicine, 2001). For in vivo studies using laboratory animals to investigate healtheffects of cigarette smoke exposure, investigators dilute the cigarette smoke andincorporate breaks from smoke exposure in their experimental design to avoid deathsof animals due to carbon monoxide toxicity (Carmines et al., 2003). It is possiblethat the highest dose of cigarette smoke tested will be determined by the carbonmonoxide in the smoke rather than the other smoke constituents. This should beconsidered when interpreting effects of smoke exposure.



9.3.2 Interpretation of data

Any testing of smoke from added ingredients should be performed with adherenceto the principles of testing, such as internal validity and good signal-to-noise ratio.Data obtained from testing cigarettes will need to be evaluated in terms of itsrelevance, importance, and biological significance. LSRO has not identified anytests that are predictive of human smokers developing cigarette smoking relateddiseases. The relevance and applicability of results from such tests to predictingeffects of smoking cigarettes with and without added ingredients on human morbidityand mortality remain matters for further clarification.

What signifies meaningful difference in smoke biological activity will depend on thespecific test. In tests involving cDNA microarray technology, changes in expressionof specific genes may be used to identify indicator genes affected by smokingcigarettes. A doubling of gene expression is often used as a standard of describingmeaningful change in such assays (De Flora et al., 2003). A decrease in geneexpression (e.g., a minimum of a 50 % decrease), may also be interpreted asmeaningful. Different criteria will apply to other tests.

LSRO will not base a conclusion about the effects of added ingredient on a singletest. The results of a battery of tests will provide the basis for a conclusion. Therole of testing the biological effects of inclusion of added ingredients in cigarettes inthe overall strategy for evaluation of added ingredients is important to consider. Ademonstration of no significant difference in biological effects of smoke fromcigarettes with and without added ingredients will not be considered to be sufficientfor demonstrating no expected change in risk of adverse health effects from smokingcigarettes. An assessment of exposure to smoke and smoke chemistry will contributeto an evaluation of the ingredient. Scientific judgment is an important component ofthe ingredient evaluation process.

9.3.3 Range of endpoints tested

Ideally, a program of testing the biological effects of smoke from cigarettes with andwithout added ingredients would assess endpoints related to all cigarette smoking-related diseases. LSRO has not identified any tests that predict the risks to humansmokers developing cigarette smoking related diseases. LSRO would also like toknow whether the added ingredient led to the development of diseases not previouslyassociated with cigarette smoking. The development of predictive tests to assesscigarette smoking-related diseases, as well as other diseases, would enhance theprocess of ingredient evaluation.

The tests discussed in this chapter are a subset of potentially useful tests. Priorinformation about the ingredient may guide the data submitter to conduct types oftests that are not specifically addressed in this chapter. A potentially useful set of



tests could measure effects of cigarette smoke with and without the added ingredienton immune system function. As described in a review by Gardner (2000) cigarettesmoke over-activates the immune system, elicits autoimmune reactions, andsuppresses the immune system. Such effects of cigarette smoke could result inhypersensitivity reactions (e.g., allergy and autoimmunity), increases in tumors, orinfections of the pulmonary system (Gardner, 2000). A data submitter could chooseto measure the effects of cigarettes with and without the added ingredient on levelsof circulating immunoglobulins or on natural killer (NK) cell activity, since cigarettesmoking has been shown to alter these parameters. Tests that assess immunesystem function are described by NTP (National Toxicology Program, 2002b).

9.3.4 Application of new technologies

As new technologies arise and tests are validated and standardized, the applicationof novel approaches to understanding the biological activity of cigarette smoke couldcontribute to evaluation of an ingredient. Efforts are underway to develop alternatemethods, such as microarrays, and genetically engineered in vitro cell systems fortesting health effects (National Toxicology Program, 2002a). Novel mouse“knockout” or “knockin” strains will be useful for studying processes such ascarcinogenesis. The next decade may see the development of a host of new“biomarkers of effect” which may provide an early indication of potential diseaseprogression. Recently, Spira and coworkers (2004) conducted gene expressionprofiling of human airway epithelial cells obtained at bronchoscopy. They assessedthe effects of cigarette smoking and smoking cessation, and variables such as age,sex, and race, on changes in expression of the “airway transcriptome.”

The National Cancer Institute’s Early Detection Research Network (EDRN) hasimplemented a five stage process for biomarker discovery, validation in distinguishingthe presence or absence of cancer, and testing markers (Reynolds, 2003). TheEDRN outlined new approaches, such as microsatellite instability, genomic, andepigenomic approaches (Reynolds, 2003).

9.4 SUMMARY

This chapter provides some examples of the kinds of tests that a data submittermight conduct to develop adequate data for LSRO to assess the potential for inclusionof added ingredients to change the risk of cigarette smoking related diseases. Thetests described in this chapter are not an immutable set to apply to all added ingredients.Whereas data about the influence of an added ingredient on the mutagenicity andcytotoxicity of cigarette smoke are advisable, any further testing could be guided byprior information about the added ingredient. In selected instances, the data submittermay see value in conducting tests not described in this chapter. However, all testsshould be conducted with adherence to sound scientific principles. Data should be



interpreted in terms of both statistical and biological significance. The refinement ofexisting tests and development of novel testing approaches will enhance the capacityto discern any risk of adverse health effects due to smoking cigarettes containing anadded ingredient. LSRO’s recommendation for a maximum inclusion level of anadded ingredient will derive from scientific judgment of an expert panel based on anaggregate of data about the effects of the added ingredient on smoke composition,the biological activity of smoke, and cigarette smoke exposure.


1010.1 INTRODUCTION

10.2 WAYS IN WHICH ADDED INGREDIENTS MAY AFFECTSMOKING BEHAVIOR10.2.1 Cigarette puffing behavior10.2.2 Frequency of consumption10.2.3 Demographic profiles of human smoking behavior

10.2.3.1 Initiation10.2.3.2 Smoking status10.2.3.3 Pack-years/cigarette-years10.2.3.4 Cessation10.2.3.5 Relapse

10.2.4 Smokers’ consumer behavior10.2.4.1 Purchasing behavior10.2.4.2 Brand selection10.2.4.3 Brand loyalty

10.3 CLINICAL STUDIES OF HUMAN SMOKING BEHAVIOR10.3.1 Proposed clinical study of human smoking behavior

and exposure10.3.2 General considerations for clinical studies of human

smoking behavior

10.4 ADDITIONAL MEASURES OF SMOKING BEHAVIOR ANDEXPOSURE10.4.1 Questionnaires10.4.2 Recording and analysis of cigarette puffing behavior10.4.3 Biomarkers of exposure10.4.4 Chemical analysis of cigarette butts10.4.5 Summary of factors that may be modified by added ingredients

10.5 SUMMARY

HUMAN CIGARETTE SMOKING BEHAVIOR



10.1 INTRODUCTION

This report describes an approach to assess whether the risk of adverse healtheffects from smoking cigarettes with an added ingredient is likely to differ from therisk of adverse health effects of smoking cigarettes without that ingredient. A sponsorwho requests an evaluation of an ingredient by LSRO will be asked to providepublished and unpublished data that show that neither the added ingredient, nor itspyrolysis or pyrosynthesis products detectably: (a) transfers into smoke, (b) changesthe physics, chemistry, or biological activity of that smoke, or (c) changes exposureto cigarette smoke substances by changing smoking behavior. A demonstration ofthe above criteria will be used as the basis for LSRO describing a maximum limit forinclusion of the added ingredient in the cigarette. This chapter concerns thedevelopment of data relevant to the third criterion: the application of behavioral teststo determine whether an added ingredient changes human exposures to cigarettesmoke.

Studies of human smoking behavior of cigarettes with and without added ingredientsare an important component of LSRO’s added ingredient evaluation process. Addedingredients or their pyrolysis products could escape detection of transfer, changes insmoke composition and some biological effects, yet alter human smoking behavior.Furthermore, ingredients are added to cigarettes for purposes including, but notlimited to, decreasing the irritating quality and harshness of cigarette smoke(Hoffmann et al., 2001) and enhancing tobacco flavor and the aroma that emanatesfrom a pack of cigarettes (Fisher, 1999). Cocoa and fruit extracts influence thebasic taste of cigarettes (Tobacco Science Research Chemists, 2003) and mentholimparts a cooling sensation to smokers (Eccles, 1994) which masks the harshnessof cigarette smoke (Perfetti et al., 1993). Inclusion of such ingredients, whichenhance the smoking experience, could lead to changes in smoking behavior, forexample, by contributing to an increase in the number of cigarettes smoked per dayor depth of inhalation of smoke. Epidemiological studies have shown an associationbetween cigarette smoking behavior and morbidity and premature mortality ofsmokers (National Institutes of Health, 1997). Whether ingredients added tocigarettes change exposure to substances in cigarette smoke and ultimately, risks ofmorbidity and mortality from cigarette smoking-related diseases by altering cigarettesmoking behavior is not known.

10HUMAN CIGARETTE SMOKING BEHAVIOR

Human Cigarette Smoking Behavior � 105


This chapter describes ways in which added ingredients may change smokingbehavior, outlines the possibility of a clinical study of the effects of added ingredientson smoking behavior that may be applied to meet the criterion for exposureassessment, and describes other measures of cigarette smoking behavior.

10.2 WAYS IN WHICH ADDED INGREDIENTS MAYAFFECT SMOKING BEHAVIOR

Added ingredients could affect smoking behavior through multiple mechanisms. Onpuffing a cigarette, a smoker inhales an array of cigarette smoke compounds, whichmay bind to central and peripheral neuroreceptors and alter smoking behavior(Benowitz, 2001). The presence of an added ingredient could alter the pharmacologyof substances in cigarette smoke and their physiological effects on the smoker.Organoleptic factors such as smell and taste influence smoking behavior (Huber etal., 1990; Jager et al., 1996; Westman et al., 1996). In addition, smokers may cometo expect pleasurable effects from smoking, for example, improved mood andenhanced concentration (Warburton, 1987). Expectations of such effects may changesmoking behavior, for example, by changing the frequency of smoking.

As described in Chapter 9, studies of the biological effects of exposure to cigarettesmoke have involved the automated delivery of defined amounts of tobacco smoketo animals using smoking machines or smoke delivery devices. Human smokers,however, are known to vary their smoking patterns, for example, in response tochanges in cigarette nicotine yield (Herning et al., 1981; Kozlowski et al., 1982;1988). Regular smokers of the same brand of cigarettes absorb widely varyingamounts of smoke chemicals on smoking that brand of cigarette (Tsujimoto et al.,1975). Smokers’ moods may also contribute to variations in smoking behavior.Smoking machines do not duplicate smokers’ behavior and will not predict ways inwhich added ingredients can affect smoking behavior (Gori & Lynch, 1985; Hoffmannet al., 2001). Because tactile, sensory, and olfactory cues have a large effect on theway people smoke, any study of added ingredients and behavior should includehuman subjects (Huber et al., 1990). Ways in which human smokers may individuallyor as a group alter their smoking behavior in response to the inclusion of addedingredients are described below.

10.2.1 Cigarette puffing behavior

The process of smoking a cigarette has been described as generally occurring intwo stages: a puff of cigarette smoke is taken into the mouth (with no or minimalinhalation into the airways and lungs) sometimes followed by inhalation of the smoke(Huber et al., 1990). Measures of cigarette puffing behavior (also known as puffingtopography) include the number of puffs per cigarette, puff duration, puff volume,puff frequency/puff interval, inhalation frequency, retention time in the lung, depth



of inhalation, flow rate, and total puff volume. Ratios such as puff volume to numberof puffs, puff volume per cigarette, and other measures have also been used todescribe smoking behavior (Bridges et al., 1990).

Depth and duration of inhalation influence the extent of exposure to tobacco smokesubstances (Adams, 1976; Huber et al., 1990) and are regarded as major contributorsto tobacco smoking related diseases (Hammond, 1966; Hammond et al, 1976).Inhalation of cigarette smoke typically increases smoke absorption (Gori et al., 1986;Zacny et al., 1987). Smokers differ in the extent to which they inhale cigarettesmoke and range from non-inhalers to breath holders (Huber et al., 1990). Tobinand Sackner (1982) recorded inhalation volumes of 270 - 1990 mL and breath-holding durations of 2.0 - 10.6 seconds.

Many studies report that smokers alter their puffing patterns on switching fromsmoking high yield cigarettes to smoking low yield cigarettes. More intense smokingof a cigarette occurs when ‘tar’ and nicotine levels are reduced (Hoffmann et al.,2001) apparently in an attempt to ensure delivery of a stable level of nicotine to thebody. Some studies show 100 % compensation for reduced yield cigarettes, whileothers show ranges close to 0 % (Scherer, 1999). Inhalation testing has shown thatsmoking cigarettes lower in ‘tar’ and nicotine result in inhalation of more ‘tar’,nicotine, carbon monoxide (CO), and carcinogens than one might expect from theirreduced presence in the cigarette (Hoffmann et al., 2001).

Determining whether nicotine, CO, hydrogen cyanide, ‘tar’, some other substance,or some combination of the substances in the smoke of cigarettes is compensatedfor is difficult (Pritchard & Robinson, 1996; Scherer, 1999). It has been proposedthat external sensory factors, other behavioral determinants or affective componentsin smoke influence compensation in order to satisfy a need from cigarettes (Huberet al., 1990). Gender differences in compensation have been recorded (Battig etal., 1982). Compensation remains an important, if poorly understood, feature ofsmoking behavior (Adams, 1976).

Changes in cigarette composition can change individual smoking behavior (Stepney,1981) when measured by biomarkers or by puff topography (Herning et al., 1981).Cigarette type affects puff volume, puff duration, frequency of puffs, and the numberof puffs per cigarette (Adams, 1976). Thus, smokers adapt their smoking behaviorto the type of cigarette in order to satisfy a need from cigarettes (Huber et al.,1990). Puff profile frequency, duration, and volume have been described as threeaspects of smoking topography found useful in smoking and behavior studies (Bridgeset al., 1990; Waters et al., 1996).



10.2.2 Frequency of consumption

If an added ingredient increases the appeal of cigarettes, a smoker may increase thefrequency of smoking cigarettes. Frequency of cigarette consumption is an easilymeasured variable (e.g., collect and count butts over a given time period) andinformation about consumption can be readily elicited in questionnaires or in interviews.The number of cigarettes consumed per day is reasonably constant irrespective ofcigarette yield (Battig et al., 1982; Garfinkel, 1979; Wald et al., 1981). However,assessing the number of cigarettes smoked per day, as reflected by the number ofbutts collected, and noting residual cigarette butt weight and length supplementsother estimates of smoking exposure and would serve as a useful tool in studies ofthe influence of added ingredients on smoking behavior.

10.2.3 Demographic profiles of human smoking behavior

Demographic profiles of smoking behavior may include a consideration of age (Biglanet al., 1995), current smoking status (Gritz et al., 1996), smoking cessation (Rennard& Daughton, 2002), and relapse of smoking. Age, gender, and smoker status mayinfluence smoking behavior (Battig et al., 1982; Martonen et al., 2002). Ethnicvariations in behavioral responses to some added ingredients have been proposed(Pickworth et al., 2002).

The roles of external sensory factors (i.e., tactile, oral, and olfactory) in smokingsatisfaction are little understood, though they are known to be important. Likewise,behavioral determinants such as self-image, cognition, and interpersonal actions canaffect smoking behavior (Huber et al., 1990). The role of added ingredients in thismatrix is not well studied.

10.2.3.1 Initiation

Most smokers report initiation of smoking as youths or at the latest, young adults(ages 18 - 22). There is evidence that an individual who has not begun to smoke byhigh school graduation is not likely to initiate smoking at a later age (Gilpin et al.,1994). Initiation of smoking is strongly influenced by tobacco advertising andparticularly, by the distribution of clothing displaying cigarette advertising (U.S.Department of Health and Human Services, 2001). Increases in numbers of smokerswill affect demographic profiles in the future to a considerable degree (Rennard &Daughton, 2002). Because exposure is a function of time, age of smoking initiationis, therefore, an important part of understanding exposure patterns in populations(Byrd & Cothern, 2000).

10.2.3.2 Smoking status

Smoking status refers to whether an individual is a current smoker, a former smoker,or has never smoked. Another category is that of the intermittent smoker, or one



who stops smoking (cessation) and begins again (relapse) in cyclical fashion. Whetheradded ingredients have differential effects on individuals of different smoking statusappears not well studied.

10.2.3.3 Pack-years/cigarette-years

Pack-years (or cigarette-years) is a measure used to quantify a patient’s smokinghistory, and to indicate exposure to cigarette smoke. It is calculated as the numberof packs of cigarettes smoked per day multiplied by the number of years the individualhas smoked. Determination of pack-years depends on retrospective self-reporteddata and does not accurately characterize individual smoking exposure (Bridges etal., 1990). Since measurements of pack-years are entrenched in the clinical andepidemiological literature, it is useful (and simple as a part of other studies) to elicitsuch information. Pack-years provide a rough estimate of exposure. It supportslinkage between clinical histories and experimental studies. To accurately characterizesmoke exposure, investigators can supplement such approximations with other data.

10.2.3.4 Cessation

Smoking cessation leads to health improvements for some diseases (Rennard &Daughton, 2002; Wiencke, 1999). Demographic studies can illuminate factorsinfluencing cessation by focusing on smokers who are trying to quit. Whether anyadded ingredient influences smokers’ ability to quit smoking has not been thoroughlyinvestigated.

10.2.3.5 Relapse

Relapse refers to cessation of smoking followed by the resumption of a smokinghabit. Questionnaires and in-depth interviews can provide information about relapsebehavior. Such studies would be retrospective and self-reported, thus recall biaswould be a significant factor affecting any conclusions developed.

10.2.4 Smokers’ consumer behavior

Smokers’ consumer behavior includes an understanding of purchasing rate, brandselection, and brand loyalty.

10.2.4.1 Purchasing behavior

Consistent with the information that youths and children are sporadic smokers, maturesmokers purchase cigarettes at a higher rate. The price of cigarettes also influencespurchases (U.S. Department of Health and Human Services, 2001). When theprice of a pack of cigarettes increases, the number of young people who are able topurchase it declines. Whether and at what age smokers purchase cigarettes withspecific added ingredients at different rates is not known.



10.2.4.2 Brand selection

Advertising highly influences brand selection, and generally, this process is associatedwith the initiation of smoking. Fowles (2001) proposed that substances affectingnicotine delivery, taste, odor, and membrane irritation are likely to affect selection ofa regular brand of cigarette. Once a brand is selected, there is strong brand loyaltyalthough well conceived advertising campaigns can influence a smoker’s brand choice(Johnston et al., 1999). Knowing whether the presence of an added ingredientinfluences initial or later choice of cigarette brand would yield information about theeffect of inclusion of an added ingredient on the appeal of a cigarette.

10.2.4.3 Brand loyalty

Knowing whether smokers of a particular brand of cigarette are less likely to switchto a different brand of cigarette, for example, one without the added ingredientwould also provide information about the appeal of cigarettes with added ingredients.Sidney and others (1989) report that regular smokers of non-mentholated cigarettesare more likely to switch to mentholated cigarettes than regular smokers ofmentholated cigarettes are likely to switch to non-mentholated cigarettes.

10.3 CLINICAL STUDIES OF HUMAN SMOKINGBEHAVIOR

LSRO thinks that the effect of added ingredients on exposure to cigarette smokeshould be determined. LSRO encourages a flexible approach to generatinginformation about the influence of added ingredients on human smoking behavior.No single methodology is adequate to describe how a change in cigarette compositionchanges smoking behavior and thus, smoke exposure.

10.3.1 Proposed clinical study of human smoking behavior andexposure

A clinical study could assess smoking behavior by counting cigarettes smoked perday and exposure to cigarette smoke substances by measuring biomarkers of cigarettesmoke exposure. Using a crossover study design, study participants would berandomly assigned to two groups. One group would be allotted cigarettes withoutthe added ingredient of interest and the other group allotted cigarettes with theadded ingredient. Participants would be instructed to smoke as frequently as theydesired, but only the cigarettes assigned to them, and to retain all butts of smokedcigarettes. The number of cigarettes smoked daily would be determined for eachstudy participant and biomarker concentration(s) (e.g., cotinine) in blood or urine(which has the advantage of being non-invasive) would be measured. (See Section10.4.3.) Study participants would then be allowed to smoke the other kind of cigarettead libitum for the same amount of time they smoked the first test cigarettes. The



number of cigarettes smoked per day and concentrations of biomarkers would againbe determined. Statistical analysis of data would determine whether there was asignificant difference between the number of cigarettes smoked per day andbiomarkers of cigarette smoke exposure measured for each individual.

10.3.2 General considerations for clinical studies of humansmoking behavior

LSRO has identified considerations generally applicable to studies of human smokingbehavior. Prior knowledge about the added ingredient, (e.g., whether the addedingredient transfers into smoke) the planned parameters in the study, and the expectedvariations in those measurements are important considerations. The degree ofinvasiveness and risk of adverse effects to study participants from acquiring thesemeasurements are of further concern. For these reasons, institutional review board(IRB) oversight will assist the studies. Study participants would provide written,informed consent prior to being allowed to participate.

The voluntary participation and ethical treatment of clinical study participants areimportant attributes of a study. Clear descriptions of the processes of study participantrecruitment, selection (including the rationale for inclusion and exclusion criteria),study participant retention, and the representative subject population arerecommended. The elimination of bias in measurement and confounding (e.g.,blinding and appropriate control measurements) are important objectives. Whenpossible, double-blind experiments should be conducted. The advantages of particularstudy designs (e.g., crossover designs), the statistical power of the experimentaldesign, and techniques to be used for data analysis are additional considerations.Insurance coverage for the study as well as compensation of study participantsshould be considered. The benefits and disadvantages of study participants residingin a testing facility versus having subjects report to the facility at defined times arean additional issue. In addition, LSRO recommends the development of a systemfor the management and statistical analysis of data generated from studies.

10.4 ADDITIONAL MEASURES OF SMOKINGBEHAVIOR AND EXPOSURE

10.4.1 Questionnaires

Questionnaires to elucidate smoking history and patterns of smoking can provideuseful information. Information collected could include number of cigarettes smokedper day, opportunities to smoke (e.g., time and place), brand of cigarettes smoked,number of years of smoking, information about social or environmental stimuli thatassist or encourage smoking, age at initiation of smoking, circadian patterns (i.e.,daily patterns) of smoking, and other information pertinent to the particular studydesign. Such questionnaires provide an opportunity to delve into the psycho-social



and external sensory factors associated with different added ingredients (Huber etal., 1990; Stepney, 1981). Questionnaires may provide data for calculation of pack-years and provide insight into effects of smoking behavior dependent on varyingdurations of exposure (Lando, 1983; Lorillard, 1983).

10.4.2 Recording and analysis of cigarette puffing behavior

Smoking topography can be mechanically recorded using a pneumotachograph withcomputerized recording of various puffing topography variables. This provides anexternal estimate of individual variation in one factor relevant to smoking intensity(Bridges et al., 1990). Some studies have shown a relationship between nicotineabsorption and puffing topography measures. Herning and others (1983) showcorrelations with nicotine absorption and number of puffs, R = 0.28; mean puffvolume, R = 0.37; and mean puff duration, R = -0.58. Bridges and coworkers(1990) studied the ability of puffing topography parameters to predict smoke exposure.The investigators determined the relationships between mean number of puffs/cigarette, interpuff interval, duration and volume/puff, total puff duration/cigarette,and puff volume/cigarette on blood levels of nicotine, cotinine, carboxyhemoglobin,and thiocyanate for men smoking their regular cigarettes ad libitum.

There were consistent variations in puffing topography measures in the population.Of these variables, measured in 108 subjects, interpuff interval covaried withbiomarkers of cigarette smoke exposure (Pearson’s R = -0.296, p < 0.01), meanvolume/puff and mean puff volume/cigarette (R = -0.190 and -0.222, p < 0.05,respectively) also showed covariation. When data analysis was restricted to smokersof the same cigarette brand (n = 31), plasma nicotine significantly associated withinterpuff interval (R = -0.438, p < 0.05), total puff duration/cigarette (R = 0.400, p <0.05), and total puff volume/cigarette (R = 0.424, p < 0.05). Statistically significantcorrelation was observed between plasma cotinine and interpuff interval,(R = -0.398, p < 0.05). Correlations between plasma cotinine and total puff duration/cigarette (R = 0.320, p < 0.1) and plasma cotinine and total puff volume/cigarette(R = 0.316, p < 0.1) approached statistical significance. Mean number of puffs/cigarette approached significance (p < 0.1). Correlations of serum thiocyanate andcarboxyhemoglobin with puffing topography measures did not approach significance.Bridges and coworkers (1990) propose that variations in baseline measurements ofsmoke exposure measures, inhalation patterns, metabolism, and clearance ofcompounds may contribute to lack of correlation between exposure measures andpuffing topography measures.

The tendency of subjects to change puffing behavior when aware of being monitoredcomplicates studies of cigarette smoking behavior in clinical settings. Smokersincrease number of puffs per cigarette approximately 20 %, when they know thatthey are being observed (Comer & Creighton, 1978; Schulz & Seehofer, 1978).



Individuals maintain consistent smoking patterns when smoking cigarettes of thesame type (Bentrovato et al., 1995).

Höfer and others (1992) studied the roles of cigarette yield, cigarette consumption,sex, and inhalation and puffing behavior on the ability to predict smoke exposure asmeasured by plasma cotinine and nicotine, and respiratory CO. They sampled plasmanicotine and cotinine and respiratory CO at various times post-smoking and undervarying smoking conditions. These studies indicated that cigarette yield, smoker-described inhalation, cigarettes smoked/day, and cigarettes smoked on theexperimental day better predicted plasma nicotine and cotinine absorption than COin expired air. However, Höfer and coworkers (1992) found that cigaretteconsumption, nicotine yield, gender, inhalation, and puffing topography measures didnot account for all of the variation in smoke exposure measures. Therefore,biomarkers, such as nicotine, cotinine, and CO are important indicators of changesin exposure.

10.4.3 Biomarkers of exposure

The adverse health outcomes associated with cigarette smoking have long latencies;biomarkers provide information about recent exposure to cigarette smoke. Levelsof nicotine, cotinine, or thiocyanate in serum, plasma, urine or saliva, as well as bloodcarboxyhemoglobin, and expired air CO can reflect exposure of individuals tocigarette smoke (Huber et al., 1990; Jarvis et al., 1987; U.S. Department of Healthand Human Services, 2001). Women are likely to smoke fewer cigarettes per daythan men, and generally smoke their first cigarette of the day later in the day than domale smokers (Royce et al., 1997). Genetics also influences smoking behavior(Koopmans et al., 1999). Because of this, information on the actual individual bodyabsorption of smoke chemicals and their metabolites is necessary. Affective conditionssuch as major clinical depression also contribute to smoking behavior (Glassman etal., 1990). For now, consideration of affective conditions would complicate researchwithout greatly improving the applicability of the findings.

The array of chemicals in smoke is enormous, and it is impractical to measure all ofthem. Particular added ingredients may present novel chemicals in smoke and newtests for absorption of these chemicals may eventually be necessary to fully assesstheir effects on smoking behavior (Eccles, 1994; Hoffmann et al., 2001) . Somebiomarkers of exposure to tobacco smoke have been used in various studies.Examples are given below.

Nicotine is minimally absorbed through mucous membranes in the oral cavity, thenasal cavities, and to a greater extent, through bronchial and pulmonary surfaces(Gori et al., 1986). The half-life of nicotine ranges from 1 to 4 hours and averages2 hours (Benowitz et al., 1982a). Intra-subject nicotine levels vary widely over aday and primarily reflect exposure from cigarettes smoked close to the time of



sampling of the body fluid (Benowitz et al., 1982b; Galeazzi et al., 1985). Nicotineconcentration may serve as a better marker of acute exposure to cigarette smokethan of long term exposure to cigarette smoke.

Cotinine, the primary metabolite of nicotine, has a half-life of 16 - 20 hours (average18 hours) (Benowitz et al., 1982b) and is maintained at steady levels throughout asmoking day. Though individual variations in nicotine metabolism and cigarette typecan both influence nicotine and cotinine concentrations, cotinine provides ameasurement for understanding the nicotine dose (Secker-Walker et al., 1997).Nicotine and cotinine may be measured in serum, saliva, and urine and serve asspecific biomarkers of tobacco smoke exposure. Measurement of other nicotineurinary metabolites (e.g., trans-3'-hydroxycotinine, cotinine-N-glucuronide andothers) in addition to nicotine and cotinine may provide additional information aboutindividual nicotine uptake (Byrd et al., 1994).

Carbon monoxide is absorbed through the lungs, and thus reflects the depth andduration of smoke inhalation (Bentrovato et al., 1995). CO attaches firmly to redblood cells, so measuring levels of carboxyhemoglobin in the blood estimates COabsorption. The half-life of CO ranges from 2 to 5 hours and averages 4 hours(Benowitz, 1983). Because CO levels build up during the day, sampling at the endof the smoking day is required to assess daily exposure to cigarette smoke. CO inexhaled air may also serve as a useful biomarker of smoke exposure. CO is notspecific for tobacco smoke, it is also found in automobile emissions. Gender, level ofphysical activity, time since last cigarette smoked and individual rates of absorptionand elimination can influence CO concentration (Benowitz, 1983; Shields, 2002).

Other biomarkers may also be useful in particular situations, (e.g., ‘tar’ andthiocyanate). ‘Tar’ absorption might predict cancer, thus ‘tar’ has public healthimplications (Yamasaki & Ames, 1977). Urine collected from regular smokers oflow ‘tar’ cigarettes and smokers who switch to low ‘tar’ cigarettes for an experimentcan be subjected to standardized microbial assays to evaluate the influence of ‘tar’level on urine mutagenicity. Factors such as basal metabolic rate, the activity levelof the enzymes for mutagen metabolism, and mutagen inactivation affect the abilityof urinary mutagenicity to reflect ‘tar’ intake (Kriebel et al., 1985).

Thiocyanate, the metabolic product of hydrogen cyanide, has a half-life ofapproximately 14 days. Time of sampling affects measurement of thiocyanateminimally (Benowitz, 1983). Thiocyanate measured in serum or saliva can providean indication of exposure to tobacco smoke in the weeks preceding any experiments,thus checking on an individual’s recall bias. Thiocyanate is not a specific marker oftobacco smoke exposure. Cyanides in beer and many foods contribute to thiocyanatelevels in the body (Bottoms et al., 1982). Although thiocyanate may allow distinctionbetween smokers and non-smokers, its usefulness as a quantitative measure ofcigarette smoke exposure within a shorter time frame is limited (Benowitz, 1983)



10.4.4 Chemical analysis of cigarette butts

The physical relics of smoking, cigarette butts, consisting of the filter and remainderof the tobacco, provide further insight into a smoker’s exposure to chemicals.Investigators can determine the length of the remaining tobacco rod and chemicallyanalyze the filter portion of the tobacco for collected/remaining ‘tar’ and nicotine.Chemical analysis of butts of cigarettes with added ingredients can provide a physicalmeasure for validating smoker recall on questionnaire studies.

10.4.5 Summary of factors that may be modified by addedingredients

Added ingredients in cigarettes may increase smokers’ exposure to smoke substancesby altering the number of cigarettes smoked (measured by counting cigarette butts)or through changing cigarette puffing behavior, which increase exposure to smokesubstances, as determined by measuring biomarkers. According to several studies,smokers do not significantly alter the number of cigarettes when smoking cigaretteswith different yields of nicotine (Adams, 1976; Battig et al., 1982; Creighton &Lewis, 1978). It is not known whether the total number of cigarettes smoked bysmokers will change after the addition of non-tobacco ingredients to cigarettes. Ifthe added ingredient affects cigarette yield, differences in puff profile may be observed(Adams, 1976; Herning et al. 1981; Huber et al., 1990). Such changes may bereflected as altered levels of biomarkers. Smokers will adjust puffing behavior todeliver what they desire in nicotine, CO, or other chemical or sensory factors. Addedingredients that restrict nicotine availability may result in behavior that increasessmokers’ exposure to CO and ‘tar’ without appreciably decreasing their exposureto nicotine (Herning et al., 1981).

Questionnaires may elicit information about affective components such as mood,sensory satisfaction, and level of pleasure in the smoke (Huber et al., 1990). Inaddition, information about smoking opportunities may provide useful information,since a change in the number of cigarettes smoked may not be due to the presenceof an added ingredient, but a change in a smoker’s opportunity to smoke. Biomarkersshould not be significantly affected, if reports about smokers changing behavior toachieve a desired “dose” of nicotine are true. However, if smokers compensate forother substances, the inclusion of objective biomarkers in study protocols may provenecessary.

10.5 SUMMARY

Information about whether an added ingredient elicits a detectable change in humansmoke exposure may be ascertained through studies of human smoking behavior.Conceivably, added ingredients could change exposure to smoke substances viavarious mechanisms, including changes in the number of cigarettes smoked per day,



the way in which the cigarette is smoked, initiation or cessation of cigarette smoking,and absorption of smoke chemicals. LSRO has described an approach to studyingthe effects of inclusion of added ingredients in cigarettes on human exposure tocigarette smoke by counting the cigarettes smoked and measuring levels of exposure.Such information could provide a baseline for understanding the effect of an addedingredient on human smoking behavior separate from indications of consumerpreference. This information is critical to determine any change in risk of adversehealth effects of smoking cigarettes conferred by an added ingredient by increasingexposure to tobacco smoke substances. Additional approaches to studying humansmoking behavior might provide further insights about exposure to smoke substanceswhen individuals smoke cigarettes with added ingredients.


1111.1 INTRODUCTION

11.1.1 Rationale11.1.2 Experimental process

11.2 ASSEMBLING EXISTING DATA

11.3 SELECTION OF TEST METHODS11.3.1 Chemical fate of the added ingredient in cigarette smoke

11.3.1.1 Ingredient transfer into smoke11.3.1.2 Smoke composition

11.3.2 Exposure and dosimetry11.3.2.1 Exposure11.3.2.2 Human smoking behavior11.3.2.3 Kinetics

11.3.3 Biological effects

11.4 EXPERT EVALUATION11.4.1 Detectable change11.4.2 Relative risk11.4.3 Potential Conclusions

11.4.3.1 Inadequate data11.4.3.2 Maximum level of ingredient11.4.3.3 Ingredient not recommended

11.5 SUMMARY

THE EVALUATION OF INGREDIENTSADDED TO CIGARETTES

The Evaluation of Ingredients Added to Cigarettes � 117


11.1 INTRODUCTION

Ingredients added to cigarettes could adversely affect the mortality and morbidity ofsmokers by different mechanisms. The previous chapters have presented acomprehensive program to test added ingredients for their potential adverse effecton human health. The uniform application of all tests to all ingredients is not aworthwhile exercise for either scientific purposes or the public health. This chapterreviews the development of test data, typically by a manufacturer using addedingredients. It outlines the evaluation process and relevant information for inclusionto the package of data submitted to LSRO for review.

11.1.1 Rationale

The relative risk of adverse health effects from the addition of an ingredient can beevaluated through comparison of cigarettes containing the ingredient to identicallymanufactured cigarettes lacking the ingredient. The net change, if any, reveals theeffects of the ingredient within a cigarette smoke matrix. The desired goal is that inclusionof the added ingredient does not change the relative risk of adverse health effects.

Where the results from balanced and diverse tests are not able to identify significantdifferences between cigarettes with and without the ingredient, LSRO does notexpect the risk of existing adverse health effects to change. Three scientific criteriawere developed that would, in LSRO’s view, provide a demonstration that thepresence of an added ingredient does not change the relative risk of adverse humanhealth effects. These criteria are not meant to impose inflexibility on the process,but to stimulate innovation. Data submitted for evaluation should aim to demonstratethat, in the presence of the added ingredient:

(a) neither the added ingredient nor a pyrolysis product of the addedingredient detectably transfers into smoke in such a way that smokersare subject to a change in adverse health effects,

(b) addition of the ingredient does not change the physics, chemistry, orbiological activity of smoke significantly, and

(c) addition of the ingredient does not change exposure to cigarette smokethrough altered human smoking behavior.

11THE EVALUATION OF INGREDIENTSADDED TO CIGARETTES



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However, should the data presented for an evaluation not comply with one or moreof the three scientific criteria, a demonstration of no change in relative risk of adversehuman health effects becomes a more involved process. Two options have beenproposed in this report to address this outcome: (a) reduce the maximum amount ofthe ingredient added per cigarette and retest in relation to these three criteria or (b)directly demonstrate that relative risk of adverse human health effects does notchange through more extensive biological, chemical, and/or exposure (dosimetry)testing. Although LSRO has not provided specific guidelines as to how to achievethis goal, it is suggested that additional standard and more extensive biological testswould provide additional information.

11.1.2 Experimental process

Should an added ingredient not meet the scientific criteria described, LSRO has nopreconceived test regime that would demonstrate an absence of change in the relativerisk of adverse human health effects. However, the previous chapters in this reportoutlined an approach for demonstrating an unchanged relative risk of adverse healtheffects, if only to show that such testing is possible.

Figure 11.1 A packet of information submitted to LSRO for review will consist of both existingpublic information and data from ingredient-specific testing. Evaluation of all relevant informationwill lead to one of three outcomes: a) an upper limit on the amount of ingredient to add to a cigarettewhere no change in the relative risk of adverse health effects is likely, b) the ingredient would not berecommended for addition to cigarettes where data shows the potential for associated health effects,and c) the absence of sufficient data will lead LSRO to detail what further information is required.



Figure 11.1 presents a potential overall flow of information. The following sectionsdiscuss this flow chart in more detail, including testing of added ingredients, submissionof relevant added ingredient data, and evaluation of the information. Appendix Dalso illustrates this evaluation process by applying the scientific criteria to somehypothetical added ingredients.

11.2 ASSEMBLING EXISTING DATA

A party submitting an ingredient to LSRO for review, would usually assemble apackage of relevant information to demonstrate that inclusion of the added ingredient(up to some maximum amount per cigarette) does not change the relative risk ofadverse human health effects of cigarette smoking according to the three scientificcriteria given in Section 11.1.1 above. (See Chapter 3.)

The assembled data package likely will include a mixture of the current worldwideliterature, data from ingredient testing, and other information relevant to the ingredient.The data submitter can assist LSRO’s review by organizing the information towardLSRO’s scientific criteria in the form of a report. If an added ingredient fails tomeet one or more of the three scientific criteria, then the submitted report willinclude additional sections detailing data showing the absence of change in the relativerisk of adverse human health effects.

In a preliminary section of a report, data submitters can include citations to theworldwide scientific literature relevant to the ingredient and preferably documenttheir search of the literature, including databases and search terms. The searchshould aim for comprehensiveness. LSRO usually will conduct an independentsearch, which will replicate and test the data submitter’s search process for missingcitations. Where possible, an assessment of an added ingredient will utilize all relevantknowledge about the toxicology of the substance. This preliminary section shouldalso include a description of the ingredient. (See Chapter 5.)

In addition to published studies, the data submitter may have access to unpublishedstudies. When a data submitter includes unpublished data, LSRO will request all ofthe primary data from the study, not only summaries or interpretation of the study.In addition, potential data submitters should note that LSRO conducts reviews in anopen forum and generates public reports. LSRO can use unpublished, non-confidential, data in its reviews. Where necessary, LSRO will obtain independentexpert reviews of previously unpublished data and make the reviews publicly availableas part of the report.

In addition to the submission of a literature search, definition of the ingredient, andthe existing data for an added ingredient, a data submitter may prefer to state anopinion about the interpretation of these data relative to LSRO’s objectives. In



these cases, LSRO will agree or disagree with the data submitter’s interpretation.The data submitter should include data relevant to LSRO’s scientific criteria, so thatthe data can be subjected to an independent evaluation. Where access to the scientistsproducing data may help the evaluation, LSRO will arrange for such a discussion.

11.3 SELECTION OF TEST METHODS

As mentioned above, not all tests are recommended for all ingredients. Chapters 6through 10 of this report provide examples of test methods to illustrate an approachto determine the effect of an added ingredient. However, LSRO recognizes thatother approaches may also be used to fulfill the intent of the scientific criteria. Thefollowing section summarizes LSRO’s evaluation process.

11.3.1 Chemical fate of the added ingredient in cigarette smoke

The chemical fate of an ingredient burned in a cigarette can generate data relevantto two of LSRO’s scientific criteria. Chapter 6 provided examples of experimentsand the relevant data that would allow for a determination of ingredient transfer intosmoke (criterion a) as well as any change in the chemical composition of smoke(criterion b). Depending on the outcomes, further testing may be appropriate.

An evaluation of the effects of an added ingredient on smoke chemistry forms anintegral part of LSRO’s scientific criteria. In the absence of detectable transfer ofthe ingredient, or some pyrolysis product of the ingredient not usually found in cigarettesmoke, LSRO will redirect its attention to the questions of smoke composition.Detection of transfer or a change in chemical composition may dictate further testingto establish that relative risk of adverse human health effects has not changed. Thesections below summarize the considerations and implications of each outcome.

11.3.1.1 Ingredient transfer into smoke

Relatively volatile ingredients in cigarettes may not decompose during combustion.These ingredients vaporize and condense ahead of the burning zone through distillation,and smokers may inhale them (Baker, 1999). Ingredients that distill (transfer intact)also change the chemical composition of cigarette smoke. Chemical substancesabsorbed in the lung have the potential to change the adverse health effects associatedwith cigarette smoking.

Less volatile added ingredients may partially distill, but they will also be subject tosome decomposition or pyrolysis, forming pyrolysis products, which subsequentlytransfer in cigarette smoke. These pyrolysis products can also react with othersmoke components to form still other products through pyrosynthesis.



Therefore, an ingredient burned in a cigarette could transfer intact, could generatepyrolysis products which transfer, or could form new substances throughpyrosynthesis, which transfer. Except for products which are chemically identicalto the pyrolysis products of tobacco, LSRO will estimate the per cigarette and potentiallifetime exposure, and with additional information, the per cigarette and potentiallifetime dose to a smoker of the new substance(s) not previously present in thesmoke.

The amount of ingredient transfer and the lifetime exposure to cigarette smokeallows for an estimation of lifetime dose of transferred added ingredient or pyrolysis/pyrosynthesis product. Pack-years are a measure used to quantify a person’s smokinghistory. The approach is a crude measure of exposure, calculated as the number ofpacks of cigarettes smoked in a day multiplied by the number of years the personhas smoked. People smoke cigarettes differently. Depth and inhalation differ andinfluence exposure to tobacco smoke. Compensation also occurs (Benowitz, 2001;Scherer, 1999). However, there is scant data and high uncertainty of the extent towhich smokers compensate and for what.

11.3.1.2 Smoke composition

If transfer of a new substance in the smoke inhaled by a smoker does not occur,LSRO will want to know about the composition of smoke in relation to key indicatorcompounds of smoke.

LSRO’s Phase One report (2004) noted that scientists have identified more than4,000 chemical substances in cigarette smoke (Green & Rodgman, 1996). In achemical analysis to establish similarity between paired cigarettes, with and withoutan added ingredient, there is no need to examine all of these substances. Doing sowould waste resources and add confusion. Instead, LSRO intends for a data submitterto submit data about the mean and variance of the mean, for some carefully selectedsubstances in smoke from test pair cigarettes, but should include water, nicotine,carbon monoxide (CO), total mass, ‘tar’, and one or more nitrogen-containingsubstances.

Methods to study change in smoke chemistry provide the most sensitive meanspresently available to look for change in a comparison of a cigarette with the addedingredient and the same kind of cigarette without the added ingredient. LSRO alsoanticipates that these methods will improve as research suggests ways to monitorthe physical characteristics of smoke.

Ingredients added to cigarettes may thermally decompose and may not transfer intosmoke. Decomposition products may also transfer into cigarette smoke withoutcausing a significant change in smoke composition (e.g., causing a change whichfalls within the normal variance associated with cigarette smoke). This can occur



when a pyrolysis product of the ingredient appears in smoke in approximately thesame proportion of added weight as the proportion resulting from combusted tobacco.In either case the addition of the added ingredient did not result in a significantchange in smoke chemistry and no basis exists to differentiate the risks from pairedtest cigarettes.

In the absence of a change in smoke chemistry, either by transfer or a change inchemical composition, it is unlikely that testing to evaluate the effects of exposure tothe added ingredient, as described in Chapter 8, would reveal a measurable effect.However, the presence of the added ingredient could alter a smoker’s exposure tocigarette smoke through changing the number of cigarettes smoked or the mannerin which the cigarettes are smoked. Therefore, it remains necessary to demonstratethat exposure to cigarette smoke does not change in the presence of the addedingredient. (See Chapters 7 and 10.)

Where an added ingredient affects smoke chemistry either by transfer or a changein smoke composition, a reduction in the amount of ingredient per cigarette to diminishthis change in smoke, may not be feasible. Further testing can demonstrate thataddition of the ingredient is unlikely to change the relative risk of adverse healtheffects. Relevant testing proposed in this report includes a description of thepharmacokinetics of inhaling the ingredient, or its pyrolysis product, as well asidentification of any active metabolites of the ingredient. (See Chapter 8.) If smokecomposition changes detectably, then the biological effects of cigarette smoke mayalso change. Chapter 9 details the kinds of test data which may prove useful inestablishing that the added ingredient does not change the relative risk of adversehuman health effects.

11.3.2 Exposure & dosimetry

The inclusion of an added ingredient could change the smoker’s exposure to specificcomponents of the smoke or to whole smoke through a change in human smokingbehavior. In addition, in the case where the added ingredient or its pyrolysis/pyrosynthesis products transfer into smoke, then the smoker’s exposure, throughinhalation, to the ingredient itself also becomes a consideration as this change couldlead to an adverse health effect.

Tests which provide an estimate of dose and exposure provide important informationin determining a level of an ingredient that can be added to a cigarette, which wouldresult in no change in the risk of adverse effects.

11.3.2.1 Exposure

The presence of an added ingredient could cause an alteration in exposure to wholesmoke. Through the comparison of biomarkers such as cotinine or carboxyhemoglobin



from individuals smoking cigarettes with or without added ingredients, the changes inexposure levels can be calculated. (See Chapters 7 and 10.)

11.3.2.2 Human smoking behavior

Whether the effects of the added ingredients on the chemical, physical, or biologicalproperties of smoke are detectable using current analytical methods does not ruleout the possibility that the inclusion of an ingredient may cause a change in humanbehavior. LSRO is only interested in ingredient-dependent changes in human smokingbehavior which result in a change in exposure to cigarette smoke. That is, to smokea different number of cigarettes or to smoke the same number of cigarettes in adifferent way (i.e., inhale deeper or retain the smoke longer).

Clinical tests which specifically examine behavioral consequences of added ingredientsin cigarettes will show important effects of the added ingredient and the potential toincrease the risk of smoking-related diseases through increased exposure. Informationon the effects of the added ingredients on human smoking behavior would berecommended for all ingredients submitted for review. Chapter 10 outlined someimportant scientific criteria for testing the effects of added ingredients on humansmoking behavior including important considerations for conducting a clinical study.

11.3.2.3 Kinetics

Should an added ingredient not meet one or all of LSRO’s scientific criteria, LSROhas proposed two options for a data submitter. One of these options is to directlydemonstrate that although a significant change in one of these tests has occurred,this does not result in a change in the relative risk of adverse human health effects.In a case where the added ingredient or its pyrolysis/pyrosynthesis products transferinto smoke at a biologically relevant level, the smokers’ exposure, through inhalationof the ingredient itself, becomes a consideration as this could lead to an adversehealth effect. (See Chapter 8.) Tests which provide an estimation of dose andexposure to an added ingredient inhaled within smoke provide important informationin determining a level of an ingredient that can be added to a cigarette that wouldresult in no change in the risk of adverse human health effects. (See Chapter 7.)

Transfer of an added ingredient or a pyrolysis product may change the compositionof cigarette smoke. Transfer of an ingredient into cigarette smoke also means thatthe smoker is exposed to, and may absorb the ingredient. Calculation ofpharmacokinetic parameters allows a relationship between dose and exposure to bedescribed. For an ingredient that transfers intact into smoke, measurement of thesubstance in a biological fluid provides important information about exposure to theingredient. Kinetic parameters provide information on the rate and extent ofabsorbance of an ingredient as well as a time course of exposure through adetermination of clearance and half-life. Although no biomarkers of exposure have



been identified for the vast majority of ingredients added to cigarettes, the detectionand measurement of metabolites could provide an estimate of exposure.

Information provided through pharmacokinetic studies will add to the data on theeffects of the added ingredient and contribute to the determination of relative risk.Kinetic studies, such as those outlined in Chapter 8, would not be expected to provideuseful data if the ingredient did not cause a change in smoke chemistry or biologicalactivity.

11.3.3 Biological effects

The second criterion includes an investigation of whether the presence of an addedingredient changes the physics, chemistry, or biological activity of cigarette smoke.The biological effects of cigarette smoke can be investigated through the applicationof a battery of biological tests. Although no model of the human health effects ofcigarette smoking exists, Chapter 9 outlines biological tests as examples, which mayprovide indications of an adverse health effect. Testing for potential biological effectsof added ingredients will add to an assessment of relative risk of a human healtheffect.

LSRO considers comparisons of the mutagenicity and cytotoxicity of smoke fromcigarettes with and without added ingredients to be advisable for all added ingredients.Testing of cigarette smoke for reversion potency in strains of Salmonella used inthe Ames test (with and without metabolic activation by liver extracts) that areresponsive to cigarette smoke condensate is stated in Chapter 9 as a relevant testfor mutagenicity. International Conference on Harmonisation (ICH) guideline S2B(1997) states:

It is appropriate to assess genotoxicity in a bacterial reverse mutationtest. This test has been shown to detect relevant genetic changes andthe majority of genotoxic rodent carcinogens.

The cytotoxicity of smoke from cigarettes with and without an added ingredientmay be compared using an assay such as the neutral red cytotoxicity assay in amammalian cell line. A measure of cytotoxicity is an important factor in understandingthe mechanism of action of chemicals on cells and tissues and may reflect potentialadverse health effects. Cytotoxicity is thought to play an important role in a numberof pathological processes, including carcinogenesis, atherosclerosis, and inflammation(Adler, 1986; Cooper et al., 1979; Ramos & Cox, 1987; Reitz, 1987; Ross, 1986;Swenberg, 1989). LSRO emphasizes that this is a minimum, but balanced battery oftests for a determination of biological activity. In the case that the added ingredientor a pyrolysis product transfers into the smoke or significantly changes the chemicalcomposition of smoke, then specific biological effects related to inhalation of theingredient may occur. In this case, standard, but more extensive biological testing,



(examples of which are given in Chapter 7) will allow for a determination of theabsence of change in relative risk.

11.4 EXPERT EVALUATION

An expert panel will evaluate all the information submitted for review using LSRO’sscientific criteria set forth in Chapter 3. Briefly summarized below are some importantconsiderations for an ingredient review, as well as some of the potential outcomes ofan evaluation...1

11.4.1 Detectable change

In its Phase One report (2004), LSRO described a “no detectable change” approach,which would reduce an ingredient in a cigarette below a level where investigatorsdetected change in any of a representative group of tests. If investigators cannotreasonably detect differences between cigarettes with and without an ingredient inthese tests, no reason would exist to expect a change in adverse human healtheffects.

However, LSRO cannot reach a conclusion of no change in relative risk due to asingle negative test result. New methods with increased sensitivity may arise whichcan subsequently detect change. Implementation of a diverse testing strategy(physical, chemical, biological, and behavioral) reduces the probability that a falsenegative result will influence estimation of the upper limit. Validated tests with goodsignal-to-noise characteristics will provide a more accurate value of an upper limitfor an ingredient, associated with no change.

The major portion of a cigarette is tobacco. There is inherent variability betweendifferent tobacco species, as well as different tobacco plants within a species. Thisnatural variability will be observed in most, if not all of the tests used, such that acomparison of identically constructed cigarettes will show a degree of difference(e.g., in the levels of smoke components).

For a change in the composition of cigarette smoke (signal) to be significant andmeaningful, it would have to exceed the variance in smoke composition associatedwith identical cigarettes. Standardizing cigarette smoke through defining the rangeof measurements for different components across different experiments allows fora definition of “smoke.” This variation defines the noise. LSRO concludes that thedefinition of the variance in measurements of indicator substances in typical cigarettesis a research need. A change in smoke composition which falls outside the variationinherent in cigarette smoke means that the smoke can be considered different cigarettesmoke. It has the potential to cause additional adverse human health effects.



11.4.2 Relative risk

The approach explained in this report depends on the relative risk of adverse healtheffects (Life Sciences Research Office, 2004). This review is not debating therisks of cigarette smoking, but the effects of adding one or more ingredients. Acriterion of relative risk based on a comparison of cigarettes with the added ingredientto an otherwise identical cigarette without the ingredient removes the considerationof background effects related to cigarette smoking. The net change, if any, revealsthe effects of the ingredient within the cigarette smoke matrix. The desired goal isthat inclusion of the added ingredient does not change the relative risk of adversehealth effects.

LSRO would not expect a change in the relative risk of adverse health effects associatedwith cigarette smoking when its three scientific criteria are met. These demonstratethat an ingredient does not detectably transfer into smoke, change the physics, chemistry,or biological activity of smoke, or alter human exposure to whole smoke. LSRO willregard any detectable change in smoke, including an increase in the level of an indicatorsubstance, as potentially adverse. Circumstances may arise where investigators cannotreduce an ingredient added to cigarettes below a point of no detectable change andachieve a useful purpose with the ingredient. An investigation of the relative risk ofadverse health effects where detectable change occurs is a different matter thanachieving compliance with the three scientific criteria outlined here.

A direct demonstration of an unchanged relative risk, ideally, would involveepidemiological studies of smokers of cigarettes with or without added ingredients.However, completion of such studies would take a long time, would require a largeinvestment of resources, and would be confounded by a large number of variables andchanging compositions of cigarette additives. For ingredients which cannot meet thethree scientific criteria, LSRO proposes that investigators try to demonstrate thatrelative risk of human health effects will not change by more extensive experimentation.

11.4.3 Potential conclusions

After review of all submitted information, LSRO will reach one of three conclusions.Appendix D illustrates the evaluation process and gives hypothetical examples ofsituations where each of these conclusions may be reached. Conclusions will bepublished based on the data available following the strategy laid out in Chapter 3 ofthis report.

11.4.3.1 Inadequate data

Should the reviewers evaluate the data and find gaps within the information provided,such that no final conclusion on the effect of the ingredient on the relative risk ofadverse health effects can be drawn, an initial report will be compiled that detailsspecific additional data that are required to reach a decision.



11.4.3.2 Maximum level of ingredient

A review of the submitted data and literature may reveal a scientific rationale for amaximum level of an ingredient added to a cigarette, where no change in the risk ofadverse health effects is expected. If all the tests were carried out with the ingredientat a certain level, then that would be the upper limit on the ingredient. If a range ofconcentrations of ingredients were tested, the maximum level would be the level atwhich there were no observed significant differences compared to the cigarettewithout the ingredient. This observation would correspond to the criterion of nochange in any of the screening tests applied to the ingredient.

11.4.3.3 Ingredient not recommended

LSRO will make a decision based on the available data. The test data reviewed bythe expert evaluation committee may highlight the potential for the ingredient tochange the relative risks of adverse health effects. A full report detailing what ledthe panel to reach this conclusion will be published showing the raw data and thecalculations performed. In these cases, scientific testing would reveal differencesbetween cigarettes with and without added ingredients. The conclusion would bethat the added ingredient changed the relative risk of smoking and, therefore, addsto potential harm.

11.5 SUMMARY

The aim of this report is to provide the data submitter with sufficient information onthe kinds of data for an evaluation LSRO would make. An outline of specific testingmethodology is also provided. However, LSRO means to encourage flexibility andinnovation.

The conclusions reached from a scientific evaluation can only be based on the dataavailable for review. The raw data, from which the conclusions were drawn, togetherwith the analysis and evaluation of the data, will be published in the form of anLSRO report. In the case that an expert panel was unable to reach a conclusion ofthe effect of an added ingredient on the adverse health effects of cigarette smoking,the report will detail what further tests would be required.

The extent to which conclusions drawn from an evaluation of an ingredient added tocigarettes from a single cigarette type can be applied to cigarettes in general, assumesthat test cigarette tobacco does not alter either the response of an ingredient to burningor the signal-to-noise ratio of an analytical method. If the test cigarette containedBurley tobacco and the experimenter changes to Maryland tobacco, test signals fromaddition of an ingredient might change. If a change in the tobacco substrate alteredthe nature or magnitude of changes in the smoke matrix, the logic of testing to detectthe absence of detectable change would still prevail, if the change in the tobacco



substrate did not change the ingredient-signal relationship. A unique signal is likely toremain unique regardless of the tobacco. For example, menthol transfer can onlycome from the addition of menthol; theobromine from the addition of cocoa.

Similarly, differential changes in the levels of substance in the smoke, given a changein the tobacco substrate, would not necessarily invalidate experiments to set themaximum level of ingredients added to cigarettes. Chemical testing to determinewhether the tobacco substrate alters the relationship between the ingredient addedand indicator substances produced lies well within the state-of-the-art.

Some published studies have investigated the variation in smoke between differentbrands of cigarettes which contain different blends of tobacco. Swauger andcoworkers (2002) compared cigarettes marketed between 1995 and 2000. Nineteenmainstream smoke components were tested including nicotine, ‘tar’, and CO.Constituent levels were found to be consistently proportional to ‘tar’ yields in thedifferent brands. The authors suggest that design differences do not significantlyaffect the composition of smoke in relation to ‘tar’. Chepiga and coworkers (2000)also found a correlation between mainstream smoke ‘tar’ delivery and mainstreamsmoke constituent levels. Even so, data submitted for an ingredient where a singletobacco type was used, do not directly address the possibility of a different combustionpathway or different effect of an added ingredient on smoke composition in a differenttobacco background.

The idea that cigarette smoking induces premature mortality of humans has stronglyinfluenced LSRO’s development of scientific criteria. (See Chapter 3.) However,some scientists believe that a particular test yields information relevant to the adversehuman health effects of cigarette smoking, LSRO cannot necessarily rely on thesetests. A test that showed no change in rodent lung cancer, might fail to reveal alarge increase in cardiovascular effects.

Epidemiology has provided the only accurate description of the range of adversehealth effects, which contribute to the premature mortality associated with cigarettesmoking. An adequate epidemiology study that compares the adverse human healtheffects of smoking cigarettes with and without an added ingredient could providedefinitive evidence of the health consequences of inclusion of the added ingredient.However, initiation of an epidemiological study, comparing the effects of cigaretteswith and without added ingredients is not conducive to this evaluation of ingredients.Therefore, LSRO will base its conclusions on the relative risk of adding ingredientsto cigarettes, on the scientific data available from a diverse testing program, and theinformation available in the scientific literature. This statement should not detractfrom the value of epidemiology data that, where available, may be an integral part ofan evaluation, for example menthol (Brooks et al., 2003; Carpenter et al., 1999;Kabat & Hebert, 1991; Sidney et al., 1995).


1212.1 INTRODUCTION

12.2 KINDS OF INFORMATION12.2.1 Literature summaries and peer reviews12.2.2 Smoke composition

12.2.2.1 Variation in smoke composition12.2.2.2 Standardization of techniques for cigarette smoke

component analysis12.2.2.3 Identification and isotopic tracing of unique

components of complex mixtures12.2.2.4 Development of novel techniques and refinement

of smoke composition analysis techniques12.2.2.5 Mechanisms underlying change in yields of

biologically active smoke components12.2.3 Exposure

12.2.3.1 Exposure to whole smoke12.2.3.2 Uptake of smoke chemicals12.2.3.3 Interactions between added ingredients and other

smoke components12.2.3.4 Smoke physics

12.2.4 Smoke exposure from altered human behavior12.2.4.1 Categorization of smokers12.2.4.2 Increased incorporation of crossover designs in

clinical studies12.2.4.3 Within-subject variations in cigarette smoking

behavior12.2.4.4 Smoking initiation and brand choice12.2.4.5 Identification of biomarkers12.2.4.6 Development of non-invasive approaches for

assessing the effects of added ingredients onexposure

RESEARCH OPPORTUNITIES


12.2.4.7 Comparisons between calculated estimates ofinhaled substances and biomarker back-calculations

12.2.4.8 Biological effects in animal models12.2.5 Biological effects in humans

12.2.5.1 Identification of mechanisms underlying smoking-related premature mortality and morbidity

12.2.5.2 Menthol and effects on development of non-cancerhealth outcomes

12.2.5.3 Racial disparities in adverse health effects of smoking12.2.5.4 Gender and adverse health effects of smoking12.2.5.5 Epidemiological studies

12.3 SUMMARY

Research Opportunities � 131


12.1 INTRODUCTION

The previous chapters describe a strategy for ingredient evaluation, the logicunderlying the selection of scientific criteria for testing added ingredients, and thekinds of tests considered to be useful for generating data about an added ingredient.In the process of developing scientific criteria for testing added ingredients, LSROhas identified information that would contribute to the process of ingredient evaluation.The intent of this chapter is to describe the kinds of information that would proveuseful in developing the data for testing specific ingredients added to cigarettes.

12.2 KINDS OF INFORMATION

12.2.1 Literature summaries and peer reviews

A sponsor soliciting a review of an added ingredient by LSRO will be asked tosubmit information about the added ingredient in the form of a report. The reportmay include a review of the relevant scientific literature and both published andunpublished data. In an attempt to ensure analysis of all relevant data about theadded ingredient, LSRO will issue a public call for data about the added ingredientand conduct an independent review of the relevant scientific literature and analysisof available data. This composite of information will provide the scientific basis fora recommendation of an inclusion level of the ingredient.

Wayne and Connolly (2004) described internal cigarette industry data, made publicas a result of the Master Settlement Agreement (1998), as a previously unminedsource of information about the effects of cigarette mentholation on human perceptionof smoke and smoking behavior. Wayne and Connolly suggest the analysis of thisdata for useful information about the effects of menthol and other ingredients. Theidentification and utilization of all sources of available data by the data submitter andthe submission of unpublished data by other investigators to LSRO are importantresearch components and represent an important research need. Such informationwould buttress the rigor of the LSRO ingredient evaluation process. All information,including unpublished data submitted to LSRO used in an evaluation of an addedingredient, will be made public in the form of a report.

12RESEARCH OPPORTUNITIES




12.2.2.1 Variation in smoke composition

The ability to detect statistically significant differences between smoke of similarcigarettes, with and without an added ingredient of interest, hinges in part on thevariability in measurements of substances in smoke from “identically manufactured”cigarettes. The variability in the analytical techniques used to measure yields ofsubstances in smoke of a cigarette (e.g., the University of Kentucky 2R4F referencecigarette) can influence the interpretation of data when cigarettes with and withoutadded ingredient(s) are compared. Baker and coworkers (2004a) measured andcompared yields of 44 smoke analytes from test cigarettes containing addedingredients and control cigarettes, lacking added ingredients. Analysis of the datainitially identified statistically significant differences in yields of analytes from testversus control cigarettes. When the within-group variation of analyte yields fromtest and control cigarettes was compared to the variation of smoke componentyields from a reference cigarette measured over an extended period of time , somedifferences between test and control cigarettes were no longer considered statisticallysignificant (Baker et al., 2004a; Rickert & Wright, 2002). The consistent use ofKentucky reference cigarettes, as a means to test variability of techniques formeasuring smoke chemistry and biological activity, represents an important researchcomponent and research need.

Variations in smoke composition can also arise from differences in the laboratoriesthat measure smoke constituents. Laboratories tend to use their own internallyvalidated analytical methods. Purkis and others (2003) studied the inter-laboratoryvariation in the yields of 44 smoke components from the same cigarettes and identifiedas much as a 10-fold difference in amounts of smoke analytes measured by sevenlaboratories. The minimization of inter-laboratory variation in smoke analytemeasurements would enhance comparability of data. The development of approachesfor reducing inter-laboratory variation represents a research opportunity.

12.2.2.2 Standardization of techniques for cigarette smokecomponent analysis

Worldwide standards exist for measuring total particulate matter (TPM), nicotine,water, carbon monoxide, and benzo(a)pyrene, but not for other smoke components(International Organization for Standardization, 1995; 1999; 2000a; 2000b;CORESTA, 2004). Since LSRO does not mandate the measurement of specificsmoke substances to determine whether inclusion of the added ingredient changesthe chemical fingerprint of the smoke, manufacturers may choose to track smokechemicals for which there are no standardized, universally utilized techniques. Theapplication of validated, universally applied approaches to measuring substances insmoke would improve the ability to make comparisons between different sets ofdata and reduce the inter-laboratory variation in data. The Coopération Pour Les



Recherches Scientifiques Relative au Tobac (CORESTA) organized a task force in1999, with the goal of developing techniques to measure cigarette smoke substances.Their work will improve the standardization of methods. The Tobacco Act ReportingRegulation of the Government of Canada (2004) identified specific procedures forcigarette smoke generation and analysis of smoke components for cigarettes.

12.2.2.3 Identification and isotopic tracing of uniquecomponents of complex mixtures

As described in Chapter 6, detectable differences between cigarettes with and withoutadded ingredients are likely to arise from unique component(s) of a complex mixture(e.g., theobromine in cocoa powder) and not elements likely to pyrolyze to the samechemicals obtained from burning tobacco. The identification of the most toxic orpharmacologically active components of a mixture would allow isotopic labeling ofthe component(s) and the identification of pyrolysis products from these ingredients.This might better characterize the relationship between individual components ofmixtures and smoke composition.

12.2.2.4 Development of novel techniques and refinement ofsmoke composition analysis techniques

This report provides examples of techniques for analyzing smoke from cigaretteswith and without added ingredients. The refinement of existing methods and thedevelopment of new methods to assess changes in smoke composition would improveability to determine whether the presence of an added ingredient results in changesin smoke chemistry. Research aimed at achieving this goal would enhance smokechemistry studies.

12.2.2.5 Mechanisms underlying change in yields ofbiologically active smoke components

Rustemeier and coworkers (2002) compared the yields of smoke analytes fromcigarettes without added ingredients and from three groups of cigarettes, each groupcontaining a different combination of added ingredients. The authors identifiedsignificant differences in concentrations of biologically active smoke compounds(e.g., hydrogen cyanide, formaldehyde, and cadmium) between the four cigarettegroups. They suggest the need for additional studies to investigate the mechanismsunderlying the increased concentration of some biologically active smoke components.



12.2.3 Exposure

12.2.3.1 Exposure to whole smoke

Many studies of the biological effects of cigarette smoke exposure involved exposuresto cigarette smoke condensate. Since whole cigarette smoke includes some volatileand semi-volatile compounds that are not found in cigarette smoke condensate (WorldHealth Organization, 1986), testing the biological activity of only fractions of smokewill not reveal the effect of all smoke components. Additional studies involving invivo and in vitro exposures to whole smoke would enhance understanding of theeffects on smoke exposed individuals. Massey and coworkers (1998) reported thatmost in vitro approaches involving cell cultures utilized only cigarette smokecondensate. The authors noted that it would be useful to know the effects of exposureof cells to whole smoke.

Cigarette smoke is a dense, dynamic aerosol (Baker, 1999). Some smoke constituentspartition between the vapor and the particulate phases of smoke in a pattern influencedby time, temperature, and dilution of smoke (Baker, 1999; Pankow, 2001). Thetiming between smoke generation and exposure of cells, tissues, or animals to thesmoke is an important aspect of testing cigarette smoke. According to Ritter andcoworkers (2003), exposure to fresh cigarette smoke, with minimal chemical andphysical changes, is an important criterion for exposure measures.

Smokers inhale a mixture of mainstream, sidestream, and environmental tobaccosmoke. Environmental tobacco smoke consists of exhaled mainstream smoke andaged sidestream smoke. Exposure to mainstream smoke alone might not result inchanges that would be seen with the mixture of smoke that smokers inhale.

Ritter and coworkers (2003) summarized approaches to expose cells to aerosols.They described aerosol exposure to cells submerged in culture medium, as well asalternating exposure to culture medium and aerosol. In addition, they outlined an air/liquid exposure technique in which human bronchial epithelial cells were cultivatedand exposed to aerosols on the apical side and culture medium on the basal side(Ritter et al., 2003). The use of cultures of human cells makes the extrapolation ofdata between species unnecessary and allows for assessment of differences insusceptibility of respiratory tract cells to the effects of inhaled substances (Pauluhn& Mohr, 2000). Improvements in the ability to expose cells and tissues in vitro andanimal models in vivo in smoking studies to cigarette smoke and validation of suchtechniques would contribute to acquisition of data about added ingredients.



12.2.3.2 Uptake of smoke chemicals

Additional research is needed to characterize the influence of menthol and otheringredients on nicotine availability to the smoker. Wayne and Connolly (2004) haveidentified the need for extended studies about the effects of inhaling smoke frommentholated cigarettes. Sloan and coworkers (1993) reported that l-menthol lozengesincrease the duration of breath-holding. Whether this phenomenon occurs in smokersof mentholated cigarettes is not known. If menthol in cigarettes allows smokers tohold their breath longer, this behavior could increase their uptake of substances insmoke (Clark et al., 2004).

Studies aimed at characterizing the effect of menthol on carbon monoxide levelshave generated inconsistent results. While some groups report higher carbonmonoxide exposure on smoking menthol cigarettes (Ahijevych et al., 1996; Clark etal., 1996; Jarvik et al., 1994), others do not report a difference in carbon monoxideexposure from smoking menthol vs. non-menthol cigarettes (McCarthy et al., 1995).Jarvik and coworkers (1994) measured higher mainstream smoke carbon monoxideyields from menthol cigarettes than from non-menthol cigarettes. Further studies onthe effects and mechanisms of menthol on carbon monoxide exposure are needed(Wayne & Connolly, 2004). Additional data are needed to determine the effect ofmenthol and other added ingredients on the uptake of substances in smoke. (SeeAppendix C, Peter Lee presentation.)

12.2.3.3 Interactions between added ingredients and othersmoke components

As discussed in Chapter 6, Gager and coworkers (1971b) described detection ofacetonitrile in cigarette smoke as indicative of pyrosynthesis reactions between sugars,decomposition products of sugars, and nitrogenous compounds. Furthermore,interactions between substances in smoke may influence smokers’ experience ofsmoking a cigarette containing the added ingredient. Wayne and Connolly (2004)summarize the effects of other added ingredients on the “perception and strength ofmenthol cigarettes.”

12.2.3.4 Smoke physics

Cigarette smoke is a complex aerosol composed of a large number of particles thattypically have a diameter of much less than 10 µm (Hollander & Stober, 1986).Particle size, density and shape, breathing pattern, and lung geometry influence totaland regional deposition of cigarette smoke particles (Schultz et al., 2000). Whetherthe inclusion of added ingredients affects factors related to deposition of cigarettesmoke substances is not known.



12.2.4 Smoke exposure from altered human behavior

12.2.4.1 Categorization of smokers

Investigators do not apply consistent criteria to the categorization of smoking statusof epidemiological or clinical study participants. Leffondre and coworkers (2002)identified varied definitions of current and former smokers. A clearer definition ofcutoff points, for the categorization of former smokers in scientific publications,would permit better delineation of the effects of exposure and cigarette design oncigarette smoke exposure. Consistency in the definition of terms used to describesmoking status would allow for comparisons of results between studies.

Smokers change their smoking behavior to attempt to maintain a given level ofnicotine delivery to the systemic circulation. While smoking cigarettes with lowernicotine levels, smokers can theoretically change their behavior (e.g., puff profile) inorder to extract enough nicotine to compensate for the lower exposures. However,the phenomenon of compensation has been reported as varying from 0 %, that is, nocompensation, to 100 %. Because of this range, nicotine may be the only substancefor which smokers compensate. Other factors may be tactile stimulation, odors, ororganoleptic elements. More careful studies of compensation that include informationon categorization of smokers (e.g., as inhalers or non-inhalers), nicotine biomarkers,personality tests, sensory satisfaction questionnaires, and other information wouldprove useful to establish the basis of compensation.

12.2.4.2 Increased incorporation of crossover designs inclinical studies

Multiple studies have identified marked inter-subject variation in smoking behavior.Crossover designs involve the exposure of study participants to sequences oftreatments in order to study differences between the effects of the specific treatments(Senn, 2002). Each study participant serves as his or her own control (Senn, 2002).The increased incorporation of a crossover design in studies of human smokingbehavior would decrease one source of variation, require fewer subjects, and improveinterpretation of results. Clark and coworkers (2004) identified additional crossoverstudies of human smoking behavior as a research need.

12.2.4.3 Within-subject variations in cigarette smoking behavior

Different smokers may change their smoking behavior for different reasons. Smokerscan change smoking behavior because they think that the cigarettes smoked in onephase of an experiment differ from the cigarettes smoked in another phase of theexperiment. Incorporating a study element that identify individuals that change theirsmoking behavior, not because of changes of the kind of cigarette, but instead,because of a perceived change in the type of cigarette, could provide informationabout the range of smoking behavior for a study participant.



12.2.4.4 Smoking initiation and brand choice

LSRO’s scientific criteria for testing added ingredients do not include an evaluationof whether added ingredients contribute to smoking initiation. However, if an addedingredient were found to increase the appeal of cigarette smoking to such an extentthat non-smokers start smoking because of the presence of the ingredient, theingredient would be increasing exposure. Whether the positive descriptions of smokingcigarettes described in advertisements for menthol cigarettes influence smokinginitiation among adolescents is not known (Clark et al., 2004). Menthol is the onlycigarette additive that is directly marketed to the public and about which consumersmake deliberate purchasing decisions. According to Fowles (2001), there exists apaucity of published studies that describe the relationship between added ingredientsand cigarette brand choice. Clark and coworkers (2004) also note a need forinformation about the choice of menthol cigarettes by sub-groups within the smokingpopulation (i.e., reasons that smokers provide for choosing to smoke mentholcigarettes).

Wayne and Connolly (2004), in their review of internal tobacco industry researchdocuments, identify the following needs for additional research on menthol: evaluationof menthol cigarette smoke perception, its influence on smoking behavior, and smokingpatterns, and its pharmacological effects (i.e., central nervous system effects ofmenthol).

12.2.4.5 Identification of biomarkers

Nicotine intake can be calculated from cotinine concentrations. (See Chapter 7.)Each 100 ng/mL of plasma cotinine at steady state reflects a daily intake of 8 mg ofnicotine (Benowitz & Jacob, III, 1994). Other groups have begun to measure othernicotine metabolites in order to better characterize nicotine exposure, as well asother biomarkers of smoke exposure (Byrd et al., 1994; Kinser et al., 2001). Theapplication of this more in-depth characterization of smoke exposure would enhanceassessment of exposure to smoke.

12.2.4.6 Development of non-invasive approaches forassessing the effects of added ingredients on exposure

The development of additional non-invasive methods for measuring substances thatsignal a change in smoke exposure would contribute to the process of evaluatingadded ingredients. As an illustration, a sponsor could investigate the levels ofmetabolites of carcinogens in urine to reflect changes in exposure to smoke substancesdue to the presence of added ingredients. A significant difference between urinarymetabolites of carcinogens measured after smoking cigarettes with an addedingredient compared to smoking a nearly identical cigarette without an addedingredient indicates a change in exposure. Metabolites of carcinogenic substanceshave been measured in urine [e.g., trans, trans-muconic acid and S-phenyl



mercapturic acid, 1- and 2-napthol, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol,glucuronides of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol, and other substances](Hecht, 2002). These metabolites are generally present in large enough quantities inurine to be measured, and urine is produced in a sufficient quantity to provide asufficiently large sample of the metabolites. These metabolites indicate the presenceof substances in the body and reflect exposure to “smoke” or “the substance insmoke.”

Montuschi and Barnes (2002) describe a dearth of non-invasive methods for assessingairway inflammation. Approaches generally applied in these assessments includeinvasive techniques, such as bronchoalveolar lavage and bronchial biopsies, or semi-invasive methods, such as sputum induction. The analysis of exhaled breathcondensate represents a simple, non-invasive approach that investigators have appliedto assessing changes in the biochemistry and the inflammation of airways (Hunt,2002). Many technical issues surround the breath analysis approach, includingvalidation of the procedure and establishment of standardized sample collection(Montuschi & Barnes, 2002). These issues will require further resolution beforeusing this technique to determine whether added ingredients in cigarettes result in anincrease in lung inflammation.

12.2.4.7 Comparisons between calculated estimates of inhaledsubstances and biomarker back-calculations

Back-calculations from measurements of biomarkers of exposure can be used assurrogate indicators of exposure to specific smoke substances (Benowitz, 1999).However, the characterizations of the sensitivities of these biomarkers are incomplete.Further evaluation of the sensitivities of biomarkers, which could reflect difference(s)in smoke exposure, is warranted. Investigators could calculate nicotine exposurebased on urinary cotinine with allowances made for individual differences in nicotinemetabolism. In addition to tracing the metabolites of an added ingredient in urine,biomarker levels after smoking cigarettes with and without an added ingredientshould indicate potential differences in smoke exposure, given the sensitivity andvariances of the method.

12.2.4.8 Biological effects in animal models

The development and application of predictive animal models of cigarette smokingrelated disease will enhance the process of ingredient evaluation. Animal models ofcigarette smoking related diseases have proved elusive. Coggins (2002) reportedno significant increases in the kinds of respiratory tract tumors associated withhuman cigarette smokers after exposure of rats, mice, hamsters, dogs, or non-humanprimates to very high concentrations of mainstream cigarette smoke. Animals andhuman smokers inhale cigarette smoke differently (Institute of Medicine, 2001).Often, investigators have to dilute the smoke and incorporate breaks from smoke



exposure into their experimental paradigm in order prevent deaths of rodents fromcarbon monoxide-related toxicity. Animal models for cigarette smoking relateddiseases will prove useful for evaluating added ingredients.

12.2.5 Biological effects in humans

12.2.5.1 Identification of mechanisms underlying smoking-related premature mortality and morbidity

The mechanisms underlying the development of cigarette smoking related diseasesthat result in the premature mortality and morbidity of cigarette smokers are largelyunknown. Further research to identify such mechanisms would contribute to thedevelopment of predictive tests for adverse health effects of cigarette smoking.Bos and coworkers (2002) noted a need for further exploration of the mechanismsunderlying sensory irritation as well as an evaluation of the ability of sensory tractirritation to predict risk of adverse human health effects.

12.2.5.2 Menthol and effects on development of non-cancerhealth outcomes

Clark and coworkers (2004) noted that some data characterized whether smokingmentholated cigarettes increased the risk of developing lung cancer. Additionalresearch is needed to identify the influence of smoking menthol cigarettes on therisk of developing other cigarette smoking-associated diseases.

Wayne and Connolly (2004) identified a lack of research on the potential centralnervous system (CNS) effects of menthol. In a recent review, the authors describedcases in the literature of apparent menthol addictions (Kennedy, 1973). Reports bythe British Committee on Safety of Medicines (1964 - 1981) of CNS effects ofmenthol include effects such as confusion, psychoses, and visual disturbances(R.J. Reynolds Tobacco Company, 1986). Additional research on the effects ofmenthol in cigarettes on the CNS is highly desirable.

12.2.5.3 Racial disparities in adverse health effects of smoking

Whether Black smokers’ preference for smoking menthol cigarettes contributes tothe health differences between Black Americans and European Americans has notbeen fully addressed (Clark et al., 2004). These health disparities exist, even thoughdaily cigarette consumption for Black smokers is lower than for European Americansmokers (Caraballo et al., 2004; Clark et al., 1996; Djordjevic et al., 2002; Kabat &Hebert, 1994; Wagenknecht et al., 1990). Additional research to identify thosefactors contributing to these health disparities is needed.



12.2.5.4 Gender and adverse health effects of smoking

Additional research could clarify whether the risk of adverse health effects fromcigarette smoking differs between men and women. Some groups report an increasedrisk of development of lung cancer for women with cigarette smoke exposuressimilar to men (Harris et al., 1993; Risch et al., 1993; Zang & Wynder, 1992).Other studies fail to identify a difference in the risk of lung cancer for men andwomen (Bach et al., 2003; Kreuzer et al., 2000; Osann et al., 1993; Perneger,2001; Prescott et al., 1998).


Four recent epidemiological studies demonstrated no increased risk of lung cancerfrom menthol cigarette smoking (Brooks et al., 2003; Carpenter et al., 1999; Kabat& Hebert, 1991; Tsujimoto et al., 1975). (See Appendix C; Peter Lee presentation.)According to Clark and coworkers, (2004), limitations of such studies include aninsufficient number of subjects and subjects that may not have smoked mentholcigarettes exclusively. In addition, smokers could choose to switch the brand ofcigarettes that they smoke. The latency in the development of cigarette smokingrelated diseases provides an additional challenge for conducting epidemiological studiesabout the adverse health effects of smoking cigarettes.

Although LSRO does not propose that data submitters initiate epidemiological studiesabout added ingredients in cigarettes and cigarette smoking-related diseases, newand existing epidemiological studies that examine this subject represent importantcomponents of an added ingredient review.

Epidemiological studies identified the relationship between cigarette smoking andcigarette smoking-associated diseases (DeGeorge et al., 1997). One approach todiscerning a relationship between added ingredients and incidence of cigarettesmoking-related diseases is to determine if smoke from cigarettes made today differsignificantly from the cigarettes smoked by study participants of historically archivedepidemiological studies. Hoffmann and Hoffmann (2001) described many changesin cigarette design between 1950 and 1975. These changes include an increase inaverage nitrate content of cigarettes from 0.5 % in the 1950s and 1960s to1.25 - 1.50 % today which is associated with the presence of reconstituted tobaccoin cigarettes (Spears, 1974; U.S. Department of Health and Human Services, 1989).Ingredients added to cigarettes are also likely to have changed over the lifetimes ofsmokers.

Further insights into added ingredients in cigarettes and the smoke they produce willprovide clues to biomarkers. This knowledge would support understanding of therelationship between added ingredients and smoking behavior. For cigarettescontaining added ingredients already on the market, demographic studies could answer



questions about gender, age, and ethnic acceptance of such products. Informationon marketing issues such as the distribution and availability of the new added ingredientproducts would be important for use in analyzing demographic and/or generalpopulation changes. Sampling of a population for biomarkers of exposure may benecessary. Although LSRO considers studies of initiation, cessation, or relapsebeyond the scope of data generation for data submitters, submission of informationin the scientific literature and in population surveys about smoking initiation, personaluse patterns, cessation attempts, and maintaining abstinence may contribute tounderstanding the link between added ingredients and cigarette smoking behaviors.

One approach to studying cessation is to identify smokers who are attempting to quitsmoking and determine if brand makes a difference in their success. Anotherapproach might be to conduct a cross-sectional study to find out how often peopletry to quit, and link the frequency of attempts to brands of cigarettes smoked. Suchsurveys might contrast with similar information gathered on smokers who havesuccessfully quit and remained non-smokers for 5 years or longer. Demographicstudies can illuminate factors influencing cessation by focusing on smokers who aretrying to quit.

Long-term epidemiological studies that investigate whether smokers of cigaretteswith added ingredients are more likely than smokers of cigarettes without suchingredients to develop cigarette smoking-related diseases, experience acceleratedrates of progression of such diseases, or develop diseases previously not associatedwith cigarette smoking would also provide insight. Perhaps most useful for continuingresearch on smoking patterns and added ingredients would be a coordinated effortto develop a database of research projects and outcomes. This could forestallduplicative research, lead to better study design, and, thus, provide relevant informationfaster, and be less expensive and resource intensive (Institute of Medicine, 2001).

The emerging field of molecular epidemiology, which is generally divided intomolecular dosimetry and susceptibility studies, will make important contributions tothe ingredient evaluation process. Molecular dosimetry involves the measurementof biological markers of internal dose (e.g., nicotine, cotinine, biologically effectivedose, and DNA adducts in individuals). Susceptibility studies assess relationshipsbetween genetic variants that can change the relationships between exposure to asubstance and disease (Furberg & Ambrosone, 2001). Genomic techniques arelikely to contribute to assessments of whether added ingredients modify risk ofdiseases associated with cigarette smoking.



12.3 SUMMARY

This chapter outlines research that might determine whether the inclusion of addedingredients in cigarettes is likely to change the risk of adverse human health effectsfrom cigarette smoking. Much information would contribute to evaluating the risksthat may be associated with specific ingredients. Information about variations insmoke composition and approaches to minimize inter-laboratory variations in smokeanalytes can contribute to the process of generating meaningful new data aboutadded ingredients. Identification of unique components of mixtures of addedingredients and exploration of mechanisms underlying changes in biologically activesmoke substances will also contribute to the development of relevant data.Standardization of techniques to be applied to the measurement of smoke analyteswould also enhance the process.

Exposure of cells, tissues, and organisms to fresh, whole smoke will contribute to ameaningful assessment of the overall effects of various smoke components. Additionalresearch about uptake of smoke chemicals would also enhance development ofdata about the influence of added ingredients and exposure to smoke substances.Development of techniques to assess the potential for an added ingredient to affectsmoke physics would further improve the process of evaluating the added ingredient.

Assessment of smoke exposure by characterization of cigarette consumption willbe enhanced by improving standardized methods for categorization of smokers andincreasing crossover designs in clinical studies. Biomarkers that provide a betterestimate of smoke exposure and its biologically relevant effects would also enhancestudies of risk of adverse health effects due to added ingredients.

Improved characterization of mechanisms underlying cigarette smoking relateddiseases would enhance knowledge about added ingredients. Whether menthol incigarettes is involved in health disparities between Black and European Americansis another research need. The assessment of the relationship between biologicaltests and epidemiological results would also be valuable (Life Sciences ResearchOffice, 2004).

The distribution or electronic posting of this PDF file is strictly prohibited without thewritten permission of the Life Sciences Research Office.

1313.1 SCIENTIFIC CRITERIA

13.2 SIGNIFICANCE OF CHANGE

13.3 SCIENTIFIC JUDGMENT

13.4 RELEVANCE OF SCIENTIFIC DATA TO ADVERSE HUMANHEALTH EFFECTS13.4.1 Epidemiological studies13.4.2 Appropriate test substance

13.5 LIMITATIONS

13.6 SUMMARY

CONCLUSIONS



13.1 SCIENTIFIC CRITERIA

This report describes scientific criteria for the evaluation of ingredients added tocigarettes with the aim of eliminating ingredients which change the risk of smoking,and setting an upper limit on the amount of an ingredient where no change in therelative risk of smoking cigarettes would be expected. LSRO’s scientific criteriastate that if an ingredient, or a pyrolysis product of the ingredient, does not detectably:(a) transfer to smokers, (b) change the physics, chemistry, or biological activity ofsmoke, and (c) change human exposure to cigarette smoke by modifying humanbehavior, then, in LSRO’s view addition of the ingredient to cigarettes will be unlikelyto change the relative risk of smoking. LSRO does not claim that these criteria arethe only ones applicable to such evaluation, only that these criteria rest on scientificknowledge.

Transfer of an added ingredient, or a pyrolysis/pyrosynthesis product, into cigarettesmoke changes smoke compared to that from a cigarette without the ingredient.What smokers inhale has changed. Addition of the ingredient to the cigarette,therefore, has the potential to change the adverse human health effects associatedwith cigarette smoking.

A change in the chemical composition of smoke, in the absence of transfer, is also achange in cigarette smoke inhaled by a smoker. A change in the biological activityof cigarette smoke caused by the addition of an ingredient can be an indication ofthe potential to produce an altered human health effect. LSRO recommends thatdata submitters characterize smoke though balanced chemical, biological, and physicaltests. The effect of an added ingredient on the physics of smoke was not discussedin detail in this report because of a paucity of data in this area. However, should aningredient affect the size of particles in the smoke, this in turn will affect the sitesand efficiencies of particle impaction in the lung and introduce the possibility of aneffect on human health.

In evaluating ingredients added to cigarettes, we are attempting to eliminate additional(new) adverse human health effects. An important part of LSRO’s scientific criteriais therefore a demonstration that an added ingredient does not cause a change inexposure to cigarette smoke. An ingredient which causes smokers to smoke more

13CONCLUSIONS

Conclusions � 145


cigarettes or smoke more intensely would not be acceptable, independent from effects,if any, on consumer preference. A brief description of a clinical study to observeany effects added ingredients may have on human smoking behavior relating toexposure was included in this report. (See Chapter 10.)

In LSRO’s view, compliance with these three scientific criteria provides sufficientevidence that a specific ingredient would not be expected to change the relative riskof cigarette smoking. Appendix D presents hypothetical examples of the applicationof the three criteria in practice. For ingredients that do not meet these criteria,LSRO sees two ways to proceed either:

(a) reduce the amount of ingredient, retest, and demonstratecompliance with the three scientific criteria, or

(b) provide more in-depth support for the proposition that addition of theingredient does not change the relative risk of adverse human healtheffects.

Not meeting the three scientific criteria will force potential data submitters to conductadditional testing to demonstrate the absence of changed relative risk. Informationobtained from scientific analysis to meet the scientific criteria should provide a submitterwith a rationale for selecting further relevant and effective tests. A demonstrationof the absence of change in relative risk of adverse health effects is a complexprocess, and providing specific guidance is difficult. LSRO describes hypotheticalexamples of relevant data in Appendix D, if only to illustrate that such an approachis feasible.

An evaluation should not exclude relevant information. Prior information or data,which alone seemingly have little significance, can add to a review in the context ofa larger body of information. The results from a pyrolysis experiment alone, wherean added ingredient was pyrolyzed in isolation, would add little to an evaluation.However, the experiment has some predictive capability in determining the potentialbehavior of an ingredient in a burning cigarette. This information can be used todesign better smoke chemistry studies.

The evaluation of each ingredient will require case-by-case expert judgment. LSROintends for the application of the scientific criteria in this report to avoid inconsistenttreatment of similar ingredients, maximize transparency of the process, and supportthe establishment of an evidence-based consensus.

13.2 SIGNIFICANCE OF CHANGE

A comparison of cigarettes with and without an added ingredient should define thesignificance of change. The first approach scientists use is statistical. The description



of a statistical inference should state the model used, including an explanation of thestatistical analysis, reasons for the use of a particular model, assumptions, and conductof the analysis. The data submitter should take steps to ensure that the statisticalmethods are relevant to the change from an added ingredient. Statistical outliers arenot always biological outliers. A “significant” statistical test (p < 0.05) does notalways indicate biological significance and vice-versa (U.S. Food and DrugAdministration 2001).

In general, a 15 - 20 % deflection in the total amount of a substance detected insmoke might be determined to have biological significance. However, measurementtechniques may not allow a precise detection of this extent of change, within onestandard deviation. Alternatively, a 20 % change in the amount of some substancethought not to have any biological effect may prove precisely detectable in a statisticalsense, but biologically meaningless. LSRO debated this topic extensively. LSROhas not set precise numerical criteria for significance, but suggests that a datasubmitter attempt to relate any observed changes to biological effect and obtainconsistency in the use of the term “significant.”

Where change is observed in a smoke component, in a comparison of cigaretteswith and without an added ingredient, any evaluation of statistical significance shouldconsider both the variation of the test method and the variation imposed by themanufacturing process. Inclusion of a third, reference cigarette, for example aUniversity of Kentucky 2R4F reference cigarette, will allow for comparison tohistorical data for the reference cigarette, and thus, verify that the test methodperforms adequately (Tobacco and Health Research Institute, 2002). Tracking thedata obtained with the Kentucky 2R4F allows a determination of whether a changefalls outside the normal range for a specific assay and smoke component.

Once a data submitter calculates statistical significance, a change in the smokeinduced by an ingredient which falls outside of normal experimental variationassociated with cigarette smoke will merit attention. Smokers are potentially inhalingsomething different. The next important question is whether this change is biologicallysignificant over a potential lifetime of smoking.

The scientific criteria present an integrative approach, which includes assessing theeffects of an added ingredient on biological activity and exposure. A statisticallysignificant change in smoke chemistry may not have an associated biological effect.A biologically significant change means that the change confers a biological activityand may indicate a change in the adverse human health effects of smoking.

In the absence of statistically significant change in any of the tests carried out tomeet the scientific criteria, the ingredient will not have changed the properties ofsmoke and, thus, will not change the adverse human health effects or relative risk ofcigarette smoking.

Conclusions � 147


13.3 SCIENTIFIC JUDGMENT

LSRO sees the evaluation of ingredients added to cigarettes as a scientific process.This means that the specified effects of added ingredients on cigarettes can bemeasured and the measurements are derived from current validated techniques,that different laboratories can use to reproduce each others’ results.

In the same way, a scientific approach applies to the evaluation of submitted data.LSRO cannot restrict the use of an ingredient in a cigarette. Instead, LSRO actsto provide and make public the conclusions of expert deliberations based onsubmitted data.

Generation of a decision tree to illustrate LSRO’s overall approach, as presented inthis report, would be complex. Many different scenarios exist with each addedingredient. Instead, decisions made during an evaluation are demonstrated throughcase studies of hypothetical added ingredients. Appendix D describes the applicationof LSRO’s initial approach to some ingredients added to cigarettes. Appendix D ismeant to assist the reader in understanding the overall approach by outlining itsapplication to some hypothetical substances.

Development of scientific criteria for the evaluation of ingredients added to cigaretteswill be an evolving process as LSRO accumulates practical experience. Asingredients are evaluated, it is likely that the criteria will be constantly modified andupdated as ingredient reviews highlight necessary amendments and additions.

13.4 RELEVANCE OF SCIENTIFIC DATA TOADVERSE HUMAN HEALTH EFFECTS

13.4.1 Epidemiological studies

Epidemiological studies such as the American Cancer Society’s (ACS) CancerPrevention Study I and II (CPS-I and CPS-II) define the adverse human healtheffects of cigarette smoking (Thun et al., 1997). Lung cancer, cardiovasculardiseases, and chronic obstructive pulmonary disease are major causes of prematuremortality associated with cigarette smoking. Although not specified as part of thesescientific criteria, an adequate epidemiological study that compares the adversehuman health effects of smoking cigarettes with and without an added ingredientwould most likely provide definitive evidence of the health consequences of inclusionof the added ingredient.

LSRO can see little value in a data submitter initiating new epidemiology studies ofingredients, particularly for ingredients added to cigarettes at undetectably low levels,for this evaluation process. Human epidemiological studies usually have lengthydurations. Cigarette-associated human diseases have long latencies. Diverse human



populations, different living conditions and environments, and variable backgroundrates of diseases, all limit quantitative epidemiological observations. Susceptibilityfactors, such as predisposing genetic traits, age, ethnicity, gender, or nutritional status,vary in human populations (Perera, 1997). Exposures from previous epidemiologicalstudies seldom provide adequate information to attribute incidence to an ingredientin a cigarette because smokers change brands, brands change ingredients, smokingmay often be intermittent, and almost none of these changes track to ingredients.

Smoke inhaled from cigarettes consumed during epidemiological studies (e.g., CPS-I and CPS-II) caused the diseases observed in those studies. If cigarettes withoutan added ingredient do not generate smoke distinguishable from the CPS cigarettes,LSRO has no reason to expect that the adverse human health effects of the testcigarettes would differ from those seen in the CPS-I and CPS-II studies. Ifinvestigators could not reasonably detect differences between cigarettes with andwithout an ingredient, no reason would exist to expect a change in adverse humanhealth effects. The anticipated effects are those previously observed inepidemiological studies. Thus, the cigarettes consumed in these studies define theranges of “indicator substances” characteristic of cigarettes. In making this statement,LSRO is aware that few studies of smoke from cigarettes typically consumed inCPS-I and CPS-II are currently available. The definition of the variance of indicatorsubstances in typical cigarettes is a research need. (See Chapter 12.)

The measure of smoking typically used by epidemiologists to quantify a person’ssmoking exposure history is pack-years. This exposure estimate is a crude measure,calculated as the average number of packs of cigarettes smoked in a day multipliedby the number of years a person has smoked. This approach has been criticized asit multiplies together two aspects of smoking (duration and amount) that may makedifferent contributions to the risk of disease from smoking. However, age adjustedpack-years is the commonly used measure, and it allows for some comparison ofeffects seen in different studies.

A distinction should be made between epidemiology studies and human clinical studies.Controlled human studies, which compare cigarettes, can provide valuable, potentiallypredictive information in a relatively short period of time that could in theory relateto specific ingredients. Clinical studies might reflect the effects of an ingredient onhuman smoking behavior (Chapter 10), as well as support estimates of smokeexposure (Chapter 7) and dosimetry (Chapter 8) of an added ingredient or specificcomponents of smoke.

13.4.2 Appropriate test substance

LSRO has not found animal models that reflect the adverse human health effects ofcigarette smoking, as observed epidemiologically. Although not wholly predictive ofhuman health effects, application of a diverse battery of experiments can potentially

Conclusions � 149


identify some effects. Experimental systems should analyze the appropriate testsubstance. (See Chapter 5.) Smokers inhale fresh whole smoke. Cigarette smokecondensate does not contain some volatile and semi-volatile compounds present inwhole smoke (World Health Organization, 1986). Witschi and coworkers (1997a)also found a significant toxicity associated with the gas phase. Therefore, testingthe particulate phase alone may miss some important effects.

Thomas and Vigerstad (1989) cite many early reports of exposing animals to wholecigarette smoke, in contrast to fractions of the smoke. Historically, investigatorspreferred rodents, including rats, mice, hamsters, and guinea pigs (Coggins, 1998;Witschi et al., 2000; Wright & Churg, 2002; Yamato et al., 1996). Witschi (1997b;2004) attempted to replicate the effects of whole smoke by subjecting strain A/Jmice for five months through whole-body exposure to a mixture of mainstream andsidestream smoke. Testing whole smoke in a relevant animal model might identify apotential adverse health effect associated with an added ingredient. Such datawould become important in evaluating potential changes in relative risk, if theingredient transfers, changes the physics, chemistry, or biological activity of smoke,or alters human exposure. However, LSRO does not understand how the endpointWitschi measures relates to human epidemiological studies.

No chemical components of smoke explain the human health effects seen in smokers.However, a significant change in the presence of smoke substances, which relate tothe addition of an ingredient, could signal a change in the potential for disease.

LSRO declines to identify specific components of smoke that would warrant particularattention. Chapter 6 outlined an approach to identify changes induced by addition ofan ingredient on key substances in smoke, which included nicotine, ‘tar’, water,carbon monoxide, and other substances. Internationally validated techniques areavailable and are widely used for these components (International Organization forStandardization, 1995; 1999; 2000a; 2000b). The potencies of the identifiedcarcinogens in cigarette smoke do not sum to the potency of cigarette smoke (Fowleset al., 2000; Menzie Cura & Associates, 1999).

13.5 LIMITATIONS

LSRO’s scientific criteria only apply to the adverse effects of added ingredients tothe smokers themselves. The criteria do not apply to the adverse effects of ingredientsadded to cigarettes on non-smokers, for example through the inhalation ofenvironmental tobacco smoke or the potential effect of added ingredients on thedeveloping fetus of a smoking mother. This emphasis does not discount the importanceof these issues. (See Appendix F.) However, the potential effects of addedingredients on non-smokers are not part of the scope of this report.



LSRO lacks adequate experimental data to determine the extent to which conclusionsmade from an evaluation of an ingredient added to cigarettes from a single cigarettetype can be applied when the tobacco substrate changes. Ingredients or pyrolysisproducts that transfer into smoke introduce a new substance. A unique signal is likelyto remain unique regardless of the kind of tobacco (e.g., menthol transfer can onlycome from the addition of menthol, or theobromine transfer from the addition of cocoa).

However, for the second criterion, which includes a change in smoke composition,tobacco substrate becomes more of a problem. At a particular exposure, an ingredientmight not change smoke composition, given one tobacco, yet the same ingredientcould change smoke composition when added to a different tobacco. Chemicaltesting to determine whether the tobacco substrate alters the relationship betweenthe ingredient added and indicator substances produced, lies well within the state-of-the-art. Such studies could generate conclusions about the effects of an ingredient.LSRO will limit conclusions to the cigarettes tested, unless additional information isavailable that demonstrate that similar effects on smoke composition are seenregardless of tobacco substrate.

13.6 SUMMARY

There are limits to the ability of many conventional toxicological tests, especiallyanimal studies, to discern the effects of low concentrations of a substance in acomplex mixture. However, in LSRO’s view, application of a diverse, integrativetesting program incorporating current and novel methodologies can inform of thepotential adverse health effects of adding ingredients to cigarettes. No single testpredicts the range of health effects associated with cigarette smoking.

LSRO is not setting a guideline about the relative risk of smoking. The intent of thisreport is to demonstrate LSRO’s approach to testing ingredients. LSRO seeks toencourage the development of methods to evaluate ingredients added to cigarettes.

LSRO has not followed standard regulatory approaches, like FDA’s Redbook 2000:Toxicological Principles for the Safety Assessment of Food Ingredients (1993).The Redbook 2000 toxicology principles do not address the background toxicity ofsmoke or the inhalation route of administration. Therefore, Redbook 2000 cannotapply directly to this evaluation. Further, Redbook 2000 contemplates testing ofpotential food additives in isolation. LSRO’s approach calls for testing within thematrix of cigarette smoke. For a test mentioned in this report, for which Redbook2000 guidance exists, the same principles apply after the method is adapted toinhalation as the route of exposure.

LSRO’s objective is not to seek additives that make cigarettes safer; rather LSROseeks to demonstrate whether an ingredient changes the adverse human health

Conclusions � 151


effects of cigarette smoking. Conclusions will be drawn from the data available forevaluation, leading to the outcomes described in Chapter 11. Submission of datawhich proves insufficient or inconclusive, will lead LSRO to return the submissionand explain what additional testing is necessary to complete the evaluation. Evaluationof all of the data, initially submitted and later requested, will lead to a conclusion thatthe data submitter might prefer not to use the ingredient or that the ingredient has ascientific rationale permitting the use in cigarettes up to some stated amount withouta change in adverse health effects. LSRO will publish a report stating the conclusionof an evaluation.


Abdel-Rahman, S. M. & Kauffman, R. E. (2004) The integration of pharmacokineticsand pharmacodynamics: understanding dose-response. Annu. Rev. Pharmacol.Toxicol. 44: 111-136.

Adams, P. I. (1976) Changes in personal smoking habits brought about by changesin cigarette smoke yield. In: Proceedings of the 6th International Tobacco ScientificCongress, Tokyo, November 14-20, 1976, pp. 102-108.

Adler, K. B. (1986) Mucin secretion by explants of respiratory tissue in vitro. In: InVitro Models of Respiratory Epithelium. (Schiff, L. J., ed.) Boca Raton, FL: CRCPress, pp. 27-50.

Ahijevych, K., Gillespie, J., Demirci, M. & Jagadeesh, J. (1996) Menthol andnonmenthol cigarettes and smoke exposure in black and white women. Pharmacol.Biochem. Behav. 53: 355-360.

Alarie, Y. (1981) Toxicological evaluation of airborne chemical irritants and allergensusing repiratory reflex reactions. In: Proceedings of the Inhalation Toxicologyand Technology Symposium. (Sponsored by Upjohn Company, Kalamazoo,Michigan, October 23-24, 1980). (Leong, B. K. J., ed.) Ann Arbor, MI: Ann ArborScience, pp. 207-231.

Andersson, G., Vala, E. K. & Curvall, M. (1997) The influence of cigaretteconsumption and smoking machine yields of tar and nicotine on the nicotine uptakeand oral mucosal lesions in smokers. J. Oral Pathol. Med. 26: 117-123.

Anonymous. (1998) Master Settlement Agreement. Available at http://caag.state.ca.us/tobacco/index.htm. Accessed 9-8-2004.

Applegate, M. L., Moore, M. M., Broder, C. B., Burrell, A., Juhn, G., Kasweck, K.L., Lin, P. F., Wadhams, A. & Hozier, J. C. (1990) Molecular dissection of mutationsat the heterozygous thymidine kinase locus in mouse lymphoma cells. Proc. Natl.Acad. Sci. U. S. A. 87: 51-55.

ASTM International. (2004) Annual Book of ASTM Standards. Available at http://www.astm.org. Accessed 3-3-2004.

Auletta, A. E., Dearfield, K. L. & Cimino, M. C. (1993) Mutagenicity test schemesand guidelines: U.S. EPA Office of Pollution Prevention and Toxics and Office ofPesticide Programs. Environ. Mol. Mutagen. 21: 38-45.

14LITERATURE CITATIONS

Literature Citations � 153


Avalos, J. T., Lawlor, T. E., Fowler, K. W., Bombick, B. R. & Doolittle, D. J. (2001)A comparison of the effectiveness of Salmonella strains for detecting mutagenicityof a cigarette smoke condensate. Environ. Mol. Mutagen. 37 Suppl 32: 19.

Bach, P. B., Kattan, M. W., Thornquist, M. D., Kris, M. G., Tate, R. C., Barnett, M.J., Hsieh, L. J. & Begg, C. B. (2003) Variations in lung cancer risk among smokers.J. Natl. Cancer Inst. 95: 470-478.

Baker, R. R. (1999) Smoking chemistry. In: Tobacco. Production, Chemistry andTechnology. (Davis, D. L. & Nielsen, M. T., eds.) Malden, MA: Blackwell ScienceInc., pp. 398-439.

Baker, R. R. & Bishop, L. J. (2004) The pyrolysis of tobacco ingredients. J. Anal.Appl. Pyrol. 71: 223-311.

Baker, R. R., Pereira da Silva, J. R. & Smith, G. (2004a) The effect of tobaccoingredients on smoke chemistry. Part I: Flavourings and additives. Food Chem.Toxicol. 42: 3-37.

Baker, R. R., Pereira da Silva, J. R. & Smith, G. (2004b) The effect of tobaccoingredients on smoke chemistry. Part II: Casing ingredients. Food Chem. Toxicol.42: 39-52.

Bartsch, H., Caporaso, N., Coda, M., Kadlubar, F., Malaveille, C., Skipper, P., Talaska,G., Tannenbaum, S. R. & Vineis, P. (1990) Carcinogen hemoglobin adducts, urinarymutagenicity, and metabolic phenotype in active and passive cigarette smokers. J.Natl. Cancer Inst. 82: 1826-1831.

Battig, K., Buzzi, R. & Nil, R. (1982) Smoke yield of cigarettes and puffing behaviorin men and women. Psychopharmacology (Berl) 76: 139-148.

Benowitz, N. L. (1983) The use of biologic fluid samples in assessing tobaccosmoke consumption. In: Research Monograph No. 48. National Institute of DrugAbuse. Rockville, MD: U.S. Department of Health and Human Services, pp. 6-26.

Benowitz, N. L. (2001) Compensatory smoking of low-yield cigarettes. In: RisksAssociated with Smoking Cigarettes With Low Machine-Measured Yields ofTar and Nicotine. Smoking and Tobacco Control. Monograph No. 13. (Burns,D. M., Benowitz, N. L. & Amacher, R. H., eds.) Bethesda, MD: National Institutesof Health, National Cancer Institute, NIH Pub. No. 02-5074, pp. 39-64.

Benowitz, N. L. (1998) Nicotine Safety and Toxicity. New York: Oxford UniversityPress.

Benowitz, N. L. (1999) Biomarkers of environmental tobacco smoke exposure.Environ. Health Perspect. 107 Suppl 2: 349-355.

Benowitz, N. L. & Jacob, P., III (1994) Metabolism of nicotine to cotinine studied bya dual stable isotope method. Clin. Pharmacol. Ther. 56: 483-493.



Benowitz, N. L. & Jacob, P., III (2000) Effects of cigarette smoking andcarbon monoxide on nicotine and cotinine metabolism. Clin. Pharmacol. Ther.67: 653-659.

Benowitz, N. L. & Jacob, P., III (1984) Daily intake of nicotine during cigarettesmoking. Clin. Pharmacol. Ther. 35: 499-504.

Benowitz, N. L. & Jacob, P., III (1993) Nicotine and cotinine eliminationpharmacokinetics in smokers and nonsmokers. Clin. Pharmacol. Ther.53: 316-323.

Benowitz, N. L., Jacob, P., III, Denaro, C. & Jenkins, R. (1991) Stable isotopestudies of nicotine kinetics and bioavailability. Clin. Pharmacol. Ther. 49: 270-277.

Benowitz, N. L., Jacob, P., III, Jones, R. T. & Rosenberg, J. (1982a) Interindividualvariability in the metabolism and cardiovascular effects of nicotine in man. J.Pharmacol. Exp. Ther. 221: 368-372.

Benowitz, N. L., Kuyt, F. & Jacob, P., III (1982b) Circadian blood nicotineconcentrations during cigarette smoking. Clin. Pharmacol. Ther. 32: 758-764.

Benowitz, N. L., Kuyt, F., Jacob, P., III, Jones, R. T. & Osman, A. L. (1983) Cotininedisposition and effects. Clin. Pharmacol. Ther. 34: 604-611.

Benowitz, N. L., Porchet, H. & Jacob, P., III (1989) Nicotine dependence andtolerance in man: pharmacokinetic and pharmacodynamic investigations. Prog. BrainRes. 79: 279-287.

Benowitz, N. L., Porchet, H. & Jacob, P., III (1990) Pharmacokinetics, metabolism,and pharmacodynamics of nicotine. In: Nicotine Psychopharmacology: Molecular,Cellular, and Behavioral Aspects. (Wonnacott, S., Russell, M. A. H. & Stolerman,I. P., eds.) New York: Oxford University Press, pp. 112-157.

Bentrovato, B., Porter, A., Youssef, M. & Dunn, P. J. (1995) Variations in tar,nicotine and carbon monoxide deliveries obtained by smokers of the same brand. In:Proceedings of the CORESTA Smoke and Technology Groups Meeting, Vienna,Austria. pp. 151-165.

Biglan, A., Duncan, T. E., Ary, D. V. & Smolkowski, K. (1995) Peer and parentalinfluences on adolescent tobacco use. J. Behav. Med. 18: 315-330.

Bombick, B. R., Avalos, J. T., Putnam, K. P., Bowman, D. L., Mabe, J. B., Fowler,K. W., Bombick, D. W., Morgan, W. T., & Doolittle, D. J. (2001) Comparativestudies on the genotoxic and cytotoxic potential of mainstream smoke condensatefrom menthol and non-menthol cigarettes which burn or primarily heat tobacco. In:55th Tobacco Science Research Conference Program Booklet and Abstracts.Available at http://legacy.library.ucsf.edu/tid/uvy51c00. Accessed 10-23-2002.



Bombick, B. R., Murli, H., Avalos, J. T., Bombick, D. W., Morgan, W. T., Putnam,K. P. & Doolittle, D. J. (1997a) Chemical and biological studies of a new cigarettethat primarily heats tobacco. Part 2. In vitro toxicology of mainstream smokecondensate. Food Chem. Toxicol. 36: 183-190.

Bombick, B. R., Murli, H., Bombick, D., Morgan, W., & Doolittle, D. J. (1995)Comparative studies on the genotoxic potential of mainstream smoke condensatefrom cigarettes which burn or primarily heat tobacco. [Abstract presented at TheInternational Congress of Toxicology VII, July 2-5, 1995]. Available at http://legacy.library.ucsf.edu/tid/usb81c00. Accessed 3-19-2004.

Bombick, D. W., Ayres, P. H. & Doolittle, D. J. (1997b) Cytotoxicity assessment ofwhole smoke and vapor phase of mainstream and sidestream smoke from threeKentucky reference cigarettes. Toxicol. Methods 7: 177-190.

Bombick, D. W., Ayres, P. H., Putnam, K., Bombick, B. R. & Doolittle, D. J. (1998a)Chemical and biological studies of a new cigarette that primarily heats tobacco. Part3. In vitro toxicity of whole smoke. Food Chem. Toxicol. 36: 191-197.

Bombick, D. W., Bombick, B. R., Ayres, P. H., Putnam, K., Avalos, J., Borgerding,M. F. & Doolittle, D. J. (1997c) Evaluation of the genotoxic and cytotoxic potentialof mainstream whole smoke and smoke condensate from a cigarette containing anovel carbon filter. Fundam. Appl. Toxicol. 39: 11-17.

Bombick, D. W., Putnam, K. & Doolittle, D. J. (1998b) Comparative cytotoxicitystudies of smoke condensate from different types of cigarettes and tobaccos. Toxicol.In Vitro 12: 241-249.

Borenfreund, E. & Puerner, J. A. (1985) Toxicity determined in vitro by morphologicalalterations and neutral red absorption. Toxicol. Lett. 24: 119-124.

Bos, P. M., Busschers, M. & Arts, J. H. (2002) Evaluation of the sensory irritationtest (Alarie test) for the assessment of respiratory tract irritation. J. Occup. Environ.Med. 44: 968-976.

Bottoms, S. F., Judge, N. E., Kuhnert, P. M. & Sokol, R. J. (1982) Thiocyanate anddrinking during pregnancy. Alcohol Clin. Exp. Res. 6: 391-395.

Bridges, R. B., Combs, J. G., Humble, J. W., Turbek, J. A., Rehm, S. R. & Haley, N.J. (1990) Puffing topography as a determinant of smoke exposure. Pharmacol.Biochem. Behav. 37: 29-39.

Brodie, B. B., Axelrod, J., Cooper, J. R., Gaudette, L., La Du, B. N., Mitoma, C. &Udenfriend, S. (1955) Detoxication of drugs and other foreign compounds by livermicrosomes. Science 121: 603-604.

Brooks, D. R., Palmer, J. R., Strom, B. L. & Rosenberg, L. (2003) Menthol cigarettesand risk of lung cancer. Am. J. Epidemiol. 158: 609-616.



Brown, B., Kolesar, J., Lindberg, K., Meckley, D., Mosberg, A. & Doolittle, D.(1998) Comparative studies of DNA adduct formation in mice following dermalapplication of smoke condensates from cigarettes that burn or primarily heat tobacco.Mutat. Res. 414: 21-30.

Brown, R. P., Delp, M. D., Lindstedt, S. L., Rhomberg, L. R. & Beliles, R. P. (1997)Physiological parameter values for physiologically based pharmacokinetic models.Toxicol. Ind. Health 13: 407-484.

Buchholz, B. & Mueller, C. (2002) Isotopic tracing of fuel components in combustionproducts. Available at http://www.ott.doe.gov/motor_fuels.shtml. Accessed3-11-2004.

Byrd, D. M. & Cothern, C. R. (2000) Introduction to Risk Analysis. Rockville,MD: Government Institutes.

Byrd, G. D., Davis, R. A., Caldwell, W. S., Robinson, J. H. & deBethizy, J. D.(1998) A further study of FTC yield and nicotine absorption in smokers.Psychopharmacology (Berl) 139: 291-299.

Byrd, G. D., Robinson, J. H., Caldwell, W. S. & deBethizy, J. D. (1994) Nicotineuptake and metabolism in smokers. Rec. Adv. Tob. Sci. 20: 5-31.

Calafat, A. M., Polzin, G. M., Saylor, J., Richter, P., Ashley, D. L. & Watson, C. H.(2004) Determination of tar, nicotine, and carbon monoxide yields in the mainstreamsmoke of selected international cigarettes. Tob. Control 13: 45-51.

Caporaso, N., Landi, M. T. & Vineis, P. (1991) Relevance of metabolic polymorphismsto human carcinogenesis: evaluation of epidemiologic evidence. Pharmacogenetics1: 4-19.

Caraballo, R., Giovino, G. & Pechacek, T. (2004) Self-reported cigarette smokingvs. serum cotinine among U.S. adolescents. Nicotine Tob. Res. 6: 19-25.

Carmines, E. L. (2002) Evaluation of the potential effects of ingredients added tocigarettes. Part 1: Cigarette design, testing approach, and review of results. FoodChem. Toxicol. 40: 77-91.

Carmines, E. L., Gaworski, C. L., Faqi, A. S. & Rajendran, N. (2003) In uteroexposure to 1R4F reference cigarette smoke: evaluation of developmental toxicity.Toxicol. Sci. 75: 134-147.

Carpenter, C. L., Jarvik, M. E., Morgenstern, H., McCarthy, W. J. & London, S. J.(1999) Mentholated cigarette smoking and lung-cancer risk. Ann. Epidemiol. 9:114-120.

Cashman, J. R., Park, S. B., Yang, Z. C., Wrighton, S. A., Jacob, P., III & Benowitz,N. L. (1992) Metabolism of nicotine by human liver microsomes: stereoselectiveformation of trans-nicotine N′-oxide. Chem. Res. Toxicol. 5: 639-646.



Cashman, J. R., Perotti, B. Y., Berkman, C. E. & Lin, J. (1996) Pharmacokineticsand molecular detoxication. Environ. Health Perspect. 104 Suppl 1: 23-40.

Chepiga, T. A., Morton, M. J., Murphy, P. A., Avalos, J. T., Bombick, B. R., Doolittle,D. J., Borgerding, M. F. & Swauger, J. E. (2000) A comparison of the mainstreamsmoke chemistry and mutagenicity of a representative sample of the US cigarettemarket with two Kentucky reference cigarettes (K1R4F and K1R5F). Food Chem.Toxicol. 38: 949-962.

Cholerton, S., McCracken, N. W. & Idle, J. R. (1993) Sources of inter-individualvariability in nicotine pharmacokinetics. In: Nicotine and Related Alkaloids:Absorption, Distribution, Metabolism and Excretion. (Gorrod, J. & Wahren, J.,eds.) New York: Chapman & Hall, pp. 219-245.

Clark, P., Gardiner, P., Djordjevic, M., Leischow, S. & Robinson, R. (2004) Mentholcigarettes: setting the research agenda. Nicotine Tob. Res. 6 Suppl 1: S5-S9.

Clark, P. I., Gautam, S. & Gerson, L. W. (1996) Effect of menthol cigarettes onbiochemical markers of smoke exposure among black and white smokers. Chest110: 1194-1198.

Clive, D., Johnson, K. O., Spector, J. F., Batson, A. G. & Brown, M. M. (1979)Validation and characterization of the L5178Y/TK+/- mouse lymphoma mutagenassay system. Mutat. Res. 59: 61-108.

Clive, D., McCuen, R., Spector, J. F., Piper, C. & Mavournin, K. H. (1983) Specificgene mutations in L5178Y cells in culture. Mutat. Res. 115: 225-251.

Coggins, C. R. (2002) A minireview of chronic animal inhalation studies withmainstream cigarette smoke. Inhal. Toxicol. 14: 991-1002.

Coggins, C. R., Haroz, R. K., Lam, R. & Morgan, K. T. (1982a) The tumourigenicityof smoke condensates from cigarettes containing different amounts of Cytrel®, asassessed by mouse skin painting. Toxicology 23: 177-185.

Coggins, C. R. E. (1998) A review of chronic inhalation studies with mainstreamcigarette smoke in rats and mice. Toxicol. Pathol. 26: 307-314.

Coggins, C. R. E., Lam, R. & Morgan, K. T. (1982b) Chronic inhalation study inrats, using cigarettes containing different amounts of Cytrel® tobacco supplement.Toxicology 22: 287-296.

Cole, J., Muriel, W. J. & Bridges, B. A. (1986) The mutagenicity of sodium fluorideto L5178Y [wild-type and TK+/- (3.7.2c)] mouse lymphoma cells. Mutagenesis 1:157-167.

Combes, R. D., Stopper, H. & Caspary, W. J. (1995) The use of L5178Y mouselymphoma cells to assess the mutagenic, clastogenic and aneugenic properties ofchemicals. Mutagenesis 10: 403-408.



Comer, A. K. & Creighton, D. E. (1978) The effect of experimental conditions onsmoking behavior. In: Smoking Behaviour. Physiological and PsychologicalInfluences. (Thornton, R. E., ed.) London: Churchill Livingstone, pp. 76-86.

Connolly, G. N., Wayne, G. D., Lymperis, D. & Doherty, M. C. (2000) How cigaretteadditives are used to mask environmental tobacco smoke. Tob. Control 9: 283-291.

Cooper, J. A., Saracci, R. & Cole, P. (1979) Describing the validity of carcinogenscreening tests. Br. J. Cancer 39: 87-89.

CORESTA. (2004) CORESTA Recommended Method No. 58. Determination ofbenzo(a)pyrene in cigarette mainstream smoke - gas chromatography - massspectrometry method. Available at http://www.coresta.org/Recommended_Methods/CRM_58.pdf. Accessed 5-26-2004.

Corrigall, W. A., Coen, K. M. & Adamson, K. L. (1994) Self-administered nicotineactivates the mesolimbic dopamine system through the ventral tegmental area. BrainRes. 653: 278-284.

Creighton, D. E. & Lewis, P. H. (1978) The effect of different cigarettes on humansmoking patterns. In: Smoking Behaviour. Physiological and PsychologicalInfluences. (Thornton, R. E., ed.) London: Churchill Livingstone, pp. 289-300.

Dalbey, W. E., Nettesheim, P., Griesemer, R., Caton, J. E. & Guerin, M. R. (1980)Chronic inhalation of cigarette smoke by F344 rats. J. Natl. Cancer Inst.64: 383-390.

Davies, H. M. & Vaught, A. (1990) The Reference Cigarette. Lexington, KY: TheUniversity of Kentucky Printing Service.

Davies, R. F., Lee, P. N. & Rothwell, K. (1974) A study of the dose response ofmouse skin to cigarette smoke condensate. Br. J. Cancer 30: 146-156.

Davis, S. G., Law, C. K. & Wang, H. (1999) Propyne pyrolysis in a flow reactor: Anexperimental, RRKM, and detailed kinetic modeling study. J. Phys. Chem. A 103:5889-5899.

De Flora, S., Balansky, R., Scatolini, L., Di Marco, C., Gasparini, L., Orlando, M. &Izzotti, A. (1996) Adducts to nuclear DNA and mitochondrial DNA as biomarkers inchemoprevention. IARC Sci. Publ. 291-301.

De Flora, S., D’Agostini, F., Balansky, R., Camoirano, A., Bennicelli, C., Bagnasco,M., Cartiglia, C., Tampa, E., Longobardi, M. G., Lubet, R. A. & Izzotti, A. (2003)Modulation of cigarette smoke-related end-points in mutagenesis and carcinogenesis.Mutat. Res. 523-524: 237-252.

Dearfield, K. L., Auletta, A. E., Cimino, M. C. & Moore, M. M. (1991) Considerationsin the U.S. Environmental Protection Agency’s testing approach for mutagenicity.Mutat. Res. 258: 259-283.



Degawa, M., Stern, S. J., Martin, M. V., Guengerich, F. P., Fu, P. P., Ilett, K. F.,Kaderlik, R. K. & Kadlubar, F. F. (1994) Metabolic activation and carcinogen-DNAadduct detection in human larynx. Cancer Res. 54: 4915-4919.

DeGeorge, J. J., Ahn, C. H., Andrews, P. A., Brower, M. E., Choi, Y. S., Chun, M.Y., Du, T., Lee-Ham, D. Y., McGuinn, W. D., Pei, L., Sancilio, L. F., Schmidt, W.,Sheevers, H. V., Sun, C. J., Tripathi, S., Vogel, W. M., Whitehurst, V., Williams, S. &Taylor, A. S. (1997) Considerations for toxicology studies of respiratory drug products.Regul. Toxicol. Pharmacol. 25: 189-193.

Diamond, G. A. (1986) Selection bias and the evaluation of diagnostic tests. [Author’sreply]. J. Chronic. Dis. 39: 359-360.

Djordjevic, M. V., Melikian, A., Szeliga, J., Stellman, S. D., Chen, S., Muscat, J. E.& Richie, J. P., Jr. (2002) Puffing characteristics and dosages of mainstream smokecomponents among Black and White smokers of regular and mentholated cigarettes.[Poster presentation at the First Conference on Menthol Cigarettes, March 21-22,2002, Atlanta, GA].

Doolittle, D. J., Lee, C. K., Ivett, J. L., Mirsalis, J. C., Riccio, E., Rudd, C. J.,Burger, G. T. & Hayes, A. W. (1990) Comparative studies on the genotoxic activityof mainstream smoke condensate from cigarettes which burn or only heat tobacco.Environ. Mol. Mutagen. 15: 93-105.

Douglas, C. G., Haldane, J. S. & Haldane, J. B. S. (1912) The laws of combinationof hæmoglobin with carbon monoxide and oxygen. J. Physiol. (Lond. ) 44: 275-304.

Doull, J., Frawley, J. P., George, W., Loomis, T., Squire, R. A., & Taylor, S. L.(1998) A safety assessment of ingredients added to tobacco in the manufacture ofcigarettes. Available at http://legacy.library.ucsf.edu/tid/vbd21c00. Accessed 7-7-2003.

Doull, J., Frawley, J. P., George, W. J., Loomis, T. A., Squire, R. A. & Taylor, S. L.(1994) Cigarette Ingredients. A Complete List and Background. Winston-Salem,NC: R.J. Reynolds Tobacco Company.

Eccles, R. (1994) Menthol and related cooling compounds. J. Pharm. Pharmacol.46: 618-630.

ECLIPSE Expert Panel (2000) A safer cigarette? A comparative study. A consensusreport. Inhal. Toxicol. 12: 1-48.

Federal Trade Commission. (1967) FTC to begin cigarette testing. News releaseAugust 1, 1967. Available at http://www.pmintl.com/tobacco_issues/link55.html.Accessed 12-12-2001.

Feyerabend, C. & Russell, M. A. (1979) Improved gas chromatographic methodand micro-extraction technique for the measurement of nicotine in biological fluids.J. Pharm. Pharmacol. 31: 73-76.



Fisher, P. (1999) Cigarette manufacture. 11A. Tobacco blending. In: Tobacco.Production, Chemistry and Technology. (Davis, D. L. & Nielsen, M. T., eds.)Malden, MA: CORESTA, Blackwell Science, Inc., pp. 346-352.

Fowles, J. (2001) Chemical Factors Influencing the Addictiveness and Attractivenessof Cigarettes in New Zealand. Prepared as Part of a New Zealand Ministry ofHealth Contract for Scientific Services.

Fowles, J., Bates, M. & Noiton, D. (2000) The Chemical Constitutents in Cigarettesand Cigarette Smoke: Priorities for Harm Reduction. A Report to the New ZealandMinistry of Health. Epidemiology and Toxicology Group, Porirua, New Zealand.

Freedman, D. A., Gold, L. S. & Lin, T. H. (1996) Concordance between rats andmice in bioassays for carcinogenesis. Regul. Toxicol. Pharmacol. 23: 225-232.

Freund, K. M., Belanger, A. J., D’Agostino, R. B. & Kannel, W. B. (1993) Thehealth risks of smoking. The Framingham Study: 34 years of follow-up. Ann.Epidemiol. 3: 417-424.

Furberg, A. H. & Ambrosone, C. B. (2001) Molecular epidemiology, biomarkersand cancer prevention. Trends Mol. Med. 7: 517-521.

Gabrielsson, J. & Bondesson, U. (1987) Constant-rate infusion of nicotine andcotinine. I. A physiological pharmacokinetic analysis of the cotinine disposition, andeffects on clearance and distribution in the rat. J. Pharmacokinet. Biopharm.15: 583-599.

Gabrielsson, J. & Gumbleton, M. (1993) Kinetics of nicotine and its metabolites inanimals. In: Nicotine and Related Alkaloids: Absorption, Distribution,Metabolism and Excretion. (Gorrod, J. & Wahren, J., eds.) New York: Chapman& Hall, pp. 181-196.

Gager, F. L., Jr., Nedlock, J. W. & Martin, W. J. (1971a) Tobacco additives andcigarette smoke. I. Transfer of D-glucose, sucrose, and their degradation productsto the smoke. Carbohydr. Res. 17: 327-333.

Gager, F. L., Jr., Nedlock, J. W. & Martin, W. J. (1971b) Tobacco additives andcigarette smoke. II. Organic, gas-phase products from D-glucose and sucrose.Carbohydr. Res. 17: 335-339.

Galeazzi, R. L., Daenens, P. & Gugger, M. (1985) Steady-state concentration ofcotinine as a measure of nicotine-intake by smokers. Eur. J. Clin. Pharmacol.28: 301-304.

Gardner, D. E. (2000) Tobacco smoke. In: Pulmonary Immunotoxicology. (Cohen,M. D., Zelikoff, J. T. & Schlesinger, R. B., eds.) Boston: Kluwer AcademicPublishers, pp. 387-409.



Garfinkel, L. (1979) Changes in the cigarette consumption of smokers in relation tochanges in tar/nicotine content of cigarettes smoked. Am. J. Public Health69: 1274-1276.

Gaworski, C. L., Dozier, M. M., Gerhart, J. M., Rajendran, N., Brennecke, L. H.,Aranyi, C. & Heck, J. D. (1997) 13-week inhalation toxicity study of menthol cigarettesmoke. Food Chem. Toxicol. 35: 683-692.

Gaworski, C. L., Dozier, M. M., Heck, J. D., Gerhart, J. M., Rajendran, N., David,R. M., Brennecke, L. H. & Morrissey, R. (1998) Toxicologic evaluation of flavoringredients added to cigarette tobacco: 13-week inhalation exposures in rats. Inhal.Toxicol. 10: 357-381.

Gaworski, C. L., Heck, J. D., Bennett, M. B. & Wenk, M. L. (1999) Toxicologicevaluation of flavor ingredients added to cigarette tobacco: skin painting bioassay ofcigarette smoke condensate in SENCAR mice. Toxicology 139: 1-17.

Gelal, A., Jacob, P., III, Yu, L. & Benowitz, N. L. (1999) Disposition kinetics andeffects of menthol. Clin. Pharmacol. Ther. 66: 128-135.

Gilpin, E. A., Lee, L., Evans, N. & Pierce, J. P. (1994) Smoking initiation rates inadults and minors: United States, 1944-1988. Am. J. Epidemiol. 140: 535-543.

Glassman, A. H., Helzer, J. E., Covey, L. S., Cottler, L. B., Stetner, F., Tipp, J. E. &Johnson, J. (1990) Smoking, smoking cessation, and major depression. JAMA264: 1546-1549.

Goldstein, L. S., Weyand, E. H., Safe, S., Steinberg, M., Culp, S. J., Gaylor, D. W.,Beland, F. A. & Rodriguez, L. V. (1998) Tumors and DNA adducts in mice exposedto benzo[a]pyrene and coal tars: implications for risk assessment. Environ. HealthPerspect. 106 (Suppl 6): 1325-1330.

Gori, G. B. (1977) Toward Less Hazardous Cigarettes. Report 3. The Third Setof Experimental Cigarettes. National Cancer Institute, Smoking and HealthProgram. Publication No. (NIH)-77-1280. Bethesda, MD: National Institutes ofHealth.

Gori, G. B. (1974) Proceedings of the tobacco smoke inhalation workshop onexperimental methods in smoking and health research. Publication No. (NIH)75-906. Washington, DC: U.S. Department of Health, Education and Welfare.

Gori, G. B. (1980) Toward Less Hazardous Cigarettes. Report 4. The Fourth Setof Experimental Cigarettes. National Cancer Institute, Smoking and HealthProgram. Washington, DC: U.S. Department of Health, Education and Welfare.

Gori, G. B., Benowitz, N. L. & Lynch, C. J. (1986) Mouth versus deep airwaysabsorption of nicotine in cigarette smokers. Pharmacol. Biochem. Behav.25: 1181-1184.



Gori, G. B. & Lynch, C. J. (1985) Analytical cigarette yields as predictors of smokebioavailability. Regul. Toxicol. Pharmacol. 5: 314-326.

Goto, K., Maeda, S., Kano, Y. & Sugiyama, T. (1978) Factors involved in differentialGiemsa-staining of sister chromatids. Chromosoma 66: 351-359.

Gourlay, S. G. & Benowitz, N. L. (1997) Arteriovenous differences in plasmaconcentration of nicotine and catecholamines and related cardiovascular effectsafter smoking, nicotine nasal spray, and intravenous nicotine. Clin. Pharmacol.Ther. 62: 453-463.

Granella, M., Priante, E., Nardini, B., Bono, R. & Clonfero, E. (1996) Excretion ofmutagens, nicotine and its metabolites in urine of cigarette smokers. Mutagenesis11: 207-211.

Green, C. R. & Rodgman, A. (1996) The Tobacco Chemists’ Research Conference:A half century forum for advances in analytical methodology of tobacco and itsproducts. Rec. Adv. Tob. Sci. 22: 131-304.

Green, J. D., Chalmers, J. & Kinnard, P. J. (1989) The transfer of tobacco additivesto cigarette smoke. Beitr. Tabakforsch. Int. 14: 283-288.

Gritz, E. R., Nielsen, I. R. & Brooks, L. A. (1996) Smoking cessation and gender:the influence of physiological, psychological, and behavioral factors. J. Am. Med.Womens Assoc. 51: 35-42.

Gross, S. B. (1981) Regulatory guidelines for inhalation toxicity testing. In: InhalationToxicology. (Leong, B. K., ed.) Ann Arbor, MI.: Ann Arbor Science, pp. 279-298.

Guerin, M., Maddox, W. L. & Stokely, J. (1974) Tobacco smoke inhalation exposure:concepts and devices. In: Proceedings of the Tobacco Smoke InhalationWorkshop on Experimental Methods in Smoke and Heatlh Research. DHEWPub. No. (NIH) 75-906. (Gori, G. B., ed.) Washington, DC: U.S. Department ofHealth, Education and Welfare, pp. 31-48.

Gundisch, D. (2000) Nicotinic acetylcholine receptors and imaging. Curr. Pharm.Des. 6: 1143-1157.

Gutcho, S. (1972) Menthol flavor. In: Tobacco Flavoring Substances and Methods.Park Ridge, NJ: Noyes Data Corporation, pp. 2-24.

Haggard, H. W. & Greenberg, L. A. (1941) Concentration of menthol in smokefrom mentholated cigarettes. Arch. Otolaryngol. 33: 711-716.

Hammond, E. C. (1966) Smoking in relation to the death rates of one million menand women. Natl. Cancer Inst. Monogr. 19: 127-204.

Hammond, E. C., Garfinkel, L., Seidman, H. & Lew, E. A. (1976) “Tar” and nicotinecontent of cigarette smoke in relation to death rates. Environ. Res. 12: 263-274.



Harllee, G. C. & Leffingwell, J. C. (1979) Casing materials—cocoa (Part 1). Availableat http://legacy.library.ucsf.edu/tid/swo73d00. Accessed 2-13-0002.

Harris, R. E., Zang, E. A., Anderson, J. I. & Wynder, E. L. (1993) Race and sexdifferences in lung cancer risk associated with cigarette smoking. Int. J. Epidemiol.22: 592-599.

Health Canada Tobacco Control Programme. (2004) Tobacco reportingregulations. Available at http://www.hc-sc.gc.ca/hecs-secs/tobacco/legislation/prop_may_36b.html. Accessed 3-25-2004.

Healy, M. J. (1968) The disciplining of medical data. Br. Med. Bull. 24: 210-214.

Hebert, J. R. & Kabat, G. C. (1989) Menthol cigarette smoking and oesophagealcancer. Int. J. Epidemiol. 18: 37-44.

Hecht, S. S. (1999) Tobacco smoke carcinogens and lung cancer. J. Natl. CancerInst. 91: 1194-1210.

Hecht, S. S. (2002) Human urinary carcinogen metabolites: biomarkers forinvestigating tobacco and cancer. Carcinogenesis 23: 907-922.

Hecht, S. S., Rivenson, A., Braley, J., DiBello, J., Adams, J. D. & Hoffmann,D. (1986) Induction of oral cavity tumors in F344 rats by tobacco-specificnitrosamines and snuff. Cancer Res. 46: 4162-4166.

Heck, J. D., Gaworski, C. L., Rajendran, N. & Morrissey, R. L. (2002) Toxicologicevaluation of humectants added to cigarette tobacco: 13-week smoke inhalationstudy of glycerin and propylene glycol in Fischer 344 rats. Inhal. Toxicol.14: 1135-1152.

Henderson, R. F. (1995) Strategies for use of biological markers of exposure. Toxicol.Lett. 82-83: 379-383.

Hengen, N. & Hengen, M. (1978) Gas-liquid chromatographic determination ofnicotine and cotinine in plasma. Clin. Chem. 24: 50-53.

Henningfield, J. E., London, E. D. & Benowitz, N. L. (1990) Arterial-venousdifferences in plasma concentrations of nicotine after cigarette smoking. JAMA263: 2049-2050.

Herning, R. I., Jones, R. T., Bachman, J. & Mines, A. H. (1981) Puff volumeincreases when low-nicotine cigarettes are smoked. Br. Med. J. (Clin. Res. Ed.)283: 187-189.

Herning, R. I., Jones, R. T., Benowitz, N. L. & Mines, A. H. (1983) How a cigaretteis smoked determines blood nicotine levels. Clin. Pharmacol. Ther. 33: 84-90.



Höfer, I., Nil, R., Wyss, F. & Bättig, K. (1992) The contributions of cigarette yield,consumption, inhalation and puffing behaviour to the prediction of smoke exposure.Clin. Investig. 70: 343-351.

Hoffmann, D., Djordjevic, M. V. & Brunnemann, K. D. (1995) Changes in cigarettedesign and composition over time and how they influence the yields of smokeconstitutents. J. Smoking-Related Dis. 6: 9-23.

Hoffmann, D. & Hoffmann, I. (2001) The changing cigarette: chemical studies andbioassays. In: Risks Associated With Smoking Cigarettes With Low Machine-Measured Yields of Tar and Nicotine. Smoking and Tobacco Control.Monograph No. 13. (Burns, D. M., Benowitz, N. L. & Amacher, R. H., eds.)Bethesda, MD: National Institutes of Health, National Cancer Institute, NIH Pub.No. 02-5074, pp. 159-192.

Hoffmann, D., Hoffmann, I. & El-Bayoumy, K. (2001) The less harmful cigarette:A controversial issue. A tribute to Ernst L. Wynder. Chem. Res. Toxicol.14: 767-790.

Hoffmann, D., Melikian, A. A. & Brunnemann, K. D. (1991) Studies in tobaccocarcinogenesis. IARC Sci. Publ. 482-484.

Hoffmann, D., Wynder, E. L., Rivenson, A., LaVoie, E. J. & Hecht, S. S. (1983)Skin bioassays in tobacco carcinogenesis. Prog. Exp. Tumor Res. 26: 43-67.

Hollander, W. & Stober, W. (1986) Aerosols of smoke, respiratory physiology anddeposition. Arch. Toxicol. Suppl 9: 74-87.

Hozier, J., Sawyer, J., Moore, M., Howard, B. & Clive, D. (1981) Cytogeneticanalysis of the L5178Y/TK+/- leads to TK-/- mouse lymphoma mutagenesis assaysystem. Mutat. Res. 84: 169-181.

Huber, G. L., Mahajan, V. K. & Rubin, A. H. (1990) Theoretical and experimentallyquantifiable determinants of tobacco smoking behavior for the development ofsuccessful smoking cessation strategies. Cancer Detect. Prev. 14: 505-514.

Hughes, J. R., Hecht, S. S., Carmella, S. G., Murphy, S. E. & Callas, P. (2004)Smoking behaviour and toxin exposure during six weeks use of a potential reducedexposure product: Omni. Tob. Control 13: 175-179.

Hunt, J. (2002) Exhaled breath condensate: an evolving tool for noninvasive evaluationof lung disease. J. Allergy Clin. Immunol. 110: 28-34.

Institute of Medicine (2001) Clearing the Smoke. Assessing the Science Basefor Tobacco Harm Reduction. (Stratton, K., Shetty, P., Wallace, R. & Bondurant,S., eds.) Washington, DC: National Academy Press.



International Agency for Research on Cancer. (1985) Acetaldehyde. In: IARCMonographs on the Evaluation of Carcinogenic Risks to Humans. AllylCompounds, Aldehydes, Epoxides and Peroxides. Volume 36. Paris: InternationalAgency for Research on Cancer, pp. 101-132.

International Agency for Research on Cancer. (1995) Furan. In: IARC Monographson the Evaluation of Carcinogenic Risks to Humans. Dry Cleaning, SomeChlorinated Solvents and Other Industrial Chemicals. Volume 63. Paris:International Agency for Research on Cancer, pp. 393-407.

International Agency for Research on Cancer. (1982) Benzene. In: IARCMonographs on the Evaluation of Carcinogenic Risks to Humans. SomeIndustrial Chemicals and Dyestuffs. Volume 29. Paris: International Agency forResearch on Cancer, pp. 93-148.

International Agency for Research on Cancer. (1987) Acetaldehyde (Group 2B).In: IARC Monographs on the Evaluation of Carcinogenic Risks to Humans.Overall Evaluations of Carcinogenicity: An Updating of IARC MonographsVolumes 1-42. Supplement 7. Paris: International Agency for Research on Cancer,pp. 77-78.

International Agency for Research on Cancer. (2004) IARC classifies formaldehydeas carcinogenic to humans. [Re: Monograph 88. Formaldehyde, 2-butoxyethanoland 1-tert-butoxy-2-propanol (In press)]. Available at http://monographs.iarc.fr/.Accessed 8-10-2004.

International Conference on Harmonisation. (2004) International Conference onHarmonisation. Available at http://www.ich.org/UrlGrpServer.jser?@_ID=276&@_TEMPLATE=254. Accessed 3-22-2004.

International Conference on Harmonisation Steering Committee. (10-27-1994a)ICH Harmonized Tripartite Guideline. Pharmacokinetics: Guidance for RepeatedDose Tissue Distribution Studies. S3B. Available at http://www.ich.org/. Accessed10-23-2003.

International Conference on Harmonisation Steering Committee. (10-27-1994b) ICHHarmonised Tripartite Guideline. Genotoxicity: Note for Guidance on Toxicokinetics:The Assessment of Systemic Exposure in Toxicity Studies. Available at http://www.ich.org/pdfICH/s3a.pdf. Accessed 10-17-2002.

International Conference on Harmonisation Steering Committee. (7-19-1995) ICHHarmonised Tripartite Guideline. Guidance on Specific Aspects of RegulatoryGenotoxicity Tests for Pharmaceuticals. Available at http://www.ich.org/pdfICH/s2a.pdf. Accessed 10-17-2002.



International Conference on Harmonisation Steering Committee. (7-16-1997) ICHHarmonised Tripartite Guideline. Genotoxicity: A Standard Battery for GenotoxicityTesting of Pharmaceuticals. Available at http://www.ich.org/pdfICH/s2b.pdf.Accessed 10-17-2002.

International Organization for Standardization. (2000c) Routine Analytical Cigarette-Smoking Machine — Definitions and Standard Conditions. Report No. ISO3308:2000. Geneva, Switzerland: International Organization for Standardization.

International Organization for Standardization. (1995) Cigarettes—Determinationof carbon monoxide in the vapour phase of cigarette smoke—NDIR method.Report No. ISO 8454:1995. Geneva, Switzerland: International Organization forStandardization.

International Organization for Standardization. (1999) Cigarettes—Determinationof water in smoke condensates—Part 1: Gas-chromatographic method. ReportNo. ISO 10362-1:1999. Geneva, Switzerland: International Organization forStandardization.

International Organization for Standardization. (2000a) Cigarettes—Determinationof nicotine in smoke condensates—Gas-chromatographic method. Report No.ISO 10315:2000. Geneva, Switzerland: International Organization for Standardization.

International Organization for Standardization. (2000b) Cigarettes—Determinationof total and nicotine-free dry particulate matter using a routine analyticalsmoking machine. Report No. ISO 4387:2000. Geneva, Switzerland: InternationalOrganization for Standardization.

Jacob, P., III & Benowitz, N. L. (1993) Pharmacokinetics of (S)-nicotine andmetabolites in humans. In: Nicotine and Related Alkaloids: Absorption,Distribution, Metabolism and Excretion. (Gorrod, J. & Wahren, J., eds.) NewYork: Chapman & Hall, pp. 197-218.

Jager, W., Nasel, B., Nasel, C., Binder, R., Stimpfl, T., Vycudilik, W. & Buchbauer,G. (1996) Pharmacokinetic studies of the fragrance compound 1,8-cineol in humansduring inhalation. Chem. Senses 21: 477-480.

Jarvik, M. E., Tashkin, D. P., Caskey, N. H., McCarthy, W. J. & Rosenblatt, M. R.(1994) Mentholated cigarettes decrease puff volume of smoke and increase carbonmonoxide absorption. Physiol. Behav. 56: 563-570.

Jarvis, M. J., Tunstall-Pedoe, H., Feyerabend, C., Vesey, C. & Saloojee, Y. (1987)Comparison of tests used to distinguish smokers from nonsmokers. Am. J. PublicHealth 77: 1435-1438.

Jenkins, R. W., Jr., Newman, R. H. & Chavis, M. K. (1970) Cigarette smokeformation studies. II. Smoke distribution and mainstream pyrolytic composition ofadded 14C-menthol (U). Beitr. Tabakforsch. 5: 299-301.



Johnston, L. D., O’Malley, P. M., Bachman, J. G., & Schulenberg, J. E. (1999)Cigarette brand preferences among adolescents. Monitoring the future occasionalpaper 45. Available at http://legacy.library.ucsf.edu/tid/jhd55c00. Accessed3-10-2003.

Kabat, G. C. & Hebert, J. R. (1991) Use of mentholated cigarettes and lung cancerrisk. Cancer Res. 51: 6510-6513.

Kabat, G. C. & Hebert, J. R. (1994) Use of mentholated cigarettes and oropharyngealcancer. Epidemiology 5: 183-188.

Kaplan, E. L. & Meier, P. (1958) Nonparametric estimation from incompleteobservations. J. Am. Stat. Assoc. 54: 457-481.

Kato, S., Bowman, E. D., Harrington, A. M., Blomeke, B. & Shields, P. G. (1995)Human lung carcinogen-DNA adduct levels mediated by genetic polymorphisms invivo. J. Natl. Cancer Inst. 87: 902-907.

Kemp, P. M., Sneed, G. S., George, C. E. & Distefano, R. F. (1997) Postmortemdistribution of nicotine and cotinine from a case involving the simultaneousadministration of multiple nicotine transdermal systems. J. Anal. Toxicol.21: 310-313.

Kennedy, J. E. (1973) Biological activity of menthol and mentholated cigarettes.Available at http://legacy.library.ucsf.edu/tid/npb33f00. Accessed 4-1-2004.

Kiefer, J. E. & Touey, G. P. (1967) Filtration of tobacco smoke particles. In: Tobaccoand Tobacco Smoke. (Wynder, E. L. & Hoffmann, D., eds.) New York, NY:Academic Press Inc., pp. 545-575.

Kiefer, J. H., Zhang, Q., Kern, R. D., Yao, J. & Jursic, B. (1997) Pyrolysis of aromaticazines: Pyrazine, pyrimidine, and pyridine. J. Phys. Chem. A 101: 7061-7073.

Kinser, R. D., Carchman, R. A., Leyden, D. E., Sanders, E. B., Stavvert, R., Tricker,A. R., von Holt, K. & Walk, R.-A. (2001) Selection of biomarkers of exposure fora population study of U.S. adult smokers to cigarette smoke. [Poster presented at9th International Congress of Toxicology, July 8-12, 2001 Brisbane, Australia and atBiomarkers for Tobacco Exposure: Application to Clinical and EpidemiologicalStudies, Minneapolis, MN October 25-26, 2001.] Toxicology 164: 41.

Klaassen, C. D. (2001) Casarett & Doull’s Toxicology. The Basic Science ofPoisons. (Klaassen, C. D., ed.) 6th ed. New York: McGraw-Hill Companies, Inc.

Koopmans, J. R., Slutske, W. S., Heath, A. C., Neale, M. C. & Boomsma, D. I.(1999) The genetics of smoking initiation and quantity smoked in Dutch adolescentand young adult twins. Behav. Genet. 29: 383-393.

Kozlowski, L. T., Pope, M. A. & Lux, J. E. (1988) Prevalence of the misuse ofultra-low-tar cigarettes by blocking filter vents. Am. J. Public Health 78: 694-695.



Kozlowski, L. T., Rickert, W. S., Pope, M. A., Robinson, J. C. & Frecker, R. C.(1982) Estimating the yield to smokers of tar, nicotine, and carbon monoxide fromthe ‘lowest yield’ ventilated filter-cigarettes. Br. J. Addict. 77: 159-165.

Kreuzer, M., Boffetta, P., Whitley, E., Ahrens, W., Gaborieau, V., Heinrich, J., Jockel,K. H., Kreienbrock, L., Mallone, S., Merletti, F., Roesch, F., Zambon, P. & Simonato,L. (2000) Gender differences in lung cancer risk by smoking: a multicentre case-control study in Germany and Italy. Br. J. Cancer 82: 227-233.

Kriebel, D., Henry, J., Gold, J. C., Bronsdon, A. & Commoner, B. (1985) Themutagenicity of cigarette smokers’ urine. J. Environ. Pathol. Toxicol. Oncol. 6:157-169.

Kyerematen, G. A., Taylor, L. H., deBethizy, J. D. & Vesell, E. S. (1987) Radiometric-high-performance liquid chromatographic assay for nicotine and twelve of itsmetabolites. J. Chromatogr. 419: 191-203.

Lando, H. A. (1983) Data collection and questionnaire design: smoking cessation inadults. NIDA Res. Monogr. 48: 74-89.

Langone, J. J., Gjika, H. B. & Van Vunakis, H. (1973) Nicotine and its metabolites.Radioimmunoassays for nicotine and cotinine. Biochemistry 12: 5025-5030.

Lave, L. B. & Ennever, F. K. (1990) Toxic substances control in the 1990s: are wepoisoning ourselves with low-level exposures? Annu. Rev. Public Health 11: 69-87.

Leffondre, K., Abrahamowicz, M., Siemiatycki, J. & Rachet, B. (2002) Modelingsmoking history: a comparison of different approaches. Am. J. Epidemiol.156: 813-823.

Lena, C. & Changeux, J. P. (1993) Allosteric modulations of the nicotinic acetylcholinereceptor. Trends Neurosci. 16: 181-186.

Life Sciences Research Office (2004) Review of Ingredients Added to Cigarettes.Phase One: The Feasibility of Testing Ingredients Added to Cigarettes. (Byrd,D. M., ed.) Bethesda, MD: Life Sciences Research Office.

Lorillard. (1983) Consumer Attitude Study. [Presentation]. Available at http://legacy.library.ucsf.edu/tid/qwh41e00. Accessed 3-10-2003.

Lugton, W. G. D., Dyas, B. J., & Binns, R. (1978) Human volunteer smoking studieson mentholated cigarettes. BAT Report No. RD.1581 Restricted. Available at http://legacy.library.ucsf.edu/tid/ckm10f00. Accessed 12-30-2002.

Martonen, T. B. (1992) Deposition patterns of cigarette smoke in human airways.Am. Ind. Hyg. Assoc. J. 53: 6-18.

Martonen, T. B., Quan, L., Zhang, Z. & Musante, C. J. (2002) Flow simulation inthe human upper respiratory tract. Cell Biochem. Biophys. 37: 27-36.



Martonen, T. B. & Schroeter, J. D. (2003) Risk assessment dosimetry model forinhaled particulate matter: II. Laboratory surrogates (rat). Toxicol. Lett. 138: 133-142.

Massachusetts Department of Public Health. (2001) Massachusetts Tobacco ControlProgram. 105 CMR 655.000: Testing and reporting constituents of cigarette smoke.Available at http://www.state.ma.us/dph/mtcp. Accessed 3-13-2002.

Massey, E., Aufderheide, M., Koch, W., Lodding, H., Pohlmann, G., Windt, H., Jarck,P. & Knebel, J. W. (1998) Micronucleus induction in V79 cells after direct exposureto whole cigarette smoke. Mutagenesis 13: 145-149.

Matsuka, N., Furuno, K., Eto, K., Oishi, R. & Gomita, Y. (1997) Effects of cigarettesmoke inhalation on plasma diltiazem levels in rats. Methods Find. Exp. Clin.Pharmacol. 19: 173-179.

McCann, J., Choi, E., Yamasaki, E. & Ames, B. N. (1975) Detection of carcinogensas mutagens in the Salmonella/microsome test: assay of 300 chemicals. Proc. Natl.Acad. Sci. U. S. A. 72: 5135-5139.

McCarthy, W. J., Caskey, N. H., Jarvik, M. E., Gross, T. M. & Rosenblatt, M. R.(1995) Menthol vs nonmenthol cigarettes: Effects on smoking behavior. Am. J. PublicHealth 85: 67-72.

McGehee, D. S. & Role, L. W. (1995) Physiological diversity of nicotinic acetylcholinereceptors expressed by vertebrate neurons. Annu. Rev. Physiol. 57: 521-546.

Meckley, D., Hayes, J. R., Van Kampen, K. R., Mosberg, A. T. & Swauger, J. E.(2004) A responsive, sensitive, and reproducible dermal tumor promotion assay forthe comparative evaluation of cigarette smoke condensates. Regul. Toxicol.Pharmacol. 39: 135-149.

Menzie Cura & Associates. (1999) Estimating Risk to Cigarette Smokers FromSmoke Constituents in Proposed “Testing and Reporting of Constituents ofCigarette Smoke” Regulations. [Report Prepared for Massachusetts TobaccoControl Program]. Chelmsford, MA: Menzie Cura Associates, Inc.

Miller, G. E., Jarvik, M. E., Caskey, N. H., Segerstrom, S. C., Rosenblatt, M. R. &McCarthy, W. J. (1994) Cigarette mentholation increases smokers’ exhaled carbonmonoxide levels. Exp. Clin. Psychopharmacol. 2: 154-160.

Miller, L. G. (1990) Cigarettes and drug therapy: pharmacokinetic andpharmacodynamic considerations. Clin. Pharm. 9: 125-135.

Mitchell, A. D., Auletta, A. E., Clive, D., Kirby, P. E., Moore, M. M. & Myhr, B. C.(1997) The L5178Y/tk+/- mouse lymphoma specific gene and chromosomal mutationassay. A phase III report of the U.S. Environmental Protection Agency Gene-ToxProgram. Mutat. Res. 394: 177-303.



Montuschi, P. & Barnes, P. J. (2002) Analysis of exhaled breath condensate formonitoring airway inflammation. Trends Pharmacol. Sci. 23: 232-237.

Moore, M. M., Clive, D., Hozier, J. C., Howard, B. E., Batson, A. G., Turner, N. T.& Sawyer, J. (1985) Analysis of trifluorothymidine-resistant (TFTr) mutants ofL5178Y/TK+/- mouse lymphoma cells. Mutat. Res. 151: 161-174.

Müller, L., Röper, W., Dixon, M., Seeman, J. I. & Carmines, E. (2000) Commentary:It ain’t necessarily so. Beitr. Tabakforsch. Int. 19: 51-54.

Muscat, J. E., Richie, J. P., Jr. & Stellman, S. D. (2002) Mentholated cigarettes andsmoking habits in whites and blacks. Tob. Control 11: 368-371.

Nakajima, M., Kuroiwa, Y. & Yokoi, T. (2002) Interindividual differences in nicotinemetabolism and genetic polymorphisms of human CYP2A6. Drug Metab. Rev.34: 865-877.

Nakajima, M., Yamamoto, T., Nunoya, K., Yokoi, T., Nagashima, K., Inoue, K., Funae,Y., Shimada, N., Kamataki, T. & Kuroiwa, Y. (1996) Role of human cytochromeP4502A6 in C-oxidation of nicotine. Drug Metab. Dispos. 24: 1212-1217.

Nasel, C., Nasel, B., Samec, P., Schindler, E. & Buchbauer, G. (1994) Functionalimaging of effects of fragrances on the human brain after prolonged inhalation.Chem. Senses 19: 359-364.

National Institutes of Health. (1997) Monograph 8: Changes in Cigarette-RelatedDisease Risks and Their Implications for Prevention and Control. Bethesda,MD: National Institutes of Health, National Cancer Institute.

National Toxicology Program. (2002a) NTP Chemical Repository. D,L-Menthol.Available at http://ntp-server.niehs.nih.gov/cgi/iH_Indexes/ALL_SRCH/iH_ALL_SRCH_Frames.html. Accessed 11-14-2002.

National Toxicology Program. (2001a) The micronucleus test. Available at http://ntp-server.niehs.nih.gov/htdocs/Overviews/MicronucleusDoc.html. Accessed 3-4-2004.

National Toxicology Program. (2001b) The Salmonella mutagenicity test. Availableat http://ntp-server.niehs.nih.gov/htdocs/Overviews/SaDoc.html. Accessed 3-4-2004.

National Toxicology Program. (2002b) National Toxicology Program. Available athttp://ntp-server.niehs.nih.gov/. Accessed 11-14-2002.

Oberdorster, G. (1996) Significance of particle parameters in the evaluation ofexposure-dose-response relationships of inhaled particles. Inhal. Toxicol.8 Suppl: 73-89.

Organization for Economic Cooperation and Development. (1981) OECD Guideline413, subchronic inhalation toxicity: 90-day study. In: OECD Guidelines for TestingChemicals. Paris: Organization for Economic Cooperation and Development.



Organization for Economic Cooperation and Development. (1997) OECDguideline for testing of chemicals. Bacterial reverse mutation test. Available athttp://www.oecd.org/dataoecd/18/31/1948418.pdf. Accessed 7-15-2004.

Organization for Economic Cooperation and Development. (2004) Chemical testing:OECD guidelines for testing of chemicals - sections 1-4. Available at http://www.oecd.org/document/22/0,2340,en_2649_34377_1916054_1_1_1_1,00.html.Accessed 3-22-2004.

Osann, K. E., Anton-Culver, H., Kurosaki, T. & Taylor, T. (1993) Sex differences inlung-cancer risk associated with cigarette smoking. Int. J. Cancer 54: 44-48.

Ott, W. R. & Roberts, J. W. (1998) Everyday exposure to toxic pollutants. Sci. Am.278: 86-91.

Pankow, J. F. (2001) A consideration of the role of gas/particle partitioning in thedeposition of nicotine and other tobacco smoke compounds in the respiratory tract.Chem. Res. Toxicol. 14: 1465-1481.

Pankow, J. F., Barsanti, K. C. & Peyton, D. H. (2003) Fraction of free-base nicotinein fresh smoke particulate matter from the Eclipse “cigarette” by 1H NMRspectroscopy. Chem. Res. Toxicol. 16: 23-27.

Pauluhn, J. & Mohr, U. (2000) Inhalation studies in laboratory animals—currentconcepts and alternatives. Toxicol. Pathol. 28: 734-753.

Pavanello, S., Simioli, P., Carrieri, M., Gregorio, P. & Clonfero, E. (2002) Tobacco-smoke exposure indicators and urinary mutagenicity. Mutat. Res. 521: 1-9.

Perera, F. P. (1997) Environment and cancer: who are susceptible? Science278: 1068-1073.

Perfetti, T. A., Hopp, R., Borschke, A. J., Reid, J. R., Borgerding, M. F., Wilson,S. A. & Best, F. W. (1993) Recent advances in tobacco science: Highlights ofcurrent research on tobacco and tobacco chemistry. 47th Tobacco Chemists’ ResearchConference. Menthol and design of mentholated cigarettes. 18 October 1993. Rec.Adv. Tob. Sci. 19: 1-206.

Perneger, T. V. (2001) Sex, smoking, and cancer: a reappraisal. J. Natl. CancerInst. 93: 1600-1602.

Perry, P. & Wolff, S. (1974) New Giemsa method for the differential staining ofsister chromatids. Nature 251: 156-158.

Peto, R., Darby, S., Deo, H., Silcocks, P., Whitley, E. & Doll, R. (2000) Smoking,smoking cessation, and lung cancer in the UK since 1950: combination of nationalstatistics with two case-control studies. Br. Med. J. 321: 323-329.



Peto, R., Pike, M. C., Armitage, P., Breslow, N. E., Cox, D. R., Howard, S. V.,Mantel, N., McPherson, K., Peto, J. & Smith, P. G. (1977) Design and analysis ofrandomized clinical trials requiring prolonged observation of each patient. II. Analysisand examples. Br. J. Cancer 35: 1-39.

Pickworth, W. B., Moolchan, E. T., Berlin, I. & Murty, R. (2002) Sensory andphysiologic effects of menthol and non-menthol cigarettes with differing nicotinedelivery. Pharmacol. Biochem. Behav. 71: 55-61.

Plowchalk, D. R., Andersen, M. E. & deBethizy, J. D. (1992) A physiologicallybased pharmacokinetic model for nicotine disposition in the Sprague-Dawley rat.Toxicol. Appl. Pharmacol. 116: 177-188.

Pocock, S. J., Geller, N. L. & Tsiatis, A. A. (1987) The analysis of multiple endpointsin clinical trials. Biometrics 43: 487-498.

Pottenger, L. H., Penman, M., Moorec, N. P., Pristone, R. A. J. & Thomas, M.(2004) Biological significance of DNA adducts: summary of discussion expert panel.Regul. Toxicol. Pharmacol. 39: 403-408.

Prescott, E., Osler, M., Hein, H. O., Borch-Johnsen, K., Lange, P., Schnohr, P. &Vestbo, J. (1998) Gender and smoking-related risk of lung cancer. The CopenhagenCenter for Prospective Population Studies. Epidemiology 9: 79-83.

Pritchard, W. S. & Robinson, J. H. (1996) Examining the relation between usual-brand nicotine yield, blood cotinine concentration and the nicotine- “compensation”hypothesis. Psychopharmacology (Berl) 124: 282-284.

Province of British Columbia. (2001) Tobacco testing and disclosure regulationdraft. Available at http://www.qp.gov.bc.ca/statreg/reg/T/TobaccoSales/282_98.htm#scheduleC. Accessed 4-22-2004.

Purkis, S. W., Hill, C. A. & Bailey, I. A. (2003) Current measurement reliability ofselected smoke analytes. Beitr. Tabakforsch. Int. 20: 314-324.

R.J.Reynolds Tobacco Company. (1986) Summary of data on menthol. Available athttp://legacy.library.ucsf.edu/tid/rxb84d00. Accessed 4-1-2004.

Ramos, K. & Cox, L. R. (1987) Primary cultures of rat aortic endothelial and smoothmuscle cells: I. An in vitro model to study xenobiotic-induced vascular cytotoxicity.In Vitro Cell Dev. Biol. 23: 288-296.

Reitz, R. H. (1987) Role of cytotoxicity in the carcinogenic process. In: NongenotoxicMechanisms in Carcinogenesis Banbury Report 25. (Butterworth, B. E. & Slaga,T. J., eds.) Cold Spring Harbor, New York: Cold Spring Harbor Laboratory,pp. 107-117.



Rennard, S. I. & Daughton, D. M. (2002) Smoking cessation. In: ChronicObstructive Lung Diseases. (Voelkel, N. F. & MacNee, W., eds.) Hamilton, Ontario:BC Decker, Inc., pp. 332-340.

Reynolds, T. (2003) Validating biomarkers: early detection research network launchesfirst phase III study. J. Natl. Cancer Inst. 95: 422-423.

Rickert, W. S. & Wright, W. (2002) The stability of yields of Canadian mandatedanalytes from the Kentucky reference cigarette 1R4F: a time series analysis. Paperpresented at CORESTA Congress, New Orleans, USA. Programme and AbstractsBooklet, no. ST26.

Risch, H. A., Howe, G. R., Jain, M., Burch, J. D., Holowaty, E. J. & Miller, A. B.(1993) Are female smokers at higher risk for lung cancer than male smokers?A case-control analysis by histologic type. Am. J. Epidemiol. 138: 281-293.

Ritter, D., Knebel, J. W. & Aufderheide, M. (2003) Exposure of human lung cells toinhalable substances: a novel test strategy involving clean air exposure periods usingwhole diluted cigarette mainstream smoke. Inhal. Toxicol. 15: 67-84.

Robinson, D. E., Balter, N. J. & Schwartz, S. L. (1992) A physiologically basedpharmacokinetic model for nicotine and cotinine in man. J. Pharmacokinet.Biopharm. 20: 591-609.

Rodgman, A. (2002) Some studies of the effects of additives on cigarette mainstreamsmoke properties. II. Casing materials and humectants. Beitr. Tabakforsch. Int.20: 279-299.

Roemer, E. & Hackenberg, U. (1990) Mouse skin bioassay of smoke condensatesfrom cigarettes containing different levels of cocoa. Food Addit. Contam. 7: 563-569.

Roemer, E., Stabbert, R., Rustemeier, K., Veltel, D. J., Meisgen, T. J., Reininghaus,W., Carchman, R. A., Gaworski, C. L. & Podraza, K. F. (2004) Chemical composition,cytotoxicity and mutagenicity of smoke from US commercial and reference cigarettessmoked under two sets of machine smoking conditions. Toxicology 195: 31-52.

Roemer, E., Tewes, F. J., Meisgen, T. J., Veltel, D. J. & Carmines, E. L. (2002)Evaluation of the potential effects of ingredients added to cigarettes. Part 3: In vitrogenotoxicity and cytotoxicity. Food Chem. Toxicol. 40: 105-111.

Ross, R. (1986) The pathogenesis of atherosclerosis—an update. N. Engl. J. Med.314: 488-500.

Royce, J. M., Corbett, K., Sorensen, G. & Ockene, J. (1997) Gender, social pressure,and smoking cessations: the Community Intervention Trial for Smoking Cessation(COMMIT) at baseline. Soc. Sci. Med. 44: 359-370.

Russell, M. A. & Feyerabend, C. (1978) Cigarette smoking: a dependence on high-nicotine boli. Drug Metab. Rev. 8: 29-57.



Rustemeier, K., Stabbert, R., Haussmann, H.-J., Roemer, E. & Carmines, E. L.(2002) Evaluation of the potential effects of ingredients added to cigarettes. Part 2:Chemical composition of mainstream smoke. Food Chem. Toxicol. 40: 93-104.

Sato, S., Ohka, T., Nagao, M., Tsuji, K. & Kosuge, T. (1979) Reduction in mutagenicityof cigarette smoke condensate by added sugars. Mutat. Res. 60: 155-161.

Sawyer, J., Moore, M. M., Clive, D. & Hozier, J. (1985) Cytogenetic characterizationof the L5178Y TK+/-3.7.2C mouse lymphoma cell line. Mutat. Res. 147: 243-253.

Sawyer, J. R., Moore, M. M. & Hozier, J. C. (1989) High-resolution cytogeneticcharacterization of the L5178Y TK+/- mouse lymphoma cell line. Mutat. Res.214: 181-193.

Scherer, G. (1999) Smoking behaviour and compensation: a review of the literature.Psychopharmacology (Berl) 145: 1-20.

Schievelbein, H. (1982) Nicotine, resorption and fate. Pharmacol. Ther. 18: 233-248.

Schlotzhauer, W. S. (1978) Fatty acids and phenols from pyrolysis of cocoa powder,a tobacco product flavorant. Tob. Sci. 22: 1-2.

Schlotzhauer, W. S., Severson, R. F. & Martin, R. M. (1986) The contributionof sucrose esters to tobacco smoke composition. Beitr. Tabakforsch. Int.13: 229-238.

Schmeltz, I. & Schlotzhauer, W. S. (1968) Benzo[a]pyrene, phenols and other productsfrom pyrolysis of the cigarette additive, (d,1)-menthol. Nature 219: 370-371.

Schmeltz, I., Wenger, A., Hoffmann, D. & Tso, T. C. (1979) Chemical studies ontobacco smoke. 63. On the fate of nicotine during pyrolysis and in a burning cigarette.J. Agric. Food Chem. 27: 602-608.

Schneider, N. G., Olmstead, R. E., Franzon, M. A. & Lunell, E. (2001) The nicotineinhaler: clinical pharmacokinetics and comparison with other nicotine treatments.Clin. Pharmacokinet. 40: 661-684.

Schranz, H. W. & Sewell, T. D. (1996) Statistical and dynamical behaviour in theunimolecular reaction dynamics of polyatomic molecules. J. Molecular Structure(Theochem) 368: 125-131.

Schultz, H., Brand, P. & Heyder, J. (2000) Particle deposition in the respiratorytract. In: Particle-Lung Interactions. (Gehr, P. J. & Heyder, J., eds.) New York,NY: Dekker, pp. 229-290.

Schulz, W. & Seehofer, F. (1978) Smoking behaviour in Germany — the analysis ofcigarette butts (KIPA). In: Smoking Behavior. Physiological and PsychologicalInfluences. (Thornton, R. E., ed.) London: Churchill Livingstone, pp. 259-276.



Secker-Walker, R. H., Vacek, P. M., Flynn, B. S. & Mead, P. B. (1997) Exhaledcarbon monoxide and urinary cotinine as measures of smoking in pregnancy. Addict.Behav. 22: 671-684.

Senn, S. (2002) Cross-over trials in clinical research. In: Statistics in Practice2nd ed. Chichester, England: John Wiley & Sons Ltd.

Shields, P. G. (2002) Tobacco smoking, harm reduction, and biomarkers. J. Natl.Cancer Inst. 94: 1435-1444.

Shields, P. G., Caporaso, N. E., Falk, R. T., Sugimura, H., Trivers, G. E., Trump, B.F., Hoover, R. N., Weston, A. & Harris, C. C. (1993) Lung cancer, race, and aCYP1A1 genetic polymorphism. Cancer Epidemiol. Biomarkers Prev. 2: 481-485.

Sidney, S., Tekawa, I. & Friedman, G. D. (1989) Mentholated cigarette use amongmultiphasic examinees, 1979-86. Am. J. Public Health 79: 1415-1416.

Sidney, S., Tekawa, I. S., Friedman, G. D., Sadler, M. C. & Tashkin, D. P. (1995)Mentholated cigarette use and lung cancer. Arch. Intern. Med. 155: 727-732.

Silbaugh, S. A. & Horvath, S. M. (1982) Effect of acute carbon monoxide exposureon cardiopulmonary function of the awake rat. Toxicol. Appl. Pharmacol.66: 376-382.

Simons, F. E., Becker, A. B., Simons, K. J. & Gillespie, C. A. (1985) Thebronchodilator effect and pharmacokinetics of theobromine in young patients withasthma. J. Allergy Clin. Immunol. 76: 703-707.

Sinclair, N. M., Frost, B. E. & Mariner, D. C. (1998) Factors related to nicotinephysio-chemistry and retention in human smokers. [Presented at the 1998 CORESTACongress, Brighton, UK]. Coresta Inf. Bull. 167.

Slaga, T. & Nesnow, S. (1985) Sencar mouse tumorigenesis. In: Handbook ofCarcinogen Testing. (Milman, H. A. & Weisburger, E. K., eds.) Park Ridge, NJ:Noyes Publications, pp. 230-250.

Sloan, A., De Cort, S. C. & Eccles, R. (1993) Prolongation of breath-hold timefollowing treatment with an L-menthol lozenge in healthy man. J. Physiol. (Lond.)473: 53P.

Smith, C. J., Bombick, D. W., Ryan, B. A., Morgan, W. T. & Doolittle, D. J. (2000)Urinary mutagenicity in nonsmokers following exposure to fresh diluted sidestreamcigarette smoke. Mutat. Res. 470: 53-70.

Spears, A. W. (1974) Effect of manufacturing variables on cigarette smokecomposition. [Presented at the CORESTA Symposium, Montreux-Suisse, 22-27,September 1974.] Coresta Inf. Bull. 74: 65-78.



Spira, A., Beane, J., Shah, V., Liu, G., Schembri, F., Yang, X., Palma, J. & Brody,J. S. (2004) Effects of cigarette smoke on the human airway epithelial celltranscriptome. Proc. Natl. Acad. Sci. U. S. A. 101: 10143-10148.

Stavanja, M. S., Ayres, P. H., Meckley, D. R., Bombick, B. R., Pence, D. H.,Borgerding, M. F., Morton, M. J., Mosberg, A. T. & Swauger, J. E. (2003)Toxicological evaluation of honey as an ingredient added to cigarette tobacco. J.Toxicol. Environ. Health A 66: 1453-1473.

Steele, R. H., Payne, V. M., Fulp, C. W., Rees, D. C., Lee, C. K. & Doolittle, D. J.(1995) A comparison of the mutagenicity of mainstream cigarette smoke condensatesfrom a representative sample of the U.S. cigarette market with a Kentucky referencecigarette (K1R4F). Mutat. Res. 342: 179-190.

Stellman, S. D., Chen, Y., Muscat, J. E., Djordjevic, M. V., Richie, J. P., Jr., Lazarus,P., Thompson, S., Altorki, N., Berwick, M., Citron, M. L., Harlap, S., Kaur, T. B.,Neugut, A. I., Olson, S., Travaline, J. M., Witorsch, P. & Zhang, Z. F. (2003) Lungcancer risk in white and black Americans. Ann. Epidemiol. 13: 294-302.

Stepney, R. (1981) Would a medium-nicotine, low-tar cigarette be less hazardous tohealth? Br. Med. J. 283: 1292-1296.

Stimpfl, T., Nasel, B., Nasel, C., Binder, R., Vycudilik, W. & Buchbauer, G. (1995)Concentration of 1,8-cineol in human blood during prolonged inhalation. Chem. Senses20: 349-350.

Stotesbury, S. J., Digard, H., Willoughby, L. & Couch, A. (1999) The pyrolysis oftobacco additives as a means of predicting their behaviour in a burning cigarette.Beitr. Tabakforsch. Int. 18: 147-163.

Stotesbury, S. J., Willoughby, L. J. & Couch, A. (2000) Pyrolysis of cigarette ingredientslabelled with stable isotopes. Beitr. Tabakforsch. Int. 19: 55-64.

Svensson, C. K. (1987) Clinical pharmacokinetics of nicotine. Clin. Pharmacokinet.12: 30-40.

Swauger, J. E., Steichen, T. J., Murphy, P. A. & Kinsler, S. (2002) An analysis of themainstream smoke chemistry of samples of the U.S. cigarette market acquiredbetween 1995 and 2000. Regul. Toxicol. Pharmacol. 35: 142-156.

Swenberg, J. A. (1989) Carcinogenic responses of the nasal passages. In: Conceptsin Inhalation Toxicology. (McClellan, R. O. & Henderson, R. F., eds.) New York:Hemisphere Publishing, pp. 299-311.

Theophilus, E. H., Pence, D. H., Meckley, D. R., Higuchi, M. A., Bombick, B. R.,Borgerding, M. F., Ayres, P. H. & Swauger, J. E. (2004) Toxicological evaluation ofexpanded shredded tobacco stems. Food Chem. Toxicol. 42: 631-639.



Theophilus, E. H., Poindexter, D. B., Meckley, D. R., Bombick, B. R., Borgerding,M. F., Higuchi, M. A., Ayres, P. H., Morton, M. J., Mosberg, A. T. & Swauger,J. E. (2003) Toxicological evaluation of dry ice expanded tobacco. Toxicol. Lett.145: 107-119.

Thomas, R. D. & Vigerstad, T. J. (1989) Use of laboratory animal models ininvestigating emphysema and cigarette smoking in humans. Regul. Toxicol.Pharmacol. 10: 264-271.

Thornton, R. E. & Massey, S. R. (1975) Some effects of adding sugar to tobacco.Beitr. Tabakforsch. 8: 11-15.

Thun, M. J., Day-Lally, C., Myers, D. K., Calle, E. E., Flanders, W. D., Zhu, B. P. &Namboodiri, M. M. (1997) Trends in tobacco smoking and mortality from cigaretteuse in cancer prevention studies I (1959 through 1965) and II (1982 through 1988).In: Monograph 8: Changes in Cigarette-Related Disease Risks and TheirImplications for Prevention and Control. Bethesda, MD: National Cancer Institute,pp. 305-382.

Thun, M. J., Henley, S. J. & Calle, E. E. (2002) Tobacco use and cancer: anepidemiologic perspective for geneticists. Oncogene 21: 7307-7325.

Tjalve, H., Hannsson, E. & Schmiterlow, C. G. (1968) Passage of 14C-nicotine andits metabolites into mice foetuses and placentae. Acta Pharmacol. Toxicol. (Copenh)26: 539-555.

Tobacco and Health Research Institute. (2002) Research Cigarette (2R4F).Preliminary analysis. Available at http://www.rgs.uky.edu/ca/infrastructure/centers/tobacco.html. Accessed 8-16-2002.

Tobacco Science Research Chemists (2003) The taste challenge: Maintainingsensory quality in a reduced-yield environment. [Symposium Proceedings from the57th Tobacco Science Research Conference, Norfolk, VA, Sept. 21-24, 2003]. Rec.Adv. Tob. Sci. 29: 1-99.

Tobin, M. J. & Sackner, M. A. (1982) Monitoring smoking patterns of low and hightar cigarettes with inductive plethysmography. Am. Rev. Respir. Dis. 126: 258-264.

Tricker, A. R. (2003) Nicotine metabolism, human drug metabolism polymorphisms,and smoking behaviour. Toxicology 183: 151-173.

Tsujimoto, A., Nakashima, T., Tanino, S., Dohi, T. & Kurogochi, Y. (1975) Tissuedistribution of [3H]nicotine in dogs and rhesus monkeys. Toxicol. Appl. Pharmacol.32: 21-31.

Turner, N. T. , Batson, A. G. & Clive, D. (1984) Procedures for theL5178Y/TK+/- → TK-/- mouse lymphoma cell mutagenicity assay. In: Handbook ofMutagenicity Test Procedures. (Kilbey, B. J., ed.) New York: Elsevier SciencePublishers, pp. 239-268.



UK Department of Health, Smoking Policy Unit. (2000) Cigarette yields using intensesmoking protocols. Part 1 - NFPDM, nicotine & carbon monoxide yields. Availableat http://www.doh.gov.uk/scoth/technicaladvisorygroup/yieldintense1.pdf. Accessed6-2-2003.

U.S. Congress. (2004) 15 U.S.C. Chapter 36 Sec. 1335a. - List of cigarettesingredients; annual submission to Secretary; transmittal to Congress; confidentiality.Available at http://www4.law.cornell.edu/uscode/15/1335a.html. Accessed8-16-2004.

U.S. Department of Health and Human Services. (1989) Reducing the HealthConsequences of Smoking. 25 Years of Progress. A Report of the SurgeonGeneral. Rockville, MD: U.S. Department of Health and Human Services, Centersfor Disease Control Center for Chronic Disease Prevention and Health Promotion,Office on Smoking and Health.

U.S. Department of Health and Human Services. (2001) Women and Smoking. AReport of the Surgeon General. (Ernster, V. L., Lloyd, G., Norman, L. A., McCarthy,A. & Pinto, A. L., eds.) Washington, DC: U.S. Government Printing Office.

U.S. Environmental Protection Agency. (1998b) Health Effects Test Guidelines.OPPTS 870-5915 In Vivo Sister Chromatid Exchange Assay. Report No. EPA712-C-98-235. Washington, DC: U.S. Environmental Protection Agency.

U.S. Environmental Protection Agency. (2004) OPPTS Harmonized Test Guidelines.Series 870 Health Effects Test Guidelines. Available at http://www.epa.gov/opptsfrs/OPPTS_Harmonized/870_Health_Effects_Test_Guidelines. Accessed 3-22-2004.

U.S. Environmental Protection Agency. (1998a) 3.4 Guidance for meeting the SIDSrequirements (The SIDS guide). Available at http://www.epa.gov/chemrtk/sidsappb.pdf. Accessed 10-29-2003.

U.S. Environmental Protection Agency. (1999) Guidance for “What to Test” for theHPV Challenge. Available at http://www.epa.gov/chemrtk/w2test9.pdf. Accessed10-29-2003.

U.S. Food and Drug Administration. (1994) Drug products containing certain activeingredients offered over-the-counter (OTC) for certain uses. 21 CFR 310: 310.545.

U.S. Food and Drug Administration. (2004) Guidance for industry. Q3B impuritiesin new drug products. Available at http://www.fda.gov/cder/guidance/1317fnl.htm.Accessed 5-13-2004.

U.S. Food and Drug Administration. (2000) Guidance for industry. Analyticalprocedures and methods validation. Chemistry, manufacturing, and controlsdocumentation. [Draft guidance]. Available at http://www.fda.gov/cber/gdlns/methval.pdf. Accessed 10-24-2003.



U.S. Food and Drug Administration. (1982) Toxicological Principles for the SafetyAssessment of Direct Food Additives and Color Additives Used in Food. RedbookI. Washington, DC: U.S. Food and Drug Administration.

U.S. Food and Drug Administration. (1993) Toxicological Principles for the SafetyAssessment of Direct Food Ingredients. Draft. Redbook II. Washington, DC:U.S. Food and Drug Administration.

U.S. Food and Drug Administration. (2001) Toxicological Principles for the SafetyAssessment of Direct Food Ingredients. Redbook 2000. Washington, DC: U.S.Food and Drug Administration.

Van Duuren, B. L. & Goldschmidt, B. M. (1976) Cocarcinogenic and tumor-promotingagents in tobacco carcinogenesis. J. Natl. Cancer Inst. 56: 1237-1242.

Vanscheeuwijck, P. M., Teredesai, A., Terpstra, P. M., Verbeeck, J., Kuhl, P.,Gerstenberg, B., Gebel, S. & Carmines, E. L. (2002) Evaluation of the potentialeffects of ingredients added to cigarettes. Part 4: subchronic inhalation toxicity.Food Chem. Toxicol. 40: 113-131.

Vineis, P., Alavanja, M., Buffler, P., Fontham, E., Franceschi, S., Gao, Y. T., Gupta,P. C., Hackshaw, A., Matos, E., Samet, J., Sitas, F., Smith, J., Stayner, L., Straif,K., Thun, M. J., Wichmann, H. E., Wu, A. H., Zaridze, D., Peto, R. & Doll, R.(2004) Tobacco and cancer: recent epidemiological evidence. J. Natl. CancerInst. 96: 99-106.

Vogel, J. S. & Turteltaub, K. W. (1992) Biomolecular tracing through acceleratormass spectrometry. Trends Anal. Chem. 11: 142-149.

Vogel, J. S., Turteltaub, K. W., Finkel, R. & Nelson, D. E. (1995) Accelerator massspectrometry. Anal. Chem. 67: 353A-359A.

Waddell, W. J. & Marlowe, C. (1976) Localization of nicotine-14C, cotinine-14C, andnicotine-1'-N-oxide-14C in tissues of the mouse. Drug Metab. Dispos. 4: 530-539.

Wagenknecht, L. E., Cutter, G. R., Haley, N. J., Sidney, S., Manolio, T. A., Hughes,G. H. & Jacobs, D. R. (1990) Racial differences in serum cotinine levels amongsmokers in the Coronary Artery Risk Development in (Young) Adults study.Am. J. Public Health 80: 1053-1056.

Wald, N. J., Idle, M., Boreham, J. & Bailey, A. (1981) The importance of tar andnicotine in determining cigarette smoking habits. J. Epidemiol. Community Health35: 23-24.

Warburton, D. M. (1987) The functions of smoking. In: Tobacco Smoke andNicotine: A Neurobiological Approach. (Martin, W. R., Van Loon, G. R. & Layten,D., eds.) New York: Plenum Press, pp. 51-62.



Waters, D., Lesperance, J., Gladstone, P., Boccuzzi, S. J., Cook, T., Hudgin,R., Krip, G. & Higginson, L. (1996) Effects of cigarette smoking on theangiographic evolution of coronary atherosclerosis. A Canadian CoronaryAtherosclerosis Intervention Trial (CCAIT) substudy. CCAIT Study Group.Circulation 94: 614-621.

Wayne, G. F. & Connolly, G. (2004) Application, function, and effects of menthol incigarettes: A survey of tobacco industry documents. Nicotine Tob. Res. 6 Suppl 1:S43-S54.

Wehner, A. P., Dagle, G. E., Milliman, E. M., Phelps, D. W., Carr, D. B., Decker,J. R. & Filipy, R. E. (1981) Inhalation bioassay of cigarette smoke in rats. Toxicol.Appl. Pharmacol. 61: 1-17.

Wei, Q. & Spitz, M. R. (1997) The role of DNA repair capacity in susceptibility tolung cancer: a review. Cancer Metastasis Rev. 16: 295-307.

Westman, E. C., Behm, F. M. & Rose, J. E. (1996) Dissociating the nicotine andairway sensory effects of smoking. Pharmacol. Biochem. Behav. 53: 309-315.

Wiencke, J. K. (1999) Response to: Early age at smoking initiation and tobaccocarcinogen DNA damage in the lung. J. Natl. Cancer Inst. 91: 1423A-1423. (lett.)

Wiencke, J. K., Thurston, S. W., Kelsey, K. T., Varkonyi, A., Wain, J. C., Mark, E.J. & Christiani, D. C. (1999) Early age at smoking initiation and tobacco carcinogenDNA damage in the lung. J. Natl. Cancer Inst. 91: 614-619.

Witek, T. J., Jr. (2000) The fate of inhaled drugs: the pharmacokinetics andpharmacodynamics of drugs administered by aerosol. Respir. Care 45: 826-830.

Witschi, H. (1998) Tobacco smoke as a mouse lung carcinogen. Exp. Lung Res.24: 385-394.

Witschi, H., Espiritu, I., Maronpot, R. R., Pinkerton, K. E. & Jones, A. D. (1997a)The carcinogenic potential of the gas phase of environmental tobacco smoke.Carcinogenesis 18: 2035-2042.

Witschi, H., Espiritu, I., Peake, J. L., Wu, K., Maronpot, R. R. & Pinkerton, K. E.(1997b) The carcinogenicity of environmental tobacco smoke. Carcinogenesis18: 575-586.

Witschi, H., Espiritu, I., Uyeminami, D., Suffia, M. & Pinkerton, K. E. (2004) Lungtumor response in strain A mice exposed to tobacco smoke: some dose-effectrelationships. Inhal. Toxicol. 16: 27-32.

Witschi, H., Uyeminami, D., Moran, D. & Espiritu, I. (2000) Chemoprevention oftobacco-smoke lung carcinogenesis in mice after cessation of smoke exposure.Carcinogenesis 21: 977-982.



World Health Organization. (1986) IARC Monographs on the Evaluation of theCarcinogenic Risk of Chemicals to Humans. Tobacco Smoking. Volume 38.Lyons, Switzerland: International Agency for Research on Cancer.

World Health Organization. (2003) IPCS Environmental Health Criteria Series: No.213 Carbon Monoxide (2nd ed.). Available at http://www.who.int/pcs/ehc/summaries/ehc_213.html. Accessed 12-16-2003.

Wright, J. L. & Churg, A. (2002) A model of tobacco smoke-induced airflowobstruction in the guinea pig. Chest 121: 188S-191S.

Wynder, E. L. & Muscat, J. E. (1995) The changing epidemiology of smoking andlung cancer histology. Environ. Health Perspect. 103 Suppl 8: 143-148.

Yamasaki, E. & Ames, B. N. (1977) Concentration of mutagens from urine byabsorption with the nonpolar resin XAD-2: cigarette smokers have mutagenic urine.Proc. Natl. Acad. Sci. U. S. A. 74: 3555-3559.

Yamato, H., Sun, J. P., Churg, A. & Wright, J. L. (1996) Cigarette smoke-inducedemphysema in guinea pigs is associated with diffusely decreased capillary densityand capillary narrowing. Lab. Invest. 75: 211-219.

Zacny, J. P., Stitzer, M. L., Brown, F. J., Yingling, J. E. & Griffiths, R. R. (1987)Human cigarette smoking: effects of puff and inhalation parameters on smokeexposure. J. Pharmacol. Exp. Ther. 240: 554-564.

Zang, E. A. & Wynder, E. L. (1992) Cumulative tar exposure. A new index forestimating lung cancer risk among cigarette smokers. Cancer 70: 69-76.

Zartarian, V. G., Ott, W. R. & Duan, N. (1997) A quantitative definition of exposureand related concepts. J. Exp. Anal. Environ. Epidemiol. 7: 411-437.

Zeiger, E. (1987) Carcinogenicity of mutagens: predictive capability of the Salmonellamutagenesis assay for rodent carcinogenicity. Cancer Res. 47: 1287-1296.


AD HOC EXPERT PANEL

Alwynelle S. Ahl, Ph.D., D.V.M.Principal Scientist, Highland Rim Research OrganizationLyles, Tennessee

Carroll E. Cross, M.D.Professor, University of California, Davis, School of Medicine, Division of Pulmonaryand Critical Care MedicineSacramento, California

Shayne C. Gad, Ph.D., D.A.B.T.Principal, Gad Consulting ServicesCary, North Carolina

Donald E. Gardner, Ph.D., Fellow A.T.S.President, Inhalation Toxicology AssociatesRaleigh, North Carolina

Louis D. Homer, M.D., Ph.D.Medical Director, Holladay Park Medical Center, Legacy ResearchPortland, Oregon

Rudolph J. Jaeger, Ph.D., D.A.B.T., B.C.F.M.Principal Scientist, Environmental Medicine, Inc.Westwood, New Jersey

Robert Orth, Ph.D.President, Apis Discoveries, LLCCedar Hill, Missouri

Emanuel Rubin, M.D.Professor and Chairman, Department of Pathology, Anatomy, and Cell BiologyThomas Jefferson UniversityPhiladelphia, Pennsylvania

15STUDY PARTICPANTS

Study Particpants � 183


James L. Schardein, M.S., Fellow A.T.S.Independent ConsultantLeesburg, Florida

Thomas J. Slaga, Ph.D.President and CEO, AMC Cancer Research CenterChair, Center for Causation and PreventionDenver, Colorado

LIFE SCIENCES RESEARCH OFFICE STAFF

Michael Falk, Ph.D.Executive Director

Daniel M. Byrd, III, Ph.D., D.A.B.T.Deputy Director

Amy M. Brownawell, Ph.D.Staff Scientist

Kara D. Lewis, Ph.D.Staff Scientist

Paula M. Nixon, Ph.D.Staff Scientist

Negin P.M. Royaee, B.S.Associate Staff Scientist

Kristine K. Sasala, B.S.Associate Staff Scientist

Robin S. Feldman, B.S., M.B.A.Literature Specialist Librarian



APPENDIX A

LIFE SCIENCES RESEARCH OFFICE (LSRO)

Added Ingredients Review Ad Hoc Expert Panel:

Alwynelle S. Ahl, Ph.D., D.V.M., is Principal Scientist at Highland Rim ResearchConsulting, Inc. (HRRC). After majoring in biology and mathematics at CentenaryCollege of Louisiana, she obtained her M.S. and Ph.D. in zoology and biochemistryfrom the University of Wyoming, and a doctorate in veterinary medicine fromMichigan State University where she also served as a professor in the Departmentof Natural Science. Prior to her work at HRRC, Dr. Ahl had a distinguishedcareer at the U.S. Department of Agriculture (USDA) serving in various capacities,including Deputy Director for animal health training and Chief of Risk AnalysisSystems within USDA’s Animal and Plant Health Inspection Service. She alsoserved in the Senior Executive Service as the first Director of the USDA Office ofRisk Assessment and Cost-Benefit Analysis and as an USDA Fellow to the Centerfor the Integrated Study of Food, Animal and Plant Systems at Tuskegee Universityin Alabama. She is a fellow of the American Association for the Advancement ofScience and has served on several panels at the National Academy of Sciences.Her research interests include public policy for science and veterinary medicineand the use of risk assessment for agricultural issues involving human health, safetyand the environment, with a special interest in food-borne microbial pathogens.Dr. Ahl’s presentations and publications total more than 250 and she has editedreports from more than a dozen symposia.

Carroll E. Cross, M.D., is a pulmonary critical care physician at the University ofCalifornia at Davis School of Medicine, Division of Pulmonary and Critical CareMedicine. He graduated from Columbia College of Physicians and Surgeons in1961, completed his internship at the University of Wisconsin Hospital in 1962, hisresidency at Stanford Hospital Center in 1964, and his clinical and researchfellowship training at the University of Pittsburgh Medical Center in 1968. He wascertified in internal medicine in 1969 and in pulmonary disease in 1971. He hasbeen at the University of California, Davis since 1968, where he is currently aprofessor of medicine and physiology. Dr. Cross has published more than 200papers in such fields as air pollutants, antioxidant micronutrients, inflammatory-immune system oxidants, ozone, oxides of nitrogen, cigarette smoke, and relatedaspects of inhalation toxicology as it relates to respiratory tract diseases. He is amember of several professional organizations including the American Physiological

Appendices � 185


Society, the UK Biochemical Society, the Oxygen Society, the Mount Desert IslandBiological Laboratory, the Western Society of Physicians, and the American Societyfor Clinical Nutrition. He serves on the editorial boards of the American Journalof Clinical Nutrition and Free Radicals in Biology and Medicine, and hasserved on research review panels for the Veterans Administration, for the NationalInstitutes of Health, and for the Heart, Lung and Cancer Society Associations.

Shayne C. Gad, Ph.D., D.A.B.T., Fellow A.T.S., is Principal of Gad ConsultingServices. After majoring in chemistry and biology at Whittier College, he obtainedhis doctorate in pharmacology/toxicology from the University of Texas. As a fellowat Bushy Run Research Center, Union Carbide Toxicology Laboratory he developeda system for assessment of toxicity of polymer thermal decomposition products.Later, he worked at the Shell Development Laboratory establishing a new inhalationtoxicology research facility. As Manager of Mammalian Toxicology at AlliedCorporation, Dr. Gad was responsible for all mammalian toxicity testing includingthe operation of the entire Department of Toxicology laboratory and all externalcontract testing. Dr. Gad has also worked at G.D. Searle & Co. as Director ofToxicology and Senior Director of Product Safety and Metabolism where he usedan interdisciplinary approach to oversee safety/toxicity research programs, assistedwith the prioritization of research and development efforts, interacted with foreignfirms and regulatory agencies, and developed world wide occupational AirborneControl Objectives. He has also served as Director of Medical Affairs ProductSupport Services at Becton Dickinson and Director of Toxicology at Synergen.Dr. Gad has directed the design, conduct, writing, and filing of investigational newdrug applications, new drug applications, drug disposition studies, and medical deviceregulations. Dr. Gad’s presentations and publications total more than 300 and include29 books and 40 book chapters.

Donald E. Gardner, Ph.D., Fellow A.T.S., is President of Inhalation ToxicologyAssociates. He received his M.S. in medical microbiology from Creighton Universityand his Ph.D. in environmental health and toxicology from the University ofCincinnati. He has worked for the U.S. Environmental Protection Agency (EPA)and the Public Health Service. Following retirement from EPA, he joined the staffof Northrop/ManTech Corporation as Vice President and Chief Scientist. He heldacademic appointments at Duke University, North Carolina State University, andthe University of Massachusetts. He served on various panels of the NationalResearch Council since 1989 and for part of this time as Vice-Chairman of theCommittee on Toxicology. He is presently on the Editorial Board of the ToxicSubstance Journal and Environmental and Nutritional Interactions. AtInhalation Toxicology Associates he provides consulting services to several federalagencies, the World Health Organization (WHO), private industry, and law firms.He has published more than 225 peer-reviewed manuscripts and book chaptersand is Founding Editor of the Journal of Inhalation Toxicology. He is also a co-



editor of the Target Organ Toxicology Series and New Perspectives: Toxicologyand the Environment. The Society of Toxicology honored him with lifetimeoutstanding achievement awards in both inhalation toxicology and immunotoxicology.

Louis D. Homer, M.D., Ph.D., retired as Medical Director of Clinical Investigationand Biomedical Research at Legacy Research, Holladay Park Medical Center.He earned a Ph.D. in physiology and an M.D. from the Medical College of Virginia.He has served as Assistant and Associate Professor at Emory Universityconcentrating on physiological processes and mathematical models. Afterward, heserved as associate professor at Brown University and then moved on to becomea Research Medical Officer at the Naval Medical Research Institute concentratingon biometrics, physiology, environmental medicine, metabolic research, and kidneytransplant histocompatibility. He regularly served as a consultant to scientists ontopics such as physiology, mathematics, statistics, and computer applications. Healso has reviewed proposals and has served on site visit teams for National Instituteof Allergy and Infectious Diseases (NIAID), National Heart, Lung, and BloodInstitute (NHLBI), National Science Foundation (NSF), and the Naval MedicalResearch and Development Command. He has reviewed prospective articles forthe Journal of Theoretical Biology, Microvascular Research, American Journalof Physiology, and the Journal of Applied Physiology. His interest in usingmathematical models of physiology in his research has led him to become familiarwith a number of computer languages, numerical algorithms, iterative least-squaresestimation, and iterative maximum likelihood estimation.

Rudolph Jaeger, Ph.D., D.A.B.T., B.C.F.M., retired as full-time Research Professorof Environmental Medicine from the New York University School of Medicine. Henow serves as a part-time faculty member at NYU where he continues to teachclasses. He is Principal Scientist at Environmental Medicine, Inc., a consultingfirm specializing in consumer product evaluations, environmental health riskassessments, and industrial toxicology. Dr. Jaeger also serves CH Technologies(USA) Inc. as President and Chief Scientific Officer. CH Technologies, a scientificinstrument manufacturing company, specializes in low volume inhalation exposuresystems for pharmaceutical and infectious disease research. CH Technologiesand affiliates manufacture cigarette-smoking machines. Dr. Jaeger earned statusas a Diplomate of the American Board of Toxicology in 1980. He has beenaccredited as an Asbestos Inspector, and he is currently a Registered EnvironmentalAssessor in the State of California. He is a Certified Lead Inspector and RiskAssessor in the State of New Jersey. He is a Diplomate of the American Board ofForensic Medicine and a Board Certified Forensic Examiner. Dr. Jaeger servedon the Toxicology Information Program Committee of the Board of EnvironmentalToxicology of the National Research Council. His research interests includeinhalation toxicology, plastics and their monomers, combustion products, pulmonarypathophysiology, liver toxicity and pathophysiology, and the effects of lead andheavy metals on the developing nervous system.

Appendices � 187


Robert Orth, Ph.D., is a physical chemist at Apis Discoveries, L.L.C. He is also aconsultant to the Monsanto Company and adjunct associate professor of physicalchemistry at the University of Missouri, where he teaches undergraduate coursesin physical chemistry, instrumental analysis, and general chemistry. After obtaininghis Ph.D. from Case Western Reserve University, he completed a postdoctoralfellowship at the University of Utah researching mass spectrometry and developeda high-pressure liquid chromatography/mass spectrometry interface usingultrasound. He conducted research in secondary ion mass spectrometry and taughtat both the University of Utah and Montana State University. He held positions ofincreasing responsibility, including Research Fellow, during a 16-year career withthe Monsanto Company. He worked on problems in environmental chemistry andremediation, and in food and agricultural science. His current work at ApisDiscoveries, L.L.C. includes setting up business units for ultratrace analysis,consulting to companies submitting direct and indirect food additives to the U.S.Food and Drug Administration (FDA), and studying the analysis and remediationof organic pollutants. He has nearly 100 publications and presentations in analyticaland physical chemistry, and he is the author of many internal Monsanto publicationsand presentations. He currently holds two patents.

Emanuel Rubin, M.D., is Gonzalo E. Aponte Distinguished Professor of Pathologyand Chairman Emeritus of the Department of Pathology, Anatomy and Cell Biologyat Jefferson Medical College. He obtained a medical degree from Harvard MedicalSchool. After completing a residency at the Children’s Hospital of Philadelphia, hecontinued as a Dazian Research Fellow in pathology and as Advanced ClinicalFellow of the American Cancer Society, both at Mount Sinai Hospital in NewYork. After his fellowship Dr. Rubin spent the next fourteen years at Mount SinaiHospital’s Pathology Service with increasing responsibilities, culminating inPathologist-in-Chief. Dr. Rubin then became the Director of Laboratories at theHahnemann University Hospital. His many academic appointments include theIrene Heinz and John LaPorte Given Professor and Chairman of the Departmentat Mount Sinai School of Medicine, Professor and Chairman of the Department ofPathology and Laboratory Medicine at the Hahnemann University School ofMedicine, Adjunct Professor of Biochemistry and Biophysics at the University ofPennsylvania School of Medicine, and several appointments at Jefferson MedicalCollege, culminating in his current position. Among honors received, the Universityof Barcelona and the University of Naples named him as Doctor Honoris Causa.He was also given the F.K. Mostofi Distinguished Service Award from U.S.-Canadian Academy of Pathology and the National Institutes of Health MERITAward, which lasts until 2006. He has held many editorial positions and has servedas a consultant to many organizations. He has more than 300 publications, including13 textbooks and one CD-ROM.



James L. Schardein, M.S., Fellow A.T.S., consults on reproductive anddevelopmental toxicology issues. Early in his career, he established one of the firstpharmaceutical industry laboratories that investigated the effect of drugs on thedeveloping embryonic animal model. After 23 years of experience in thepharmaceutical sector, Mr. Schardein joined International Research andDevelopment Corporation as Director of the Reproductive and DevelopmentToxicology group, where he later was named Division Vice President. In 1992, heacquired additional responsibilities as Associate Director of Research for managingStudy Directors and oversaw the direction of all research programs at the laboratory.In 1995, he joined WIL Research Laboratories and served as Director of Researchand Senior Vice President through March 2000. He has served as consultant tomany organizations, including the National Institute of Environmental HealthSciences, the EPA, Interagency Regulatory Liaison Group, and the InternationalLife Sciences Institute. He was a member of an International Committee onHarmonisation guideline panel on reproduction. He is the author of over 150publications and two books, Drugs as Teratogens (1976) and Chemically InducedBirth Defects (1985). The latter now in its 3rd (2000) edition.

Thomas J. Slaga, Ph.D., is President and CEO of the Center for Cancer Causationand Prevention at the AMC Cancer Research Center. He completed his doctoratein physiology and biophysics at the University of Arkansas Medical Center and apostdoctoral fellowship at the McArdle Laboratory for Cancer Research at theUniversity of Wisconsin. He held positions of increasing responsibility as an AssistantMember of the Fred Hutchinson Cancer Research Center, Staff Member at theEast Tennessee Cancer Research Center, and Senior Staff Member of the SkinCarcinogenesis and Tumor Promotion and Biology Division at the Oak RidgeNational Laboratory (part of the U.S. Department of Energy). He simultaneouslyheld faculty positions at the University of Washington Medical School, the OakRidge Graduate School of Biomedical Sciences at the Texas A&M University, andat the University of Texas. Several of Dr. Slaga’s former students now work forcigarette manufacturers. Currently, his joint/adjunct positions include Interim DeputyDirector and Member of the Comprehensive Cancer Research Center and Memberof the Department of Biochemistry and Molecular Biology at the University ofColorado Health Sciences Center. He has served on many scientific advisorycommittees and editorial boards. He also reviews submissions to several scientificjournals. His research interests include mechanisms of chemical carcinogenesis,tumor promotion, and mechanisms of action of dermal anti-tumor agents. Dr.Slaga has published more than 500 publications.

Appendices � 189


Added Ingredients Review Meeting Speakers:

Lesa L. Aylward, B.S., M.S., is Managing Scientist in Exponent’s Toxicology/Human Health practice, and is based in Washington, D.C. She received her B.S.and M.S. degrees in materials science and engineering from the MassachusettsInstitute of Technology. Prior to joining Exponent, Ms. Aylward was a SeniorLife Science/Manager with BBL Sciences, providing risk assessment andlitigation support consulting services to a variety of industrial clients. Ms. Aylwardhas broad experience in exposure and risk assessment in various contexts, includinglitigation, regulatory matters, and site assessment and cleanup activities. AtExponent, with collaborating scientists, she has developed and publishednovel approaches to assessing the biologically relevant exposures to2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds, and analyzedthe effect of these methods on conclusions drawn from conventional risk assessmentmethods. She has analyzed and used physiologically-based pharmacokinetic modelsto estimate tissue-specific doses of chemical exposures. Ms. Aylward has alsoprovided detailed analysis of risk assessments for Superfund and other hazardouswaste sites involving a wide array of chemical contaminants, and conducted riskand hazard assessments focused on arsenic, replacement compounds for chlorinatedfluorocarbons, and other chemicals. Ms. Aylward has also prepared and submittedpremanufacture notifications (PMN) for new chemicals, and performed analysesfor several clients on Toxic Substances Control Act PMN and inventory registryprojects, focusing on chemical identity ascertainment, polymer exemptions, andother registry issues.

Edward L. Carmines, Ph.D., is Principal Scientist at Philip Morris, USA, where heis responsible for evaluating and approving materials used in the production andpackaging of cigarettes worldwide. Dr. Carmines received his B.S. in chemistryfrom the Hampden-Sydney College in Virginia and his Ph.D. in pharmacology/toxicology at the Medical College of Virginia, Virginia Commonwealth University.Prior to joining Philip Morris, USA, Dr. Carmines served as the Director of ProductSafety and Regulatory Affairs at the Dial Corporation; the Director of ProductSafety at L & F Products, and various positions with increasing responsibility atHoechst-Roussel Pharmaceuticals, Inc. Dr. Carmines’ research interests includeevaluation of potential effects of ingredients added to cigarettes and mammaliantoxicology with an emphasis on inhalation and developmental toxicity.

Gerd Kobal, M.D., Ph.D., is Director of Sensory Research, Worldwide ScientificAffairs, at Philip Morris, USA. He received his M.D. and Ph.D. in physiology andpharmacology at the University Erlangen-Nuremberg in Germany. Prior to joiningPhilip Morris, USA, Dr. Kobal was a professor and chair of the Department ofPsychophysiology at the University of Bramberg and as a professor of physiologypharmacology at the University Erlangen-Nuremberg. He also served as Presidentof the European Chemoreception Research Organization. Dr. Kobal’s research



interests include: pharmacology of pain, receptor pharmacology of algogenicsubstances, olfactory and brain imaging research and environmental toxicology.

Stephen I. Rennard, M.D., is a physician and Professor of Pulmonary and CriticalCare Medicine at University of Nebraska Medical Center. He graduated withhonors from Baylor College of Medicine and completed his residency in internalmedicine at Washington University’s Barnes Hospital. He then completed aresearch associateship in developmental biology and anomaly at the National Lungand Blood Institute of the National Institutes of Health. Dr. Rennard becameChief of the Pulmonary and Critical Care Medicine Section at University ofNebraska Medical Center. His research interests have focused on the inflammatoryand repair processes of lung tissues. This has included investigations in airwayepithelial cell biology, mechanisms of epithelial repair, and peribronchiolar airwaysdisease. He has more than 200 peer reviewed publications and has served on thecommittees of many organizations including the Pulmonary Subspecialty Sectionof the American Board of Internal Medicine, the Graduate Education Committee,the Council of Governors for the American College of Chest Physicians, the Societyfor Research on Nicotine and Tobacco, the Research Advocacy Committee, theLong-Term Planning Committee for the American Thoracic Society, and theBronchoalveolar Task Group of the European Respiratory Society.

John H. Robinson, Ph.D., is Senior Principal Scientist and Director of the SmokingBehavior & Physiology Division Product Evaluation Group at R.J. Reynolds TobaccoCompany. He received his B.A. in psychology from Providence College and Ph.D.in physiological psychology from the State University of New York at Stony Brook.Prior to joining R.J. Reynolds, he served as a research associate and adjunct assistantprofessor of physiology at Wake Forest University Medical School. A major focusof Dr. Robinson’s research is on the psychopharmacological effects of nicotineand human smoking behavior.

Hans-Juergen Roethig, M.D., Ph.D, F.C.P., F.F.P.M., is Director of ClinicalEvaluation, Worldwide Scientific Affairs at Philip Morris, USA. He received hisM.D. and Ph.D. from the Heinrich Heine University in Duesseldorf, Germany.Dr. Roethig’s postgraduate training includes studies in internal medicine, surgery,diabetes research, and psychiatry and neurology at the University Hospital inDeusseldorf, Germany. Prior to joining Philip Morris, USA, he served in clinicalpharmacology positions with increasing responsibility at Behringwerke AG, andHoechst AG/Hoechst-Roussel in Germany and at Hoescht Marion Roussel, Inc. inthe US. He also served as the Senior Director of the PAREXEL ClinicalPharmacology Research Unit at Georgetown University Medical Center inWashington, DC.

Robert J. Scheuplein, Ph.D., obtained his B.S. and M.S. degrees from the Universityof Miami and his Ph.D. from the University of Utah. In April 1999, Dr. Scheuplein

Appendices � 191


joined Keller and Heckman LLC as a scientific consultant. He began his scientificcareer at Harvard Medical School in 1962 as a Ph.D. physical chemist at theMassachusetts General Hospital specializing in skin physiology. He spent 17 yearsat FDA (1977-1994) where he worked in various senior scientific positions in theareas of toxicology, risk assessment, and food safety. He was Director of theOffice of Toxicology Sciences at FDA’s Center for Food Safety and AppliedNutrition and was responsible for the management of both regulatory and researchtoxicology. In this position, he was the Chief Scientist responsible for advising theDirector on the safety of food additives, color additives, contaminants, and otherconstituents in food. He served as chairman of the Center’s Cancer AssessmentCommittee and as a member of the Quantitative Risk Assessment Committee. In1992, he was appointed to the position of senior toxicologist for the Center. Hereceived several awards and citations, including the FDA Award of Merit and theDistinguished Career Award. Dr. Scheuplein is well known nationally andinternationally for his expertise in food safety and cancer risk assessment. Heserved as a consultant to the WHO Food and Agriculture Organization on riskassessment and risk management in the area of food safety. He has authored over50 publications in the areas of risk assessment, food safety, skin physiology, andskin permeation. He has prepared testimony for Congress and has testified beforeCongress on food safety, risk, dioxin, pesticides, and lead. In his career as a consultanthe has advised firms on the safety of cosmetics, environmental contaminants,compliance with FDA safety regulations, food additive safety testing, and FDApre-clearance requirements.

James D. Wilson, Ph.D., is a chemist and risk analyst with technical skills in cancermodeling, exposure assessment, and safety evaluation. He graduated A.B., cumlaude, in chemistry from Harvard College and received a Ph.D. in organic chemistryfrom the University of Washington. He held a variety of positions of increasingresponsibility during a 29-year career with the Monsanto Company, where heincreased the cost-effectiveness of health and environmental protection. As partof this effort, he developed a strategy for modernizing regulatory risk assessmentpractices and led chemical industry efforts to implement the strategy through multi-party coalitions, including federal and state government agencies, trade associations,and other non-governmental organizations. He has served as Vice-President ofthe American Industrial Health Council and President of the Society for RiskAnalysis. As Vice-President, has directed the St. Louis, MO office of Consultantsin Toxicology, Risk Assessment and Product Safety since 1996. He also serves onthe Board of Directors of Toxicology Excellence for Risk Assessment in Cincinnati,OH and as Consulting Fellow at Resources for the Future in Washington, DC. Dr.Wilson has published more than 50 technical articles and holds five patents.

Garold S. Yost, Ph.D., Fellow A.T.S., is Professor of Pharmacology and Toxicologyand Adjunct Professor of Medicinal Chemistry in the Department of Pharmacologyand Toxicology at the University of Utah, Salt Lake City. He received his Ph.D. in



organic chemistry at the Colorado State University and has taught pharmaceuticalchemistry at the University of California at San Francisco, Towson State University,MD, and Washington State University, WA. He is a member of the Society ofToxicology and is Fellow of the Academy of Toxicological Sciences. He has servedon various review committees for the National Academy of Sciences, NationalInstitute of Environmental Health Sciences, and National Cancer Institute and hasserved on the editorial boards of Toxicology and Applied Pharmacology, Journalof Toxicology and Environmental Health, and Drug Metabolism andDispostion. His research interests include: 3-methylindole-induced lung injury,cytochrome P450 gene regulation in the lung, and mechanisms of vanilloid (capsaicin)receptor-induced cell death. He has more than 120 peer-reviewed publications.

Added Ingredient Review Open Meeting Speakers:

Fawky M. Abdallah, Ph.D., is concurrently President of Fawky Abdallah Company,Inc., a provider of consulting services in leaf-blending and processing, cigarettemanufacturing technology, and sensory testing and smoke evaluation; and Presidentof ISSCORP (USA), a sales and marketing corporation. Dr. Abdallah obtained aB.S. and M.S. from the Cairo University, Cairo, Egypt, in agriculture and life sciencesand food flavors, respectively. His Ph.D. was obtained at the North CarolinaState University in sensory testing of cigarettes smoke, panel selection, training,and use. Dr. Abdallah served as Director of the Smoking and Research Laboratoryat North Carolina State University as well as various positions within the U.S.cigarette and tobacco industry in the areas of sales and technical services. He hasmade over 15 presentations and participated in over 18 seminars on cigaretteproduction and development. In addition to his consulting, Dr. Abdallah is acontributor in charge of product development at the Tobacco Reporter magazine.

Richard A. Ford, Ph.D., received his B.S. degree in chemistry from the OklahomaState University and his Ph.D. in physical organic chemistry from Wayne StateUniversity. Currently Dr. Ford is a consultant who specializes in aroma chemicalsat International Aroma Chemical Consultants. Dr. Ford has served as Presidentand Vice-President International at the Research Institute for Fragrance Materialsand as a consultant at Bernard Oser Associates and the Flavor & ExtractManufacturers’ Association. Dr. Ford is a member of various professional societiesincluding, the American Chemical Society, the Institute of Food Technologists, andthe American College of Toxicology. He has over 90 publications regarding hisresearch interests in flavorings, food additives, and fragrances.

Peter N. Lee, M.A., C.Stat., received his degrees at Exeter College, Oxford. Mr.Lee has served as Statistician and Research Coordinator at the Tobacco ResearchCouncil (now Tobacco Manufacturers Association) in London, England. Atpresent, he is an independent consultant in statistics and adviser in epidemiologyand toxicology to a number of tobacco, pharmaceutical, and chemical companies.

Appendices � 193


Mr. Lee has published more than 170 papers and three books. A number of thesedescribe statistical methods of analysis of long-term animal studies and theirapplication to numerous experiments. Others concern epidemiological studies andclinical trials, relating to lung cancer, chronic bronchitis, heart disease, stroke,Alzheimer’s disease, Parkinson’s disease, inflammatory bowel diseases, urinaryincontinence, and other diseases. Agents investigated include aluminum,polychlorinated biphenyls, asbestos, vitamin A, and other aspects of diet, personality,drugs, and even pet birds. Smoking and environmental tobacco smoke (ETS)exposure are particular interests. Two of his books concern ETS, the secondbeing a comprehensive review of the epidemiological evidence relating to mortalityin adults. The third is a reference book on international smoking statistics.

LSRO STAFF:

Amy M. Brownawell, Ph.D., is Staff Scientist at the Life Sciences Research Office.She completed her postdoctoral training in the Center for Cell Signaling at theUniversity of Virginia where she conducted research on the nuclear transportmechanisms of proteins and RNA. Dr. Brownawell obtained her Ph.D. in Cellularand Molecular Pharmacology at the University of Virginia and holds both a B.S.and M.S. in Chemistry from Georgetown University. She has co-authored thirteenpeer-reviewed journal articles and two book chapters and is a member of theAmerican Society for Cell Biology and Sigma Xi.

Daniel M. Byrd III, Ph.D., D.A.B.T., is Deputy Director of the Life SciencesResearch Office and the recent coauthor of Introduction to Risk Analysis: ASystematic Approach to Science-Based Decision Making (2000). He receivedhis B.A. and Ph.D. degrees from Yale University. He first received certificationfrom the American Board of Toxicology in 1982. Previously, Dr. Byrd taughtpharmacology and conducted independent research into the mechanisms anddosimetry of chemotherapeutic drugs at Roswell Park Memorial Institute and atthe University of Oklahoma. At the EPA he subsequently held positions in theOffice of Chemical Control, the Office of Pesticide Programs, the CarcinogenAssessment Group, and the Science Advisory Board, for which he was awarded aSilver Medal for Management and Leadership. He managed committees for threetrade associations and served as the President of Consultants in Toxicology, RiskAssessment and Product Safety, a scientific support firm, which helps clientsacquire, interpret, and use biomedical information. He is the author of more than100 regulatory documents and 40 scientific articles.

Michael Falk, Ph.D., is Director of the Life Sciences Research Office. He receivedhis Ph.D. in biochemistry from Cornell University and completed postdoctoral trainingat Harvard Medical School. He was employed in various capacities at the NavalMedical Research Institute, Bethesda, MD, supervising as many as 80 senior level



scientists. As Principal Investigator he was a key member of the Scientific AdvisoryBoard and Acting Director for the Institute. He was also Director of the WoundRepair Program and pioneered a new position as the Director of Biochemistry andCell Biology. As Director, he rescued the Septic Shock Research Program bycutting inefficiencies and increasing productivity in terms of grant funding andpublication production. He managed peer reviews and subject review panels ininfectious diseases, environmental sciences, military medicine, and other health-related fields. He reviewed research proposals for the National Science Foundation,Medical Research Council of Canada, and Office of Naval Research. As Directorof the Life Sciences Research Office, Dr. Falk manages the evaluation of biomedicalinformation and scientific opinion for regulatory and policy makers in both thepublic and private sectors. He has written white papers on infant nutrition, foodlabeling, food safety, and military dental research and has organized two internationalconferences. He concurrently works at MCF Science Consultants, providinganalysis and consultation on emerging technologies. Dr. Falk has published morethan 60 research articles, abstracts, technical reports, and presentations.

Robin S. Feldman, B.S., M.B.A., is Literature Specialist within the Life SciencesResearch Office. She is an information specialist with experience in the electronicacquisition, analysis, and management of scientific, business, and regulatoryinformation. Ms. Feldman obtained a B.S. in zoology from the George WashingtonUniversity in Washington, DC, and a M.B.A. with a concentration in science andtechnology from the University of Maryland at College Park. Previously, she workedas a biomedical research assistant at Consultants in Toxicology, Risk Assessmentand Product Safety, where she obtained and researched scientific literature forprivate and governmental clients. At the National Alliance for the Mentally Ill, shedesigned and implemented a document management and retrieval system for theBiological Psychiatry Branch of the National Institute of Mental Health and servedas Managing Editor of Bipolar Network News, a newsletter for the StanleyFoundation Bipolar Network. At Howard Hughes Medical Institute, she oversawthe implementation of the Predoctoral Fellowship program. While serving as ScienceInformation Specialist at the Distilled Spirits Council of the United States, shemanaged the installation of a local area network and participated in the developmentand maintenance of an electronic research database for the beverage alcoholindustry. As Report Coordinator at Microbiological Associates, Inc. she conductedstatistical analyses and prepared technical reports about toxicology studies usinganimal models. She served as Data Management Administrator for the NationalToxicology Program’s sponsored studies. Currently, Ms. Feldman maintains LSRO’slibrary, responds to requests for reports, and assists LSRO’s scientists in discovering,obtaining, compiling, and documenting the scientific literature required to preparereports for sponsors.

Karin French, B.S., is Associate Staff Scientist at the Life Sciences ResearchOffice. Ms. French received B.S. degrees in animal science and in cell and

Appendices � 195


molecular biology and genetics from the University of Maryland at College Park.In addition, she earned a College Park Scholars Certificate in “Science, Technology,and Society.” Ms. French worked in the Dairy Nutrition Laboratory at the university,helping Maryland dairy farmers use milk urea nitrogen (MUN) to evaluate herdprotein nutrition. She helped design and complete studies to compare and evaluatethe MUN analysis techniques used in the National Dairy Herd ImprovementAssociation laboratories.

Kara D. Lewis, Ph.D., is Staff Scientist at the Life Sciences Research Office.Dr. Lewis completed postdoctoral research at Yale University. She obtained herPh.D. in biology with a concentration in neuroscience from Clark University andgraduated summa cum laude with a B.S. degree in biology from Spelman College.Dr. Lewis conducted research on taste and smell of the fruit fly, Drosophilamelanogaster, and on the molecular mechanisms of sweet taste transductionin the blowfly, Phormia regina. She has collegiate teaching experience andthree peer-reviewed publications. She is a member of the Association forChemoreception Sciences.

Paula M. Nixon, Ph.D., is Staff Scientist at the Life Sciences Research Office.Dr. Nixon completed her postdoctoral research at the Babraham InstituteCambridge, UK. She obtained her Ph.D. in molecular biology from the ImperialCollege, London. She graduated with an honors degree in molecular biology fromthe University of Manchester, UK. Dr. Nixon conducted research on the MEK5/ERK5 MAP kinase pathway and the role of the AP-2 transcription factors in thecontrol of gene expression in breast cancer. In addition she was involved in researchto identify the Currarino syndrome gene and the development of mitochondrialDNA profiling techniques for forensic science. She has three peer-reviewedpublications and is a member of the American Society for Biochemistry andMolecular Biology.

Negin P-M. Royaee, B.S., worked as an Associate Staff Scientist during thepreparation of the Phase One and Two reports. She was previously employed atBiophysical Society, FASEB, where she assisted in the production of theBiophysical Journal. Ms. Royaee obtained her bachelor’s degree from LakeForest College in Chicago, IL, with a major in biology and minor in art history. Ms.Royaee has volunteered at The Children’s Inn at the National Institutes of Health.Currently Ms. Royaee resides in Boston, MA, where she is working and pursuinga graduate degree at Boston University.

Kristine K. Sasala, B.S., was Associate Staff Scientist at the Life Sciences ResearchOffice. Ms. Sasala obtained her B.S. degree in biology from the University ofCincinnati, OH, with a focus on cell biology. Prior to joining the LSRO, Ms. Sasalaworked at Nerac, Inc., as Information Scientist. She has been involved in researchingthe molecular biology of osteoporosis, pharmacology of xenobiotics, and the



development of vaccinations for Brucella, in addition to graduate work inprotistology. Ms. Sasala has two peer-reviewed publications and several conferencepublications.

LSRO CONSULTANTS:

Kenneth Bailey, Ph.D., serves as a consultant in toxicology since retiring from theEPA in 1996. While at EPA, Dr. Bailey held several positions of increasingresponsibility in the Office of Pesticide Programs, Office of Toxic Substances, andOffice of Water. He was instrumental in initiating and managing the EPA part ofthe U.S. National Research Council review of fluoride and nitrate/nitrite in drinkingwater as well as investigating exposure from inhalation and dermal absorption ofdrinking water contaminants. More recently as Senior Toxicologist at the WaterResearch Centre in Buckinghamshire, UK, he was the Editor and co-author of theWorld Health Organization fluoride document for less developed countries andprovided guidance and training concerning toxicological developments in the U.S.

Robert P. Beliles, Ph.D., D.A.B.T., received his B.A. in zoology and M.S. in biologyfrom the University of Louisville and a Ph.D. in pharmacology and physiologyfrom Iowa State University. Dr. Beliles served as Director of the Office of RiskAssessment and the Office of Carcinogen Standards, at the U.S. Department ofLabor, and as Director of the Department Of Toxicology and Associate Directorfor the Department of Pharmacology and Toxicology, at Litton Bionetics, Inc. Heheld various positions within the EPA and recently as a toxicologist at the NationalCenter of Environmental Assessment and as an environmental health scientist atthe Office of Health and Environmental Assessment. Dr. Beliles is a member ofthe American Conference of Governmental Industrial Hygienists, Society ofEnvironmental Toxicology and Chemistry, Society of Toxicology, Teratology Society,and is a charter member for the Association of Governmental Toxicologists. Hehas served on the editorial board of Toxicology and Industrial Health, Journalof Regulatory Toxicology and Pharmacology, and the Registry of Toxic Effectsof Chemical Substances and authored chapters in both Patty’s Industrial Hygieneand Toxicology and Toxicology: The Basic Science of Poisons.

John G. Keller, Ph.D., is Principal for Professional Consulting Services, servingprivate sector and government clients. Dr. Keller received his B.S. in biology andchemistry at Georgetown University and a M.S. in zoology and Ph.D. in animalphysiology, biochemistry, and pathology at the St. Louis University. His 46 yearcareer has included occupational and environmental assessments, risk analysis,regulatory analysis, environmental health, and litigation support. Prior to joiningProfessional Consulting Services, Dr. Keller was Senior Health Scientist at ApexEnvironmental, Inc. where he provided technical support to private sector andgovernment clients in areas of toxicology, carcinogenesis, regulatory affairs, risk

Appendices � 197


characterization, risk management, and risk communication, and litigation support.Dr. Keller has also provided support of government activities, including managementof the preparation of more than 30 Toxicological Profiles for the Agency for ToxicSubstances and Disease Registry; he was principal author on the tin, styrene, andpyridine profiles and co-author on methylene chloride and other profiles. His researchand laboratory experience includes supervision of all types of toxicology andcarcinogenesis studies on many classes of chemicals and products, including themanagement of Carcinogenesis Bioassay Testing for the National ToxicologyProgram. Dr. Keller is active in many professional and learned societies, wasFounding Editor of Drug and Chemical Toxicology and is past Chair of the GordonResearch Conference on Toxicology. Currently, he is a member of the InternationalSociety of Regulatory Toxicology, the American Association for the Advancementof Science, the American Industrial Hygiene Association, the Society for RiskAnalysis, and the American Chemical Society. He has authored or otherwisecontributed to the preparation of more than 2,500 confidential commercial client orgovernment reports covering toxicology, pharmacology, pathology, and carcinogenicstudies.

Hazel B. Matthews, Ph.D., serves as a consultant in toxicology since retiring fromthe National Institute of Environmental Health Sciences (NIEHS) in April 2001.Dr. Matthews received B.S. degrees in zoology and plant protection from NorthCarolina State College, a M.S. in entomology from North Carolina State University,and a Ph.D. from the University of Wisconsin with a major in entomology and aminor in biochemistry. Dr. Matthews worked at NIEHS for over 30 years duringwhich time he held a variety of positions including, most recently, Head of theChemistry Section and Head of the National Toxicology Program’s Office ofNominations. His recent outside activities included a year as the Society ofToxicology’s Congressional Fellow in Washington, DC (2000-2001). Dr. Matthewshas served on a variety of national and international committees including the editorialboard of Drug Metabolism Reviews and as associate editor of Fundamental andApplied Toxicology. Dr. Matthews’ research interests at NIEHS includedinvestigations of the fate and toxicity of chemicals and the acute and chronicinteractions of chemicals and/or their metabolites with subcellular macromoleculesthat result in toxicity and/or carcinogenicity.

Earl F. Walborg, Jr., Ph.D., serves as Co-founder and President of Dermigen, Inc.,a company providing consulting and research management in tissue-based toxicologyand as Co-founder of BioChemix, Inc, a company focused on the manufacturingand marketing of natural chemicals with potential to inhibit disease processes andaging. Dr. Walborg received a B.S. degree with a major in chemistry and a minorin biology from Austin College in Sherman, Texas. He received his Ph.D. inbiochemistry from Baylor University College of Medicine at Houston. Dr. Walborg’sacademic career in biomedical research includes his appointments as Biochemistand Professor of Biochemistry at the University of Texas, M.D. Anderson Cancer



Center; Biochemist and Professor of Biochemistry at The University of TexasHealth Science Center at Houston, Graduate School of Biomedical Sciences; andAssociate Director, at the Science Park-Research Division, in Smithville, Texas.Dr. Walborg is a member of several professional societies including the Society ofToxicology, American Society of Biochemistry and Molecular Biology, and AmericanAssociation for Cancer Research. Dr. Walborg has over 80 peer-reviewedpublications and has edited two conference proceedings: Cellular Membranesand Tumor Cell Behavior and Glycoids in Disease Processes. He served onthe editorial board of the Year Book of Cancer with the responsibility in the areaof carcinogenesis.

Appendices � 199


LIFE SCIENCE RESEARCH OFFICEBOARD OF DIRECTORS:

Chair: Salvatore (Sam) J. Enna, Ph.D.University of Kansas Medical CenterKansas City, KS

Vice Chair: Taffy J. Williams, Ph.D.IMCOR Pharmaceutical Co.New Hope, PA

Treasurer: Arnold Kahn, Ph.D.University of California at San FranciscoSan Francisco, CA

Past Chair: William Heird, M.D.Baylor College of MedicineHouston, TX

Directors: Joseph F. Borzelleca, Ph.D.Virginia Commonwealth UniversityRichmond, VA

Gilbert Leveille, Ph.D.Cargill Health & Food TechnologiesWayzata, MN

Robert Newburgh, Ph.D.The Protein SocietyBethesda, MD

Terry Quill, M.S., J.D.International Society for Regulatory Toxicology and PharmacologyWashington, DC

Jacob J. Steinberg, Ph.D.Albert Einstein College of MedicineMontefiore Medical CenterBronx, NY

Secretary: Michael C. Falk, Ph.D.Life Sciences Research OfficeBethesda, MD



APPENDIX B

GLOSSARY

Abruptio placentaeThe premature separation of the placenta from the uterine wall.

AbsorptionThe transport of a substance from the site of exposure or administration, acrosscell membranes, into the bloodstream. The uptake of a substance.

AccuracyThe extent to which a measurement conforms to a standard or “true” value.

Added ingredientA non-tobacco substance added to a cigarette (e.g., flavoring, humectant).

AdditiveA substance (e.g., flavoring, preservative) added to another defined substance(e.g., cigarettes, food). In this report, “additive” is used synonymously with “addedingredient”.

AerosolLiquid and/or solid particles suspended in a gas. Aerosol particle diameters rangebetween approximately 0.002 - 100 µm.

Air-curedTobaccos (e.g., Burley and Maryland) typically dried without the application ofartificial heat. Air-cured tobaccos are generally alkaline, nitrogen rich, and aftercuring, have little or no sugar.

AneuploidA chromosome number that is not an exact multiple of the haploid number ofchromosomes.

AshSolid remains generated from burning of a cigarette, cigar, or pipe tobacco.

Biologically effective dose (BED)The amount of substance that is sufficient to elicit an effect on biological tissue.

Appendices � 201


BiomarkerA biological response variable that is indicative of exposure, and/or effect(i.e., disease initiation, disease progression, remission, death).

Biomarker of exposureA substance measured in biological tissue or fluid, following an exposure, that hasthe potential to interact with biological molecules. A surrogate measurement ofexposure.

Blended cigaretteA cigarette made from a mixture of different tobaccos.

Burley tobaccoA type of air-cured tobacco, ranging in color from light brown to reddish brown,with undetectable or very low sugar concentrations. Burley tobacco gives off acocoa-like aroma, has the ability to absorb a significant amount of casing, and is animportant component of blended tobacco cigarettes. Burley tobacco is grownmainly in Kentucky and Tennessee in the United States.

CasingA solution of added ingredients applied to tobacco to condition it for processing andimprove smoke quality.

ChromatidOne of the two daughter strands of a duplicated chromosome that are joined by asingle centromere in somatic cells during mitosis.

Chronic obstructive pulmonary disease (COPD)Group of diseases characterized by progressive, airflow limitation that is notcompletely reversible spontaneously or with treatment. Includes chronic obstructivebronchitis and emphysema.

CigaretteA rod of tobacco wrapped in paper.

Cigarette smoke condensateThe particulate phase of cigarette smoke. Includes liquid and/or solid particleswithin the smoke aerosol. (See particulate phase.)

ClearanceThe kinetic parameter relating the elimination rate of a substance to plasmaconcentration. A representation of the volume of plasma completely freed of asubstance in a specified time.



Complex mixtureA substance consisting of multiple chemical constituents.

CotinineThe primary metabolite of nicotine. Cotinine has a half-life of approximately 19hours and is used as biological marker of cigarette smoke exposure.

Crossover studyA study in which each participant is given a sequence of treatments/exposuresthat allow for assessment of differences in effects of treatments/exposures. Incrossover studies, each study participant is used as his/her own control, removingcontribution of inter-subject variations in responses.

CytotoxicityMeasurement of a chemical’s ability to damage or kill cells.

DeletionThe removal of a part of a chromosome.

DepositionProcesses determining the portion of inhaled substance remaining in the lungsfollowing exhalation.

Detection limitThe smallest concentration of a substance measurable with a particular technique.Definitions of detection limits vary with the objective and method.

Developmental toxicantAn agent causing abnormal fetal development resulting in congenital malformation.

DistillationSeparation of a liquid substance from a mixture through evaporation andcondensation.

DoseThe mass of a substance that crosses the outer most membranes following anexposure.

DosimetryThe application of scientific techniques for measuring the amount, metabolism,distribution, and elimination of a substance.

Appendices � 203


EliminationElimination of a substance from the body follows its removal from the blood, possiblyits metabolism, and finally excretion from the body. The combination of chemicaldegradation of a xenobiotic in the body and excretion by the intestine, kidneys,lungs, skin, or in sweat, expired air, milk, semen, menstrual fluid, or secreted fluids.

ExcretionProcess by which a substance or a metabolite is removed from the body withoutadditional chemical change.

EndpointA parameter measured to assess a biological effect.

Environmental tobacco smoke (ETS)Smoke consisting of aged sidestream smoke and exhaled mainstream smoke.

EpigenomicModulation of regional chromatin organization, without the alteration of thecorresponding DNA sequence, influencing gene expression (e.g., DNAmethylation).

ExposureThe concentration of a substance in contact with outer membranes of an organsystem.

Filter (cigarette)A device positioned at the butt end of cigarette, which serves as a smoke permeablemedium used to modify tobacco smoke. In the U.S., filters are usually composedof cellulose acetate fibers and encased by a wrapper.

Flue-curedTobaccos (e.g., Virginia) that are dried (cured) in tightly constructed barns withartificial heat beginning at 35°C and ending at about 75°C over a 5 to 7 day period.

Frame-shift mutationThe insertion or deletion of a number of nucleotides, not divisible by three, into acoding sequence causing an alteration in the open reading frame of the entiresequence downstream of the mutation.

Gas phaseA phase is a distinct and homogenous state of a system with no visible boundaryseparating it into parts. One property of a gas is that it fills the entire volume of thecontainer. In this report the gas phase is the tobacco smoke not readily condensablewhen passed through a filter, in contrast to the particulate phase.



Genotoxicity testsTests of the propensity of cigarette smoke, or fraction of cigarette smoke, to damagethe genetic material (DNA) of the test cell; a more general term for mutagenicitytests.

HumectantSubstance or compound added to cigarette tobacco during primary processing toretain moisture and plasticity. The principal humectants in cigarette manufacturingare glycerol and propylene glycol.

Indirect additiveA chemical substance detectable in a cigarette, introduced into cigarettes throughtreatment during the growing and handling of the tobacco, or manufacture andhandling of paper. Indirect additives have no specific purpose or functional effectin the cigarette.

IngredientSubstances added to tobacco in the manufacture or preparation of cigarettes. Someingredients such as flavors are added to the tobacco during the processing and/ormanufacturing process. Additional ingredients such as filters, paper, and rod inksare used in the product to complete the cigarette unit. Ingredients may be a singlesubstance in the case of a pure flavor, or several substances as in the case ofadhesives.

In vitro testA test that is performed on single cells or organs derived from an animal (or human),as opposed to an in vivo test that is performed on an entire living animal (orhuman). Tests performed on single-celled organisms, such as bacteria or yeast,are classified as in vitro tests.

In vivo testA test that is performed in a whole, living animal (or human), as opposed to an invitro test.

KineticsThe study of the rate of change of dose (e.g., pharmacokinetics or toxicokinetics).How rapidly the body processes a substance, specifically the rates of absorption,distribution, metabolism, and elimination of chemical substances.

Mainstream smoke (MS)Smoke drawn through the butt end of the cigarette into the mouth, as a smokerpuffs on a cigarette.

Appendices � 205


Margin of exposure (MOE)The ratio of the no-observed-adverse-effect-level (NOAEL) to the estimatedexposure dose.

Maryland tobaccoA type of light bodied, mild, air-cured tobacco. Originally grown on the sandy soilsin southern Maryland, now also grown on other countries such as Italy and Japan.

Minimal risk level (MRL)An estimate of the daily human exposure to a hazardous substance, that is likely tobe without appreciable risk of adverse non-cancer health effects, over a specifiedduration of exposure.

Maximum use level (MUL)The MUL is the maximum amount of each ingredient that may be added to cigarettetobacco.

Maximum tolerated dose (MTD)In theory, an MTD is an estimate of the maximum daily dose or exposure which aspecies can experience over a lifetime and experience no adverse effects. Inpractice, MTDs for animal species usually are estimated from experimental dataabout lifespan or inhibition of body weight gain.

MentholationThe addition of menthol to a cigarette during production. Menthol is added as a topflavoring and is applied to cut tobacco after final drying to minimize evaporation ofthe ingredient.

MicroarrayAn array of DNA or protein samples that can be hybridized with probes to studypatterns of gene expression.

Micronucleated cellDisruption of cell division, or damage to chromosomes by chemicals or radiation,affects the distribution of genetic material between two daughter nuclei during celldivision. Pieces or entire chromosomes may fail to be included in either of the twodaughter nuclei. When this occurs, the genetic material not incorporated into anew nucleus, may form its own “micronucleus” which is clearly visible with amicroscope. Micronucleated cells are therefore used as a measure of chromosomedamage and mitotic disturbances.



Mutagenesis testsTests for the propensity of cigarette smoke, smoke particulates, or smoke condensateto cause mutations in the genetic material (DNA) of the test cell. A widely usedmutagenesis test is the Ames test, which is performed on special strains ofSalmonella bacteria.

N-NitrosaminesChemical substances formed by nitrosation of secondary and tertiary amines.Tobacco-specific nitrosamines (TSNAs) are formed by the nitrosation of the majortobacco alkaloid, nicotine.

NicotineA cyclic tertiary amine composed of a pyridine and a pyrrolidine ring. It is acolorless to pale yellow, water soluble, oily, volatile, and hydroscopic liquid derivedfrom plants of the Nicotiana genus.

Nicotine free dry particulate matter (NFDPM)See definition for tar.

Nicotinic cholinergic receptor (nAChR)A receptor that binds nicotine and which has been identified in the brain,neuromuscular junctions, autonomic ganglia, and adrenal medulla. nAChRs arepentamers made up from different combinations of four subunits (α, β, γ and δ).

NoiseBackground interference due to the inherent variations associated with analyticalmethodology.

OrganolepticAffecting or involving a sense organ such as that of taste, smell, or sight.

Oriental tobaccosSun-cured tobacco predominantly grown in Turkey, Greece, and the Balkans. Thesetobaccos are derived from the main species Nicotiana tabacum; specific oils andresins from these tobaccos provide distinctive taste and aroma.

Pack-yearA unit measure of smoking exposure. One pack-year represents the consumptionof 20 cigarettes per day (one pack) for one year.

PaperCigarette paper (wrapper) is composed of inorganic filler and cellulose fibers fromwood pulp and/or flax. Cigarette paper controls smoke yield through the burnproperty and air permeability of the paper.

Appendices � 207


Particulate phaseThe solid and/or liquid phase suspended in the smoke aerosol. The particulatephase is often described as that portion of smoke retained on a Cambridge filterpad during smoke collection. However, separation of the gas and particulate phasesof smoke by a Cambridge filter is not absolute. The pad can also collect somevapor phase constituents.

Physiological-based pharmacokinetic (PBPK) modelsThe pharmacokinetic behavior of a compound in the body (absorption, distribution,metabolism, and elimination) represented by equations that describe actualphysiological processes quantitatively.

Placenta previaImplantation of the placenta over or near the cervix.

Point mutationA mutation caused by the substitution of one nucleotide by a different one. Thisresults in the DNA molecule having a change in a single base pair.

Polycyclic aromatic hydrocarbon (PAH)A class of organic compounds containing fused aromatic ring systems, formedduring incomplete combustion. PAHs include benzo(a)pyrene, fluoranthene, andpyrene.

Premature rupture of fetal membranesThe breaking apart of fetal membranes more than one hour before labor begins.

Processing aidsIngredients that facilitate the manufacturing procedures and enhance the efficiencyof cigarette production.

Puff duplicatorAn automated smoking machine that can be programmed with puff parameterswhich duplicate those of an individual human smoker.

Puff flowMainstream smoke flow from cigarette to smoker in mL per second.

Puff intervalDuration of time between the start of one puff and the next in seconds.



Puff profileA puff profile graphically represents the rate of flow of inhaled cigarette smokeduring a puff. Puff profiles are a basis to calculate puff volume (InternationalOrganization for Standardization, 2000).

Puff recorderA device to measure smoking behavior while a subject smokes. Puff recordersmeasure parameters such as puff flow and duration.

Puff volumeThe volume of smoke inhaled from one puff in mL.

PyrolysisThermal degradation of a substance, generally resulting in smaller chemicalfragments and generally occurring in a reducing, not an oxidative, atmosphere.(See pyrosynthesis and oxidation.) Pyrolysis often is used as a term to describe all(pyrolysis, pyrosynthesis, and oxidation) of the chemical processes related to thermalenergy that go on in a puffed cigarette.

PyrosynthesisFormation of additional compounds by the recombination of chemically reactivefragments arising from incomplete combustion of a parent chemical/compoundduring pyrolysis.

ReceptorA binding site on a cell’s surface. When chemicals bind to receptors, variouscellular functions are activated or inhibited.

RecombinationA bringing back together of two, previously separated parts. Recombination has aspecialized, genetic meaning in biology. “In general, any process in a diploid orpartially diploid cell that generates new gene or chromosomal combination notfound in that cell or its progenitors. At meiosis, the process that generates ahaploid product of meiosis whose genotype is different from either of the twohaploid genotypes that constituted the meiotic diploid,” (Griffiths et al., 1999).

Reconstituted sheet tobacco (RST)A processed tobacco widely used in tobacco blends made though a process likemaking paper or from slurries of small scraps not suitable for cut filler. In a typicalAmerican blended cigarette, RST constitutes 10 - 25 % of content by weight.

Appendices � 209


Reference cigaretteCigarettes prepared under controlled conditions with uniform, documented sourcetobaccos and producing standardized yields of ‘tar’, nicotine, and carbon monoxide.The Tobacco & Health Research Institute at the University of Kentucky producesamong others the 1R2 and 2R4F reference cigarettes.

Relative riskExpression of a risk in relation to another risk, (e.g., the risk of death at work isapproximately twice the risk of death from drowning).

Residence timeThe time between inhalation and exhalation, when a smoker holds smoke in therespiratory tract. Estimates of residence time vary between 4 seconds and 24seconds.

Resistance-to-draw (Draw resistance)The negative pressure (suction) applied to puff smoke through a cigarette, measuredin Pascals (Pa) (Pa = one Newton per square meter).

RetentionThe amount of smoke (and its constituents) found in the lungs at any time, includingdeposited and absorbed smoke (Brain & Valberg, 1979).

Reverse smokingA method of generating smoke by machine in which air is forced through thelighted cigarette by applying an elevated pressure to the burning end. This methodhas been used in animal exposure systems to minimize the time to deliver thesmoke to the animal (Schultz & Wagner, 1975).

RevertantsHaving reverted to the normal phenotype, usually by a second mutation: a revertantmutant.

RiskThe probability of a future loss: in this report the probability of mortality, morbidity,or adverse health effects within a stated time (Byrd & Cothern, 2000).

Sales-weightedStatistical adjustment of data about cigarette brands, such as ‘tar’contents, bytheir sales volumes.



SensitivityThe capacity to detect a signal (response) above background interference (noise).

Sensory impactThe effect of a stimulus which changes behavior dependent on sensate pathways(e.g., an attractant odor or the localized sensation in a smoker’s throat after inhaling)(Junker et al., 2001).

Sidestream smoke (SS)The smoke emitted directly into the air from the burning end of the cigarette,mostly during the smolder interval between puffs.

Signal-to-noise ratioThe ratio of the measurement to interference. Signal-to-noise ratio is a measureof the quality of information.

SmokeUsually a suspension of fine particles in air that scatters light and is visible. Cigarettesmoke contains many chemical substances in both gas and liquid state, suspendedin a dynamic aerosol created by incomplete combustion and changing both physicallyand chemically with time.

Smoke analyteA chemical constituent of cigarette smoke.

SmolderBurning slowly without acceleration by forced air.

SpecificityIn testing, the ability to detect the absence of signal when none is present.

Surrogate endpointA measurement, such as a biomarker, that can be substituted for an endpoint, suchas a disease. (See sensitivity and specificity.)

TarThe Federal Trade Commission refers to ‘tar’ as the weight in grams of the totalparticulate matter collected on a Cambridge filter minus the weight of alkaloids, asnicotine, and water (Federal Trade Commission, 1967).

Appendices � 211


Topography/TopologicalIn this report, the study of human cigarette consumption, including the intensity ofthe draw on the volume of smoke in each puff, the duration of the puff, the volumeof smoke in each puff, the intervals between puffs, the number of puffs taken percigarette, and the number of cigarettes smoked daily.

Toxicological (toxic)Related to circumstances that alter biological function, such as a change in morbidityor mortality.

ToxicologyThe study of poisons.

TumorigenicCausing tumors or cancers in laboratory animals or humans; sometimes usedsynonymously with “carcinogenic”.

Vapor phaseSee definition for gas phase.

VentilationPerforations in the filter tipping paper or paper wrapping the tobacco rod thatdilute total air flow inside the rod and reduce pressure drop; expressed as apercentage with higher percentages indicating greater ventilation.

Virginia tobaccoA dark, flue-cured tobacco.

YieldCigarette classifications based on the amounts of substances, such as ‘tar’, nicotine,or carbon monoxide, usually as produced under standard smoking conditions.



LITERATURE CITATIONS

Brain, J. D. & Valberg, P. A. (1979) Deposition of aerosol in the respiratory tract.Am. Rev. Respir. Dis. 120:1325-1373.

Byrd, D. M. & Cothern, C. R. (2000) Introduction to Risk Analysis. Rockville,MD: Government Institutes.

Federal Trade Commission. (1967) Cigarettes. Testing for tar and nicotine content.Fed. Reg. 32 (147):11178.

Griffiths, A. J. F., Gelbart, W. M., Miller, J. H. & Lewontin, R. C. (1999) Glossary.In: Modern Genetic Analysis. New York: W.H. Freeman and Company, pp.608-633.

International Organization for Standardization. (2000) Routine AnalyticalCigarette-Smoking Machine — Definitions and Standard Conditions. ReportNo. ISO 3308:2000. Geneva, Switzerland: International Organization forStandardization.

Junker, M. H., Danuser, B., Monn, C. & Koller, T. (2001) Acute sensory responsesof nonsmokers at very low environmental tobacco smoke concentrations in controlledlaboratory settings. Environ. Health Perspect. 109:1045-1052.

Schultz, F. J. & Wagner, J. R. (1975) A thirty-port smoking machine for continuoussmoke generation. Beitr. Tabakforsch. 8:53-59.

Appendices � 213


ACRONYMS

American Cancer Society ACSAmerican Society for Testing Materials ASTMAnalysis of variance ANOVAArea under the concentration time curve AUCAssociation of Official Agricultural Chemists AOACBenzo(a)pyrene BaPBiologically effective dose BEDCancer Prevention Study I CPS-ICancer Prevention Study II CPS-IICarbon dioxide CO

2

Carbon monoxide COChemical Abstract Service CASChronic obstructive pulmonary disease COPDConsumer Product Safety Commission CPSCCoopération Pour Les Recherches Scientifiques Relative au Tobac CORESTAEarly detection research network EDRNEffective dose EDEnvironmental tobacco smoke ETSFederal Trade Commission FTCFlavors and Extracts Manufacturers Association FEMAFood and Agriculture Organization of the United Nations FAOGas chromatography GCGas chromatography – mass spectrometry GC-MSGas chromatography – mass spectrometry/mass spectrometry(Gas chromatography – tandem spectrometry) GC-MS/MSGood Laboratory Practice GLPGood Manufacturing Practice GMPHigh-pressure liquid chromatography HPLCInfrared IRInstitute of Medicine IOMInstitutional Review Board IRBInternational Agency for Research on Cancer IARCInternational Conference on Harmonisation ICHInternational Programme on Chemical Safety IPCSInternational Standards Organization ISOJoint FAO/WHO Expert Committee on Food Additives JECFALethal dose LDLife Sciences Research Office, Inc. LSROMainstream smoke; Mass Spectrometry MSMargin of exposure MOEMassachusetts Department of Public Health MDPH



Maximum concentration reached Cmax

Maximum tolerated dose MTDMaximum use level MULMicrogram µgMinimal risk level MRLNanogram ngNational Institutes of Health NIHNational Toxicology Program NTPNatural killer NKNicotine free dry particulate matter NFDPMNicotinic cholinergic receptors nAChRsNo-observed-adverse-effect level NOAELNuclear magnetic resonance NMROrganization for Economic Cooperation and Development OECDParts per billion ppbParts per trillion pptPhysiological-based pharmacokinetic PBPKPolycyclic aromatic hydrocarbon PAHPotential reduced-exposure products PREPsReconstituted sheet tobacco RSTSidestream smoke SSStandard deviation SDStandard error SEThin-layer chromatography TLCThreshold of toxicological concern TTCTime when maximum concentration is reached T

max

Tobacco Science Research Conference TSRCTobacco-specific nitrosamines TSNATotal particulate matter TPMU.S. Department of Agriculture USDAU.S. Department of Health and Human Services DHHSU.S. Environmental Protection Agency EPAU.S. Food and Drug Administration FDAU.S. General Accounting Office GAOUltraviolet UVWorld Health Organization WHO

Appendices � 215


APPENDIX C

PUBLIC AND INVITED COMMENTS

The following text was presented at LSRO’s November 5, 2003, Open Meetingon the Relative Risk of Non-Tobacco Ingredients Added to Cigarettes. Withthe consent and review of the presenters, LSRO is making the content of thepresentations available to readers. LSRO did not generate the text, nor doesthe text necessarily represent LSRO’s positions.

Additives Used in the Production of Cigarettes

Fawky M. Abdallah, Ph.D.

First, I would like to thank LSRO and the organizers of this meeting for inviting meto share with you some of my thoughts and findings. My presentation discussesthe role of blending and processing. My presentation consists of these parts:

I. DefinitionsII. Manufacturing OperationsIII. BlendingIV. ProcessingV. Recent Developments

I. DEFINITIONSFor the sake of clarity, I will provide five generally-accepted definitions.

A. Cigarette Smoke

The smoke is the actual product of consumption and the cigarette is technically asmoke-delivery system.

* Smoke formation occurs in the burning cone where the processes of pyrolysisdegradation take place as well as in the thermal gradient zone ahead of theburning cone, where the processes of distillation, volatilization, sublimation,cellular eruption, and chemical interactions take place.

* The nature of smoke determined as the mainstream smoke (MS) travels inthe tobacco column, is subjected to air dilution by the cigarette paper, tipping,perforation and ventilation, where condensation and re-volatilization occur.It is important to recognize that cigarette smoke is a dynamic aerosol system,as evidenced by the different physical, chemical, and sensory properties of“fresh” smoke versus stale smoke or smoke condensate that is set in solutionfor chemical analysis.



B. Smoke Chemistry

The definition of smoke chemistry differs according to the type of investigation.

* Research and analytical chemists identify the thousands of componentsubstances in the total particulate matter (TPM), commonly called ‘tar’, andthe gaseous phase of cigarette smoke.

* Blenders work on the foundation blend and develop the extenders, casings,and flavorings that complement the blend in order to have a total blend cutrag with the target nicotine and sugars while maintaining the desired smokingqualities.

* Process engineers determine the processing parameters and cigarette designconfiguration that are required to produce cigarettes with the target ‘tar’and nicotine deliveries and acceptable smoking qualities.

C. Smoke Yield

The term “smoke yield” usually concerns the toxicologists, biologists, and workerson smoking and health, who investigate not only the deliveries of ‘tar’ and nicotinein the cigarette MS but also its content of specific harmful compounds, such asnitrosamines, benzo(a)pyrene, phenols, and carbon monoxide. Studies on smokeyield investigate the carcinogenic, co-carcinogenic, antagonistic, and other healthrisks of smoke compounds to test animals in an effort to explore health risks tohumans (smokers).

D. Smoking Qualities

* Product developers study smoking qualities in terms of the physical andchemical properties of the MS that are required to meet the target cigarette.

* Sensory analysts refer to smoking qualities as the overall sensory propertiesof the “fresh” MS as determined by flavor (taste, smell, and feel), aroma,mildness, after-taste, impact, burning qualities, etc. Sensory analysts trainand use smoking panels as a measuring device to evaluate the smokingqualities of the test cigarettes.

* Marketing specialists conduct consumer tests using focus groups and marketsurveys to determine the overall acceptability in terms of smoking qualities,price, brand identity, etc.

Appendices � 217


E. Additives

Casings, flavorings, humectants, and any other non-tobacco materials added duringcigarette manufacturing are collectively called additives.

Additives cover anything added to tobacco during processing, other than water,and to the cigarette paper, filter, and tipping, during making. Additives should meetthe regulations required by one or more pertinent authorities, such as the U.S.Food and Drug Administration (FDA), Institute of Food Technologists (IFT),American Society for Testing & Materials (ASTM), Flavor and ExtractManufacturers Association (FEMA), World Health Organization’s Joint ExpertCommittee for Food Additives (JECFA), and Council of Europe (CoE).

Since the actual product consumed is the MS and not the cigarette per se, theacceptability of certain ingredients for use in food does not necessarily meanapproval for use in smoking tobacco products such as cigarettes.

II. MANUFACTURING OPERATIONS

Manufacturing a typical American blended cigarette (ABC) is a combination of:

A. Primary operation covering blending and processing; andB. Secondary operation covering cigarette making and packing.

A. Primary Operation

The ABC primary operation requires working in four areas simultaneously:

Foundation Blend Flue-cured, Burley, and Oriental, each is a sub-blend

Extenders Reconstituted sheet, expanded cut tobacco (ECT), andcut-rolled expanded stems (CRES)

Additives Casings, flavorings, and humectants

Processing Six main stages

B. Secondary Operation

The secondary operation covers:

Cigarette Making Including tobacco column’s moisture content, weight,density, firmness, filling value, pressure drop, etc., and thecigarette configuration’s paper, porosity, filter, tipping,ventilation, perforation, etc.

Cigarette Packing Including package design and packaging materials



III. BLENDING

This part covers three main subjects.

A. Why Blending?

Because of the inherent variation among tobacco types, origins, crops, and stalkpositions, along with the incompatibility between the elements of tobacco quality,there is No Perfect Grade.

Leaf grades that have more aroma and smoke flavor usually have a slower burningrate and are high in ‘tar’ and nicotine deliveries in the mainstream smoke. Gradesthat are high in filling value are low in ‘tar’ and nicotine but have less desirablesmoking qualities. Each type is grown under certain weather conditions using avariety of cultural practices; therefore, there is no uniformity from one year’s cropto the next or from one region to another.

In principle, each grade is selected to contribute certain traits, in terms of physicalproperties, smoking qualities, and value. The quality of a particular grade is thedegree to which it possesses characteristics that are required for the target blend.

Blending know-how allows for the development of the desired ABC blend in astep-wise technique. Blending is an on-going process that requires extensiveexperience in handling the continuous changes in the quality and cost of the blendcomponents from year to year and one origin to another. The result of a goodblending is achieved when the overall quality of the blend is more desirable thanthe sum of the qualities of the individual component grades.

B. Basic Principles

There are four basic principles for blending:

1. Balance. The objective of blending is to create the desired balance betweenthe physical, chemical, and sensory properties of the component grades ineach of the sub-blends: flue-cured, Burley, and Oriental.

2. Categories. The component grades in the foundation blend are classifiedinto three categories: Full Flavor, Medium Flavor, and Filler grades.

* Full Flavor (FF) Grades: Provide desirable flavor attributes and the rightintensity of irritation. This category contributes the most to the deliveries of‘tar’ and nicotine in the mainstream smoke.

* Medium Flavor (MF) Grades: Contribute to the overall taste and, althoughthey do not possess a noticeable flavor of their own, MF grades enhance theflavor attributes of the FF grades. This category contributes moderately tothe ‘tar’ and nicotine deliveries.

Appendices � 219


* Filler Grades (FG): Increase filling power and absorptive capacity and reducecost. They contribute least to flavor and must be neutral with no off-taste orundesirable odor. This category contributes the least in terms of ‘tar’ andnicotine.

3. Criteria. There are no criteria that fit all tobacco types and grades. Thereare different criteria for each tobacco type and within type according to theorigin, crop year and stalk position. A tobacco blender works on theassumption that each of the three tobacco types in the foundation blendcontains grades that fit one or more of the three categories FF, MF, and FG.Processing plays a major role in the contribution of the sub-blends. Thecontribution of flue-cured and Oriental is largely dependent on the carefulselection of the component grades therein and to a certain extent on theprocessing of such. The contribution of Burley is dependent not only on thecomponent grades but also on the adequate casing and toasting.

4. Philosophy. The basic philosophy for a balanced, consistent and cost-effectiveblend is that of optimization between the amount and ratio of the differenttobacco types and the grades within each type as well as the cost, availability,and continuity.

C. Deciding Factors

There are five deciding factors that determine the target blend:

1. Blend chemistry Nicotine and sugars2. Physical properties Filling value and processing ability3. Smoke chemistry ‘Tar’ and nicotine deliveries4. Smoking qualities Sensory properties and burning qualities5. Economics Cost, availability, and continuity of leaf tobacco

and extenders

IV. PROCESSINGThis part of my presentation covers four subjects:

A. Primary Processing

A typical ABC primary processing operation consists of six main stages:

1. Burley Casing and Toasting2. Total Blend Casing3. Expanded Cut Tobacco (ECT)4. Cut-Rolled Expanded Stems (CRES)5. Total Blend Cutting, Drying, and Cooling6. Flavorings (Top Dressing)

As an option, a top casing can be applied right before cutting.



B. Why Casings?

Casing is an integral part of processing of any ABC product.

Ingredients. The main ingredients in the casing solution consist of sugars, licorice,cocoa, fruit concentrates, and humectants dissolved in water.

Benefits. There are four major benefits of casing:

1. Sugars improve the overall sensory quality, especially smoke flavor.2. Licorice, cocoa, and fruit concentrate contribute to the basic taste.3. Humectants improve the tobacco processing ability and increase its moisture-

holding capacity, thus extending the product’s shelf life.4. The casing’s solids act as a cost-reducer by replacing tobacco in the overall

cigarette rod, while maintaining the same puff count.

C. Why Flavorings?

Flavoring or Top Dressing is an integral part of processing of any ABC product.

Ingredients. The content of flavoring ingredients is confidential information toeach cigarette manufacturer, but it generally covers aroma chemicals, essentialoils, natural and/or synthetic extracts, and commercial flavorings.

The major Benefits of flavoring are:

1. Ameliorates off taste and negative flavor attributes usually associated withtobacco smoke

2. Generates a balanced smoke flavor matrix that complements the naturaltobacco taste and flavor

3. Imparts a “signature” pack aroma and smoke flavor that are unique to eachparticular ABC product

There is no Formula or ratio that can fit all ABC products. Each ABC productrequires a formula and ratio that complement the particular blend.

D. Role of Additives

Additives should be used with care in order to complement, not overpower, thebasic tobacco characteristics and positively contribute to processing ability, smokingqualities and safety.

The increasing demand to produce “safer” cigarettes with lower yield andacceptable smoking qualities led to efforts to develop new additives and applicationtechniques. Additives can be used as a tool to enhance the signature flavor matrixand pack aroma of the “safer” cigarettes with reduced yield.

Appendices � 221


This Rule is highly recommended for using additives:

Use a minimum number of components at lowest possible levelsthat can provide maximum positive effects on physical properties,smoking qualities and overall smoke chemistry while preserving theABC brand integrity.

V. RECENT DEVELOPMENTS

A. Sensory Testing.

Now, I would like to elaborate on the current status of sensory testing.

* There are many research studies on the chemistry and biological activity ofcigarette smoke; however, the most advanced chemical testing devices cannot“sense” the qualitative aspects of the mainstream smoke and its sensoryproperties as perceived by the smoker.

* Adequate smoking panels should be used as a measuring device parallel tothe chemical and physical testing devices now being used in productdevelopment.

B. What is ‘Tar’?

The total particulate matter (TPM) of the cigarette MS is commonly called ‘tar’ aterm that is often misleading. The ‘tar’ delivery per se does not necessarily reflectspecific sensory properties or indicate a certain safety factor.

Many ABC brands have similar ‘tar’ delivery but differ greatly in smoking qualitiesas reflected by acceptability in the market place. The wide range of acceptabilityfor the same ‘tar’ delivery presumably reflects qualitative differences in the degreeof safety, in terms of ‘tar’ and nicotine deliveries, as well as the level of harmfulcompounds such as benzo(a)pyrene, nitrosamines, phenols, CO, etc.

C. LITERATURE ON INGREDIENTS

Following are the main recent publications and ongoing studies on the evaluation ofthe health effects and relative risks of non-tobacco ingredients added during themanufacturing of cigarettes:

JTI’s Paschke et al. 50-year review (1952-2002)PM’s Carmines et al. 4 parts (2002)RJR’s Rodgman et al. 2 parts (2002)BAT’s Baker et al. on-going (2003)LSRO’s (independent) on-going (2003)



Controversies

* A large number of studies reveal scientific controversies about the risks ofcigarette ingredients.

* The controversies are due to the diversity of test materials, test conditions,and test procedures among investigators.

* The complexity of studying the role of ingredients in the health risks of cigarettesmoking is also due to the lack of:

• Standardized procedures to test the “fresh” smoke

• Ethically-accepted methodologies to employ human subjects (smokers) toparticipate in the required tests

Dr. Fawky Abdallah is the President of Fawky Abdallah Co., Inc., an internationalconsulting firm and ISSCORP (USA) a sales and marketing corporation and theauthor of Cigarette Product Development, published by Tobacco Reporter.

Appendices � 223


Use of Threshold of Concern in determining data requirementsfor the evaluation of tobacco ingredients

Richard A. Ford, Ph.D.

Use of Threshold of Concern in determining data requirements for the evaluation of tobacco

ingredients

Richard A. Ford. Ph.D.

International Aroma Chemical Consultants

As you may have gathered from my comments during the question period thismorning, I am very concerned about misdirected resources and duplication of effortin the evaluation of tobacco ingredients.

Today I’m going to talk about a method that can be used to decide which substancesof the approximately 500 known tobacco additives really need to be evaluated andwhat additional data, if any, would be necessary for that evaluation. I will presenta method to that uses structure active relationships (SAR) and exposures to determinethresholds of concern: that is, exposure levels below which there is no significantrisk of adverse effects.

Tobacco Additives

• Approximately 500 substances are used

• Approximately 70% are chemically defined substances

• Remainder are complex mixtures (natural extracts, etc.)

The figures given in this slide and subsequent ones have been derived from lookingat the marvelous databases that are now available on the web sites of R.J.R.,Phillip Morris, and other companies. There is a lot of information in these sites.



Chemical structures of tobaadditives vary widely

O

H O

A c e tic a c id

O

O

Decahydro-3a,6,6,9a-tetramethylnaphtho[

to

(Sclareolide)

Additionally, the British Columbia Department of Health has put together a databaseon additives in tobacco. The figures I present are derived from those databases. Idon’t mean to imply these figures are exact but they are adequate for illustrationpurposes. There’s approximately 500 substances—you’ve heard the number 599but I’ve heard the number has gone down in recent years so we’ll say approximately500 substances and about 70 % of those are chemically defined substances. Therest are complex mixtures, mostly natural extracts.

LSRO Plan

• Phase 1 � Feasibility

• Phase 2 � Criteria

� This presentation addresses one important aspect of criteria � determining data requirements for the evaluation of additives

• Phase 3 � Evaluation

You’ve also heard that in the LSRO plan, the first phase is feasibility and PhaseTwo covers criteria and evaluation. The points I’m making today are directly relatedto Phase Two. I plan show how you can look at structure and exposure anddetermine from that what would normally be required in terms of a database forevaluation.

Appendices � 225


The structures of the chemicals that are used as additives vary widely. Acetic acidhas a very simple structure, while sclareolide, is much more complicated—just toillustrate the variation.

Amount of available toxicological data also varies widely

• Propylene glycol – complete HEDSET including chronic inhalation studies

• Amyl octanoate – no toxicological data

Similarly, the amount of toxicological data on additives varies enormously. Propyleneglycol has a complete HEDSET — that means it fulfills the government requirementsfor basic test data.

On the other hand, there’s amyl octanoate, which has no toxicological data. Icouldn’t even find an LD50. Now propylene glycol is used at levels of greater than1 % as an additive while amyl octanoate (a simple fatty ester which would beconsidered as a structure having little topological concern) is used in less than onepart per million. Thus, the amount of toxicological data that is available on thesetwo materials makes sense.

Variation in use levels is also very large

<0.0001% to > 10%

Variation in exposure may be even larger due to

differences in volatility, etc.

Just as the variation in structure is significant, the variation in use level is also verylarge, from less than one part per million to greater than 10 %. Of course, thevariation in exposure may be even larger due to differences in volatility andcombustibility.



In fact, most additives are used are very low levels

• Approximately 48% are are used below 1 ppm

• Another approximately 22% are used at between 1 and 10 ppm

The fact is that most of the additives to tobacco are used at very low levels. Icounted roughly 48 % which are used at less than one part per million and another22 % between one and ten parts per million. That’s quite low and the resultingexposures would, of course, be quite low.

Clearly it is not practical nor necessary to require the same

degree of toxicological data for each additive

• To obtain the same amount of data on amyl octanoate as for propylene glycol could cost

several million dollars and the use of thousands of rats.Is it necessary?

I think it is clear that it is not practical nor is it necessary to require same degree oftoxicological testing on each and every additive. To obtain the same amount of dataon amyl octanoate as exists for propylene glycol would cost millions of dollars andI maintain is not necessary.

Appendices � 227


Structure and use levels (exposure) can be used to systematically to

determine the database that would normally be expected for safety

evaluation

Structure and use levels (and the resulting exposures) can be used to systematicallydetermine the database that would normally be expected for a safety evaluation. Iunderline “normally be expected” because in any evaluation scheme that hascredibility, the end decision has to be with experts. What I’m putting forward canbe only guidance to help decide what databases would normally be expected basedon chemical structures and exposures but it has been and still is used quitesuccessfully by others.

Combining exposure and structure logically

• Low exposure / innocuous structure – little concern – less need for data

• High exposure / structural alerts – high concern – significant data needed

• High exposure / innocuous structure or low exposure / structural alerts – intermediate

Combining exposure and chemical structure is quite logical in safety evaluation. Ifa substance has a very low exposure and an innocuous structure, such as amyloctanoate, there should be little concern and very little need, if any, for actualtoxicological testing. Of course, if a substance has a high exposure and it has structuralalerts, say, for carcinogenicity or other toxic endpoints, it would clearly be of highconcern and a significant amount of toxicological data would be needed. Of course,with high exposure and innocuous structure or low exposure and structural alerts,then intermediate data packages would normally be required. This is logical, andit’s really nothing new.



Such a system has been in use for years for food additives

• NAS/NRC, 1958, Insignificant levels of chemical additives in food, Food Drug Cosmetic Law J. 13: 477-479

• FDA, 1982, Toxicological principles for the safety assessment of direct food additives and coloradditives used in food. Red Book, U.S. Food and Drug Administration, Bureau of Foods, Washington DC

Such an approach has been around for many, many years in the area of foodadditives. The earliest reference I could find, and I’m not counting Paracelsushere, was the National Academy of Sciences National Research Council in 1958,which published a paper on insignificant levels of chemical additives in food. In thispublication they said, for certain types of chemical structures, there is a level infood which presents no safety concerns and no toxicological data would be neededto reach that conclusion.

The FDA was a little bit more conservative in 1982 but they still used the samepremises in their so-called red book in which, if you recall, they had three classes ofchemical structure and different data requirements depending on the combinationof the chemical structure and the exposure. They used a scheme similar to what Ishowed on the previous slide: high exposure and high structural concern results ingreater need for data, low exposure and low structural concern results in lowerneed for data.

And particularly for flavors

• Cramer, G.M., Ford, R.A. and Hall, R.L. 1978, Estimation of toxic hazard – a decision tree approach. Food Cosmet. Toxicol. 16(3): 255-276

• Munro I.C., Kennepohl E. and Kroes R. (1999) A procedure for the safety evaluation of flavouring substances Food and Chemical Toxicology, 37(2-3), 207-232

This has been particularly useful for flavors. I first got involved in applying it toflavors back in 1978 in what has become known as the FEMA decision tree. It’s asystematic way of looking at chemical structure and exposure, calculated from use

Appendices � 229


levels in food, to determine database needs. More recently (it takes a while for themessage to get around) it’s been adopted by JECFA. They published theiracceptance of what is essentially the same approach in 1999.

And has even been used to determine an exposure below

which there is no concern regardless of structure

• Federal Register, 1995, Food Additives; Threshold of Regulation for Substances Used in Food-Contact Articles. Department of Health and Human Services, Food and Drug Administration. 21 CFR Parts 5, 25, 170, 171 and 174. Docket Nos. 77P-0122 and 92N-0181

A similar concept has been written into a law by the FDA which where they haveconcluded that there is an exposure below which, regardless of the chemical structure,there is no safety concern. This has been applied to the evaluation of indirect additivessince1995: this is the de minimis concept.

This approach is now being adapted specifically for additives to tobacco

I’ve been working on an adaptation of this threshold approach for tobacco additives.



The adaptation is compatible with the food additives approaches mentioned

• Assumes thresholds of concern based on structure and structural alerts– 4 Structural categories of concern

• Combined with possible exposure via smoking tobacco– Calculated based on use levels and resulting

exposures

The adaptation is compatible with the food additives approaches mentioned. Itassumes thresholds of concern based on structure and structural alerts. There arefour structural categories. One is based on the FDA de minimis threshold forindirect additives, that is, a level below which there is no concern regardless ofstructure. That level is so low, however, that it may be impractical for tobaccoadditives. Other thresholds have been compared with possible exposures via smokingtobacco. Such exposures can be conservatively estimated based on maximum uselevels, maximum transfer from the smoke and maximum absorption into the lung.These assumptions are conservative and therefore add additional safety factors.

Additional factors taken into consideration

• Exposure somewhat more difficult to quantitate

• Inhalation is the route of exposure

• Pyrolysis must be considered

• Additives cannot be assumed to be safe based only on their occurrence as natural components of food (or status as approved food additives)

There are three factors that which must be considered in applying a method originallydesigned for food additives to tobacco additives. One, exposure is somewhat moredifficult to quantify. For example, as you have heard, there are significant differencesin smoking patterns. Second, inhalation is the route of exposure as opposed to oralfor food additives. Third, pyrolysis must be considered. Additionally, it cannot beassumed that a tobacco additive is safe based on the fact that it is GRAS or approvedas a food additive.

Appendices � 231


Use of this adaptation could make a monumental evaluation

project (covering several hundred materials) more practical and

efficient

This threshold approach, using structure and exposure in the initial evaluation, couldmake a monumental task more efficient, saving enormous amounts of effort. I’mvery concerned about how much money and effort is being focused on tobaccoadditives and as a result being taken away from the real issue, which is smokingitself. While tobacco additives are important and some of them may need to belooked at, let’s not misdirect efforts to the large number of additives that are used atlow levels and have innocuous structures.

Applies to all structurally defined organic chemical additives and

most natural mixtures

The threshold method applies to all structurally-defined organic chemical additivesand as mentioned earlier, I estimate that to be about 70 % of the total number ofadditives. It can also be used with many of the complex mixtures or natural extracts.



Allows safety evaluation (and testing) efforts to be expended where most needed – diverting

resources from those substances of very low exposures and

innocuous structures

It allows safety evaluation and testing efforts to be expended where most neededwhile not wasting resources on those many substances with very low exposureand/or innocuous structures.

LSRO is urged to ask the Panel to consider a detailed and in

depth presentation of this practical and logical approach

I encourage LSRO to consider this approach in more detail and I’d be happy topresent the details to the panel, to the staff or both. I think it would save a lot ofeffort.

Dr. Richard A. Ford is a Consultant who specializes in aroma chemicals with theInternational Aroma Chemical Consultants.

Appendices � 233


Using Epidemiological Techniques To Evaluate Possible LongTerm Health Effects Of Menthol And Other Non-TobaccoIngredients Added To Cigarettes

Peter N. Lee

USING EPIDEMIOLOGICAL TECHNIQUESTO EVALUATE

POSSIBLE LONG TERM HEALTH EFFECTSOF MENTHOL

AND OTHER NON-TOBACCO INGREDIENTSADDED TO CIGARETTES

PETER N. LEE

INDEPENDENT CONSULTANTIN EPIDEMIOLOGY AND STATISTICS

SUTTON, SURREY. UK.

I have been working in smoking and health since the mid 1960s, formerly as anemployee of the Tobacco Research Council and, since 1979, as an independentconsultant in statistics and epidemiology. Over that period, numerous epidemiologicalstudies have documented a strong relationship between smoking and variousdiseases, notably including lung cancer, coronary heart disease (CHD), and chronicobstructive pulmomary disease (COPD). In principle, these effects could arisesolely from the inhalation of combustion products of tobacco, but it is possible thatnon-tobacco ingredients added to cigarettes could be partly responsible. However,epidemiological evidence relating to possible effects of additives is extremely limited,partly because of the difficulty of obtaining such evidence.

In my talk today, I will start by summarizing conclusions that can be drawn from arecent evaluation I conducted related to menthol, supported by Japan TobaccoInternational. This review, completed in January this year, is available on mycompany’s website www.pnlee.co.uk, and my talk today will also refer to somemore recently published material.



“EPIDEMIOLOGICAL EVIDENCEON RISK OF DISEASE

RELATING TO THE USE OFMENTHOLATED CIGARETTES”

JANUARY 2003

AVAILABLE FROM WWW.PNLEE.CO.UKGO INTO “OTHER REPORTS” AND CLICK ON

DOWNLOAD: LEE2003H.PDF(72 PAGES)

I will then consider to what extent the techniques used to obtain information on theeffects of adding menthol to cigarettes can be used to obtain information on theeffects of other additives.

My review starts by summarizing various aspects of menthol, including its chemicalproperties, sources, uses, status, carcinogenicity, genotoxicity, toxicity,pharmacological effects, and case-reports of possible effects.

MENTHOL – 1CHEMICAL PROPERTIES (-)- ISOMER OF C

10H

19OH

MONOCYCLIC TERPENE ALCOHOLCOOLING ACTION ON SKIN AND MUCOSALSURFACES PRODUCES 3,4-BENZPYRENE WHENCOMBUSTED, BUT NOT MEASURABLE INMENTHOLATED CIGARETTES

SOURCES ESSENTIAL OILSPEPPERMINT OIL AND CORNMINT

MENTHOL – 2 USES MANY PRODUCTS

WORLD’S THIRD MOST IMPORTANT FLAVOURINGSTATUS GRAS

FDA APPROVED FOR FOOD USECARCINOGENICITY NOT CARCINOGENIC TO RATS OR MICE

GENOTOXICITY NOT GENOTOXIC IN VITRO OR IN VIVO

MENTHOL – 3TOXICITY VERY LOW ACUTE TOXICITYPHARMACOLOGICAL NASAL DECONGESTANT ACTIVITYEFFECTS INHIBITION OF RESPIRATORY REFLEXES

ANTITUSSIVE PROPERTIESSO MIGHT AFFECT HOW TOBACCO IS INHALED

CASE-REPORTS OFEFFECTS VERY LIMITED

Appendices � 235


Generally, this material shows that menthol, which has been widely used for manyyears, gives no reason for concern regarding its carcinogenicity, genotoxicity, oracute toxicity. With regard to the possible health effects of mentholated cigarettes,the major interest is the evidence that it may have acute effects on the mouth,nose, and respiratory tract that might affect how cigarettes are smoked.

Turning now to mentholated cigarettes rather than menthol itself, it is worth notingthat the literature relates essentially only to the USA, presumably as use is rareelsewhere. Although I do not have up-to-date exact figures, usage is probablyaround 25 %. It is much higher in Blacks than Whites, and higher in younger thanolder Blacks, with some studies reporting over 80 % of younger Black smokersuse mentholated cigarettes.

LITERATURE ONLY RELEVANT TO USA,AS USE PRESUMABLY RARER

ELSEWHERELESS THAN 3% OF MARKETUP TO 1949 THEN A RISE CURRENTLYAROUND 25%

CONFLICTING CLAIMS HEREUS SURGEONGENERAL 1989 SAYSNO ASSOCIATION

MUCH HIGHER USE BY BLACKS,ESPECIALLY IN YOUNG WOMEN (80%+)

MARKET SHARE

RELATIONSHIP TO TAR LEVEL

RACIAL DIFFERENCES

MENTHOLATED CIGARETTES

MENTHOLATED USE AMONG SMOKERS

PROPORTION WHO SMOKEAMOUNT SMOKED PER SMOKERAGE OF STARTING TO SMOKE

‘TAR’ LEVEL OF CIGARETTE SMOKED

PROBABILITY OF QUITTINGCOTININE LEVELS IN SMOKERSLUNG CANCER DEATH RATES (1980-95)

(1950-95)

BLACKS (V WHITES)

MUCH HIGHER (X 4)

SOMEWHAT HIGHER (X 1.15)LOWER (X 0.65)START LATER – BY ABOUTHALF A YEAR

HIGHER

LESSHIGHERHIGHER IN MENSIMILAR IN WOMEN

BLACKS V WHITES



One of the reasons that menthol has been studied is that US black men havesubstantially higher lung cancer rates than do US white men, despite black smokerssmoking less cigarettes per day and starting to smoke somewhat later. The suspicionhas arisen that this difference may be due to their increased use of mentholatedcigarettes. As shown in the slide, there are in fact quite a number of differencesbetween the smoking habits of US Blacks and Whites, and it is not totally clearwhat difference in risk one would expect as a result. However, it is interesting tonote that the difference in lung cancer rates between Blacks and Whites is notseen in women, with rates very similar for many years.

There are a number of experimental studies of the effect of mentholation on smokingcharacteristics. The studies are generally small and some are rather artificial. Ido not propose to go into detail here, but together they provide little clear supportfor the idea that mentholation may affect how a cigarette is smoked so as toincrease the uptake of toxic or carcinogenic smoke constituents.

Thirteen-week inhalation studies have been conducted in which rats were exposedto the smoke from mentholated or unmentholated cigarettes, but any effects seen(reversible nasal changes) related to dose of smoke and not to mentholation. Long-term animal carcinogenicity studies have not been conducted. Genotoxicity studieshave also found no difference in response to the smoking of mentholated orunmentholated cigarettes. Nor was there any evidence of central pharmacologicaleffects of mentholation in a small crossover study.

EXPERIMENTAL EVIDENCE EFFECT OF MENTHOLATION ON SMOKING CHARACTERISTICS

STUDIES MAINLY QUITE SMALL SOME USE UNUSUAL SMOKINGPROTOCOL INVOLVING PUFFING EVERY 15S

ENDPOINT EVIDENCE

NUMBER OF PUFFS 4 STUDIES SHOW INCREASE2 STUDIES SHOW DECREASE

PUFF VOLUME 2 STUDIES SHOW NO EFFECT2 STUDIES SHOW DECREASE1 STUDY SHOWS INCREASE

HEART RATE AND BLOOD PRESSURE 4 STUDIES SHOW NO EFFECT

MARKER LEVELS CONFLICTING EVIDENCE

EFFECTS OF MENTHOLATED CIGARETTES

CARCINOGENICITY IN ANIMALS NO EFFECT IN 13-WEEKRAT STUDIES

GENOTOXICITY NO EFFECT

PHARMACOLOGICAL EFFECTS NO EVIDENCE OF CENTRALEFFECTS

Appendices � 237


I now turn to the epidemiological evidence proper relating to mentholated cigarettes,starting with studies on lung cancer. Four studies have reported findings, the Brooksstudy being published since my review. Of the four studies, three were of case-control and one of prospective design. Two restricted attention to current smokersand two included former smokers. Two restricted attention to long-term smokersof 20 years or more. All the studies were of a reasonable size, involving between298 and 1,044 cases. Three studies asked questions about the brand of cigarettesmoked and inferred mentholation from that as best they could. One study asked

AUTHORS KABAT AND SIDNEY CARPENTER BROOKSHEBERT ET AL ET AL ET AL

YEAR 1991 1995 1999 2003

STUDY DESIGN CASE- PROSPECTIVE CASE- CASE-CONTROL CONTROL CONTROL

CONTROLS/ PATIENTS WITH 1979/85-1991 DRIVERS/ PATIENTSFOLLOWUP NSAD MEDICARE WITH NSADPERIOD

LOCATION NEW YORK OAKLAND, LOS ANGELES EASTERN USACHICAGO CALIFORNIA COUNTY,PHILADELPHIA (KAISER CALIFORNIADETROIT PERMANENTE)

STUDY CURRENT CURRENT EVER SMOKED EVERPOPULATION SMOKERS SMOKERS 20+Y SMOKED 20+Y

CASES 588 M 160 M 202 M 435 M456 F 138 F 135 F 208 F

SOURCE OF EACH BRAND CURRENT BRAND Q RE % OF BRANDMENTHOLATION SMOKED (AT BASELINE) LIFETIME SMOKEDDATA CIGARETTES CURRENTLY

MENTHOL AND LONGEST

EXPOSURE <1, 1-14, 15+ 0, 1-9, 10-19, 20+ 0, 1-15, 16-31, 32+ 0, 1-15, 16+INDEX FOR YEARS YEARS PACK YEARS YEARS (ALSOMENTHOL BY % USE)

RESULTS BY YES NO NO NOHISTOLOGICALTYPE OF LUNGCANCER

ADJUSTED FOR:

AGE YES YES YES YES

RACE YES YES YES YES

EDUCATION YES YES NO NO

BMI YES NO NO NO

CIGS PER DAY YES YES NO YES

INHALATION YES NO NO NO

DURATION YES YES NO YES

PACK-YEARS NO NO YES NO

YEARS QUIT NO NO YES YES

FILTER USE NO NO NO YES



directly about the proportion of lifetime cigarettes used that were mentholated. Allstudies presented results, in relation not only to ever/never use of mentholatedcigarettes, but also to a variable, in terms of years of use, pack-years of use, orproportion of lifetime use. All the analyses adjusted for age, race, and variousmeasures of exposure to cigarettes generally. Only one study reported results byhistological type of lung cancer.

LUNG CANCER META-ANALYSISFOR USE OF MENTHOLATED CIGARETTES

RELATIVE RISK (95% CI)

MEN WOMENKABAT EVER/NEVER 1.06 (0.82-1.37) 0.78 (0.57-10.8)SIDNEY USE/NO USE 1.45 (1.03-2.02) 0.75 (0.51-1.11)CARPENTER EVER/NEVER 1.00 (0.68-1.48) 0.88 (0.50-1.57)BROOKS EVER/NEVER 0.77 (0.55-1.08) 1.05 (0.72-1.55)

HETEROGENEITY CHI-SQUARED 6.84 NS 1.86 NSFIXED EFFECTS ESTIMATES 1.05 (0.89-1.23) 0.85 (0.70-1.03)COMBINED SEXES 0.97 (0.86-1.10)

Relative risk estimates and 95 % CI are shown by sex for use of mentholatedcigarettes. Overall, the results for the four studies are statistically homogeneousand show no evidence of an increased risk associated with mentholation. For thecombined data, the estimate is 0.97 with relatively tight 95 % CI of 0.86 - 1.10.

LONG TERM USE

RELATIVE RISK (95% CI)

MEN WOMENKABAT 15+ VS 0 YEARS 0.98 (0.70-1.38) 0.76 (0.53-1.16)SIDNEY 20+ VS 0 YEARS 1.59 (0.96-2.63) 0.70 (0.40-1.23)CARPENTER 32+ VS 0 PACK YEARS 1.48 (0.71-3.05) 0.41 (0.15-1.11)BROOKS 16+ VS 0 YEARS 0.91 (0.57-1.46) 1.00 (0.63-1.60)

HETEROGENEITY CHI-SQUARED 3.76 2.84FIXED EFFECTS ESTIMATES 1.11 (0.88-1.39) 0.78 (0.60-1.01)COMBINED SEXES 0.95 (0.80-1.13)

The same conclusions are reached if comparison is made between the most extremecategories studied; 15+ vs. 0 years for Kabat, 20+ vs. 0 years for Sidney; 32+ vs.0 pack-years for Carpenter and 16+ vs. 0 years for Brooks. Here the estimate is0.95 (0.80 - 1.13). Data from the Kabat study also show no evidence of an increasedrisk for any of the four major histological types of lung cancer.

Appendices � 239


POSSIBLE WEAKNESS OF THE STUDIESPRINCIPAL CONCERNS:

RELIABILITY OF DATA ON MENTHOLATION

POSSIBLE CONFLICTS BETWEEN ANSWERS ON BRANDSAND ON MENTHOLATED USE NOT DISCUSSED

APPROPRIATENESS OF ADJUSTMENT FOR DAILYCONSUMPTION/INHALATION MAY BE AFFECTED BYMENTHOL

COULD MENTHOL AFFECT QUITTING?

VERY LONG TERM DURATION NOT STUDIED

Various possible weaknesses of the four studies are discussed in detail in my review.Any concerns that may exist are mainly with the reliability of the data collected onmentholation and with proper control of aspects of smoking (daily consumptionand inhalation) that may both differ between people who choose to smokementholated cigarettes or non-mentholated cigarettes and be affected as aconsequence of switching to mentholated cigarettes. Also, the studies available sofar have not studied possible effects of very-long-term use. One issue not discussedin my review or considered in the source papers is whether mentholation mightaffect propensity to quit.

CONCLUSIONS FOR LUNG CANCER

• EVIDENCE SO FAR DOES NOT INDICATEMENTHOLATED CIGARETTES HAVE ANINCREASED RISK COMPARED TO NON-MENTHOLATED CIGARETTES.

• A POSSIBLE SMALL INCREASE CANNOTBE EXCLUDED, BUT INCREASED USE OFMENTHOLATED CIGARETTES BY BLACKSCANNOT EXPLAIN THEIR HIGHER LUNGCANCER RISK (SEEN ONLY FOR MENANYWAY).



However, I doubt whether any of these points would have affected the generalconclusion that any effect of mentholation, if it exists, must be very small. Onewould require a very clear effect of mentholation, with a relative risk of order 2.5or more to fully explain the difference in lung cancer rates between black andwhite men. As the upper 95 % CI of the estimates of risk are way short of this, theexplanation of the excess risk in black men must lie elsewhere.

EVIDENCE FOR OTHER DISEASES

AMERICAN HEALTH FOUNDATIONCASE-CONTROL STUDY (HEBERT 1989, KABAT 1994)

OESOPHAGEAL CANCER MALE 1.03 (0.39-6.89)10+ YEARS USE FEMALE 2.30 (0.93-5.72)

(NB NOT AGE ADJUSTED)

KAISER PERMANENTE PROSPECTIVE STUDY (FRIEDMAN 1998)

ALL SMOKING RELATED CANCERS MALE 0.76(0.52-1.11)(EXCEPT LUNG)

USE VS NO USE FEMALE 0.79 (0.53-1.18)

NORTH CAROLINA PROSPECTIVE STUDY IN PREGNANT WOMEN(SAVITZ 2001)

NO ADVERSE EFFECT OF MENTHOLATION ON FETAL GROWTH

Evidence relating to diseases other than lung cancer is quite limited and providesno real indication of any adverse effect of mentholation. It is interesting that dataon CHD are not available at all.

PROBLEMS FOR ADDITIVES OTHER THANMENTHOL

• SMOKERS KNOW THEY ARE SMOKINGMENTHOLATED CIGARETTES

• SMOKERS MAY NOT KNOW THEY ARESMOKING CIGARETTES WITH ADDITIVE X

• HAVE TO ASK QUESTIONS ON BRANDAND OBTAIN ADDITIVE DATA INDIRECTLY

Could one carry out similar studies related to other additives? There are considerabledifficulties in doing this. The first problem is that while, from the brand name and

Appendices � 241


the taste, smokers know whether they are smoking a mentholated or anunmentholated cigarette, they will not in general know whether their cigarettecontains other specific additives. Thus, questions such as “do you smoke,” or“how long have you smoked” cigarettes with added “X” cannot sensibly be asked.This implies that one has to obtain the information via knowledge of the brand ofcigarette that was smoked.

OBTAINING INFORMATION ON BRAND

• NEED CURRENT BRAND

• ALSO NEED PAST BRANDS

• INTERVIEWER MAY ASK TO SEE PACKET

• INTERVIEWER SHOULD HAVE LIST OFBRANDS ON MARKET FOR DIFFERENTPERIODS

• MANY BRANDS HAVE SIMILAR NAMES

This leads to two further problems. One is getting reliable information on whichbrand is smoked currently and was smoked in the past. Accuracy of recordedbrand may be assisted by requiring the respondent to show a packet of the cigarettein question – only possible for current brand – or choosing from a list of brands onthe market – which would vary depending on the period in question. The point isthat there are many versions of the same brand family and simply answering“Marlboro™” or “Merit™” is not adequate.

OBTAINING INFORMATION ON ADDITIVES GIVEN BRAND

• FROM MANUFACTURERS

• BY CHEMICAL ANALYSIS

• OBTAINING SAMPLES OF OLD CIGARETTES

WALD, DOLL, AND COPELAND (BMJ, 1981; 282:763-765)

The other difficulty is obtaining information relating to presence or level of inclusionof a given additive in a given brand.



What information is available or can be obtained from the cigarette manufacturers?My understanding is that, currently, the manufacturers release data on whethervarious additives are present (above a minimum level) in brands that are on themarket and would probably be prepared to release similar data relating to brandssmoked in the past, though such data may be less reliable. They are not generallywilling to publicly release actual levels of additives, regarding it as trade secret,though it is perhaps possible that such information could be supplied on a confidentialbasis to investigators carrying out an epidemiological study. However, given thatthe analyses of mentholated cigarettes I presented were all based simply on whetherthe brands smoked were or were not mentholated, and not on the level of inclusionof menthol, one need perhaps not worry too much about getting data on levels ofadditives.

If information cannot be obtained from the manufacturers, one can in theory acquiresamples of cigarettes on the market and then carry out one’s own chemical analysisrelating to the additives of interest. I cannot comment on the difficulties or cost ofdoing this. At first glance, it would seem possible to do this only for current cigarettes.However, it is not impossible to obtain samples of old cigarettes. I rememberclearly the study by Wald, Doll, and Copeland published in the British MedicalJournal (1981; 282:763-765) in which ‘tar,’ nicotine, and carbon monoxide levelswere reported for UK cigarettes manufactured since 1934. The sample wasobtained by an appeal on the radio by Sir Richard Doll, widely publicised in thepress, for members of the public to provide samples of old cigarettes kept as amemento or stumbled on when clearing out a cupboard or attic. Whether such atechnique would work in the USA I do not know, but it might do. However, onemust bear in mind that many additives are volatile and may not still be present aftera long time period. I leave it to others to judge whether such an approach would beat all useful or practical.

CASE-CONTROL OR PROSPECTIVE STUDIES

ADVANTAGES FOR CASE-CONTROL STUDIES• FEWER SUBJECTS TO STUDY• CHEAPER• ANSWERS MORE QUICKLY

ADVANTAGES FOR PROSPECTIVE STUDIES• INFORMATION ON RANGE OF DISEASES• LESS RELIANCE ON RECALLED INFORMATION

ON BRAND• AVOIDS RECALL BIAS

Given one can obtain information on additive levels from brand data, what is thebest epidemiological technique? Case-control studies and prospective studies are

Appendices � 243


both possible and have different advantages and disadvantages. Case-controlstudies are cheaper and more rapid, but provide data only for the disease studiedand rely heavily on the reliability of recalled information, provided by someonewho is ill, on brand smoked and other aspects of smoking. Prospective studies aremore expensive due to the much larger samples needed, and take a longer time, asone has to wait for the sample to contract the diseases of interest. However, theyprovide data for a range of diseases and rely less heavily on recalled data. Indeed,questions on brand which relate only to that currently smoked at the start of thefollow-up period, possibly supplemented by further questioning at intervals duringthe follow-up period, would provide useful data.

SAVE COSTS AND TIME!

• USE DATA FROM EXISTING STUDIES

• THAT HAVE ASKED ABOUT BRAND

FOR EXAMPLE: 3 OF 4 MENTHOL STUDIESACS CPS-II STUDY

If one does not want to go to the expense of starting a new study, and wants toavoid waiting years for the results, one useful approach may be to try to obtainaccess to existing studies that have asked questions on brand. For example, brandof cigarette has been asked in three of the four studies I cited for menthol and alsoin the American Cancer Society CPS-II prospective study involving almost1¼ million men and women.

ALTERNATIVE INTERNATIONAL APPROACH

OBTAIN DATA FOR A VARIETY OF COUNTRIES ON

1. USE OF ADDITIVES2. MORTALITY FROM SMOKING RELATED DISEASES3. SMOKING HABITS4. OTHER FACTORS

SEE IF ADDITIVES CORRELATE WITH RISK INDEPENDENTOF SMOKING HABITS AND OTHER FACTORS

An alternative, completely different approach is to try to gain information, on aninternational basis, of how use of specific additives varies from country to countryand then see how this relates to their rates of smoking-related diseases. I have adatabase which can be made available giving, for 30 countries, age-, period-, and



sex-specific data on mortality rates from lung cancer, COPD, and CHD, and onprevalence of smoking and on cigarette consumption per adult. In theory, onecould use this data to see whether additive use predicts mortality given prevalenceof smoking and consumption of cigarettes.

INTERNATIONAL APPROACH

POINTS TO CONSIDER

UNLIKELY TO BE USEFUL FOR CHD AS TOO MANY RISKFACTORSCOULD TRY IT FOR LUNG CANCER AND COPD

IMASS HAS AGE-SEX-PERIOD-SPECIFIC DATA FORMORTALITY, PREVALENCE OF SMOKING, TAR LEVEL ANDCIGARETTE CONSUMPTION PER ADULT

ECOLOGICAL APPROACH NOT AS GOOD ASEPIDEMIOLOGICAL APPROACHCOULD ONLY DETECT MAJOR EFFECTS OF ADDITIVES

This would probably only be a useful approach for diseases, such as lung cancer,where risk is dominated by smoking, and would not be useful for diseases likeCHD, where differences between countries depend on other factors such as diet.Even then, it would be quite an insensitive tool, and could only hope to detect, orrule out, major effects. Using an ecological approach, where one studies wholepopulations, is never as reliable as using an epidemiological approach.

Overall, though there are considerable difficulties in using epidemiology to evaluatepossible effects of additives, I feel it is not an impossible task. I do not propose tocomment on the validity of alternative approaches based on species otherthan man.

Peter N. Lee is an independent consultant in epidemiology and statistics in SuttonSurrey, UK.

Appendices � 245


APPENDIX D

CASE STUDY SUMMARIES FOR SOMEHYPOTHETICAL INGREDIENTS

INTRODUCTION

This appendix describes the application of LSRO’s scientific criteria. Here, LSROseeks to make its approach evident without prejudging its application to specificsubstances already in use, and to assist the reader in understanding the overallapproach by outlining its application to some hypothetical substances. None of thehypothetical cases are complex mixtures. None of the hypothetical cases involvecircumstances where postmarketing surveillance or epidemiology studies wouldbe feasible. Where relevant, these hypothetical cases explain how a data submittermight choose to respond to circumstances which do not adhere to LSRO’s initialapproach.

SUBSTANCE A

The data submitter adds substance A to a brand in amounts of 8.5 µg per cigarette(10 ppm) or less. Little is known about substance A other than its chemical properties.However, substance A has been purified, and it has a CAS #.

The data submitter obtains a preparation of substance A labeled with a stableisotope. The data submitter tests the stability and transfer of substance A in itsbrand of cigarettes at 5 ppm. Under these conditions, substance A undergoescomplete pyrolysis; none of substance A transfers in smoke in such a way that itwould reach smokers’ lungs in biologically significant amounts. Under smokingconditions, substance A burns mostly to carbon dioxide and water. The highermolecular weight pyrolysis products of substance A that are found analytically insmoke, reflect transitory, incomplete combustion. The pyrolysis products found insmoke, which might be inhaled, mimic the mass spectrometric profiles of knowntobacco combustion products.

Because no detectable transfer occurs, the data submitter persists with the initialphase of LSRO’s suggested approach. Experimental data obtained with stableisotope labeled substance A also show that the pyrolysis products of substance Afound in cigarette smoke do not significantly change the amounts of key smokeindicators (chemical composition of the smoke). The pyrolysis products ofsubstance A in smoke do not suggest toxicological concerns. They are notsubstances with known toxicological properties. The data submitter also directlyaddresses the possibilities of different combustion pathways or different effects of



substance A on smoke physics, chemistry, or biological activity with different tobaccosubstrates in different kinds of cigarettes.

Because the data submitter finds neither transfer of new substances nor changesin smoke physics, chemistry, or biological activity, the data submitter persists withthe initial phase of LSRO’s suggested approach. Separately conducted tests revealthat substance A at 5 ppm does not change the exposure of human smokers. Studysubjects did not significantly change in either the number of cigarettes consumedor their internal biomarkers of smoke exposure.

Given these results, LSRO would conclude that a scientific rationale exists not toanticipate any increase in relative risk of adverse human health effects from theaddition of the ingredient to cigarettes in an amount of 5 ppm or less to cigarettes.However, LSRO would advise the data submitter not to use substance A in amountsgreater than 5 ppm without retesting at the higher amount. (See Figure D.1.)

Appendices � 247


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SUBSTANCE B

The data submitter adds substance B to a brand of cigarettes in amounts of nomore than 8.5 µg per cigarette (10 ppm) or less. Chemical properties of substanceB are known, substance B has been purified, and it has a CAS #.

The data submitter obtains substance B labeled with a stable isotope. The datasubmitter tests the stability and transfer of substance B added to a brand of cigarettesat 5 ppm. Consistent with its vapor pressure, boiling point, and stability, substanceB resists pyrolysis under the combustion conditions in cigarettes.

Assuming a one pack per day smoker, starting to smoke at age 21 and smoking fora lifetime (ending at age 66), a hypothetical individual would smoke in excess of3 x 105 cigarettes, and each cigarette would generate approximately 500 mL ofmainstream smoke in ten 50 mL puffs (Baker, 1999). Each smoked cigarettewould burn mostly to smoke and ash. Each cigarette would contain approximately850 mg of tobacco before smoking. After combustion, it would generateapproximately 350 mg of vapor, mostly common atmospheric gases (90 - 96 % byweight) (Baker, 1999; Pankow, 2001).

Since substance B resists pyrolysis, the data submitter initially assumes that 20 %of it transfers. (The transfer of substance B is subject to experimentaldetermination.) Hypothetically, a lifetime smoker would be exposed to aconcentration as high as 2.4 x 10-5 or 0.24 ppm in smoke repeatedly with eachcigarette consumed or approximately 30 g over a lifetime. Assuming 100 %absorption, lifetime smokers might absorb 30 g of substance B, over a lifetime.The primary concern with substance B is that if it introduced a novel toxicity, therelative risks of smoking cigarettes containing substance B might increase. Onesecondary concern is that if the biological effects of substance B mimic a knownadverse human health effect of cigarette smoking, the incidence and severity ofthe disease might increase, even if the increase could not easily be detected inan epidemiological study.

Another secondary concern about substance B would relate to changes in smokecomposition. Experimentally, except for the presence of substance B, other keysubstances (nicotine, carbon monoxide, water, and nitrous oxide) in smoke do notchange their relative proportions in cigarettes containing this ingredient. SubstanceB does not change the mutagenic potency of cigarette smoke using the sameSalmonella test strains used to test the pyrolysis products of substance B byreversion. Substance B does not change the cell killing potency of cigarette smokefor cultured human lung cells. The data submitter obtains data about changes inchemical composition of the smoke from cigarettes with 5 ppm of substance B anddifferent tobacco substrates and shows that changing substrates does not createan effect of substance B on smoke composition.

Appendices � 249


In comparison to existing toxicological data about this substance, this amount oflifetime exposure to substance B might be significant in relation to chronic exposureto other substances by inhalation, because substance B interacts with receptors orcauses genetic lesions. Since substance B transfers, smoke composition haschanged, almost by definition.

The data submitter then decides not to continue with the initial phase of LSRO’ssuggested approach and the data submitter also decides that using a lesser amountof substance B in cigarettes would vitiate its use. The data submitter decides toobtain data about other uses of substance B to evaluate the potentially adversehealth effects associated with these other uses, particularly from already conductedhuman epidemiological studies.

The data submitter continues investigating substance B, in biological tests and inisolation. The test data show that the biological effects of substance B relate toreceptor binding, not to a genetic lesion, which might have been masked by thepresence of smoke. Subsequent data reveal that substance B binds to a specificfamily of receptors. From this knowledge, biological effects consistent with receptorbinding can be anticipated. Binding to these receptors might lead smokers toincrease their exposure, thus increasing their risk. However, separately conductedtests reveal that substance B at 5 ppm does not change the exposure of humansmokers in that the number of cigarettes consumed and biomarkers of cigarettesmoke exposure remain the same with and without the ingredient. The test datanegate the concern that substance B might change exposure to smoke and increasethe incidence of cigarette-associated diseases.

After review of the data, LSRO would likely conclude that a scientific rationaledid not exist for additional concerns about the human health consequences of additionof substance B to cigarettes. LSRO would not recommend additional biologicaltesting, as the genetic toxicity, receptor binding, and cell killing data do not suggesta nonspecific toxic effect. Lifetime exposure of 30 g or less is equivalent to a dailyexposure of 85 pg.

LSRO would likely conclude that a scientific rationale exists to anticipate that theprobability of any adverse human health effects from the addition of the ingredientto cigarettes in an amount of 5 ppm or less to cigarettes would be very small.However, LSRO also would advise the data submitter not to use substance B inamounts greater than 5 ppm without some retesting at the higher amount.(See Figure D.2.)



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Appendices � 251


SUBSTANCE CCustomers of the data submitter add substance C to their brands of cigarettes inamounts of no more than 85 mg (10 % by weight). Little is known about theproperties of substance C, beyond its chemical structure. It is a carbohydrate.However, scientific literature shows that substance C has been obtained in a purifiedform, and it has been synthesized. The data submitter obtains substance C withstable isotopic elements replacing various chemical elements in its structure. Additionof this substance to the cigarette at 10 % and machine smoking of cigarettesreveals that substance C undergoes complete pyrolysis. For this reason, substanceC could not transfer to smokers’ lungs in any toxicologically significant way.

Under smoking conditions, substance C burns mostly to carbon dioxide and water.Because of the large amount of substance C added, its combustion products dilutethe combustion products of tobacco. However, the addition of substance C doesnot affect the yield of smoke proportionally, because a given weight of substanceC produces pyrolysis products. Analysis of smoke from cigarettes containingdifferent amounts of substance C shows that substance C does not significantlychange the relative amounts of substances derived from tobacco, only their absoluteamounts.

Higher molecular weight pyrolysis products of substance C occur in smoke, reflectingincomplete combustion under machine smoking conditions. The intermediatepyrolysis products mimic the mass profile of some known tobacco combustionproducts, aldehydes. The altered aldehyde levels, which substance C generates,significantly distort the overall chemical composition of smoke, relative to otherwiseidentical cigarettes not containing substance C. Because of the amounts involvedand the toxicological properties of aldehydes, a concern exists about the use ofsubstance C. With the exception of the aldehydes and the dilution effects, smokecomposition changes insignificantly.

The primary concern about potential adverse human health effects of substance Crelate to increased inhalation of aldehydes in smoke. Whether these substancescontribute to the known adverse human health effects of cigarette smoking is notknown. The data submitter provides literature citations about the human healtheffects of inhaling these aldehydes in isolation (not in smoke) in the data packagesubmitted to LSRO. LSRO supplements these citations with its own literaturereview. LSRO’s review of all of the data leads to the conclusion that the increasesin aldehyde levels, related to the addition of substance C to cigarettes, are smallerthan levels which did not significantly increase adverse human health effects in theother studies.

LSRO explains its initial conclusions to the data submitter. The data submittersubsequently obtains data about how human behavior changes exposure in response



to smoking cigarettes with substance C added to 10 %. However, these data arecomplex. Subjects smoking the data submitter’s test cigarette without addedsubstance C object to its harshness and smoke less of it, whereas subjects smokingcigarettes with added substance C find their flavor less harsh, and they smokenumbers of cigarettes not detectably different from the numbers they smokedbefore testing.

Because substance C replaces substances sometimes present in tobacco, andbecause the literature about adverse human health effects of inhaled aldehydessuggests no increase in adverse health effects, LSRO probably would concludethat no scientific rationale exists to exclude the addition of substance C up to 10 %by weight. (See Figure D.3.)

Appendices � 253


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SUBSTANCE D

The data submitter adds substance D to a brand of cigarette in amounts of nomore than 8.5 µg (10 ppm). Little is known about substance D other than itschemical structure and a few physical properties. However, substance D can beobtained as a pure substance, and it has a CAS #.

The data submitter obtains substance D labeled with a stable isotope and tests itsstability and transfer at 10 ppm in cigarettes. Substance D mostly burns to carbondioxide and water. The traces of higher molecular weight pyrolysis products ofsubstance D, which do transfer in smoke, reflect incomplete combustion of substanceD, and all but two are found in tobacco combustion products of cigarettes lackingsubstance D. Other than these two products, combustion products of substance Ddo not distort the composition of smoke significantly and do not produce any concernfor adverse human health effects.

The two combustion products of substance D not found in smoke from cigaretteslacking substance D do transfer in smoke, and they have unknown toxicologicalproperties. One occurs only in trivial amounts. Lifetime exposures to this pyrolysisproduct would not elicit concern, even if it were as carcinogenic as2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) or as lethal as botulinum toxin.However, the other pyrolysis product occurs in greater amounts, and it does generatea toxicological concern. The data submitter obtains bioassay data to show that thispyrolysis product has potency in other species that is lower than a potency generatinga concern at higher levels. By comparison to the potencies of other substancesstudied in the same test species, the data submitter concludes that the addition ofsubstance D to cigarettes does not change the relative risk of smoking. LSROreviews these data and sees no reason to disagree. (See Figure D.4.)

Appendices � 255


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SUBSTANCE E

The data submitter adds substance E to a brand of cigarette in amounts of no morethan 85 µg (0.1 ppt). Substance E has an extensive body of toxicological literature,occasioned by intensive U.S. regulatory agency scrutiny. The data submitter obtainssubstance E labeled with a stable isotope and tests stability and transfer at 0.1 ppt.The substance undergoes extensive pyrolysis; most of it does not transfer intact insmoke. The data submitter submits a data package to LSRO which summarizesthe toxicological literature about substance E. LSRO conducts its own literaturereview and concurs with the data submitter’s summary.

However, one combustion product of substance E appears thermodynamicallystable. It is not found in cigarette smoke and is widely regarded as highly toxic.Setting aside all debates about the merits of the data submitter’s conclusions aboutthe toxicity of substance E and its pyrolytic breakdown products, LSRO wouldexplain the scientific and regulatory history of work on this pyrolysis product to thedata submitter and recommend that the data submitter not use substance E as aningredient in cigarette formulations. (See Figure D.5.)

Appendices � 257


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SUBSTANCE F

The data submitter adds substance F to a brand of cigarette in amounts of no morethan 0.85 µg per cigarette (1 ppm). Substance F has a moderate toxicologicalliterature associated with its use, as manufacturers of non-cigarette products haveadded it to their products (mostly foods). The data submitter obtained substanceF labeled with a stable isotope and tested its stability and transfer at 1 ppm. Thesubstance underwent minor pyrolysis; most of it transferred intact in smoke.

A scientific rationale would not exist for concerns about the adverse human healthconsequences of addition of substance F to cigarettes. Beyond the tests for smokecomposition and exposure, which do not change, LSRO would not recommendadditional biological testing, as none of the data, including mutation reversion andcell killing data, suggest a nonspecific toxic effect through a change in smokecomposition, and neither substance F nor substance(s) related to the addition ofsubstance F pass through in smoke to induce changes in exposure. A lifetimeexposure to 0.5 g or less of substance F is equivalent to a daily exposure ofapproximately 6.5 µg/day. At this exposure, literature shows that the acute andchronic no-effect levels for substance F are many thousands of times higher.(See Figure D.6.)

Appendices � 259


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SUBSTANCE G

Customers of the data submitter add substance G to their brands of cigarettes inamounts of no more than 8.5 mg (1 % by weight). Little is known about theproperties of substance G, beyond its chemical structure. It is a carbohydrate.However, scientific literature shows that substance G has been obtained in a purifiedform, and it has been synthesized. The data submitter obtains substance G withstable isotopic elements replacing various chemical elements in its structure. Additionof this substance to cigarettes at 1 % and machine smoking of cigarettes revealsthat substance G undergoes complete pyrolysis. For this reason, substance Gwould not transfer to smokers’ lungs in a toxicologically significant way.

Under smoking conditions, substance G burns mostly to carbon dioxide and water.Because of the large amount of substance G added, its presence causes a dilutionin the combustion products of tobacco, but the changes in yields are proportional tothe amount of substance G added. Thus, analysis of smoke from cigarettes containingdifferent amounts of substance G shows that substance G does not significantlychange the relative amounts of substances derived from tobacco, only their absoluteamounts. Therefore, substance G does not change smoke composition in anyovert way.

Because the data submitter finds neither transfer of new substances nor changesin smoke composition, the data submitter continues with the initial phase of LSRO’ssuggested approach. Separately conducted tests reveal that substance G at 1.0 %changes the exposure of human smokers. Study subjects smoke twice as manycigarettes, when substance G is present at 1.0 %, and their internal biomarkers ofsmoke exposure reflect this change in cigarette consumption. Apparently somesmoke substance generated in the presence of substance G transferred in an amountthat was not detected by the previously employed analytical methods but inducedchanges in smokers’ exposures.

Given these results, LSRO would conclude that a scientific rationale exists toanticipate an approximately two-fold increase in relative risk of adverse humanhealth effects from the addition of substance G to cigarettes in amounts of 1.0 %or less. The data submitter might be able to reduce the amount of substance Gadded, and through retesting, demonstrate that the reduction eliminates the problemof increased exposure to smoke.

If the data submitter could not reduce the amount of substance G added to cigarettesand obtain the properties needed from its use, LSRO would recommend furtheranalysis, initially additional studies of exposure, dosimetry, biological effects, behavior,and particularly studies to discover the cause of increased consumption and itselimination. Barring such further analysis, LSRO would recommend that the datasubmitter not use substance G in cigarettes. The preponderance of data would

Appendices � 261


have to show that smoking cigarettes containing substance G will not change relativerisk of adverse human health effects. (See Figure D.7.)


Baker, R. R. (1999) Smoking chemistry. In: Tobacco. Production, Chemistryand Technology. (Davis, D. L. & Nielsen, M. T., eds.) Malden, MA: BlackwellScience Inc., pp. 398-439.

Pankow, J. F. (2001) A consideration of the role of gas/particle partitioning in thedeposition of nicotine and other tobacco smoke compounds in the respiratory tract.Chem. Res. Toxicol. 14: 1465-1481.



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Appendices � 263


APPENDIX E

LSRO



APPENDIX F

REPRODUCTIVE AND DEVELOPMENTAL EFFECTSOF CIGARETTE SMOKE EXPOSURE

Cigarette smoking produces adverse effects on female reproductive function (Rubin,2004). Women who smoke cigarettes transition to menopause at a younger agethan women who do not smoke cigarettes. Cigarette smoking by pregnant mothershas harmful effects on their offspring, effects sometimes referred to as “fetaltobacco syndrome”. Labeling of tobacco products with respect to reproductioncarries the Surgeon General’s Warnings that “tobacco use increases the risk ofinfertility, stillbirth, and low birth-weight.” More specifically, “smoking by pregnantwomen may result in fetal injury, premature birth, and low birth-weight.”

In defining the scientific criteria for evaluating non-tobacco ingredients added tocigarettes, LSRO’s goal was to identify tests that predicted the kinds of adversehealth effects experienced by smokers. A review of the scientific literature generallyrevealed a lack of evidence of biological tests for these health outcomes. However,developmental tests suggest the possibility of predicting adverse health effects ofmaternal smoking on offspring. By extending the analysis to assess the influenceof cigarette smoking on female reproductive outcomes, a sponsor could evaluate ahealth outcome shared by human smokers and animal surrogates. To this end,LSRO has appended a description of developmental effects on offspring exposedto mainstream tobacco smoke, in utero, and concordant effects on birth-weight inanimal surrogates.

Pregnant women who smoke are at a higher risk for complications during pregnancy,as a consequence of damage to the uteroplacental unit. These complications includeabruptio placentae, placenta previa, bleeding of the uterus, and premature ruptureof fetal membranes. As a result, infants born to mothers who smoke duringpregnancy have a higher risk for perinatal mortality than infants whose mothers donot smoke during pregnancy.

Cigarette smoking during pregnancy also increases the incidence of fetuses thatare small for gestational age. Due to the retarded growth of their fetuses, pregnantwomen who smoke cigarettes are at an increased risk for giving birth to low birth-weight infants, that is, infants who weigh less than 2,500 g at birth. Infants born towomen who smoke are, on average, 200 g lighter than infants born to comparablewomen who do not smoke. As low birth-weight children mature, they experiencedelayed intellectual and emotional development, as well as continued retardationof physical growth (Rubin, 2004).

Appendices � 265


The lowered birth-weight among the issue of smoking mothers also has beendemonstrated in laboratory animals. In the earliest experimental study in moderntimes, Wistar strain rats were exposed (whole-body) in a glass jar to the smokeyield of approximately a pack of cigarettes for 1 - 3 exposures per day for up to 3minutes total from mating through gestation, delivery, and lactation (Essenberg etal., 1940). Two-thirds of the offspring were underweight at birth and/or lost weightduring weaning compared to the concurrent controls. Additionally, the mothersshowed overt clinical toxicity, and 13.5 % of their young died during the experiment,the latter in parallel with human mortality in this study. The reduction in fetal bodyweight from maternal cigarette smoke exposure, on the order of 16 - 19 %, wasreplicated in this species (rat) in other studies under variable experimental conditions(Haworth & Ford, 1972; Leichter, 1995; Reznik & Marquard, 1980; Tachi &Aoyama, 1983; Younoszai et al., 1969). Studies in two other species, laboratorymice (Peterson et al., 1981; Seller & Bnait, 1995) and rabbits (Schoeneck, 1941)demonstrated similar effects on fetal body weight from maternal exposure tocigarette smoke.

The prototype Good Laboratory Practices (GLP)-compliant and contemporarydevelopmental and reproduction studies in animals demonstrating the effects ofsmoke exposure in surrogate models were recently published (Carmines et al.,2003; Gaworski et al., 2004). In these studies, 150, 300, and 600 mg/m3 totalparticulate matter (TPM) of 1R4F reference cigarette target mainstream smokewas generated in Canon style exposure chambers and administered via nose-onlyinhalation to groups of 20 (female) or 40 (male) Sprague-Dawley strain rats restrainedin plastic tubes. Exposures were given for 2 hours/day for 2 (females) or 4 (males)weeks prior to and during mating, through gestation day 20 or lactation day 21.Sham and cage control animals were compared concurrently.

Paternal toxicity, manifested as significantly reduced body weight gain (anddecreased uterine weights maternally,) was observed at the highest exposure level(600 mg/m3). As in the earlier studies, reduced fetal body weight on the order of 7- 23 % and accompanying increased skeletal variations in fetuses of dams exposedto 300 and 600 mg/m3 cigarette smoke occurred in those taken at term. In thoseallowed to wean, there was absence of effects on developmental landmarks andneurobehavioral assessments. However, significantly (p < 0.05) reduced birth-weights ranging from 7 - 18 % at the 300 and 600 mg/m3 levels and consistentgrowth retardation at these same exposure levels on postnatal days 21 and 63compared to the controls, confirmed the developmental effect on body weights inthe term animals.

Nicotine, cotinine, and CO blood levels, identified in the parental and/or term fetusesand neonates, increased in a concentration-related manner, did not bioaccumulatewith time, and confirmed that the smoke was generated consistently. Particle size



measurements demonstrated that the smoke was respirable, and respiratory tracttissue changes from the maternal animals were consistent with those previouslyreported in nose-only studies with cigarette smoke (Vanscheeuwijck et al., 2002).Notably, and in partial contrast to human adverse effects, adult fertility, gestationallengths, fetal mortality and malformation rates, and neonatal survival were notaffected when compared to controls. The methodology used in these studies mayserve as a useful tool for cigarette evaluation in the future.

SUPPORTING BIBLIOGRAPHY

Carmines, E. L., Gaworski, C. L., Faqi, A. S. & Rajendran, N. (2003) In uteroexposure to 1R4F reference cigarette smoke: evaluation of developmental toxicity.Toxicol. Sci. 75: 134-147.

Essenberg, J. M., Schwind, J. V. & Patras, A. B. (1940) The effects of nicotineand cigarette smoke on pregnant female albino rats and their offspring. J. Lab.Clin. Med. 25: 708-717.

Gaworski, C. L., Carmines, E. L., Faqi, A. S. & Rajendran, N. (2004) In utero andlactation exposure of rats to 1R4F reference cigarette mainstream smoke: effecton prenatal and postnatal development. Toxicol. Sci. 79: 157-169.

Haworth, J. C. & Ford, J. D. (1972) Comparison of the effects of maternalundernutrition and exposure to cigarette smoke on the cellular growth of the ratfetus. Am. J. Obstet. Gynecol. 112: 653-656.

Leichter, J. (1995) Decreased birth-weight and attainment of postnatal catch-upgrowth in offspring of rats exposed to cigarette smoke during gestation. GrowthDev. Aging 59: 63-66.

Peterson, K. L., Heninger, R. W. & Seegmiller, R. E. (1981) Fetotoxicity followingchronic prenatal treatment of mice with tobacco smoke and ethanol. Bull. Environ.Contam. Toxicol. 26: 813-819.

Reznik, G. & Marquard, G. (1980) Effect of cigarette smoke inhalation duringpregnancy in Sprague-Dawley rats. J. Environ. Pathol. Toxicol. 4: 141-152.

Rubin, E. (2004) Smoking impairs female reproduction function. In: Rubin’sPathology: Clinicopathologic Foundations of Medicine. 4th ed. (Rubin, E. &Gorstein, F., eds.) Philadelphia: Lippincott Williams & Wilkins, pp. 316-319.

Schoeneck, F. J. (1941) Cigarette smoking in pregnancy. N. Y. State J. M. 410:1945-1948.

Appendices � 267


Seller, M. J. & Bnait, K. S. (1995) Effects of tobacco smoke inhalation on thedeveloping mouse embryo and fetus. Reprod. Toxicol. 9: 449-459.

Tachi, N. & Aoyama, M. (1983) Effect of cigarette smoke and carbon monoxideinhalation by gravid rats on the conceptus weight. Bull. Environ. Contam Toxicol.31: 85-92.

Vanscheeuwijck, P. M., Teredesai, A., Terpstra, P. M., Verbeeck, J., Kuhl, P.,Gerstenberg, B., Gebel, S. & Carmines, E. L. (2002) Evaluation of the potentialeffects of ingredients added to cigarettes. Part 4: subchronic inhalation toxicity.Food Chem. Toxicol. 40: 113-131.

Younoszai, M. K., Peloso, J. & Haworth, J. C. (1969) Fetal growth retardation inrats exposed to cigarette smoke during pregnancy. Am. J. Obstet. Gynecol. 104:1207-1213.



APPENDIX G

SOME PERSPECTIVES ON LIMITS

LSRO recommends that data submitters place chemical detection limits intoperspective and, when possible, submit these perspectives to LSRO as part of thedata package. This appendix outlines two approaches to obtaining such perspectives:(1) risk-informed detection limits and (2) comparisons to existing regulatorystandards for other substances. The basis of all such approaches is that lowexposures merit less concern. If the toxicological concern about a substance doesnot relate to dose, these approaches will not apply. For example, if an immuneeffect is of concern, immune reactions may not depend on dose.

The sensitivity of currently used chemical technology to detect a substance is nowin the parts per trillion (ppt) range. Therefore, a detected change, which is significantphysiologically and analytically, might occur at a level too low to cause a significantlifetime exposure or a conceivable adverse health effect from direct toxicity.

For all substances transferring into smoke, an estimate of potential lifetime exposurewill assist in developing an appropriate perspective. Even if a detected ingredient,or a pyrolysis-related product of the ingredient, transfers into smokers’ lungs, themaximum exposure to the substance may not yield a conceivable risk. Given thata change in the detected amount of a chemical substance in smoke is bothreproducible and analytically measurable, a data submitter should seek to identifyan amount so small that it could not generate a lifetime exposure associated withan adverse health effect.

For example, assume that available technology identifies a chemical substance inthe one part per billion (ppb) range [approximately one nanogram per liter (~1 ng/L)]. For ingredients that transfer and have associated chronic adverse healtheffects, it is relatively easy to calculate the potential maximum lifetime exposure.Assuming that the body does not eliminate the relevant substance(s), a lifetimesmoker (~45 years) with an exposure rate of two packs per day might inhale asmuch as ~0.3 mg of this substance in smoke during their lifetime, as shown in thefollowing calculation:

0.5 ng/cigarette (in cigarette smoke)two packs/day = 40 cigarettes/daySo, 20 ng/day in smokeGiven 365 days/yr, this exposure rate yields 7.3 x 103 ng/yr,and assuming 45 years of smoking, it also yields 3.3 x 105 ng/lifetime,which is equivalent to 0.33 mg/lifetime.

Appendices � 269


Two approaches are detailed below:

(a) One potential approach would compare the amount of the substance incigarette smoke to a “risk-informed detection limit,” a concept analogousthe U.S Food and Drug Administration’s (FDA’s) “threshold of regulation,”a risk-based approach applied to indirect food additives (Kroes et al., 2000;Rulis, 1989). FDA based this approach on an analysis of 477 chemicalcarcinogens from a carcinogenicity potency database developed by Goldand coworkers (Gold et al., 1984). FDA generated a probability distributionof these substances and used it to derive an estimate of dietary concentration,where the upper bound lifetime risk of cancer was less than 10-6 (one-in-a-million). FDA calculated this “threshold of concern” for indirect foodadditives as 0.5 ppb (Rulis, 1989). Any additive consumed at this levelwould pose a risk of less than 10-6 cancer deaths.

A data submitter could evaluate the evidence supporting a maximum riskof inhaling a chemical substance in smoke. Assumptions for the calculationof a chemical substance in smoke might include the maximum daily cigaretteconsumption (e.g., two packs a day) and a maximum absorption of thesubstance in the inhaled smoke by the smoker (e.g., 100 %). A datasubmitter could calculate a theoretical maximum daily uptake of individualingredients or their pyrolysis-related products from data obtained bychemical analysis of the smoke. A data submitter could then refine theirestimate with additional data, if it was deemed worthwhile to do so.

(b) A different approach would be to compare the amount of the substance inthe smoke to an existing regulatory standard for another substance, makingthe assumption that the first substance is fully absorbed. For this purpose,the data submitter could select a regulated substance X and determinewhether the concentration of the substance of interest, as detectedanalytically in cigarette smoke, would fall below the level of regulatoryconcern for substance X. This approach would lead to a statement like,“Even if this detected substance in the smoke were X, the concentrationwould comply with chronic health standards set by U.S. regulatoryagencies.” Some of more suitable properties of substance X might be:

(1) stability to pyrolysis,

(2) likelihood of directly transferring in smoke without reactingwith other substances, including pyrolysis products, and

(3) relative uniformity of standards across international regulatoryorganizations.

The two examples above refer to the possibility of establishing a general humanthreshold exposure, in terms of chemical concentration, for any ingredient, pyrolysis



product, or smoke constituent, below which human health is not significantly atrisk. A data submitter could apply a different approach than either of the twoexamples given above. LSRO will review the scientific reasoning for the approachtaken by the data submitter.


Gold, L. S., Sawyer, C. B., Magaw, R., Backman, G. M., de Veciana, M., Levinson,R., Hooper, N. K., Havender, W. R., Bernstein, L., Peto, R. (1984) A carcinogenicpotency database of the standardized results of animal bioassays. Environ. HealthPerspect. 58: 9-319.

Kroes, R., Galli, C., Munro, I., Schilter, B., Tran, L., Walker, R. & Wurtzen, G.(2000) Threshold of toxicological concern for chemical substances present in thediet: a practical tool for assessing the need for toxicity testing. Food Chem. Toxicol.38: 255-312.

Rulis, A. M. (1989) Establishing a threshold of regulation. In: Risk Assessment inSetting National Priorities. (Bonin, J. J. & Stevenson, D. E., eds.) New York:Plenum Press, pp. 271-278.

Index � 271


INDEX

AAbsorption, 9, 72-74, 85, 111-113, 200Added ingredient, 2-4, 8-10, 15, 17, 37,

47-52, 74-75, 77, 105, 118-120, 133,140-141, 147-148, 200

Ash, 50, 85, 200

BBiologically effective dose, 94, 140, 200Biomarker, 64-64,96-97, 101, 105, 109,

111-114, 122-123, 136-138, 140, 201

CChromosome

deletions, 93frame-shift mutation, 90-91recombination, 90

Cigarette, 201filter, 33, 38, 47, 50, 203paper, 33, 37-38, 47, 206

Cigarette smoke condensate, 89, 91-92,134, 149, 201particulate phase, 90, 91, 92, 207

Cigaretteshigh yield and/or low yield, 105mentholated, 65, 97, 109, 135, 139mentholation, 65, 97, 131, 205non-mentholated, 65, 97, 109

Clearance, 65, 76, 78, 82, 111, 201Complex mixture, 1, 15, 32, 37-38, 44-

45, 50-52, 202Cotinine, 5, 65, 74-76, 83, 97, 109, 111-

113, 122, 137-138, 141, 202Crossover study, 65, 109-110, 202Cytotoxicity, 89-90, 124, 202

DDeposition, 9, 97, 135, 202Detection limit, 41-42, 59, 202

Diseasescardiovascular disease, 9-10, 88, 147,

233chronic obstructive pulmonarydisease, 9-10, 88, 147, 201, 233lung cancer, 9, 13, 15, 88, 98, 127,

139, 147, 233stroke, 10

DNA adducts, 94-96, 140-141Dose, 4-5, 9-10, 56-58, 65, 71, 80-84, 93-

94, 120, 122, 140, 202Dosimetry, 4-5, 15, 22, 56-58, 71, 73, 82-

83, 118, 140, 148, 202

EElimination, 66, 72, 74, 76-77, 79, 82, 110,

112, 203Endpoint, 12, 87, 95, 100-101, 203Environmental tobacco smoke, 11, 39,

51, 99, 134, 149, 203Epigenomic, 101, 203Excretion, 65, 76, 95, 203Exposure, 1, 3-5, 9, 11-12, 22, 25, 27, 29,

33, 52, 56-67, 96, 98-100, 104-114, 117-118, 120-123, 125, 134, 136-138, 140,146-149, 203

FFlue-cured, 33, 203

GGas phase, 49, 67, 149, 203

Iin vitro, 88, 92-93, 101, 134, 204in vivo, 88, 92, 94, 99, 134, 204Indicator substances, 45, 53, 148

benzo(a)pyrene, 12, 42, 132carbon monoxide, 3, 20, 39, 62, 80,

97, 99, 106, 112, 114, 121, 128, 132,135, 139, 149



carboxyhemoglobin, 66-67, 99, 111-112,122

cotinine, 5, 65, 74-76, 83, 97, 109, 111-113, 122, 137-138, 141, 202

nicotine, 3, 20, 40, 46, 53, 62, 66, 72-77, 80, 83, 97, 105-106, 108, 111-112,114, 121, 128, 132, 135-137, 1 4 0 ,149, 206

nicotine free dry particulate matter,46, 206

nitrogen dioxide, 20nitrogen oxides, 3, 42, 62‘tar’, 3, 39, 41, 45-46, 73, 106, 113-

114,121, 128, 149, 210thiocynate, 97, 111-113total particulate matter (TPM), 41,

45, 49, 53, 132water, 3, 20, 38, 42, 51, 121, 132, 149

Ingredients, 204indirect additives, 8, 204anisaldehyde, 44, 48anisole, 48benzaldehyde, 32, 48cocoa, 45, 51-52, 95, 104, 127, 133,

150flavor/flavorings, 11, 38, 39, 47, 50,

78, 104humectants, 38, 42, 204isoamyl isovalerate, 47licorice, 51menthol, 11, 47-48, 65-66, 78-79, 89,

91, 98, 104, 109, 127, 135-142, 149,233-244

methyl cinnamate, 48rosemary oil, 79sugars, 48, 135vanillin, 44, 48, 50

KKinetics, 204

pharmacokinetics, 5, 58, 71-73, 77, 79-85, 123-124physiological-based pharmacokinetic(PBPK), 82-83, 207

MMaximum tolerated dose (MTD), 99,

205Maximum use level (MUL), 18, 34, 205Metabolite(s), 5, 58, 64-66, 68, 72-77, 80,

82, 84, 97, 112, 122-123, 137-138cotinine, 5, 65, 74-76, 83, 97, 109,

111-113, 122, 137-138, 141, 202Micronucleated cell, 94, 205

NNicotine, 3, 20, 40, 46, 53, 62, 66, 72-77,

80, 83, 97, 105-106, 108, 111-112, 114,121, 128, 132, 135-137, 140, 149, 206

Nicotinic cholinergic receptors(nAChRs), 73, 206

Noise, 125, 206

OOrganoleptic, 105, 136, 206

PPack-year, 57, 108, 111, 121, 206Point mutation, 90, 93, 207Polycyclic aromatic hydrocarbon (PAH),

12, 207Puff flow, 207Puff interval, 40, 105, 111, 207Puff profile, 105, 114, 136, 208Puff volume, 40, 59, 67, 105, 111, 208Pyrolysis, 2, 4, 11, 19, 21, 37, 42-52, 56,

60-61, 67-68, 71, 84-85, 91, 104, 117,120-123, 133, 144, 150, 208

Pyrosynthesis, 37, 44, 49-50, 52, 63, 71,99, 104, 120, 122-123, 135, 144, 208

RReference cigarette, 39, 46, 91, 94, 96,

132, 146, 209Relative risk, 2-4, 8, 11-13, 15, 42, 65,

68, 71, 125-126, 209de minimis risk, 25, 229-230

Retention, 68, 79, 82, 105, 209

Index � 273


SSales-weighted, 21, 39, 209Sensitivity, 51, 77, 89, 92, 95, 125, 138,

210Signal-to-noise, 23, 38, 62, 89, 101, 125,

210Sister chromatid exchange, 92-93Smoke, 210

environmental tobacco smoke (ETS),11, 39, 51, 99, 134, 149, 203

mainstream smoke (MS), 13, 16, 33,38, 42, 47, 49, 65, 69, 78, 99, 127,134, 135, 149, 204

sidestream smoke (SS), 13, 33, 39, 99,134, 149, 210

Smoking behavior, 135-138

TTests

Ames (Salmonella mutogenicityassay), 90-92, 206genotoxicity, 90-92, 204

inhalation studies, 96mammalian erythrocyte micronucleus,

94microarray, 100-101, 205mouse lymphoma, 93novel, 101sister chromatid exchange assay, 92-

93skin tumorogenicity, 95-96

Test substance, 32-33Tobacco

Burley, 33, 127, 201flue-cured, 33, 203Maryland, 127, 205Oriental, 33, 206reconstituted, 33, 140, 208Virginia, 211

Topography/Topological, 67, 105-106,111-112, 211, 225

YYield, 46, 58, 211