U.S. CONSUMER PRODUCT 

SAFETY COMMlSSlON 

IN CONSULTATION WITH THE 

U.S. DEPARTMENT OF HEALTH 

AND HUMAN S~avices 

AUGUST 1993 
Fire Safe Cigarette Act of 1990 

Under the Cigarette Safety Act of 1984 (P.L. 98.567). the Technical Study Group on Cigarette and Little 

Cigar Fire Safety CrSG) found that it is technically feasible and may be commercially feasible to develop 

a cigarette that will have a significantly reduced propensity to ignite furniture and mattresses. Furthermore, they found that the overall impact of such a cigarette on other aspects of the United States 

society and economy may be minimal. 

Recognizing that cigarette-ignited fires continue to be the leading cause of fire deaths in the United 

States, the Fire Safe Cigarette Act of 1990 (P.L. 101.352) was passed by the lOlst Congress and signed 

into law on August 10, 1990. The Act deemed it appropriate for the U.S. Consumer Product Safety 

Commission to complete the research recommended by the TSG and provide. by August 10. 1993, an 

assessment of the practicality of a cigarette fire safety performance standard. 

Three particular tasks were assigned to the National Institute of standards and Technology's Building 

and Fire Research Laboratory: 

- develop a standard test method to determine cigarette ignition propensity, 

compile performance data for cigarettes using the standard test method, and 

Conduct laboratory studies on and computer modeling of ignition physics to develop valid, 

user-friendly predictive capability. 

- 

- 

Three tasks were assigned to the Consumer Product Safety Commission: 

- design and implement a study to collect baseline and follow-up data about the characteristics of 

cigarettes, products ignited, and smokers involved in fires, 

develop information on societal costs of cigarette-ignited fires, and 

in consultation with the Secretary of Health and Human Services, develop information on changes 

in the toxicity of smoke and resultant health effects from cigarette prototypes. 

- 

- 

The Act also established a Technical Advisory Group to advise and work with the two agencies. 

This report is one of six describing the research performed and the results obtained. Copies of 

these reports may be obtained from the U.S. Consumer Produa Safety Commission. 

Washingto-, DC 20207. 
5 

Erian C. Lee 

Lakshmi C. Mishra 

U.S. Consumer Product Safety Commission 

David M. Burns 

University of California at San Diego Medical Center 

C. Gary Gairola 

Tobacco and Health Research Institute 

University of Kentucky at Lexington 

Jeffrey E. Harris 

Internal Medicine Associates 

Massachusetts General Hospital 

Dietrich Hoffman 

American Health Foundation 

Harold c. Pillsbury, Jr. 

Donald R. Shopland 

National Cancer Institute 

U.S. Department of Health and Human Services 

August 1993 

TABLE OF CONTENT8 

PART I 

TOXICITY TESTING PLAN FOR LOW IGNITION-POTENTIAL CIGARETTES 

IZ2si.e 

Health Effects Assessment Plan ....................... 1-8 

Brian C. Lee, PhD, DABT 

Chapter A 

Overview and Major Considerations in the Toxicity 

Testing of Low Ignition-Potential Cigarettes ........ Al-AZ3 

Jeffrey E. Harris, MD, PhD 

Chapter B 

Smoking Machine Parameters for Collection of Total 

Particulate Matter and Gases from Low Ignition-Potential 

Cigarettes .......................................... Bl-B7 

Harold C. Pillsbury, Jr. 

Chapter C 

Assessing Changes in Topography (Inhalation Profile) and 

Biological Effects of Tobacco Smoke in Humans ....... C1-C25 

David M. Burns, MD 

Chapter D 

Analysis of Toxic Smoke Constituents ................ D1-D38 

Dietrich Hoffmann, PhD 

Chapter E 

Short-Term Tests for the Evaluation of Cigarette Smoke 

Toxicity ............................................ El-E22 

C. Gary Gairola, PhD 

Chapter F 

In Vivo Bioassays for Carcinogenicity ............... Fl-Fl7 

Dietrich Hoffmann, PhD 

PART I1 

LIMITED TIER I AND I1 DEMONSTRATION OF THE TOXICITY TESTING PLAN 

FOR LOW IGNITION-POTENTIAL CIGARETTES 

Report and Data ...................................... 1-14 

Tobacco and Health Research Institute, American 

Health Foundation, Brian C. Lee, PhD, DABT, and 

Lakshmi C. Mishra, PhD 
CONTRIBUTORS TO PART I 

Expert Panel 

David M. Burns, MD, (University of California at San Diego 

Medical Center), Professor of Medicine 

C. Gary Gairola, PhD (Tobacco and Health Research Institute, 

University of Kentucky at Lexington), Associate Professor of 

Pharmacology and Experimental Therapeutics 

Jeffrey E. Harris, MD, PhD (Internal Medicine Associates, 

Massachussetts General Hospital) 

Dietrich Hoffmann, PhD (American Health Foundation), Associate 

Director and Chief, Environmental Carcinogenesis 

Harold C. Pillsbury, Jr., Cigarette Testing Consultant 

Federal Government 

Brian C. Lee, PhD, DABT (US Consumer Product Safety Commission), 

Toxicologist, Manager, Expert Panel 

Donald Shopland, MD, (US Department of Health and Human Services, 

National Institutes of Health, National Cancer Institute), 

Coordinator, Smoke and Tobacco Program 
CONTRIBUTORS TO PART I1 

Laboratories 

Tobacco and Health Research Institute, University of Kentucky at 

Lexington (C. Gary Gairola, PhD, Associate Professor of 

Pharmacology and Experimental Therapeutics) 

American Health Foundation (Klaus Brunnemann, MS, Section Head, 

Analytical Chemistry, and Dietrich Hoffmann, PhD, Associate 

Director and Chief, Environmental Carcinogenesis) 

Federal Government 

Brian C. Lee, PhD, DABT (US Consumer Product Safety Commission), 

Toxicologist, Contract Officer 

Lakshmi C. Mishra, PhD (US Consumer Product Safety Commission), 

Toxicologist 

Donald Shopland, MD, (US Department of Health and Human Services, 

National Institutes of Health, National Cancer Institute), 

Coordinator, Smoke and Tobacco Program 

Part I 

VOLUME 5 

PART I 

TOXICITY TESTING PLAN FOR 

LOW IGNITION-POTENTIAL CIGARETTES 

U.S. Consumer Product Safety Commission and its 

Expert Panel, in consultation with the 

US. Department of Health and Human Services 

May 5, 1993 

HEALTH EFFECTS ASSESSMENT PLAN 

Brian C. Lee, PhD, DABT 

U.S. Consumer product safety Commission 

May 5, 1993 

EXECUTIVE SUMMARY 

I. Introduction 

The Fire-Safe Cigarette Act of 1990 requires the U.S. 

Consumer Product Safety Commission (CPSC), in consultation with 

the Secretary of the U.S. Department of Health and Human Services 

(DHHS), to develop information on changes in the toxicity of 

smoke and resultant health effects of cigarettes with a reduced 

ability to start fires. The Act states that CPSC Ilshall not 

obligate more than $50,000 to develop such information." The 

Technical Advisory Group (TAG) established by the Act agreed that 

this amount precluded any significant testing of prototypes. The 

Act succeeds the Cigarette Safety Act of 1984 which established a 

Technical Study Group to examine the feasibility of developing 

cigarettes with lowered ignition potential. The Technical Study 

Group concluded it is technically feasible and may be 

commercially feasible to develop cigarettes that will have a 

significantly reduced propensity to ignite upholstered furniture 

or mattresses. 

The Act expresses a consideration for the possible 

nationwide health implications of changes resulting from the 

market substitution/entrance of low-ignition cigarette types. 

There were about 50 million smokers in the U.S. in 1991, 

according to the National Cancer Institute. The primary concern 

is that a small increase in the risk of a serious health effect, 

due to new cigarette types, could result in a great increase in 

human mortality and morbidity and thus overbalance the benefits 

that would be achieved from the reduction of fires. 

CPSC staff, in consultation with DHHS and with the 

concurrence of the TAG, decided that in view of the statutory 

$50,000 limitation, a plan must be developed for the 

toxicological work needed. CPSC convened an expert panel to 

assist in the development of the plan. The panel was composed of 

knowledgeable scientists in the field of cigarette toxicity 

testing. These members were nominated by TAG members and 

selected by the CPSC staff. 

This report discusses significant issues and recommends 

testing necessary for the comprehensive assessment of health 

effects of low-ignition potential cigarette smoke. It is not 

intended to be a detailed manual of cigarette toxicity testing, 

although some necessary technical information is presented. 
page 2 

11. General Discussion 

Several adverse health effects of serious concern are the 

basis for considering the various existing toxicity tests. 

effects include: lung and throat cancer, chronic obstructive 

lung disease, heart and vessel disease, male and female 

reproductive effects, fetal growth retardation, and 

psychophysiological addiction, as indicated in Chapter A. Not 

all of these health effects can be addressed at this time due to 

the impracticality or non-existence of adequate tests, expenses, 

or time needed for testing. Therefore, only the tests believed 

to be practical are recommended. Estimates of costs and times 

needed for testing are included in Chapters B and D-F. 

Major issues surrounding the testing include sidestream 

smoke, bases of comparisons, analytical vs. in vitro vs. in vivo 

testing, machine reflection of human smoking behavior, design or 

performance-based testing, screening paradigms, and disclosure of 

new additives or increased levels of existing additives, as 

discussed in Chapter A. Since low ignition-potential cigarettes 

might cause changes in smoking behaviors and therefore modify the 

toxicity, altered human behavior may become a significant factor 

in exposure, as discussed in Chapter C. Since the smoke is 

collected by mechanically smoking the cigarettes, the apparatus 

should be set to reflect smoking behavior as closely as 

technically feasible. 

Two methods presently exist for the mechanical smoking of 

cigarettes, as noted in Chapter B. The Federal Trade Commission 

(FTC) method, established in 1969, is used in the United States, 

and the CORESTA method (IS0 3308-1991) is mainly used in Europe. 

The FTC method is described in Chapter B and is very similar to 

the CORESTA method. Both methods analyze for tar, nicotine, 

carbon monoxide, and moisture content. 

These 

In light of present knowledge on the adverse health effects 

and toxic constituents of cigarette smoke, further testing beyond 

the Federally mandated requirements for tar, nicotine, and carbon 

monoxide levels is needed to evaluate the toxicity. Levels of 

key chemical constituents known to be associated with adverse 

health effects need to be measured, as described in Chapter D. 

Cigarette smoke is a complex mixture of more than 3,500 chemicals 

containing at least 35 known carcinogens, and analysis of a 

limited number of individual chemicals may not predict the net 

toxic effects of the smoke. In order to address certain 

conglomerative toxicities of the non-gaseous constituents, in 

vitro and animal testing are needed, as described in Chapters E 

and F. Limited whole-animal testing is necessary because of the 

complexity of the biological systems and a variety of toxic 

reactions caused by cigarette smoke. As an example, pulmonary 

inflammation testing requires intact immune, respiratory, and 

cirulatory systems to be simultaneously present. 
Page 3 

The CPSC staff recommends the following guidance plan after 

reviewing the considerations of its expert panel and DHHS. 

111. Assessment Plan 

This plan provides guidance for the development of data 

needed to evaluate the changes in toxicity associated with low 

ignition-potential cigarettes. Performance-based, rather than 

design-based, testing will be used to provide data specific to 

cigarette prototypes. A screening paradigm that requires 

acceptable performance levels by a candidate cigarette type at 

one tier of tests before proceeding with the next tier is 

recommended. This would allow early rejection of candidates 

evaluated as unacceptable. However, definition of acceptable 

levels of performance is beyond the scope of this plan and the 

direction given by the Act. Therefore, the tests are presented 

in a sequence of tiers for screening without ascribing acceptable 

levels of performance at each tier. 

Results of the recommended testing will be used to assess 

the relative toxicity of low-ignition potential cigarettes. The 

toxicity of a candidate low ignition cigarette should be compared 

to: 

1) the specific marketed brandltype intended for 

replacement, or comparable marketed brandsltypes for a non- replacement candidate, and 

2) standard reference cigarettes, such as the University of 

Kentucky standard cigarettes mentioned in Chapter E, for 

quality control. 

There are insufficient test methods and data on exposure to 

cigarette smoke and resultant effects for the direct translation 

of the results into absolute risks to humans. Since the overall 

health goal is to avoid the production of greater or perhaps new 

toxicities than that caused by existing cigarettes, a comparative 

approach of assessing toxicity is appropriate. 

Selection of the guidance plan tests assumes that no new 

additives would be present in the candidate cigarettes and that 

presently used additives would not exceed the levels in the 

current cigarettes. Since toxic effects not. considered by this 

guidance plan could also occur, it is recommended that additives 

exceeding the current maximum levels of use on a per unit weight 

of tobacco basis must be disclosed to the U.S. Department of 

Health and Human Services. Confidential business information 

status may be requested for the data disclosed. 

A. Smokina machine 

The FTC method described in Chapter B is the basis for the 

mechanical generation of smoke constituents. Puff volume, 
page 4 

frequency, and draw velocity may be modified as dictated by 

behavioral data developed from human testing (Tier 111), as 

described in Chapter C. 

results of mainstream and sidestream smokes can be shown, both 

must be separately collected and tested. 

B. DeSCriDtiOn of Tiers 

An outline of four tiers is presented in Table 1. A description 

of the tiers follows. 

Unless consistent correlation of testing 

Tier I - Analvses of chemicals 

All constituents will be reported as per unit weight of 

tobacco burned and per cigarette. Moisture, nicotine, tar (total 

particulate matter- dry), and carbon monoxide will be measured 

according to the FTC method, as described in Chapter B. Nitric 

oxide will also be measured using the detector attachment to the 

smoking machine. The gaseous phase will be analyzed for acidity, 

reductionfoxidation potential, hydrogen cyanide, volatile 

hydrocarbons, aldehydes, and volatile nitrosamines, as described 

in Chapter D. The tar will be analyzed for phenols, catechols, 

polyaromatic hydrocarbons, and tobacco-specific nitrosamines 

(Chapter D) . 

Tier I1 - In vitro tests 

The tar will be assayed for mutagenic activity with Ames' 

Salmonella test with strains TA98, 100, and 1535. The tar will 

also be assayed for malignant cell transforming activity, using 

C3HjlOTlf2 mouse embryo fibroblast cells. Both mutagenicity and 

cell transformation assays are described in Chapter E. 

Tier I11 - Human smokins behavior 

Humans are typically the last experimental tier in testing 

products with potential human health effects. An example is the 

premarket testing of new drugs. Human testing to collect 

topographical data is limited to a couple of weeks of exposure. 

Smoking behavior, including puff volume, frequency, and draw 

velocity of a selected group of human volunteers would be 

monitored, as outlined in Chapter C. Carbon monoxide (breath or 

blood) and cotinine (urinary, salivary, or blood) will serve as 

biological markers of exposure to the smoke. If the smoking 

behavior data is significantly different from the FTC smoking 

machine settings such that an increase in exposure to the 

analyzed chemicals might result, then the machine must be set to 

reflect these data before generating smoke constituents for 

further Tier I and I1 testing and then animal testing. 

Tier IV - Animal tests 
Page 5 

Inflammatory lung response to cigarette smoke in C57B1 mice 

will be assayed as described in Chapter E. Tumor formation in 

the upper respiratory tract of random-bred golden Syrian hamsters 

from inhalation exposure and the skin, lungs, and other tissues 

of Swiss albino Ha/ICR/Mil strain mice from skin painting 

exposure will be examined. These two carcinogenicity tests are 

described in Chapter F. 

All testing must conform to good laboratory practices, 

humane laboratory animal methods, and informed human consent 

procedures accepted within the scientific community. Evaluations 

of toxicity must be conducted by scientists possessing 

appropriate toxicological qualifications. 

IV. First implementation step 

Table 2 is a collection of direct cost estimates for Tiers 

I, 11, and IV. No estimates are available for Tier 111. 

Completion of all four testing tiers by successful low ignition 

potential cigarette candidates might be considered expensive 

relative to the present level of testing required by FTC ($330X 

for Tiers I, 11, and IV vs. $3.5K for FTC; Table 2). Therefore, 

a stepwise implementation of the plan is suggested. 
Table 1 

Health Effects Assessment Plan 

Outline of Tiers 

Tier I - Analyses of chemicals 

Whole smoke 

acidity (pH) 

reduction/oxidation potential 

gases 

Gas phase 

carbon monoxide 

hydrogen cyanide 

nitric oxide 

acetaldehyde 

acrolein 

proprionaldehyde 

volatile hydrocarbons 

benzene 

toluene 

1,3-butadiene 

isoprene 

N-nitrosodiethylamine 

N-nitrosodimethylamine 

N-nitrosopyrrolidine 

aldehydes 

volatile nitrosamines 

Particulate phase 

catechol 

nicotine 

phenols, as phenol 

polyaromatic hydrocarbon 

benzo (a) pyrene 

tar-FTC 

tobacco specific nitrosamines 

Nt-nitrosonornicotine 

4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone 

Tier 11- In Vitro Tests 

Salmonella mutagenicity (Ames' assay) 

mouse embryo fibroblast cell transformation assay 

Tier I11 - Human Smoking Behavior 

cotinine 

carbon monoxide 

topography 

mouse inflammatory lung response 

hamster upper respiratory tract carcinogenicity 

mouse skin painting carcinogenicity 

Tier IV - Animal Tests 
Table 2 

Estimated Direct Costs in 1993 U.S. Dollars 

per brand or prototype . 

2 

Tier 

3500 

250 

500 

350 

400 

700 

600 

800 

350 

250 

500 

500 

800 

I - Analyses of chemicals = $9,500 

FTC-required tar, nicotine, and carbon monoxide 

Whole smoke 

acidity (pH) 

reduction/oxidation potential 

gases 

Gas phase 

hydrogen cyanide 

nitric oxide 

aldehydes 

volatile hydrocarbons 

volatile nitrosamines 

N-nitrosodiethylamine 

N-nitrosodimethylamine 

N-nitrosopyrrolidine 

Particulate phase 

catechol 

nicotine 

phenols, as phenol 

benzo(a)pyrene 

tobacco specific nitrosamines 

Tier 11- In Vitro Tests = $9,350 

1850 Salmonella mutagenicity (Ames’ assay) 

7500 mouse embryo fibroblast cell transformation assay 

Tier IV - Animal Tests = $309,000 

50K mouse inflammatory lung response 

220K hamster upper respiratory tract carcinogenicity 

39K mouse skin painting carcinogenicity 

________ 

________ 

$327,850 total for Tiers I, 11, and IV 
Page 8 

A practical selection of recommended tests should comprise a 

first step in the implementation of this health effects 

assessment plan. Subsequent steps should consider the testing 

recommended by this plan. The first step should include: 

Smoke and condensate generated by machine according to the FTC 

protocol 

Tier I $5,050 

tar-FTC 

nicotine 

carbon monoxide 

whole smoke pH 

benzo (a) pyrene 

tobacco specific nitrosamines 

N'-nitrosonornicotine 

4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone 

Tier I1 $1,850 

Salmonella mutagenicity ("Ames") assay 

_______ ----___ 

$6,900 Estimated total per brand or prototype 

The rationale for selecting these tests extends beyond cost 

and time duration considerations. Levels of specific chemicals 

(Tier I) as well as an indication of the genotoxicity of the 

mixture (Tier 11) are needed. Tar, nicotine, and carbon monoxide 

are presently required by FTC. The pH of the whole smoke is 

relevant to nicotine uptake. Benzo(a)pyrene is a known animal 

and human carcinogen; however, cigarettes are not the only source 

of exposure. The tobacco-specific nitrosamines are potent animal 

carcinogens and tobacco is the only known source of human 

exposure. 

these nitrosamines. 

No data are available on the human carcinogenicity of 
Chapter A 

OVERVIEW AND MAJOR CONSIDERATIONS IN THE TOXICITY 

TESTING OF LOW IGNITION-POTENTIAL CIGARETTES 

Jeffrey Harris, M.D., Ph.D. 

Internal Medicine Associates - 2 

Wang Ambulatory Care Center 605 

Massachusetts General Hospital 

Parkman Street 

Boston, MA 02114 

21 AUg 1992 

A1 

ABSTRACT 

Both mainstream and sidestream cigarette smoke are complex 

chemical mixtures. In view of this chemical complexity, it 

should be no surprise that cigarette smoke has multiple, diverse 

effects on human health. Nor should it be unexpected that 

multiple chemicals in cigarette smoke contribute to any single 

adverse health effect. 

The diverse human health consequences of cigarette smoking 

are briefly reviewed. Many experimental laboratory models have 

been developed to study the mechanisms of cigarette smoke-induced 

disease. These laboratory models are not always convertible into 

practical, standardized test systems that quantitatively compare 

one cigarette prototype with another. In view of the 

multiplicity of health effects and mechanisms of smoke-induced 

health damage, no single test or battery of tests can capture all 

possible health endpoints. 

While analyses of smoke constituents and studies in 

laboratory animals are feasible, human epidemiological studies 

are not practical for short-term assessment of small differences 

in the toxic effects of various cigarette prototypes. Cigarette 

smoke samples for chemical analysis and biological testing need 

to be collected in a manner that approximates human cigarette 

puffing as closely as technically feasible. 

In formulating a testing plan, the CPSC essentially has two 

options: a design-based testing plan, in which individual, 

pre-selected cigarette design parameters, such as paper porosity 

or percent expanded tobacco, are systematically varied and 

tested; and a performance-based testing plan, in which complete 

cigarette prototypes, and not individual design parameters, are 

evaluated. 

Some testing protocols entail a "screening paradigm." 

Multiple tests are performed in sequence. If a prototype fails 

any particular test in the sequence, the prototype is rejected 

and no further tests are performed. Other multi-test protocols 

allow for tradeoffs among costs and benefits. An unfavorable 

result at any point along the testing sequence does not 

necessarily result in rejection. 

commercial cigarette brands is the measurement and reporting of 

"tar," nicotine and carbon monoxide in mainstream smoke by the 

Federal Trade Commission. With this exception, none of the 

toxicity tests described by the Expert Panel are routinely 

performed on existing cigarette brands by any governmental 

agency. The contents of currently marketed cigarettes are 

proprietary information. Specific additives, tobacco 

The only governmentally-mandated, health-oriented testing of 
A2 

composition, and other design features are not publicly 

disclosed. 
A3 

SCOPE OF THE EXPERT PANEL REPORT 

This report addresses: scientific aspects of the design of 

cigarette toxicity testing systems; the selection and sequencing 

of particular tests; the reliability, feasibility, and costs of 

particular tests; and the interpretation, limitations, uses and 

misuses of test results. In addition to the present Overview 

chapter, the report contains specific chapters on: 

(i) collection of smoke samples from prototype cigarettes 

for toxicity testing, by Dr. Harold Pillsbury (Chapter B); 

(ii) measuring the dosage of smoke constituents actually 

absorbed by human smokers of different cigarette prototypes, by 

Dr. David Burns (Chapter C) ; 

(iii) measuring the amounts of specific chemicals contained 

in the collected smoke, by Dr. Dietrich Hoffmann (Chapter D); 

(iv) toxicity testing in single-cell ("in vitro") systems, 

by Dr. Gary Gairola (Chapter E); 

(v) toxicity testing in whole animal ("in vivogt) systems, by 

Dr. Dietrich Hoffmann (Chapter F); and 

(vi) research needs for developing methods to collect 

additional data (Chapter G, input needed). 

The Expert Panel has not made policy recommendations. The 

Panel members did not perform any testing of prototypes in 

connection with this Report. 

SOURCES OF INFORMATION 

In preparing this Report, the Expert Panel relied upon: the 

Final Report of the Technical Study Group on Cigarette and Little 

Cigar Fire Safety under the Cigarette Safety Act of 1984 [28]; 

background papers issued in connection with the Technical Study 

Group Report [17;27]; reports issued by the National Institute 

for Standards and Technology (and its predecessor, the National 

Bureau of Standards) in connection with low-ignition potential 

cigarettes [11;18]; communications from members of the TAG, CPSC 

staff and DHHS Staff; the published scientific literature; as 

well as its own expertise and experience. No proprietary or 

confidential information was requested, offered, or considered. 

MAINSTREAM VERSUS SIDESTREAM CIGARETTE SMOKE 

Both smokers and nonsmokers can incur adverse health effects 

from the smoke of burning cigarettes. Smokers inhale mostly 
A4 

"mainstream (MS) smoke" that is drawn through the burning tobacco 

column and filter tip and exits through the mouthpiece of the 

cigarette. Nonsmokers inhale mostly "sidestream (SS) smoke" that 

is emitted into the surrounding air between puffs from the end of 

the smoldering cigarette. Sidestream smoke is the major source of 

tlenvironmental tobacco smoke (ETS)." 

compositions, the respective quantities of individual smoke 

constituents can be quite different [35, Chapt.3; 37, p.881. For 

example, in studies of nonfilter cigarettes smoked by machines, 

the yield of carbon monoxide (CO) in sidestream smoke was 2.5 to 

4.7-fold that of MS smoke, while the corresponding SS/MS ratio 

for N-Nitrosodimethylamine (NDMA), an animal carcinogen, was 20 

to 100 [35, pp.130-1311. In one compilation of toxic and 

tumorigenic agents in cigarette smoke, the SS/MS ratio ranged 

from 0.03 to 130 [14]. 

Cigarette modifications that reduce the yields of Ittar," 

nicotine and CO in mainstream smoke do not necessarily reduce the 

corresponding yields in sidestream smoke. In one study of U.S. 

commercial cigarettes, the SSfMS ratios for carbon monoxide were 

2.1 and 2.7, respectively, in two nonfilter cigarettes; 3.5 in a 

conventional filter cigarette; and 26.8 in a perforated filter 

cigarette. The SSfMS ratios for NDMA were 23.6 and 139 in the 

nonfilter cigarettes; 50.4 in the filter cigarette; and 167 in 

the perforated filter cigarette [35, p.1311. The exposure to 

sidestream smoke constituents, though, may be greatly reduced 

depending on distance from the cigarette and ventilation 

characteristics. 

While SS and MS smoke have qualitatively similar chemical 

Modifications of cigarette design intended to reduce 

ignition potential may likewise have different effects on the 

compositions of MS and SS smoke. In principle, ignition-reducing 

chemical agents added to the tobacco column or paper wrapper, 

such as metals and silicates, may transfer differently into MS 

and SS smoke. 

A number of devices have been developed to collect samples 

of SS smoke for chemical analysis [7]. However, there are no 

regularly published data on the composition of SS smoke of U.S. 

cigarette brands. By contrast, the Federal Trade Commission 

regularly publishes machine-measured yields of f@tar," nicotine 

and CO of the MS smoke of U.S. commercial cigarettes, as 

described later in this Report. Still, a testing plan for 

low-ignition potential cigarette prototypes needs to consider 

both MS and SS smoke. 

RANGE OF HUMAN HEALTH CONSEQUENCES 
A5 

Cigarette smoke (whether MS or SS) is not a homogeneous 

entity, but a complex mixture of substances. Some smoke 

components, such as CO, hydrogen cyanide and nitrogen oxides, are 

gases. Others, such as nicotine and polyaromatic hydrocarbons 

(PAH), are contained in the submicron-sized solid particles that 

are suspended in the smoke. Still others, such as formaldehyde 

and benzene, are volatile chemicals contained in the liquid-vapor 

portion of the smoke aerosol [37, p.79; 39, Chapt.141. In view 

of this chemical complexity, it should be no surprise that 

cigarette smoke has multiple, diverse effects on human health. 

Nor should it be unexpected that multiple chemicals in cigarette 

smoke contribute to any one adverse health effect. 

Among the major health effects of cigarette smoke that need 

to be considered in the development of a toxicity testing plan 

are the following: cancer; non-cancerous lung diseases; 

atherosclerotic diseases of the heart and blood vessels; and 

toxicity to the human reproductive system. 

Cancer 

Cigarette smoking causes cancers of the lung, esophagus, 

larynx, oral cavity, bladder, and pancreas in male and female 

smokers. Smoking has reported to increase the risks of cancers 

of the kidney, liver, anus, male penis, and female uterine 

cervix, as well as leukemia [13;31;37;38]. Cigarette smoking is 

far and away the major cause of lung cancer in the U.S., 

accounting for 90 percent of cases in men and 79 percent in women 

[37, p.1561. 

Numerous epidemiological studies covering the experience of 

millions of men and women over many years show that smokers' 

risks of developing cancer increase with the number of cigarettes 

smoked daily, with the lifetime duration of smoking, and with 

early age of starting smoking. Smoking cessation gradually 

reduces cancer risk [37;38]. Filter-tipped and low "tar" 

cigarettes reduce cancer risk somewhat. Cigarette smoking 

interacts with other causative agents, including alcohol, 

asbestos, certain viruses, and certain workplace exposures, in 

the development of human cancers [31;34;37]. 

Mainstream cigarette smoke contains over three dozen 

distinct chemical species considered to be tumorigenic in humans 

or animals [14; 31, pp.192-218; 37, p.861. Some of these 

chemicals are alone capable of initiating tumors in laboratory 

animals; others can promote the development of previously 

initiated cancers. As described later in this Report, 

condensates collected from cigarette smoke cause mutations and 

damage to DNA in laboratory assays of mutagenesis [12], as well 

as malignant transformation in laboratory tests of a chemical's 

ability to induce malignant changes in mammalian cells [3;8]. 
A6 

Undiluted mainstream cigarette smoke is too toxic to be 

tolerated by laboratory animals such as rodents. In long term 

experiments with diluted smoke, these animals still do not inhale 

the smoke in the same way as humans. In natural human smoking, 

the smoke is puffed in volumes of about 30 to 70 ml; the puffed 

smoked is temporarily retained in the smoker's mouth, after which 

it may be inhaled deeply into the lungs. By contrast, some 

laboratory animals breath by panting, while others are obligate 

nose breathers. Even with installation of smoke through 

artificial airways, it can be quite difficult to get the animals 

to inhale deeply, as human smokers do. Accordingly, the 

distribution and retention of smoke components in the respiratory 

systems of laboratory animals may not mimic natural human 

smoking. 

progress has been made in the design of inhalation devices that 

can expose laboratory animals, especially rodents, to diluted 

smoke for long periods. Long-term smoke inhalation regularly 

induces tumors of the larynx in Syrian golden hamsters. Direct 

installation of cigarette tar into the airways of laboratory 

animals causes lung cancers [14;31]. As discussed later in this 

Report, the most widely used experimental system is the mouse 

skin bioassay, in which cancers are induced by the repeated 

application of condensates of cigarette smoke to the shaved skins 

of mice. 

Nevertheless, as described later in this Report, significant 

Independent scientific agencies have concluded that 

environmental tobacco smoke causes lung cancer in nonsmokers 

[22;35]. SS smoke, like MS smoke, contains numerous tumorigenic 

agents. 

Non-Cancerous Luncl Diseases 

Cigarette smoking is the main cause of chronic obstructive 

lung disease (COLD), also called chronic obstructive pulmonary 

disease (COPD) [33]. Smoking accounts for 84 percent of COLD 

deaths in men and 79 percent in women [37, Chapt.31. 

COLD is a slowly progressive illness that develops after 

repeated insults to the lung over many years. In the early years 

after starting to smoke, an individual may report no symptoms. 

Even at this early stage, however, breathing tests can often 

detect abnormalities in the small, terminal airways of the lung 

[2;26;33], and these abnormalities have been directly observed in 

autopsy studies of young smokers who died suddenly [23]. For 

smokers in their twenties, there is already a dose-response 

relation between the extent of abnormal lung tests and the number 

of cigarettes smoked daily. In random population surveys, from 

17 to 60 percent of adult smokers under age 55 have detectable 

small airways dysfunction [33, pp.27-321. 
A7 

Over the course of two decades or more of smoking, a 

constellation of chronic respiratory changes develops. 

picture of chronic lung injury includes: (i) mucus 

hypersecretion, with chronic cough and phlegm; (ii) airway 

thickening and narrowing, resulting in obstruction to airflow 

during expiration; and (iii) emphysema, i.e., abnormal dilation 

of the air spaces at the end of the respiratory tree, with 

destruction of the walls lining the air sacs, resulting in 

further airflow obstruction. These changes can cause significant 

respiratory impairment, disability, and death. While individual 

patients vary in the relative contribution of these three 

changes, those with clinically severe COLD typically have all 

three. 

This 

While a minority of cigarette smokers will develop 

clinically severe COLD, some chronic deterioration in 

lungstructure or function is demonstrable in the majority of 

long-term smokers [33, Chapt.21. Some smokers show more chronic 

cough and phlegm, others more airway obstruction. In general, 

breathing function declines as a person's cumulative exposure to 

smoke, measured in pack-years, increases [6]. 

Cigarette smoke produces pathological changes in the lungs 

of smokers by a number of different mechanisms [38, pp.282-2851. 

Cigarette smoke is toxic to the small hairlike cilia that line 

the central breathing passages. These cilia, in combination with 

mucus secretions, defend against deep inhalation of foreign 

material [33, p.2791. Smoking also induces many abnormalities in 

the inflammatory and immune systems within the lung [34, p.2561. 

In particular, cigarette smoke causes inflammatory cells to 

produce an enzyme called elastase. 

breaks down elastin, an important protein that lines the elastic 

walls of the air sacs [9; 33, p.4311. Moreover, oxidants present 

in cigarette smoke can inactivate a separate protective enzyme 

called alpha-1-antitrypsin, which inhibits the destructive action 

of elastase [16; 33, p.4341. 

Researchers have produced various types of acute and chronic 

lung injury in laboratory animals exposed to cigarette smoke [33, 

pp.286,428,432,436]. But they have had difficulty inducing 

genuine emphysema from cigarette smoke alone. As in experimental 

models of cancer, the laboratory animals do not inhale the smoke 

deeply. Moreover, very long smoke exposures may be required, as 

is the case in humans. In one experimental study, hamsters 

exposed either to low doses of elastase or low doses of smoke 

alone did not develop emphysema, but the combination of low doses 

of cigarette smoke and elastase caused emphysema-like changes 

[15]. A later chapter in this Report describes a laboratory test 

for the acute inflammatory effects of cigarette smoke on the 

lung, in which mice are exposed to cigarette smoke through a 

nose-only system. 

The enzyme elastase in turn 
A8 

A large number of organic and inorganic chemicals in the 

gaseous, volatile and particulate phases of cigarette smoke 

appear to contribute to its toxicity to the respiratory system 

[33, pp.289,415], including hydrocarbons, aldehydes, ketones, 

organic acids, phenols, cyanides, acrolein, and nitrogen oxides. 

Some components contribute to the development of chronic mucus 

hypersecretion in the central airways, while others play a 

greater role in the production of small airway abnormalities and 

emphysematous injury to the peripheral air sacs [33, p.4251. As 

noted above, oxidizing agents in smoke inhibit the enzymes that 

defend against the destruction of lung elastin. 

respiratory irritation in nonsmokers, particularly in the 

children of smoking parents [33, Chapt.7; 35, p.371. Infants and 

children of smoking parents are at increased risk of acute 

respiratory infections, chronic cough and wheezing, and 

measurable declines in lung function [35, pp.38-591. These 

early-life infections can have long-term adverse effects. In 

adults passively exposed to ETS, some studies have reported 

measurable changes in lung function. Overall, the effect appears 

to be too small to implicate passive smoking alone as a cause of 

full-blown COLD [35, p.621. 

Atherosclerotic Cardiovascular Diseases 

Passive exposure to environmental tobacco smoke produces 

Cigarette smoking is a major contributing cause to coronary 

heart disease, stroke, and other atherosclerotic diseases of the 

circulatory system [32;37]. 

arterial blood vessels in virtually every part of the human body, 

including the coronary arteries that supply blood to the heart 

muscle; the aorta that carries the blood directly from the heart; 

the carotid arteries that carry blood to the brain; and the iliac 

and femoral arteries that carry blood to the legs. 

The common underlying lesion of atherosclerosis is the 

plaque, which occurs within the wall of the affected artery. As 

the plaque enlarges and matures, the artery becomes narrowed, and 

blood flow is reduced. If the narrowed artery carries blood to 

the heart, then chest pain on exertion (angina) is produced. If 

the affected artery carries blood to the leg, then calf pain on 

walking (claudication) is produced. If the affected artery 

carries blood to the brain, then transient neurological symptoms, 

such as fainting, loss of vision, movement, or speech (transient 

ischemic attacks) are produced. If the affected artery carries 

blood to a man's penis, impotence can result. 

blockage by a superimposed blood clot. If the blocked artery 

carries blood to the heart, then a heart attack (myocardial 

Atherosclerosis is a chronic disease that can affect the 

A sufficiently narrowed artery is susceptible to complete 
A9 

infarction) is produced. A blockage of an artery supplying a 

limb can produce gangrene. A blockage to the arteries supplying 

the brain can cause a stroke. 

The most important form of atherosclerosis in the U.S. is 

coronary atherosclerosis. Its manifestations, which include 

angina, heart attack, heart failure, and sudden death, are 

described by the inclusive term coronary heart disease (CHD). 

Atherosclerosis involving the arteries supplying the brain is a 

form of cerebrovascular disease (CVD). Atherosclerosis involving 

the arteries to the limbs is called peripheral vascular disease 

(PVD) . 

Atherosclerotic plaques take years to develop. The earliest 

lesion is called a fatty streak, which consists of deposits of 

cholesterol within the arterial wall. These fatty streaks can be 

observed in young people with no symptoms, and even in children. 

There is a progressive inflammatory reaction to the fatty 

deposits, and a collection of fibrous debris, muscle cells, and 

more fatty deposits is incorporated into the developing plaque. 

Cholesterol is a fatty substance that does not dissolve 

readily in water. It circulates in the blood mostly by attaching 

to specialized proteins. These cholesterol-protein complexes, 

which also contain other fatty substances, form particles of 

various sizes, which are called lipoproteins. The lipoprotein 

particles are classified by their density. There are very-low 

density lipoprotein (VLDL), intermediate density lipoprotein 

(IDL), low-density lipoprotein (LDL) and high-density lipoprotein 

(HDL) particles. 

The fundamental event in the initiation of a fatty 

cholesterol deposit appears to be the transfer of LDL particles 

from the blood across the inner lining (endothelium) of the 

arterial wall. This transfer may require prior injury to the 

inner lining of the artery, in order to expose the raw surface to 

LDL transfer. When a person's blood cholesterol is measured, the 

amount that is specifically attached to LDL is called the 

LDL-cholesterol, or popularly the "bad cholesterol." 

On the other hand, HDL particles work in the opposite 

direction, removing cholesterol from LDL and transporting it back 

to the liver. Because of this reverse-transport function of HDL, 

the amount of cholesterol attached to HDL is popularly termed the 

"good cholesterol. It 

In epidemiological studies of humans, certain measurable 

personal characteristics have been consistently found to be 

predictors of the risks of atherosclerotic disease. These 

predictors are sometimes called risk factors. For example, male 

gender is a risk factor for coronary heart disease. This does 

not mean that maleness per se causes CHD. Still, the fact that 
A10 

women have lower rates of CHD, and that their risk of CHD 

increases after menopause, indicates that sex hormones are 

important in the development of the disease. Likewise, elevated 

blood pressure is a risk factor for CHD (and for strokes). 

Again, this does not mean that hypertension per se causes CHD. 

However, higher pressures in the arterial system tend to damange 

the inner lining (endothelium) of arteries, thus contributing to 

the development of plaque formation, arterial narrowing and 

blockage. Because atherosclerosis entails a sequence of 

pathological events over an extended period of time, it is to be 

expected that multiple environmental agents and personal 

characteristics can affect the course of the disease. 

In numerous epidemiologic studies of millions of people, 

cigarette smokers have been found to have higher rates of heart 

attack, sudden death, and other manifestations of CHD. They also 

have higher rates of stroke, peripheral vascular disease, and 

other atherosclerotic lesions [32;37;39]. In a study of over one 

million people followed during 1982-1986, currently smoking men 

had a 94 percent greater risk of CHD than lifelong nonsmokers; 

while currently smoking women had a 78 percent greater risk. In 

smokers under age 65, men had a 181 percent greater risk, and 

women a 200 percent greater risk [37, Chapt.31. 

factor" for CHD because smokers' CHD rates are found to be higher 

even when other risk factors such as gender, blood pressure, and 

cholesterol level are taken into account. It is sometimes called 

a "modifiable risk factor" because one can reduce or stop 

smoking. While smoking obviously cannot be a cause of CHD in 

someone who never smoked, it can be an important contributor to 

CHD in a smoker. Among 540 thousand deaths from CHD in the U.S. 

in 1905, an estimated 115 thousand would not have occurred but 

for the presence of cigarette smoking [37]. 

process by several different mechanisms [38, p.1921. Cigarette 

smoking affects cholesterol metabolism. 

been observed to have lower HDL-cholesterol levels [41]; and 

smoking cessation raises HDL-cholesterol [25]. In animal models, 

cigarette smoke can damage the inner lining of blood vessels, 

thus enhancing the transfer of LDL and the development of 

underlying plaques [19;42]. Cigarette smoking can also affect the 

blood clotting system, including the adherence of blood platelets 

to the lining of arterial blood vessels [24;32] and the formation 

of blood clots that block a narrowed artery. Cigarette smoke can 

also cause spasm of the coronary arteries. 

Cigarette smoking is sometimes called an "independent risk 

Cigarette smoke appears to enhance the atherosclerotic 

Smokers have repeatedly 

Many chemical components of cigarette smoke have been 

implicated in the development of atherosclerotic disease. 

Nicotine, the major psychoactive component of smoke, causes 

powerful changes in heart rate and blood circulation. Nicotine 

...... 
All 

appears to cause injury to the arterial lining [19;42]. Carbon 

monoxide in cigarette smoke binds to the hemoglobin in red blood 

cells, thereby-reducing the oxygen- carrying capacity of the 

blood. Hydrogen cyanide, nitrogen oxides, and chemical 

components of cigarette Ittar" have also been implicated [32]. 

Oxidants in cigarette smoke may also promote plaque formation 

Cisarette Smokins and Human Reproduction 

Cigarette smoking adversely affects sexual and reproduct 

function in women in a number of different ways. 

ve 

Cigarette smoking appears to impair female fertility [l; 5; 

21; 30, p.2351. Among the possible mechanisms are direct 

toxicity to female eggs, interference with motility in the female 

reproductive tract, and alterations in immunity that predispose 

female smokers to infections that block the Fallopian tubes [4]. 

Maternal cigarette smoking has serious adverse effects on 

the outcome of pregancy. These include: retarded fetal growth; 

low birthweight; spontaneous abortion; certain complications of 

pregancy, labor and delivery, such as bleeding during pregnancy 

and prolonged premature rupture of membranes; and infant death 

[30, p.188; 37, p.71; 38, Chapt.8; 39, Chapt.81. Direct nicotine 

toxicity has been suggested as a mechanism for spontaneous 

abortion 138, p.3721. While a smoking-induced reduction in 

maternal weight gain contributes to fetal growth retardation [30, 

p.202; 401, the evidence points to oxygen starvation of the fetus 

and placenta as important factors. Carbon monoxide in cigarette 

smoke can cross the placenta and bind to the hemoglobin in fetal 

blood. Smoking causes constriction of the umbilical arteries, 

impairing placental blood flow. Nicotine, which also crosses the 

placenta, can have a number of toxic effects on the fetus [30, 

p.2291. Cyanide, another component of cigarette smoke, has also 

been implicated. 

Currently smoking women enter nonsurgical menopause about 

one to two years earlier than nonsmokers [38, p.3971. Heavy 

smokers experience an even earlier menopause than light smokers. 

This effect has important consequences for women's health, 

because the rates of osteoporosis and atherosclerotic 

cardiovascular diseases increase after menopause. One proposed 

mechanism for early menopause is that polyaromatic hydrocarbons 

(PAH) in smoke are directly toxic to ovarian follicles [20]. 

Cigarette smoking may also affect male reproductive 

performance. In a number of studies, men who report impotence 

(i.e., the inability to maintain an erection sufficient for 

intercourse) were more likely to be cigarette smokers. This 

association between smoking and impotence is particularly common 

among men who have high blood pressure or diabetes, and appears 

to be a consequence of increased atherosclerotic disease in the 
A12 

blood vessels supplying the genitalia, rather than an effect on 

sexual drive. 

Nicotine as a vsvchoactive druq 

The psychoactive drug in cigarette smoke is nicotine. 

Cigarette smoking is a highly controlled form of selfadministration of this drug. Nicotine use is self- reinforcing. 

Attempts to stop smoking lead to craving, withdrawal symptoms, 

and high rates of relapse [36]. 

RESEARCH MODELS VERSUS STANDARDIZED TEST SYSTEMS 

As the foregoing brief review indicates, there are many 

laboratory and animal models of the mechanisms of cigaretteinduced human toxicity, and there are many methods of studying 

the health effects of smoking in humans. However, not all of 

these models and methods are easily converted into inexpensive, 

practical, standardized tests that quantitatively compare one 

cigarette prototype with another. 

THE MULTIPLICITY OF TESTING PROTOCOLS 

It is unlikely that any battery of standardized, practical 

tests will be able to gauge all important dimensions of human 

cigarette toxicity. Exhaustive testing of every conceivable 

dimension of toxicity is a "bottomless pit. From the scientific 

standpoint, there will necessarily be some stopping point to 

testing. 

At present, there exists a wide range of testing protocols, 

reflecting different dimensions of human toxicity. 

protocols will be considered in detail in later sections of this 

Report. In general, tests of cigarette toxicity include: 

(1) Chemical and physical analyses of MS and SS smoke 

collected by smoking machines under standardized conditions. 

These tests include quantitative measurement of known smoke 

constituents, qualitative analyses for new chemicals, and studies 

of particle size distribution. 

(2) Studies of the dosage of specific smoke constuents 

actually received by human smokers or by nonsmokers exposed 

passively to environmental tobacco smoke. 

fractions of smoke on individual cells and tissues. The 

individual cells can be single-cell organisms, such as bacteria. 

They can be cells extracted from a specific organ of an animal 

These testing 

(3) Laboratory tests of the effects of whole smoke or 
A13 

and preserved in tissue culture. Tests that do not entail 

exposure to an entire living animal are called in vitro tests. 

(4) Laboratory tests of the effects of smoke or smoke 

fractions in whole animals. These include short-term tests to 

study specific mechanisms of disease or to assess acute toxicity, 

and long-term tests to assess the effects of chronic exposure. 

The multiplicity of human health endpoints, as well as the 

wide range of available tests, means that a particular cigarette 

prototype may appear more toxic in some tests, equally toxic in 

other tests, and less toxic in still others. 

EPIDEMIOLOGY 

Human epidemiological studies play a central role in 

generating and testing hypotheses about causation of disease; in 

identifying groups of people who at higher or lower risks of 

disease; in estimating quantitatively the risks of specific 

diseases in relation to different levels of toxic exposure; and 

in evaluating the effects of preventive measures. 

Epidemiological studies are more limited in assessing the 

differences in the toxic effects of various types or brands of 

cigarettes. For example, to determine whether brand "A" causes 

less lung cancer than brand l'B," a researcher would have to 

identify and compare long-term smokers exclusively of brand "A" 

with long-term smokers of brand "B" alone. If the expected 

differences in cancer rates are small, then large numbers of 

long-term smokers of each brand need to be identified. 

Epidemiologic methods are impractical for testing the 

comparative effects of prototype cigarettes that have not already 

been marketed and smoked by consumers. 

ABSOLUTE RISK VERSUS RELATIVE RISK 

Human epidemiology can be used to estimate quantitatively 

the risk of specific diseases to human smokers. For example, in 

a study of smoking practices and mortality rates among 1.2 

million U.S. adults followed during 1982-1986, about 0.8 percent 

of current male smokers aged 65 or more died of lung cancer each 

year [37, p.1431; while the comparable annual lung cancer death 

rate was about 0.04 percent among men aged 65 or more who never 

smoked. These quantitative risk estimates are often termed 

"absolute risks." The fact that the continuing smokers' risk of 

lung cancer was 20-fold that of nonsmokers is an expression of "relative risk." 

Estimating absolute risks from nonhuman toxicity studies is 

much more complicated. For example, the smoke from prototype 
A14 

cigarette "Z" might contain 0.05mg of benzo(a)pyrene (BaP), a 

known carcinogen, while the smoke from a control cigarette might 

contain 0.02mg of BaP. To estimate human lung cancer risks from 

these data alone would require a number of assumptions relating 

the dose of BaP to the incidence lung cancer in humans. 

Toxicity studies can give estimates of relative risk, but 

applying these estimates directly to humans requires caution. 

While prototype "Zg1 had 2.5-fold as much BaP as the control 

cigarette, we cannot automatically conclude that their relative 

risks of lung cancer in humans is 2.5. The relative 

concentrations of benz(a)anthracene, another carcinogen in the 

"polyaromatic hydrocarbon" group, might be higher or lower. 

Estimating relative risks from toxicity studies entails combining 

estimates from different sources [8]. 

HUMAN BEHAVIORAL RESPONSES ARE IMPORTANT IN TESTING 

Testing plans require samples of cigarette smoke, which can 

then be analyzed chemically or biologically. The results of such 

testing may hinge critically on the method of collecting the 

sample. Smoke samples from cigarettes are generally collected 

from smoking machines, not from living smokers. As discussed in 

more detail later in this Report, it is important that such 

samples be collected in a manner that mimics human smoking as 

closely as is technically feasible. 

The study of the ways in which humans consume cigarette 

smoke is called smoking topography. Many variables are involved, 

even in the smoking of a single cigarette: the intensity of the 

draw on the column of smoke during a single puff; the duration of 

the puff; the volume of smoke in each puff; the intervals between 

puffs; and the number of puffs taken per cigarette. These 

variables, as well as other physiological factors, affect the 

actual dosages of smoke constituents that are inhaled, absorbed, 

and retained in the smoker's body. The study of the actual 

dosages of smoke constituents received by human smokers is called 

smoking dosimetry. 

intensity, duration and volume, as well as inter- puff intervals 

and puffs per cigarette, vary among human smokers. Accordingly, 

no protocol for machine-based collection of cigarette smoke can 

accurately mimic all human smoking. Toxicity testing of 

machine-collected smoke samples may not accurately gauge a 

particular smoker's risk, but rather an average or representative 

smoker's risk. 

No two humans smoke cigarettes exactly the same way. Puffing 

Toxicity testing ordinarily requires a uniform method of 

collecting smoke samples. To compare the *'tar," nicotine and CO 

yields of the smoke of prototype f'X'l with those of a control 
A15 

cigarette, one uses the same smoking machine to smoke both 

cigarettes under the same conditions according to the same 

protocol. For example, under the current FTC protocol, a smoking 

machine takes one puff each minute. The puff volume is 35ml; and 

the puff duration is 2 seconds. As described lqter in this 

Report, the smoking machine continues to take puffs on the test 

cigarette until a pre-specified butt length is achieved. 

Different cigarette prototypes or design modifications may 

affect the ways that people smoke cigarettes. This can 

complicate the choice of test conditions for collecting smoke 

samples from prototype cigarettes. For example, when cigarette 

prototype "Xt' is smoked by machines under standard FTC 

conditions, the amount of nicotine in the smoke may appear to be 

reduced. But human smoking topography may show that smokers 

actually take deeper puffs on prototype "X" than the 35 ml-puffs 

taken by the smoking machines. Human dosimetry may further show 

that the amount of nicotine actually absorbed from prototype "X" 

is not reduced. Alternatively, prototype "Y" may contain more "tar" per machine-smoked puff. But human topography may show 

that smokers take fewer puffs on that prototype, so that the 

total yield of "tar" per cigarette is not increased. 

For these reasons, human smoking topography and dosimetry 

may need to be a part of cigarette testing for increased 

toxicity. 

DESIGN-BASED TESTS VERSUS PERFORMANCE-BASED TESTS 

Section 2(c) of the Fire Safe Cigarette Act of 1990 mandates 

the development of information on "changes" in toxicity of smoke 

and resultant health effects of cigarette "prototypes." Such 

information can be acquired by design- based testing, which 

assesses the effects of a specific, known modification (or a 

combination of modifications) in cigarette design. 

The "tar,f' nicotine and CO analyses of "Series 1" and 

I'Series 2" experimental cigarettes performed by the National 

Bureau of Standards (NBS) are examples of design-based testing 

[ll, Tables 2-5, 2-6, 2-7, 3-10, 3-11, and 3-12]. In Series 1, 

for instance, NBS analyzed five different dimensions of cigarette 

design: tobacco leaf composition (burley vs. flue cured); 

tobacco density (decreased by tobacco expansion); paper porosity; 

the presence of citrate additive to the cigarette wrapping paper; 

and the circumference of the tobacco column. Experimental 

cigarettes were produced that contained modifications in one or 

more of these design dimensions. The modified cigarettes could 

then be compared to each other and to control cigarettes with no 

modifications. By such comparison, NBS estimated that lower 

tobacco density decreased the lltarll yield per smoke puff; while 
A16 

low paper permeability increased "tar" yield per puff 111, Table 

3-12]. 

The NBS Series-1 tests did not encompass all possibilities 

in design-based testing. A specific newchemical additive could 

be incorporated into a test cigarette, whose smoke would be 

compared with that of a control cigarette that is otherwise 

identical. In the NBS Series 1, the experimental cigarettes were 

all filter- tipped, with the individual tows, plug wraps, and 

plasticizer levels selected by the participating cigarette 

manufacturers [ll, p.331. The effects of lower-porosity wrapping 

paper could also have been assessed in nonfilter cigarettes, or 

perforated filter-tip cigarettes. This might be important if the 

presence or type of filter affected ignition propensity [11, 

P. 651 

From the scientific standpoint, design-based testing is 

advantageous when there is a limited practical range of cigarette 

design modifications, and when such design modifications are 

publicly known. For example, if changes in tobacco packing 

density and paper permeability were the only feasible design 

modifications under consideration, and if the proposed methods of 

tobacco expansion and paper manufacture were specifically 

disclosed, then the effects of such design changes could be 

assessed. However, if a specific cigarette prototype entailed 

tobacco expansion combined with proprietary changes in tobacco 

leaf composition, cigarette paper, and filter design, then 

designed-based testing may be impractical. 

individual cigarette prototypes-- not design technologies-- are 

assessed. Such testing may be more appropriate when there are 

many different cigarette prototypes, each with complex design 

changes, and when the specific changes are proprietary or not 

fully disclosed. NBS's analyses of patented cigarettes [ll, 

Table 3-14] more closely resembles performance-based testing. In 

that case, inventors submitted their own prototypes, along with 

unmodified control cigarettes. While NBS appears to have tested 

these patented prototypes for ignition propensity only, analyses 

of "tar," nicotine and CO in such patented cigarettes would 

constitute performance-based testing. 

The alternative is performance-based testing, in which 

Accordingly, in design-based testing, information might be 

acquired on the effects of changes in paper porosity on smoke 

carbon monoxide. By contrast, in performance-based testing, 

information is acquired on the CO delivery of prototype "X." 

control cigarette. As in the NBS testing of patented cigarettes, 

the smoke of prototype "X" could be compared to a control 

cigarette that incorporates none of the proposed modifications. 

But this alternative is not necessarily so simple. Prototype stXsB 

In performance-based testing, there is no unique or natural 
A17 

could include modifications designed to: (i) reduce ignition 

potential; (ii) reduce smoke toxic constituents; and (iii) 

improve consumer acceptability. A comparable control cigarette 

may be unmodified in one or all of these dimensions. Such 

controls may not correspond to any currently marketed cigarette 

brand. Alternatively, the prototype "X" could be compared to 

other existing marketed cigarettes; to another prototype "Y;" or 

to pre-set standard cigarette. Thus, in performance-based 

testing, one could conclude that prototype "Z" delivered more or 

less nicotine than any other prototype; than the average marketed 

cigarette; or than some value set by a public or private 

standard-setting body. 

TESTING PROTOCOLS: SCREENING VERSUS TRADEOFFS 

Testing is expensive and time-consuming. Accordingly, most 

testing protocols entail a sequence of tests. The order of 

testing is usually influenced by the cost and time required. If 

human subjects are involved, then risk and ethical considerations 

are important. 

For example, in the screening of environmental agents for 

their carcinogenic potential (e.g., under the Toxic Substances 

Control Act), bacterial mutagenesis and other short-term tests 

for genotoxicity are performed first. After that, whole animal 

exposure studies of acute toxicity may be considered. 

Thereafter, longer term whole-animal studies of carcinogenicity 

may be undertaken. In the screening of investigational new 

drugs, human studies are undertaken only after laboratory and 

whole animal studies are completed. 

Some testing protocols entail a "screening paradigm." If a 

substance or product fails any particular test in the sequence, 

the product is rejected and no further tests are performed. For 

example, in the testing of cigarette prototypes, analytical 

studies of smoke components might be performed initially, 

followed by short-term mutagenicity studies, followed then by 

long-term bioassays of carcinogenicity in animals, followed by 

studies of smoke dosimetry in humans. 

In the screening paradigm, a cigarette prototype "Zl9 that 

initially yielded an excess of carcinogenic polyaromatic 

hydrocarbons (PAH) might be rejected, and no further testing 

performed. Alternatively, if cigarette prototype "Q" yielded no 

excess of toxic compounds on chemical analysis, then testing of 

prototype "Q" would proceed to the next level. 

In contrast to screening protocols, other testing protocols 

allow for tradeoffs among costs and benefits. A positive test at 

any point along the testing sequence does not necessarily result 

in rejection. For example, prototype "R" may have performed 
A18 

exceptionally in tests of low- ignition potential, but it yielded 

an excess of tobacco-specific nitrosames in chemical analysis. 

Such a finding might not lead to automatic rejection of prototype 

"R.lf Instead, testing would continue, and the positive analytical 

test result would be weighed against other evidence. 

Conservatively designed protocols may be appropriate when 

the potential adverse health effects of a new product or new 

design are more important than its potential benefits for fire 

safety. On the other hand, if a relatively small increase in 

or nicotine delivery is to be gauged against a major 

reduction in ignition potential, then some form of cost-benefit 

analysis will be required. 

SPECIAL ASPECTS OF CIGARETTE PROTOTYPE TESTING 

The only governmentally-mandated, health-oriented testing of 

While USDA and DHHS may conduct research programs on 

the finished cigarette product is the measurement and reporting 

of tar, nicotine and carbon monoxide by the Federal Trade 

Commission. 

the health effects of smoking, no other federal or state agency 

is currently required to perform tests for toxicity on various 

brands of marketed cigarettes. With the exception of 

standardized machine measurements of v'tar,vl nicotine and CO, none 

of the toxicity tests described by the expert panel are routinely 

performed on existing cigarette brands by any governmental 

agency. 

that are not in currently marketed cigarettes. Such additives may 

have qualitatively different health effects than those discussed 

above. Neither performance-based nor design-based testing solves 

the problem of evaluating the health effects of new, undisclosed 

cigarette additives. For example, if a new inorganic compound, 

such as a metal salt, were added to the cigarette tobacco, then 

one might have supplement the test battery with additional 

studies of acute or chronic toxicity to kidney, liver and other 

organs. In cases where new additives are involved, and not 

merely a quantitative change in existing design parameters, 

disclosure of contents is required for adequate toxicity testing. 

Some low-ignition protype cigarettes may contain additives 

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11. Gann RG, Harris RH Jr, Krasny JF, Levine RS, Hitler HE, 

12. Gairola CG. Genetic effects of fresh cigarette smoke 

in Saccharomyces cerevisiae. Mutation Research 1982; 

102:123-136. 

13. Garfinkel L, Bofetta P. Association between 

smoking and leukemia in two American Cancer Society prospective 

studies. Cancer 1990; 65:2356-2360. 

14. Hoffmann D, Hecht SS. Advances in tobacco 

carcinogenesis. In: Handbook of Experimental Pharmacology 1990; 

94 (I) : 63-102. 
A2 0 

15. Hoidal JR, Niewoehner DE. Cigarette smoke inhalation 

potentiates elastase-induced emphysema in hamsters. American 

Review of Respiratory Diseases 1983; 127:478-481. 

of the protease-antiprotease hypothesis. American Review of 

Respiratory Disease 1985; 132:417-433. 

literature review with commentary. Report No. 6, Technical Study 

Group, Cigarette Safety Act of 1984. Washington DC: U.S. 

Consumer Product Safety Commission, June 1987. 

16. Janoff A. Elastases and emphysema: current assessment 

17. Krasny JF. Cigarette ignition of soft furnishings-- a 

18. Krasny JF, Gann RG. Relative propensity of selected 

commercial cigarettes to ignite soft furnishing mockups. Report 

NBSIR 86-3421, U.S. National Bureau of Standards, Gaithersburg 

MD, June 1986. 

effects of hypertension and nicotine on injury- induced 

myointimal thickening. Surgery 1987; 102:409-415. 

destruction by polycyclic aromatic hydrocarbons in mice. 

Toxicology and Applied Pharmacology 1980; 53:249-259. 

gametogenesis to implantation. Environmental Research 1982; 

19. Krupski WC, Olive GC, Weber CA, Rapp JH. Comparative 

20. Mattison DR. Morphology of oocyte and follicle 

21. Mattison DR. The effects of smoking on fertility from 

28:410-433. 

22. National Research Council. Environmental tobacco 

smoke. Measuring exposure and assessing health effects. 

Washington, D.C.: National Academy Press, 1986. 

changes in the peripheral airways of young cigarette smokers. 

New England Journal of Medicine 1974; 291:755-758. 

smoking and platelet adhesion. British Journal of Haematology 

25. Rabkin SW. Effect of cigarette smoking cessation on 

23. Niewoehner DE, Kleinerman J, Rice DB. Pathologic 

24. Pittilo RM, Clarke JM, Harris D, et al. Cigarette 

1984; 58:627-632. 

risk factors for coronary atherosclerosis: a control clinical 

trial. Atherosclerosis 1984; 52:173-184. 

26. Seely JE, Zuskin E, Bouhys A. Cigarette smoking: 

objective evidence for lung damage in teenagers. Science 1971; 

172 : 741-743. 

27. Sumner DA, Greenberg D, Lago AM, Shannon JA, Oster G, 

Ford GT, Brown JP, Calfee JE. Economic sector data for modeling 
A2 1 

the impact of less ignition-prone cigarettes. Report NO. 6, 

Technical Study Group, Cigarette Safety Act of 1984. Washington 

DC: U.S. Consumer Product Safety Commission, October 1987. 

28. Technical Study Group on Cigarette and Little Cigar 

Fire Safety. Toward a less fire-prone cigarette: Final report of 

the Technical Study Group on Cigarette and Little Cigar Fire 

Safety. Washington D.C.: U.S. Government Printing Office, 

October 1987. 

29. U.S. Consumer Product Safety Commission. First annual 

report to Congress under the Fire Safe Safe Cigarette Act of 

1990. Washington, D.C.: U.S. Consumer Product Safety 

Commission, October 10, 1991. 

30. U.S. Department of Health and Human Services, Public 

Health Service. The health consequences of smoking for women: a 

report of the Surgeon General. Washington, D.C.: U.S. 

Government Printing Office, 1980. 

31. U.S. Department of Health and Human Services, Public 

Health Service. The health consequences of smoking -- cancer: a 

report of the Surgeon General, 1982. DHHS Publication No. 

(PHS)82-50179. Washington, D.C.: U.S. Government Printing 

Office, 1982. 

32. U.S. Department of Health and Human Services, Public 

Health Service. The health consequences of smoking -- 

cardiovascular disease: a report of the Surgeon General, 1983. 

DHHS Publication No. (PHS)84-50204. Washington, D.C.: U.S. 

Government Printing Office, 1984. 

33. U.S. Department of Health and Human Services, Public 

Health Service. The health consequences of smoking -- chronic 

obstructive lung disease: a report of the Surgeon General, 1984. 

DHHS Publication No. (PHS)84-50205. Washington, D.C.: U.S. 

Government Printing Office, 1984. 

34. U.S. Department of Health and Human Services, Public 

Health Service. The health consequences of smoking -- cancer and 

chronic lung disease in the workplace: a report of the Surgeon 

General, 1985. DHHS Publication No. (CDC)85-50207. Washington, 

D.C.: U.S. Government Printing Office, 1985. 

35. U.S. Department of Health and Human Services, Public 

Health Service. The health consequences ofinvoluntary smoking: 

a report of the Surgeon General, 1986. DHHS Publication No. 

(CDC)87-8398. Washington, D.C.: U.S. Government Printing 

office, 1987. 

36. U.S. Department of Health and Human Services, Public 

Health Service. The health consequences of smoking -- nicotine 
A2 2 

addiction: a report of the Surgeon General. DHHS Publication No. 

(CDC)88-8406. Washington, D.C.: U.S. Government Printing 

Office, 1988. 

37. U.S. Department of Health and Human Services, Public 

Health Service. 

25 years of progress: 

DHHS Publication No. (CDC)89-8411. Washington, D.C.: U.S. 

Government Printing Office, 1989. 

Health Service. The health consequences of smoking cessation: a 

report of the Surgeon General, 1990. DHHS Publication No. 

(CDC)90-8416. Washington, D.C.: U.S. Government Printing 

Office, 1990. 

39. U.S. Department of Health, Education, and Welfare, 

Public Health Service. Smoking and health: a report of the 

Surgeon General. DHEW Publication No. (PHS)79-50066. Washington, 

D.C.: U.S. Government Printing Office, 1979. 

Teratology 1985; 32:473-481. 

Reducing the health consequences of smoking -- 

a report of the Surgeon General, 1989. 

38. U.S. Department of Health and Human Services, Public 

40. Werler MM, Pober BR, Holmes LB. Smoking and pregnancy. 

41. Willett W, Hennekens CH, Castelli WP, et al. Effects of 

cigarette smoking on fasting triglyceride, total cholesterol, and 

HDL-cholesterol in women. American Heart Journal 1983; 

105: 417-421. 

42. Zimmerman M, McGeachie J. The effect of nicotine on 

aortic endothelium: a comparative study. Atherosclerosis 1987; 

63 : 33-41. 

GLOSSARY AND ABBREVIATIONS 

benzo(a)pyrene (BaP): a carcinogenic chemical in cigarette 

smoke, a member of the class of polyaromatic hydrocarbons. 

benzo(a)anthracene: a carcinogenic chemical in cigarette smoke, 

a member of the class of polyaromatic hydrocarbons. 

carbon monoxide (CO): a gas found in cigarette smoke. 

condensate: the portion of whole smoke that condenses upon 

passage of the smoke through a cold trap. 

dosimetry of smoking: study of the actual dosages of smoke 

constituents inhaled, absorbed and retained by human smokers. 

environmental tobacco smoke (ETS): mostly sidestream smoke, but 

also exhaled mainstream smoke, as well as some gaseous and 
A2 3 

vapor-phase constituents of smoke that diffuse through the 

cigarette paper wrapper into the surrounding air. 

genotoxicity tests: 

smoke particles, or smoke condensate to damage the genetic 

material (DNA) of the test cell; a more general term than 

mutagenicity tests. 

mainstream (MS) smoke: smoke that is drawn through the burning 

tobacco column and filter tip and exits through the mouthpiece of 

the cigarette. 

"in vitro" test: a 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 (or 

human). Tests performed on primitive single-celled organisms, 

such as bacteria or yeast, are classified as "in vitro" tests. 

"in vivotq test: a test that is performed in a whole, living 

animal (or human), as opposed to an "in vitro" test. 

mutagenesis tests: tests for the propensity of cigarette smoke, 

smoke particulates, or smoke condensate to cause mutations in the 

genetic material (DNA) of the test cell. A widely used 

mutagenesis test is the Ames test, which is performed on special 

strains of the Salmonella bacterium. 

N-Nitrosodimethylamine (NDMA): an animal carcinogen. 

particulate phase: the portion of cigarette smoke that is 

trapped by a standard Cambridge filter at room temperature. 

polyaromatic hydrocarbons: a class of carcinogenic chemicals 

found in cigarette smoke. An example is benzo(a)pyrene. 

sidestream (SS) smoke: smoke that is emitted into the 

surrounding air between puffs from the end of the smoldering 

cigarette. 

topography of smoking: study of the ways that humans consume 

cigarette smoke, including the intensity of the draw on the 

column of smoke during a single puff; the duration of the puff; 

the volume of smoke in each puff; the intervals between puffs; 

the number of puffs taken per cigarette; and the number of 

cigarettes smoked daily. 

tumorigenic: causing tumors or cancers in laboratory animals or 

humans; used synonymously here with "carcinogenic." 

vapor phase: the gaseous and vaporizable chemicals in cigarette 

smoke that pass through a standard Cambridge filter at room 

temperature. 

tests of the propensity of cigarette smoke, 

Chapter B 

SMOKING MACHINE PARAMETERS FOR COLLECTION OF TOTAL 

POTENTIAL CIGARETTES 

PARTICULATE MATTER AND GASES FROM LOW IGNITIONHarold C. Pillsbury, Jr 

13009 Magellan Ave. 

Rockville, MD 20853 

Under contract to the U.S. Consumer Product Safety Commission 

#CPSC-S-92-5472 

March 14, 1993 

B1 

Introduction 

The official Federal Trade Commission method (FTC, 1969, 

1979, 1980) to determine tar, nicotine, and carbon monoxide in 

cigarette smoke is presented as a basis for the method of 

collecting total particulate matter and gases from low-ignition 

potential cigarettes. The FTC method is very similar to the 

Centre de Coop6ration pour les Recherches Scientifiques au Tabac 

(CORESTA) Standard Method (CORESTA, 1968) used in Europe. One of 

these two methods or a slight modification thereof is used in all 

countries that test cigarettes. 

Differences between the FTC and the CORESTA methods are 

small. The environmental rooms under the CORESTA method are 

maintained at 22 f3 OC and 55-65% relative humidity. The FTC 

method requires conditions of 75 ?2 OF (23.9 ?1.1 "C) and 

relative humidity of 60 +2%. Under the FTC method, cigarettes 

are smoked to a butt length of 23 mm or the overwrap plus 3 mm, 

whichever is longer. Using the CORESTA method, cigarettes with a 

filter length exceeding 15 mm are smoked to the length of filter 

plus 8 mm and cigarettes with extra long filter tips are smoked 

to the length of the tipping plus 3 mm. 

The Filtrona-400 smoking machine used in the CORESTA method 

requires an increased draft over the burning cigarette, whereas 

the FTC method does not. The increased draft is not encountered 

under normal smoking conditions. It causes the cigarette to burn 

faster, reducing the number of puffs and lowering the yield of 

total particulate matter (TPM) and gases. This air flow over the 

cigarette is needed to match the results of the Filtrona smoking 

machine to that of the Borgwaldt smoking machine. 

and Borgwaldt are the only two existing commercial manufacturers 

of smoking machines. 

The Filtrona 

The following is a summary of the FTC protocol. 

Materials and Methods 

1) Environmental Room: A room where cigarette conditioning and 

smoking is conducted. The room should be maintained at 75 f2 OF 

(23.9 fl.1 "C) and 60% f2% relative humidity. 

2) Smoking Machine: The cigarette smoking machine should be 

similar to the Filtrona machine used by FTC (Pillsbury, 1969). 

This machine can smoke 20 cigarettes at one time, one in each 

port. Each port can be fitted with a filter holder and filter 

pad for the collection of TPM. Gases pass through the pad and 

are collected in specially designed plastic bags (Filtrona). 
B2 

3) Smoke Collection Trap: The polyacrylic plastic holders 

(Wartman, 1959) can be obtained from the manufacturer of the 

smoking machine (Filtrona). 

4) Filter Disks: Filter disks (pads) are made from a fiberglass 

sheet pre-cut to a diameter of approximately 44 mm. The filters 

collect at least 99.9% of all particles larger than 0.3 um in 

diameter (Ogg, 1964). The filter disks fit into plastic holders. 

The particles collected on the pad are referred to as total 

particulate matter (TPM). 

5) Standard solutions: 

A) Extraction solution: This solution contains extractant 

and internal standards- 2-propanol containing 1 mg anethole 

(p-propenylanisole, 1-methoxy-4-propenylbenzene) per mL as 

an internal standard for nicotine and 20 mg ethanol per mL 

as an internal standard for water. 

B) Moisture content: Standards are prepared by adding 

measured amounts of water into measured volumes of 

extraction solution. A standard curve is constructed from 

the ratio of peak heights of the water to the peak height of 

ethanol against the amount of water added to the extraction 

solution, after correcting with a solvent blank. 

C) Nicotine: A stock solution contains 2.500 g nicotine in 

100 mL of extraction solution. Working solutions are made 

from 1, 2, 3, 4, and 5 mL of the stock solution diluted and 

brought to 100 mL volume with extraction solution. A 

standard curve is constructed as with moisture content. 

6) Carbon monoxide: Gases from the plastic bags in the smoking 

machine are passed into an infrared detector. The detector is 

calibrated using a carbon monoxide gas standard. 

7) Gas chromatograph: 

A) Moisture content analysis: The 6 ft x 1/8" (1.8 m x 0.32 

cm) diameter column is packed with 80-100 mesh porous polymer 

(Porapak Q). Operating temperature for the column is set at 200 

OC, injection port at 240 OC, and thermal conductivity detector 

at 210 OC. The helium carrier gas flow is about 100 mL per 

minute. 

B) Nicotine analysis: The 6 ft x 1/8" (1.8 m x 0.32 cm) 

diameter column is packed with 2% KOH and 10% polyethylene glycol 

(Carbowax 20M) on 45-60 mesh acid washed diatomaceous earth. The 

column temperature is set at 165 OC, and the injection port and 

the flame ionization detector are set at 200-250 OC. Helium 

carrier gas flow is about 40 mL per minute. 
B3 

8) 9tMonitorvg cigarettes: These are cigarettes with known tar, 

nicotine and carbon monoxide yields. Monitor cigarettes serve as "standards" to ensure that the smoking machine is operating 

properly. True standard reference cigarettes are mentioned in 

Dr. Gairola's chapter on Short-term Toxicity Tests. No fewer 

than four ports should be used for monitors per 20 port machine 

on each run. 

9) Run: This is a complete smoking of 100 cigarettes- five of 

the same type in each of the 20 ports (4 monitor and 16 test 

cigarettes). 

Samples 

Cigarette quantities: A minimum of 150 cigarettes and 

preferably 200 cigarettes of each type are needed for the FTC 

specified tests. This would ensure that at least 100 cigarettes 

of each type were succesfully smoked for one run. Typically, 

some test pads are discarded due to cigarette lighting failures, 

port leaks, or other technical problems. 

Sample preparation,and selection: Store all cigarette samples 

and monitors in an environmental room or chamber for not less 

than 24 hours before marking or smoking. Cigarettes should 

remain in the environmental room until they are smoked. Select 

only cigarettes without physical damage. Cigarettes should be 

marked to either a butt length of 23 mm or the overwrap plus 3 

mm, whichever is longer. The insertion depth of about 9 nun is 

also marked. Mark the perforations for easy identification by 

the technician during placement into the holder. The 

perforations must not be occluded or compressed by the holder 

since this would affect the smoke yield. 

Machine Smoking of Cigarettes 

1) Puff volume: 35 mL f 0.5 mL 

2) Puff duration: 2 sec f 0.2 sec, measured under actual 

machine smoking conditions. Resulting draw velocity is about 

17.5 mL per sec. 

3) Puff frequency: One puff per 60 sec f 1 sec. 

Weigh the filter assembly to the nearest 0.05 mg and connect 

it to the smoking machine so that the cigarette and filter 

assembly are held horizontally. Test the smoking apparatus and 

filter assembly for leaks. Insert a cigarette through the hole 

in the rubber membrane until the butt end is inserted 

approximately 9 nun, such that the butt end does not contact the 

filter disk. Light the cigarette at the beginning of the first 
B4 

puff. Smoke five cigarettes per pad. If the cigarettes are very 

low in tar, more cigarettes may be smoked per pad providing the 

pad does not wet through. The cigarettes should be protected 

from drafts, other than normal convection, during smoking. 

Results 

After five or more cigarettes are smoked in each port, each 

pad is extracted with the extraction solution. The extracted 

material is analyzed for moisture content and nicotine levels. 

Other extracted materials, such as described in the Analyses 

chapter, may also be analyzed. Part of the gas phase, which has 

been accumulated in the bag, is passed through an infrared 

detector for the determination of carbon monoxide. Although not 

required by FTC, nitric oxide may be measured by a 

chemiluminescent detector designed by Filtrona, specifically for 

the smoking machine. The gas may also be analyzed for substances 

indicated in Dr. Hoffman's Analysis chapter. Tar, nicotine and 

carbon monoxide are reported as mg per cigarette. 

TPM (total particulate matter): Immediately after smoking the 

cigarettes disconnect the filter assembly from the smoking 

machine. Record the weight gain of the filter assembly to the 

nearest 0.05 mg and divide this by the number of cigarettes 

smoked to determine TPM per cigarette. 

Extraction: Immediately after weighing, place the filter pad in 

a dry, rubber-stoppered 25 mL flask. Wipe out the filter 

assembly with one-fourth of an unused pad and place this into the 

flask. Add 10.0 mL of extraction solution and shake for 30 

minutes. 

Water: A 1-10 UL aliquot of the extract is withdrawn through the 

stopper and injected into the chromatograph. Compare the 

resulting peak against the standard curve to determine the 

moisture content. 

Nicotine: A 1-10 UL aliquot of the extract is withdrawn through 

the stopper and injected into the chromatograph. Compare the 

resulting peak against the standard curve to determine the 

nicotine content. 

Carbon Monoxide: The gaseous phase collected in the plastic bag 

is passed through an infrared detector for the determination of 

carbon monoxide. 

Tar: Tar is the TPM minus the water and nicotine. This is 

sometimes referred to as "FTC tar" to distinguish it from other 

definitions of l'tar'l. 
B5 

Discussion 

The Filtrona smoking machine can be modified by installing a 

collection funnel at each port to collect sidestream smoke, which 

may have different constituent levels (Johnson, 1973; see also 

discussion in Dr. Harris‘ Overview chapter). Filtrona’a 8-port 

smoking machine is more easily modified than the 20-port model. 

The filter assembly can be replaced with a cold trap if this 

technique for collecting condensate is desired. 

There may be a wide range of variability in smoking 

behaviors due to cigarette design, physiological, psychological, 

and pharmacological factors (see Dr. Burns’ Topography chapter; 

Guyatt, 1989a,b; Kolonen, 1991, 1992; Nil, 1989; Zacny, 1988). 

Although the present testing methods are designed to produce 

comparative results, the smoking machine could be set up as 

closely as technically feasible to reflect future data on smoking 

behavior. The machine has sufficient range to acommodate 

possible changes, for example, puff frequency from one puff per 

minute to six puffs per minute or volume from 20 mL to 50 mL 

puffs. The draw velocity would also change since it is related 

to the frequency and volume. 

A high degree of replicability for tar, nicotine, and carbon 

monoxide was found in parallel testing between FTC and most other 

laboratories over a period of 20 years. Unfortunately, all the 

data from these tests were destroyed when FTC closed the 

laboratory in 1987. Attached to this chapter is a typical graph 

from my files that illustrates the close correlation of tar 

levels found by the FTC and a private laboratory. 

Cost 

The approximate cost of machine smoking and analyses for 

tar, nicotine, and carbon monoxide for one run with a 20-port 

machine would be $3,000-$4,000 (4 monitor ports + 16 test ports = 

20 monitor cigarettes + 80 test cigarettes, minimum). The 

current capacity of the Tobacco Institute Laboratory is six runs 

per day. 

Recommendations 

The FTC method should be used as the basis for the smoking 

machine setup in the collection of gases and total particulate 

matter for low ignition-potential cigarette testing. The 

apparatus and methodology is adaptable to changes that may be 

indicated by new and future data on human smoking behavior and 

smoke exposure. The FTC method is replicable and wellestablished among the US industry. 
B6 

References 

CORESTA Standard Method No.10 (Sept. 1968) 

Federal Trade Commission, Federal Register Vo1.45 No.134 

pp 46483-46487 (Thursday July 10, 1980) 

Federal Trade Commission, Federal Register V01.44 No.13 

pp 3777 (Thursday Jan. 18, 1979) 

Federal Trade Commission, J. Assoc. Official Analytical Chemist, 

VOl. 52, NO. 3 1969 

Guyatt, Andrew R.; Kirkhan, Andrew J.T.; Baldry, Andrew G:; 

Dixon, Michel and Cumming, Gordon "How Does Puffing Behavior 

Alter During the Smoking of a Single Cigarette?" Pharmacology 

Biochemistry and Behavior Vol. 33 pp 189-195 (1989a) 

Guyatt, A. R.; Kirkham,A. J. T.; Mariner, D. C.; Baldy, A. G. and 

Cumming, G. "Long-Term Effects of Switching to Cigarettes With 

Low Tar and Nicotine Yield" Psychopharmacology (Berlin) 99 pp 80- 

86 (198933) 

Johnson, W. R.; Hale, R. W.; Nedlock,J. W.; Geubbs, H. J.and 

Povell, D. H. "The Distribution of Products Between Mainstream 

and Sidestream Smoke" Tobacco Science Vol. 17 pp 141 (1973) 

Kolonen, Sakar; Tuomisto, Jouko; Puustinen, Pekka and Airaksinen, 

Mauno M. "Smoking Behavior in Low-Yield Cigarette Smokers and 

Switchers in Natural Environment" Pharmacology Biochemistry and 

Behavior Vol. 40 pp 170-180 (1991) 

Kolonen, Sakar; Tuomisto, Jouko; Puustinen, Pekka and Airaksinen, 

Mauno M. "Puffing Behavior During the Smoking of a Single 

Cigarette in a Naturalistic Environment" Pharmacology 

Biochemistry and Behavior Vol. 41, pp 701-706 (1992) 

Nil, Rico and Battig, Karl "Separate Effects of Cigarette Smoke 

Yield and Smoke Taste on Smoking Behavior" 

Vol. 99 pp 54-59 (1989) 

Psychopharmacology 

Ogg,C.L., J. Assoc. Official Analytical Chemist 47, pp 365-362 

(1964) 

Pillsbury, H.C., Bright, C.C., O'Connor, K.J. and Irish, F.W. J. 

Assoc. Official Analytical Chemist 52, pp 458-462 (1969) 

Wartman, et al, Analytical Chemistry 31 pp 1705-1709 (1959) 
B7 

Wegner, John R. and Thaggard, Neil A. "Gas-Liquid 

Chromatographic Determination of Nicotine Contained on Cambridge 

Filter Pads: Collaborative Study" Vol. 62 No. 2 (1979) 

Zacny, James P.and Stitzer, Maxine L. "Cigarette Brand-Switching: 

Effect on Smoke Exposure and Smoking Behavior" J. Pharmacology 

and Experimental Therapeutics Vol. 246 No. 2 pp 619-627 (1988) 
AIR FLOW VELOCITY MEASUREMENT 

3 The VMDlOO provides a clear 

digital display enabling easy 

measurement of time 1 averaged air flow velocity. 

0 Printed velocity 

measurements and graphical plot of values against time. 

3 TSI 1640 measurement probe 

specially designed for 

maximum accuracy of low 

velocity air flows. 

a Omnidirectional probe 

measures velocity as 

independently of direction as 

possible. ~ ~~ 

0 Traceable calibration. 

e Calibration matched units. 

TSI 1640 and VMDlOO 

(send mte* I$ optlonau 

~~ ~ 

The combination of the "SI 1640 and VMDlOO completely supports 

the measurement requirement detailed in IS0 3308 (1991) Annex A, 

and CORESTA recommended Method No.25. 

The TSI 1640 was selected as the most suitable measurement device 

available for the measurement of air flow velccities in the smoke 

hood and extraction/ducting systems of smoking equipment. 

The VMDlOO Digitiser has been specially designed to be 

programmed with the calibration coordinates of the TSI 1640 

providing accurute digital meawements of air flow velocity. 

____ .~ ~~ ~ . .. 
TSI 1640 Omnidirectional 

Air Flow Velocity Meter 

The Model TSI 1640. Omnidirectional Air Velocity Meter wcn 

selected for the measurement of air flow velocities in the smoke 

hood and extraction/ducting system for the following reasons: 

a It is a battery powered, portable unit, with mains bcmery charger. 

a The TSI 1640 scale ranges me: -. .~ 

0.00 - 300 mmsl For Smoke Hood Velocities. 

250 - l2O0 --' FO~ Extracion/Ducting velocities. 1000 - 3000 m-1 1 

0 The probe is qxcially designed for maximum amacy 

0 Omnidirectional probe designed to measure velocity CLS independently 

0 Provides signal averaging. The thermal capacitance of the copper 

0 The accuracy of measurement is +/- 2% of full scale deflection. 

0 Each sensor is individually culibrated with a system that has been 

0 A certificate of traceability to the National Institute of Standards & 

of low velocity flows. 

of direction as possible. 

sensor kull approximates a lime constant of about 2s. 

verified using Laser Doppler Vel-em. 

Technology, Maryland, (USA), is supplied with each probe. 

VMD 100 Airflow Measurement 

Digitiser/Results Plotter. ~ ~~~~ ~ ~~ ~~~~ ~ 

~~ ~~ 

0 purpose designed to interface directly with the cmalcgue output 

0 Calibration coordinates can be reprogrammed by user. 

9 The TSI 1640 is specially calibrated in a TSI wind tunnel. The calibration 

of the TSI 1640. 

coordinates obtained are programmed into the VMD 100 which 

provides a clear and simple -lay of flow velocity in mms-1. 

0 The flow integration time in the VMD 100 is selectable from 10 to 

120s in 10s steps. 

0 A serial output printer port is provided which gives a formatted report of 

elapsed time. average velocity and a graphical plot of flow vmiation. 

0 Mains powered 110V: 60Hz & 22OV; 50HZ 

Product Ordering Description/Code: 

TSI 1640 (1 1OV; 60HZ) - Stock Code: 64054 

751'1640 (22OV: 50HZ) - Stock Code: 64053 

VMD 100 - Stockcode: 91580 

rWmr .A Teleohone' IO9081 372716 Teleohone 18041 275-7850 
SM342 Eight Channel Harmonised 

Smoking Machine 

SM342 shown wth vopour phase attachment (COM302) 

mounted on top of the ut. 

Conform to IS0 3308: 

199 1 and Coresta 

Methcds. 

Simple adjustment of 

flow at cigarette level 

with single ball. valve. 

Easy installation and 

operation - no need 

for special lccboratory 

enclosures. 

Extended butt length adjustment. 

Automatic lighting 

sequence - ideal for 

routine smoking. 

The Filtrona Model SM342 is an eight channel smoking machine 

designed for the collection of pcuticullae mattex from dgarette 01 

cigar smoke and the collection of vapour phase. 

This instrument is designed to have minimum dead volume for 

enhanced vcrpour phase mecrsurement; Le. total or puff by puff 

measurement of CO, or puff by pulf measurement of CO and/or NO 

Volume, duration and frequency of puff can be vaxied to suit 

individual requirements. It has a motor driven lighter ignition system. 

SM304 UpGrade Packages are avQilQble to convert exisling SM302's 

in the field to the new harmonised standard. 

-.-

FIDUS FIDUS FIDUS 

- - - 

FIDUS --- 
SM342 Smoking Machire 

Specification for SM342 (w.U SMZC~ ^. ~ 

~~ ~~~~ .~ ~~ ~ ~ ~ ~~~~~~~ ~ ~~~~ 

Number of smoking channels: a 

Type: Restricted smokmg clrcuit comprising separate 

volumetric displacement pump and change- over solenoid. 

Collection media: 

Porhculate matter: 

Vcrpour phase: 

Glcm tibre pad (Cambridge filter) 

Collection bags (one per channel uslng COM 302) 

md witable analysers co~eded to 

sumpling system. 

Not more than Sml per channel. measured from 

the front face of the mlet port to the top of 

the synnge. 

Normally set to 35 ml. 

(vcmable over the range 20-40 ml) 

Normally set to 2.0s 

(variable from 1.6 to 6.0s). 

Normally set ai 1 per 60s 

(vcniable from 10 to 99%). 

Dead volume: 

Puff volume: 

putf durahon: 

putf frequency: 

Sample Range: 

Cigarettes: 

Cigars: 

Length: 

Diameter: 4.5-9.5 mm 

Length: 

Diameter: 

Mcdns services (operating voltages): 

Dimensions (bench area required): 

66-120 mm (85 mm bum hgth). 

66120 mm (85 m hm length). 

With altemahve hokkr. any dmmeter 

110/115/220/240 V; 50pXlHZ. 

upto 19 mm. 

Width: 1500 mm 

Depth: 850 mm 

Net weight installed: 100 Kg. ~ ~~~ ~ _..____~ ~~~ ~ 

Filtrona Instruments & Automatton Lid. 

Denbigh Road, Blerchley. 

Milton Keynes MKl 1DH. England. 

Telephone: 109081 372716 

Telex: 82429 Tdkm 7109564151 

Fax: IO9081 373976 F?r I-) 7431 108 

HONG KONG SINGAPORE Ilk” 

Fiitrana Innrurnentr & Amornmon Lld 

3706 Hong Lono P82a 

Ki 1rievment CXL. 

7m. ‘Mtepine Road 

miad. VA 23237. USA 

TeIme: 18041 275-7850 

Fiitrona In~tru~ts~SUmrn~m~u~ 

9 Psnang Road Fiiirona Inarumentr E. ~,~~o~t,~~ Vm Cam Tone 2 
SM400 Twenty Channel Harmonised 

Smoking Machine 

0 Conforms to ISO3308-1991 

and CORESTA Methods. 

0 Improved operator access 

by the use of a smoking 

bcn which moves forward 

for easy cottoning and 

loading. 

0 Computer controlled, hgh 

torque motor drive system produces excellent pulf profiles. 

With the new design of 

hood and versatile 

ducting arrangement, the 

air flow at the cigarette 

position is adjustable to 

meet the new IS0 

standards. 

0 SM400 upgrade packages 

are available for the 

earlier SM350 and SM300 

Smoking Machines. 

The Harmoniscrtion Task Force of the CORESTA Smoke Study 

Group has been working to develop one set of standard methods 

which may be used worldwide. 

Part of the work has been concerned with the control and 

standardiscrtion of the air flow at the cigarette smoking position. 

and FILTRONA'S pahcipa-hon in this work has led to the 

introduction of a new smoking machine - Model SM400. -- r 

FlDUS FlDUS 

-E 
SM400 Smoking Machine 

PRODUCT CODE DESCRIPl7ON 

SM400 Harmonised smoking machine complete 

with new style (SMK40 1) 'operator friendly' 

CF Adjustors. 

SM435 Up-grade package for SM350 

SM430 @Upgrade puckage for SM300 

Both up-grcde packages consist of: 

- SM400 Harmonised Smoke Hood. 

- Extraction Ducting System. 

- First Stage Extraction Fan. 

- CF Adjustor (old style) Retaining Kit 

- Air Velocity Setting Jig. 

- Installation Instructions. 

- Air Flow Setting Procedures. 

OPTIONS: 

SMK40 1 New butt length and eccentricity adjustors 

(set of 20). for SM350 smoking bars only 

(93mm cotton pillars/micro switch arms.) 

TSI 1640 Air Velocity Meter. 

VMD 100 Air Velocity Digitiser and Plotter 

The installation of a new smoking bar 

is recommended. 

Note 1: 

Filtrana Instruments &Automation Ltd. Fidus Instrument Carp. 

Denbigh Road. Bletchley. 7400. Whitepine Road 

Milton Keynes MK1 IDH, England. Richmond, VA 23237. USA 

Telephone: 10908l372716 Telephone: 18041 2757850 

Telex: 82429 Telex: 710-9560151 

Fax: 109081 373976 Fax: 18041 743-1108 

HONG LONG SllYiPlrnRE ,TALI 

Filtro~ lnruumsnii & Autmutrn Lm 

3706 Haq KM(1 Fm. 

186- I91 Cmwl ROXlWCn :%EWd! 821M B-nm 

now x- - 0913 It.* 

Tale- 54e469l rew.-:ffi33631rn Fax 5 530318 

Fi- lrnrrvmnn & A.~lomdlion LIO Wtrona ~nsti~monts & Avroma!,cn TAL A 

v* Car*, io.rc 2 

rasma. 824 42~98 

SUI 5535323 Fax 824 E4817 -~ ~ "__.^ - ~ .... 
Nitric Oxide Analyser 

Cigarette Smoke 

Nitric oxide is a physiologically important 

constituent of the vapour phase of cigarette 

the subjectof Mure legislation. However, 

one of the problems of isolating and. 

that it reacts rapidly with othersmoke 

constituents and atmospheric oxygen to ghre 

other oxides of nitrogen. It is therefore . 

essential that samples are analysed on a 

puff-by-puff basis and are diluted with an 

inert gas in order to prevent secondary reactions and interference from other smoke 

constituerts. 

* Designed to work with Filtrona Model 302 

* Can be U& with other pist-pe~ed 

*Puff.bWxlffdilutlorrwithnitrogen preve 

* Measurement by chemil 

Smoking Machine 

. 

.' smoke, andits measurement may become smoking machinawith low deackvolume. 

secondary reactions ' analysing nitricoxidsin cigarette smoke1 ,/ 

which is specficfw NO and 

does not require skilled operators 

a wide range of NO concentrations 

* Highsensitivity photomultiplier measures 

, Automatic 

* Built-in ozone eenemtor 
The measurement technique used is 

chemiluminescence, which is a widely 

accepted method for the analysis of nitric 

oxide in cigarette smoke. The principle is t 

react the sample of cigarette smoke with 

ozone and to observe the photoemission 

using a photo-multiplier tube behind a dark- red optical filter. This reaction has a direct 

relationship to the quantity of nitnc oxide 

Method at operatiom 

NOAsl~developed by British-American recorder, andthe readings am compared 

-TobaoazCompany in consultation with other with those obtained fromstandard mixtures 

UK tobaax companies, is designed for of nitric oxide irr nitrogen-Thii gives the 

automatic sampling of vapour phase with concentration (voiumetricparts per million) of 

Filtrona Model 302 &channel Smoking nitric oxide, and a smplecalculatcon is used 

Machine It can also be used manually with tu convee to ddivety in p g per cigarette. 

By attachment of a simple valving system, sane other piston-operated smoking 

that part of the vapour phase not required machines. 

After each puff, the vapour phase is for NO analysis can be transferred to a 

exhausted from the smoking machine into a Filtrona CO analyser, for simultaneous 

gas sampling valve in the analyser. Nitrogen analysis of NO and CO of the same sample. 

is fed into the sampling valve and sweeps 

the sample into the reaction cell. Ozone is Note that this instrument must be used with 

there added to the sample, and the photo- a chart recorder. This must have a response 

multiplier tube measures the resulting photc- time of better than 0.6 seconds for full-scale 

emission. Results are displayed on a chart deflection. 

present in thttsmoke Thaphotmultiplier 

signal is procassed and disprayed on a chart 

recorder: 

Interference from other smoke constituents, 

and secondary reactions, are avoided by 

dilution of the smoke sample with a large 

volume of nitrogen before reaction with the 

ozone. 

9 

Linearity * 1% 

Accuracy I f 1% of full Scale after calibration 

Reproducibillty f 1% 

Nitrogen supply pressure (external) 

Oxygen supply pressure 

Gas connections ] 1/8 inch Swageiok 

Electrical supply 

4 bar (60 psi) (Nitrogen to be supplied by user) 

1.4 bar (20 psi) (Oxmen to be supplied by user) (external) 1 - 

I 220-240V 50Hz supply (standard); versions for other supplies 

Specifications 

Range 

Attenuation settings 

Sensitlvity [ 1 VPM NO 

1 up to 5000 volumetric parts per million (VPM) 

1 1, 2. 5, 10, 20, 50 (low, medium and high range for each setting) 

Weight 

output 

Standard Equipment 

20kg 

An external socket is provided for connecting to a lmVtast-response chart recorder. This can be supplied as an optional exba (see below) 

All electrical and mechanical fittings needed toconnect NOA 100 to a 

Filtrona E-channel Smoking Machine (SM 302) am supplied. 

1 at extra cost 

Dlmensions (mm) I Width 483 (rack or case mounting) Depth 495 Height 223 I 

Rltrona lnsttuments 6 lvranabon . Ltd., Denbigh Road, BI 

Milton Keynes. M* En@and. 

Telepnone: (0908) 72716 Tder 82429 
Correlation Between Tar Levels Recorded in FTC Test and a Private Laboratory 
Chapter C 

ASSESSING CHANGES IN TOPOGRAPHY (INHALATION PROFILE) 

AND BIOLOGICAL EFFECTS OF TOBACCO SMOKE IN HUMANS 

David M. Burns, MD 

University of California at San Diego Medical Center 

Under Contract to the U.S. Consumer Product Safety Commission 

YCPSC-s-92-5475 

16 Mar 1993 

c1 

INTRODUCTION 

The most useful approaches in the evaluation of differences 

in risks between presently marketed and low-ignition-potential 

cigarettes focus on chemical analyses of the cigarette smoke, 

measures of smoke absorption, and assays in biological systems. 

Because of practical constraints, human epidemiologic studies 

would be of very limited practical value. Although the ideal 

database would derive from prospective human epidemiologic 

studies, at least twenty years of observation would be needed to 

collect sufficient data on seriously adverse health effects. 

Also, if small differences in toxicity are expected, then large 

sample populations would be needed. 

The toxicity of cigarette smoke is a function of the toxic 

constituents present in the smoke, the levels of the constituents 

in the smoke, and the dosage of the constituents to the smoker. 

Thus these data must also be collected for the evaluation of the 

relative risks of low ignition-potential cigarettes in comparison 

to current brands. 

Smoke production, as quantified by tar yield, varies 

substantially among the current cigarette brands. It also varies 

for a single brand when different patterns of inhalation are 

used. Moreover, the relative concentrations of toxic 

constituents also vary with brand of cigarette and pattern of 

inhalation, at least as measured by tar and nicotine yield. 

These differences are related to the health risks among different 

brands of cigarettes (DHHS 1981). Since it is possible that 

ignition-potential reducing desi9ns in cigarette manufacturing 

might quantitatively and qualitatively alter the smoke produced, 

there is concern that the health risks might be increased. 

The inhalation profile of a smoker as he or she smokes a 

cigarette is termed "topography". Ignition-potential reducing 

designs may alter the topography in ways that lead to greater 

inhalation and retention of the smoke. Differences in the depth 

and pattern of inhalation may change the amount of smoke that is 

deposited and retained in the airway. Therefore, machine 

generation of smoke from low ignition-potential cigarettes for 

testing should reflect the human patterns of inhalation for the 

specific brand of cigarette. This will ensure that the smoke 

being tested is similar in composition to that being inhaled by 

human smokers. 

Assessment of differences in the risks of smoking low 

ignition-potential cigarettes in comparison to current brands of 

cigarettes should address: 

- Differences in chemical composition of mainstream and 

sidestream smoke produced by these cigarettes 
c2 

- Differences in the amount of the mainstream and sidestream 

smoke produced by these cigarettes 

- Differences in the amount of smoke inhaled and retained by 

smokers, and 

tested in biological systems. 

- Toxicity of the smoke produced by these cigarettes, as 

FACTORS INFLUENCING SMOKE YIELD AND COMPOSITION 

Different brands of cigarettes currently manufactured in the 

US vary markedly in yields of tar and nicotine (DHHS 1981) when 

smoked using the standard puff profile developed by the Federal 

Trade Commission (Chapter B). When the same brand of cigarette 

is smoked using different puff profiles, the yields of tar and 

nicotine also vary substantially (Zacny 1992). Individuals 

smoking the same brand of cigarette may inhale using markedly 

different patterns (Nil 1989). When smokers of a high yield 

cigarette switch to a lower yield cigarette, their inhalation 

pattern often changes (Kolonen 1991; Woodman 1987; Guyatt 1989). 

Interactions have been demonstrated between the yield of a 

cigarette and the pattern with which the smoker smokes the 

cigarette (DHHS 1988; Benowitz 1983; Kolonen 1991; Hofer 1992). 

These changes in yields and smoking patterns should be considered 

during the evaluation of the health effects. 

A number of the manufacturing changes under consideration in 

the effort to reduce the ignition potential of cigarettes (e.g., 

higher porosity paper, less densely packed tobacco, different 

tobacco blends) may alter the amount and chemical composition of 

the smoke produced (Gann 1991), potentially changing its 

toxicity. The same changes in cigarette manufacturing processes 

may also alter the pattern of inhalation of the cigarette 

(Bridges 1990; Kolonen 1991; Armitage 1988). This, in turn, may 

change the chemical composition of the smoke (Kozlowski 1988; 

Fischer 1989), influence the retention of toxic and carcinogenic 

compounds from the smoke in the lungs of smokers (Zacny 1992; 

Hofer 1991; Battig 1982; Bridges 1986), and alter the composition 

and toxicity of the environmental tobacco smoke (Adams et a1 

1985). 

A single set of machine smoking parameters, such as the 

current FTC protocol (Chapter B), could be followed for the 

generation of cigarette smoke for testing. However, this single 

set would ignore possible differences in patterns of smoking (and 

resultant constituent yield) of low-ignition potential 

cigarettes. For example, a drop in the draw resistance of a 

cigarette may lead to a puff volume greater than that specified 

in the FTC protocol. The larger puff volume could then lead to a 

deeper inhalation of the smoke and a greater fraction of the 

total particulate matter being deposited in the lung. 
c3 

The complexity of the interaction of smoke yield and pattern 

of inhalation suggests that chemical and biological approaches 

are needed. Chemical analyses of the differences in whole smoke 

exposure of the smoker can be assessed from measures of the 

amount and composition of the smoke produced when cigarettes are 

smoked by machine using a variety of inhalation patterns, and 

these quantitative estimates can be compared to measures of the 

absorption of smoke constituents obtained from human smokers of 

these brands of cigarettes. 

Biological assays of relative carcinogenicity and toxicity 

of the smoke produced by low ignition-potential cigarettes can be 

accomplished using a combination of chemical analytic techniques 

to measure the relative yields of individual compounds produced 

by different cigarettes and bioassay techniques to assess the 

relative toxicity of the smoke produced. 

The toxicity of cigarette additives is of concern. A new 

additive, or its pyrolysis products, could increase the known 

toxicity of the smoke. It might also cause toxicities that are 

qualitatively different than those presently associated with 

cigarette smoke. Direct toxicity testing of additives and their 

combustion products should also be required. 

The following section describes how smoking patterns can be 

measured and explores what is known about the variation in 

topography of smoking among smokers of the same type of 

cigarette, among smokers of cigarettes with different yields and 

among those who switch to cigarettes with different yields. A 

subsequent section defines what is known about the absorption of 

smoke constituents; and finally, an approach will be recommended 

for use in assessing the changes in risks and exposures that may 

occur with implementation of the proposed technologies to reduce 

the ignition potential of cigarettes. 

PATTERN OF SMOKING 

The first step in the process of assessing the relative risk 

of low ignition-potential cigarettes would be to establish how 

the patterns of smoking differ for low ignition-potential 

cigarettes compared to current cigarette brands. The major 

determinants of disease risks from smoking are the duration of 

smoking and the intensity of smoke exposure (DHHS 1982; 1983; 

1984). Several measures of the intensity of smoke exposure 

correlate with increased disease risks, including: number of 

cigarettes smoked per day (DHHS 1982; 1983; 1984), depth of smoke 

inhalation (DHHS 1982), and tar and nicotine content of the 

cigarette (DHHS 1981). Each of these measures of intensity of 

exposure might change when a smoker switches from smoking 

conventional brands of cigarettes to low ignition-potential 

cigarettes. 
c4 

Topographical characteristics, such as puff volume, draw 

rate, puff duration, and draw pressure differ between smokers and 

may alter the composition of the smoke. Smokers differ in the 

number of puffs per cigarette, length of time between puffs, 

depth of inhalation, and holding of the puff in the mouth before 

inhaling (Nil 1986; Guyatt 1989; Bridges 1990; Russell 1982; 

Battig 1982; DHHS 1988). Ignition potential-reducing changes in 

the blend or amount of tobacco in a cigarette, packing density of 

the cigarette, and porosity of the paper wrapper are changes 

under consideration (Gann 1988) and may also alter the 

topography. 

The major purpose of examining topography when smoking low 

ignition-potential cigarettes is to determine appropriate smoking 

profiles for machine smoking of these cigarettes. Once the range 

of smoking topography is established for each brand of low 

ignition-potential cigarette, then the yields and chemical 

composition of mainstream and sidestream smoke likely to be 

generated by human smokers can be estimated. 

Measurement of Smoking Pattern 

Number of cigarettes smoked per day is typically estimated 

by self-report (by the smoker) through an interview or a 

questionnaire. This measure has been shown to be closely 

correlated with risks of serious disease (DHHS 1982; DHHS 1983, 

DHHS 1984). 

Inhalation depth has also been assessed by self-report and 

is associated with disease risk in most, but not all, 

epidemiologic studies (DHHS 1982). A number of other methods 

have also been used to estimate depth of inhalation including 

measures of chest wall motion with strain gauges, impedance, 

magnetometers and whole body or inductance plethysmography (DHHS 

1988). Depth of inhalation has also been assessed by measurement 

of blood carboxyhemoglobin to estimate exposure of the lung 

alveolar surface to the carbon monoxide in smoke (Herling 1988). 

Draw characteristics of the topography have been measured 

using a variety of techniques including self-report and third 

person observation (Hofer 1991). The most common approach has 

been to use a flowmeter attached to the butt end of the cigarette 

as it is smoked (Creighton 1978; Puustinen 1987). This device 

allows direct measurement of the flow of smoke drawn into the 

mouth, draw pressure, and flow duration. The flow rate is 

integrated to calculate puff volume. The inter-puff interval is 

calculated from the time between periods of flow. The limitation 

of using a flowmeter is that it is placed between the smoker and 

the cigarette and may affect the pattern of smoking. 

Variation in the Pattern of Smoking with Existing Cigarettes 
c5 

There is a marked variation in the number of cigarettes 

smoked per day by different smokers. Some individuals smoke less 

than one cigarette per day and others smoke 60 or more cigarettes 

per day (Pierce and Hatziandreu 1989; Burns and Pierce 1992). 

The day-to-day variation in number of cigarettes smoked per day 

by an individual smoker is much smaller than the variation among 

different smokers in the population, particularly for those who 

smoke every day. The number of cigarettes smoked per day varies 

somewhat with age, gender, and socioeconomic factors. There is 

also some variation according to racial grouping. Black smokers 

tend to report fewer cigarettes smoked per day than white 

smokers, and Hispanic smokers are more likely to be occasional 

smokers. 

An extensive presentation of the variation in smoking 

topography is presented in the Surgeon General's report on 

nicotine dependence (DHHS 1988), and the variation in the 

topography of smoking as measured in published studies is 

presented in Table 1. There is relative uniformity in the mean 

values for the measures of smoking topography across these 

studies; but there is a substantial variation in the measures of 

smoking topography among individual smokers (Nil 1986; Guyatt 

1989; Bridges 1990; Russell et al.; Battig 1982). This variation 

among individual smokers is defined by the standard deviation of 

the measured values presented in Table 1, and it is evident that 

there is a wide variation in the pattern of inhalation among 

individual smokers. The variation in pattern of smoking is much 

less for two cigarettes smoked by the same smoker (Battig 1982), 

suggesting that it was differences between smokers in the way 

that they smoked, rather than differences in the way a specific 

smoker smoked sequential cigarettes, that produced the variation 

in smoking topography found by these studies. 

The pattern of smoking also varies with the tar and nicotine 

yield of the cigarette smoked. Some smokers of lower yield 

cigarettes clearly compensate for the lower yield of these 

cigarettes by altering the way they smoke these cigarettes to 

increase the absorption of nicotine. Their nicotine absorption 

levels are substantially higher than would be predicetd from the 

nicotine yields generated from machine smoking (Benowitz 1983; 

Kolonen 1991; Hofer 1992). This compensation is largely 

accomplished by changes in puff volume and f'requency, rather than 

by increasing the number of cigarettes smoked per day (Kolonen 

1991; Battig 1982; Bridges 1990). 

A number of investigators have examined changes in the 

topography when smokers switch to lower yield cigarettes, both 

immediately and after a period of regular smoking of these 

cigarettes. When smokers switch to a cigarette that has a lower 

yield than their regular brand, there is an immediate 

compensation in the smoking topography with an increase in puff 

volume and puff frequency (Kolonen 1991; Woodman 1987; Guyatt 
C6 

1989). However, the change in puff frequency can revert to the 

original rate within a few weeks or months (Guyatt 1989). In the 

studies that measured nicotine absorption, there was a fall in 

amount of nicotine absorbed when the smoker switched to a lower 

yield cigarette, but the magnitude of the fall was much smaller 

than would be predicted from the machine smoking results. 

manufacturing processes which affect the yield of the cigarette 

can influence smoking topography. Many of the changes appear to 

be a compensatory repsonses intended maintain nicotine absorption 

from cigarettes with a lower nicotine yield, such as increased 

puff volume and inhalation depth. Topography alterations may 

also result from changes in draw resistance (Guyatt 1988), filter 

perforations (Kozlowski 1988), and possibly other differences in 

manufacturing technology. 

Existing data clearly indicate that differences in cigarette 

MARKERS OF THE DOSE ABSORBED BY THE SMOKER 

Chemical analyses can quantify the several thousand 

constituents present in tobacco smoke (Chapters B and D), but it 

is neither practical nor technically possible to measure human 

absorption of each of the several thousand smoke constituents. 

Therefore, evaluation of the relative toxicities of the smoke 

produced by low ignition-potential cigarettes will need to rely 

heavily on selected chemical analyses and biological (in vitro 

and in vivo) toxicity tests of the whole smoke and its 

components. Measures of absorption can be used predominantly as 

markers of whole smoke exposure. 

Biochemical markers, such as nicotine and carbon monoxide, 

can quantitatively estimate the amount of smoke absorbed by the 

smoker and effectively integrate the sometimes competing effects 

of differences in cigarette manufacturing and resultant changes 

in smoking topography. 

estimate of the differences in exposure/dose resulting from 

smoking different brands of cigarettes. If switching to low 

ignition brands leads to a greater number of cigarettes being 

smoked per day, a deeper inhalation, or a change in the 

topography of smoking, it may lead to greater smoke absorption; 

these would increase the risk of those adverse health effects 

summarized in Chapter A. A number of biochemical markers have 

been used to quantify the amount of smoke inhaled and retained by 

the smoker (IARC 1986, DHHS 1988). 

marker to estimate the absorption of whole smoke is influenced by 

several factors. First is the quantitative relationship between 

an increase in the level of the marker and the amount of the 

smoke absorbed by the smoker. Second is the specificity of the 

marker for the absorption of tobacco smoke as compared to other 

Biochemical markers produce a unifying 

The accuracy and utility of using a single biochemical 
c7 

sources of the marker. Third is the direct quantitative 

relationship between the absorption of the marker and the 

absorption of other toxic constituents in the smoke. And 

finally, there is the precision of the measurements. 

Biochemical markers of absorption can be used under non- experimental conditions that represent more typical smoking 

behavior. Biochemical markers are advantageous since they can be 

sampled after a period of smoking under non-experimental 

conditions approximating normal behavior. This may be less 

disruptive to the smoking behavior than topographical measures 

taken durinq smoking by attaching instrumentation to the 

cigarette or to the human smoker. 

Carbon Monoxide 

Carbon monoxide (CO) is a toxic gas phase constituent of 

tobacco smoke and is a useful measure of the depth of inhalation 

of cigarette smoke. The CO produced is dependent on the amount 

of oxygen available to the burning tobacco. However, other 

factors, such as density of the tobacco, paper porosity, puff 

volume, and draw rate, may influence the amount of CO produced 

and reduce the correlation between CO and particulate matter 

(IARC 1986). Therefore, CO can not be predicted on the basis of 

particulate matter (or tar) yield. 

CO binds reversibly to hemoglobin (the oxygen-carrying 

protein in the blood), and is cleared from the body primarily by 

exhalation. The concentration of carbon monoxide in the blood 

can be determined as the partial pressure of CO in the expired 

air or as the level of carboxyhemoglobin in the blood (DHHS 1988; 

IARC 1986). The blood and breath levels of CO vary markedly 

during a smoker's day due to the intensity of smoking in the 

hours immediately before the measurement and the short 4-hour 

half-life of CO in the blood. 

The single point-in-time measurement of blood or breath CO 

is very precise, but may not represent the total daily smoke 

absorption due to its short half-life, the background sources of 

CO and endogenous production of CO (Woodward 1991). However, 

the change in blood or breath CO level after smoking a single 

cigarette can represent the smoke absorbed and may be a better 

measure of the depth of inhalation of the cigarette smoke than 

serum nicotine or cotinine levels. Since CO is absorbed almost 

exclusively by the lung, it is a more specific measure of smoke 

inhalation than nicotine, which is also absorbed through the oral 

mucosa (Herling 1988). CO is also a marker for exposure to the 

gas phase constituents of smoke since it is present in only the 

gas phase. 

Nicotine and Cotinine Levels 
C8 

Concentrations of nicotine and its longer half-life 

metabolite, cotinine, can be measured in the blood, saliva and 

urine to very low levels (DHHS 1988; IARC 1986; Anderson 1991; 

Benkirane 1992; Etzel, 1990; Feyerabend 1990; Weinhold 1987). 

The major non-tobacco sources of nicotine are nicotine patches 

and gums used as adjuncts to smoking cessation. Nicotine is not 

produced by the body and is found in only trace amounts in plants 

or foods other than tobacco (DHHS 1990). The contribution of 

environmental tobacco smoke to the cotinine level of most regular 

smokers is less than one percent (DHHS 1986; Watts 1990). As a 

result, nicotine and cotinine levels are highly specific to 

tobacco smoke exposure. 

Tar and nicotine yields are well correlated for most current 

brands of cigarettes (DHHS 1981). Therefore, nicotine or 

cotinine levels can be used to estimate the amount of particulate 

matter (I'tarl') absorbed and retained by a smoker. Cotinine is 

the preferred measure of nicotine absorption because of its 

longer 20-30 hour half-life in the blood (Benowitz 1983; IARC 

1986). Cotinine is also less influenced by active smoking or 

environmental tobacco smoke than salivary nicotine levels. 

Salivary, blood, or urinary levels of cotinine can represent 

the average daily dose of particulate phase constituents 

absorbed. This is important because the bulk of the carcinogenic 

activity in tobacco smoke is in the particulate phase (IARC 

1986). Cotinine may also be useful for determining the net 

effect of changes that have opposing effects on smoke absorption, 

for example a cigarette with a lower tar and nicotine yield that 

is inhaled more deeply. 

Thiocyanate 

Hydrogen cyanide is found in the gas phase of cigarette 

smoke and is metabolized to thiocyanate by the body. Thiocyanate 

can be measured in plasma, urine, and saliva (IARC 1986). 

Although thiocyanate has an extremely long half-life 

(approximately 2 weeks), there are a number of foods that 

influence thiocyanate levels, including leafy vegetables and 

nuts. Dietary sources make the independent contribution of 

tobacco smoke to the thiocyanate level difficult to determine. 

Therefore, thiocyanate is an imprecise and poorly reproducible 

quantitative marker for smoke absorption. 

Other Compounds 

metabolic products of tobacco smoke constituents, have been used 

as measures of smoke absorption including N-nitrosoproline and 

thioethers (IARC 1986). However, none of these measures appear 

to offer any advantages in estimating smoke absorption over the 

three measures described above. 

A number of other constituents of tobacco smoke, or 
c9 

TESTING FOR TOXIC EFFECTS OF THE SMOKE ABSORBED BY THE SMOKER 

A number of short-term toxic effects of human tobacco smoke 

inhalation and retention can be measured. Some are postulated to 

be early or intermediate steps leading toward seriously adverse 

health effects (Chapter A). These effects include reduced serum 

high-density to low-density lipoprotein ratios, increased 

platelet aggregation, acute bronchial reactivity to smoke 

inhalation, small airway dysfunction in the lung, and greater 

mutagenicity of the urine. Measurement of these effects might 

possibly define the degree of damage being done by the smoke in a 

way that would integrate the effects of dose of exposure and 

biologic susceptibility. In addition, they might also detect 

pathophysiological toxicities that would not be evident from 

chemical analyses or markers of whole smoke exposure. 

The limitations of these tests include their variability of 

measurement, the relatively long period of time required for 

smoking behavior to produce measurable change, and the degree of 

scientific uncertainty concerning their role in the 

pathophysiology of smoking-related disease. In addition, the 

general population varies widely in the levels of most of these 

effects. Due to the large variation and possibly small relative 

changes, a large group of volunteers may be needed to attain 

statistical significance. 

COMPARISON OF LOW IGNITION-POTENTIAL CIGARETTES 

The ideal comparison "standard" for the low ignition- potential candidate brands would be existing cigarette brands 

tested under the same protocol. This would allow comparison of 

the toxicity of the candidate brands with the range of toxicities 

for the currently marketed brands. 

One approach is to compare the risk estimates for the 

candidate brand to the range of risk estimates for brands 

currently on the market. For example, low ignition-potential 

cigarettes with risk estimates that are within the range of those 

for currently marketed brands might be assumed to be no more 

hazardous than current brands of cigarettes. Risk estimates for 

current brands of cigarettes would be obtained by performing the 

same tests used on the low ignition-potential candidates. This 

approach avoids the problem of defining an acceptable standard 

for low ignition-potential cigarettes. 

A variant of this approach would be to require no increase 

in the sales-weighted average risk for all of a given cigarette 

manufacturer's products when a low ignition brand of cigarettes 

is marketed. This allows the manufacturer to adjust the mix of 

their brands to avoid an overall increase in risk. These 
c10 

approaches assume that existing brands of cigarettes will be 

allowed in the market even though they may have higher ignition 

potential. 

Another approach, which does not assume that existing brands 

will continue to be marketed, compares a candidate brand with the 

one it was intended to replace or duplicate in the marketplace or 

the one from which it was developed. This approach would ensure 

that a new cigarette brand would not increase the average risk of 

cigarette smoking. However, it may not always be possible to 

pair a candidate brand with an existing brand. 

SOURCES OF INCREASED HAZARDS 

The major measurable increases in the hazard of smoking low 

ignition-potential cigarettes compared to current cigarettes are 

likely to come from differences in the: 

1. cigarette manufacturing process or tobacco used that lead 

to a greater total yield of mainstream or sidestream smoke, 

2. pattern of smoking that lead to a greater total yield of 

mainstream or sidestream smoke, 

3. chemical composition of mainstream and sidestream smoke 

produced that lead to more toxic or carcinogenic smoke, 

4. pattern of smoking that lead to greater absorption of 

smoke, and 

5. additives to the cigarette that increase the toxicity or 

add new toxicities to the smoke. 

Changes in toxicity could be evaluated for each of the many 

different possible designs that could reduce the ignition 

potential of cigarettes. However, this approach would be both 

impractical and wasteful since the many different 

ignition-potential reducing strategies would generate a 

prohibitively large number of combinations to be tested. The 

vast majority of the combinations would also be unlikely to be 

used in commercial cigarettes. Thus, it is only necessary to 

examine those combinations of low-ignition-potential changes that 

cigarette manufacturers present as potential commercial brands. 

RECOMMENDATIONS 

Generation of information to assess the risks of low 

ignition-potential cigarettes is product research and toxicity 

testing, a responsibility most appropriately belonging to the 
c11 

cigarette manufacturers. The data should be generated by the 

manufacturer when contemplating the introduction of a new brand 

or replacement of an existing one. In addition, a new low 

ignition-potential cigarette is likely to combine designs to 

reduce the ignition potential with those to improve the taste or 

other marketing characteristics of the cigarette. It is the 

combination of all changes in a low ignition-potential cigarette, 

not just those that reduce the ignition potential, that determine 

its relative toxicity. Therefore, these cigarettes will need to 

be examined on a brand-by-brand (product performance) basis 

rather than on a manufacturing change-by-change (product design) 

basis. The following data should be collected on a candidate 

cigarette brand: 

1. Smokina topoqraohy 

The number of cigarettes smoked per day, puff volume, puff 

duration, puff interval, maximal puff inspiratory pressure 

and flow, and number of puffs per cigarette should be 

measured in an experimental group of smokers of the brand 

after they have acclimatized to smoking the low ignitionpotential brand. 

2. Smoke yield and composition 

The range of mainstream and sidestream concentrations of the 

compounds listed in Table 3 of Chapter D should be assessed 

for smoke produced by machine smoking the brand using a 

range of smoking topographies that correspond to those 

observed for that specific brand. 

3. Additives 

A complete list of additives and the concentrations used, as 

well as their likely pyrolysis products, should be disclosed 

for each brand. Confidential Business Information status 

may be requested to protect proprietary information. 

4. Inhalation and retention of smoke 

The change in CO level that results from smoking a single 

cigarette of the brand should be measured in acclimitized 

smokers as a marker for acute exposure. Cotinine levels 

should be measured in acclimatized smokers of the brand as a 

marker for average daily exposure. 

5. Toxicity of the smoke produced 

The toxicity of the mainstream and sidestream smokes 

produced by machine smoking each brand of cigarettes should 

be evaluated using the approaches described in Chapters E 

and F of this report. 

Testing Sequence 
c12 

The sequence of testing for the evaluation of toxicity and 

carcinogenicity of low-ignition-potential brands of cigarettes 

should include the following stages for mainstream and sidestream 

smokes : 

I. Initial Evaluation 

1. Machine testing 

Initial evaluation of each candidate brand before testing in 

humans, should include analyses of CO, nicotine, tar, and each of 

the other constituents of tobacco smoke listed in Table 3 in 

Chapter D using the FTC method (Chapter B) to generate the smoke. 

substantially greater amounts of the compounds listed in Table 3 

of Chapter D, then testing would proceed using a range of smoking 

topographies observed for current brands of cigarettes. 

Topographies reported by several studies for current cigarettes 

(Table 1) are graphically presented in a puff volume / puff 

duration matrix (Table 2 of this chapter). The central cells in 

this matrix are the most commonly reported values from the 

studies in table 1 and the values for the FTC method. Tar, 

nicotine and CO yields should be measured under the smoking 

conditions specified by each shaded and unshaded cell (15 mljl 

sec through 90 m1/3 sec) to reflect the range of observed human 

topographies. 

should then be analyzed using smoke generated according to the 

topographies indicated by the shaded cells of Table 2 in this 

chapter (except for the FTC protocol, which has already been 

conducted). These represent the most common and the extremes of 

reported topographies. The reason for testing under conditions 

reflecting the extremes is to examine the effects on yield of 

using rapid and slow puffing, and large and small puff volumes. 

It is under these conditions that unexpected changes in smoke 

yield and composition are most likely to occur. The smoke 

consitutents should also be analyzed for any cell that resulted 

in a tar level that was substantially higher than the average for 

the entire matrix. Smoke constituents should be reported as a 

fraction of the tar generated, for example nicotine/g of tar. 

If the smoke produced using the FTC method did not yield 

The toxic chemical constituents in Table 3 of Chapter D 

If the ranges of the constituents generated using the 

smoking profiles defined by this matrix is within or below the 

range for existing commercial brands of cigarettes, the candidate 

cigarette may proceed to the biological testing stage. If one or 

more of the constituents exceeded the range for current brands of 

cigarettes, a risk benefit analysis which includes the other 

constituents and the reduction in ignition potential may decide 

whether to reject the cigarette or to proceed with biological 

testing. 
C13 

2. Biological testing 

Biological testing, described in Chapters E and F, can be 

conducted utilizing mainstream and sidestream smoke generated 

using the FTC method and the topographies specified by any cell 

in the matrix in Table 2 where the ratio of toxic constituents to 

tar content is statistically significantly greater than the mean 

value for all of the tested cells (see section of machine testing 

above). The results from the tests of the low ignition brands 

can be compared with the range of the results available from 

testing of existing commercial brands. An increase in the 

toxicity of the candidate brand over the range of results for 

currently marketed brands may be considered an increase in the 

health hazard attributable to the candidate brand. 

3. Additives 

A complete list of the levels of additives in the candidate 

cigarette brand and the probable identities of associated 

pyrolysis products should be reported. If the toxicity data on 

these additives and pyrolysis products are not available, 

additional toxicity testing should be performed. 

11. Characterization of topography in humans 

Topography studies in humans may be conducted when the 

initial evaluation of the candidate brand indicates it may be no 

more hazardous than current commercial brands of cigarettes 

(considering both disease risks and ignition potential). Human 

studies would define the actual topography of smoking that occurs 

with a new low ignition-potential cigarette. It would also 

collect data on the associated markers of smoke absorption, which 

are needed for determination of human risk. 

Volunteers should be selected to represent a balanced 

gender, socioeconomic, and ethnicfracial distribution. 

Additionally, the group should be selected to equally represent 

smokers who smoke the full range of the number of cigarettes per 

day of cigarette brands with nicotine yields (FTC method) in the 

top, middle and lowest thirds of the current brands of cigarettes 

by market share. In order to be confident that there is adequate 

representation of each of these factors in the study, at least 

200 smokers should be selected for each study group. 

Data collected before and after switching to the candidate 

brand include smoking topography, cigarettes smoked per day, 

urinary cotinine, and CO levels in the breath before and after 

smoking a candidate cigarette. The volunteers would be allowed 

to acclimatize to the candidate brand for two to three weeks. 

Mean values of the markers of smoke absorption that exceed the 

range reported for current cigarettes or increases in mean levels 
C14 

that result from switching to the prototype brand would be 

evaluated for toxicity concerns. 

should then be compared to the matrix in Table 2 of this chapter. 

If the results are significantly above or below the ranges in the 

table, then chemical analysis and toxicity testing must be 

repeated using the topographies that were outside the matrix. 

The range of smoking topography observed in the groups 
C15 

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c2 1 

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C25 

Table 2 

I Matrix for Machine Measurement of Tohaeco Smoke Yield 

Chapter D 

Analysis of Toxic Smoke Constituents 

Dietrich Hoffnann, Ph.D. 

American Health Foundation 

Valhalla, New York 10595-1599 

Prepared for the U.S. Consumer Product Safety Commission under contract 

CPSC-s-92-5473 

March 14, 1993 

Contents 

Introduction 

I. Toxic Agents in Tobacco Smoke 

11. Smoking Conditions 

111. Analysis of Individual Smoke Constituents 

1. Total Particulate Matter (TPM-dry) 

2. pH of Mainstream Smoke 

3. Redox Potential of Cigarette Smoke 

VAPOR PHASE 

4. Carbon Monoxide 

5. Nitrogen Oxides (NO,) 

6. Hydrogen Cyanide 

7. Volatile Hydrocarbons 

8. Aldehydes 

9. Volatile N-Nitrosamines (VNA) 

PARTICULATE MATTER 

10. Nicotine 

11. Phenols 

12. Catechols 

13. Polynuclear Aromatic Hydrocarbons (PAH) 

14. Tobacco-Specific N-Nitrosamines (TSNA) 

IV. Postscript 

Acknowledgements 

References 

Tables 

Figure Legend 

Figures 

Glossary 

Cost Estimates 

Introduction 

The chemical analytical evaluation of several key 

constituents of cigarette smoke is a useful predictor of 

potential toxic andfor tumorigenic activity of the combustion 

products of cigarettes. 

for the determination of select known toxic and tumorigenic 

agents in tobacco smoke as well as the standardized analytical 

procedures that can be applied to the evaluation of the vapor 

phase and particulate matter of cigarette smoke. 

This chapter describes the methodology 

Although sidestream smoke (SS) of cigarettes is a major 

contributor to environmental tobacco smoke, and the emission of 

SS constituents from prototype cigarettes may raise some health 

concerns, this chapter does not specifically address the overall 

systematic analytical elucidation of sidestream smoke 

composition, yet it highlights some compounds in SS that are 

relevant to health concerns. 

The burning of cigarettes generates mainstream smoke (MS) 

during puff drawing and SS during smoldering between puffs. The 

physicochemical nature of these smoke types is dependent upon 

factors such as the type of tobacco, the temperatures prevailing 

during puff-drawing (860-9OO0C) and smoldering (50O-65O0C), the 

reducing atmosphere that is characteristic of the burning cone, 

and the physical design of the cigarette (e.g., length, diameter, 

filter tip, and type of cigarette paper). These different 

parameters also influence the ignition propensity of a cigarette. 

The 400-500 mg of mainstream smoke that are freshly emerging 

from the mouthpiece of a cigarette are an aerosol that contains 

about 1~10'~ particles per milliliter in the vapor phase (1). The 

range in diameter is 0.1-1.0 pm with a mean of about 0.2 pm. 

About 95% of the MS effluent of a nonfilter cigarette is 

comprised of 400-500 individual gaseous compounds with nitrogen, 

oxygen and carbon dioxide as major constituents. Until now, at 

least 3500 individual compounds have been identified in the 

particulate matter (Figure 1; 2, 3). 

For chemical analysis, MS is arbitrarily separated into 

vapor phase and particulate phase. Individual compounds of which 

more than 50% appear in the vapor phase of fresh MS are 

considered volatile smoke constituents; all others are 

particulate phase components (Figure 1). Tables 1 and 2 list the 

major types of compounds identified and their estimated 

concentrations in the smoke of one nonfilter cigarette (4). All 

data were derived from machine-smoking of cigarettes under 

standardized laboratory conditions (5). [Machine smoking does 

not necessarily reflect the range of human smoking behavior 

characteristics (6-8, Topography chapter).] 
D2 

Tables 1 and 2 do not contain information about the presence 

of agricultural chemicals and pesticides that originate from the 

residue of such compounds on the tobacco (9). These residues 

differ in respect to chemical nature, concentrations, and type of 

agricultural chemicals used in the various tobacco-growing 

countries, and they may vary from season to season (10). The 

list also lacks information on flavor additives, because the 

nature and composition of such agents remain trade secrets. One 

exception among flavor additives is menthol of which up to 500 

ug/cigarette may be found in MS (11). 

Tobacco is known to contain at least 30 metals (12). In 

general, less than 1% of the metals is transferred from the 

tobacco into the MS of a cigarette (13). The levels of these 

elements are very low and thus are not listed in Table 2. 

However, the formulation of cigarettes with lower ignition 

propensity may include metallic additives. Therefore, it may be 

advisable to consider the analysis of added metals if they are 

suspected of having biological significance. 

I. Toxic Asents in Tobacco Smoke 

Hundreds of studies have been concerned with the chemical 

nature and quantitative aspects of toxic and tumorigenic agents 

in the mainstream smoke of cigarettes. Table 3 lists those toxic 

agents that have been most extensively studied and are considered 

to be major contributors to the toxicity of the smoke (14-16). 

This listing is not complete; however, the concentrations of all 

of these compounds give sufficient indication of the overall 

toxicity and tumorigenicity of the MS of a particular cigarette 

relative to a control cigarette, or to other commercial 

cigarettes. The possible presence and effects of additives to 

the tobacco or paper and their corresponding combustion products 

in the MS should be considered. The determination of such 

additives and their combustion products in the MS and SS may 

require specific methods. 

The yields of particulate matter in undiluted SS are 1.3-1.9 

times higher than those in the MS of cigarettes. About 30-40 

compounds have been identified and quantified in the particulate 

matter of SS. Nicotine levels in undiluted SS are 2-4 times 

higher than in MS. However, SS yields of aromatic amines exceed 

those in MS 20-to-30-fold. Undiluted SS also contains remarkably 

high levels of ammonia and of N-nitrosodimethylamine and 

N-nitrosopyrrolidine (Table 4). The greater release of the trace 

metals into SS might point toward increased releases of metallic 

additives into environmental smoke. 

SS which is generated during smoldering of the cigarette, is 

the major contributor to environmental tobacco smoke (ETS) (or 

secondhand smoke). Minor contributions to ETS are made by vapors 
D3 

diffusing through the cigarette paper, smoke escaping from the 

mouthpiece or burning cone, and exhaled smoke. Table 5 presents 

some of the data for toxic agents reported in indoor 

environments. 

11. Smokins Conditions 

The analysis of most of the individual smoke compounds 

requires 20 cigarettes or less. Cigarettes should be selected, 

prepared, and smoked individually by the standard smoking 

conditions established by the Federal Trade Commission in 1969 as 

modified in 1979 (22), described in Chapter B, unless human data 

(Chapter C) indicates otherwise. Additionally, cigarettes within 

f 20 mg of the average weight of 200 cigarettes should be 

selected for analysis. In the case of filter cigarettes, draw 

resistance must be within f 5% of the average of the 

weight-selected cigarettes (14). Quantitative assessment of most 

of the individual smoke compounds requires 20 cigarettes or even 

less. 

When analyses require 20 cigarettes or less, piston-type 

smoking machines (Chapter B), such as the 20-channel Phipps and 

Bird smoker, should be employed (23). When the analysis requires 

more than 20 cigarettes, a constant-volume-constant-time smoking 

machine, such as the Borgwaldt-20 smoker with rotating head, may 

be preferred (Chapter B). [The latter is available in the US 

through International Planters Corp., Borgwaldt Division, P.O. 

Box 24505, Richmond VA, 23224; phone 804-230-0011]. The machines 

with rotating head are preferred because they allow flushing with 

nitrogen after each puff in procedures where avoidance of 

artifacts is important. They also require less space and are 

easy to operate. The determination of each smoke constituent 

should be completed in duplicate or triplicate. 

111. Analysis of Individual Smoke Constituents 

The undesirable effects of cigarette smoking relate to the 

exposure to toxic, ciliatoxic, tumor-initiating and 

tumor-promoting agents and to organ-specific carcinogens. The 

selection of key compounds in cigarette smoke that are to be 

determined analytically for the evaluation of the toxic and 

tumorigenic potential of cigarette smoke is guided by knowledge 

on the proven toxic and/or tumorigenic effects in bioassays 

(Tables 3 and 4). The extent to which these same agents are 

toxic and/or tumorigenic to humans is not always known but can be 

deduced from interpretation of their probable activities made by 

the International Agency for Cancer Research (IARC Monograph 

Series, Vols. 1-53; 1972-1991). 
D4 

The carcinogenicities of tobacco and tobacco smoke in 

bioassays as well as the tobacco-related cancers in human 

epidemiologic studies are dose-related. Human risk assessment 

therefore depends not only on the relative biologic potency of 

individual or total tobacco carcinogens, but also on the 

quantitative aspects of exposure as these relate to personal 

smoking habits. Since smoking of cigarettes by machines mimics 

yet never completely duplicates the smoking patterns of 

individuals, the analytical evaluation of key components of 

tobacco smoke by standardized methods provides primarily valid 

comparisons of the relative toxic and tumorigenic potential of 

one cigarette brand over another. 

It is proposed that MS should be analyzed for 14 parameters. 

These are listed below. The cited analytical methods are known 

to be reproducible within a given laboratory. However, only 

methods for total particulate matter (“tarlt), carbon monoxide, 

and nicotine are standardized. Methods for these three were 

compared in collaborative studies between laboratories. 

Measures should be taken to avoid artifacts that might occur 

during cigarette smoke analysis, e.g. aging of smoke resulting in 

the conversion of NO to NO,, nitrosamine formation during 

trapping, degradation of carbonyl chemicals by secondary 

reactions, etc. Where practical, at least two methods are 

suggested for each parameter. 

1. Total Particulate Matter (TPM-dry) 

The major carcinogenic activity of tobacco smoke resides in 

the particulate matter. Therefore, the total yield of 

particulate matter in the smoke of a given cigarette is a key 

determinant of its carcinogenic potential. However, the vapor 

phase does contain additional toxins and tumorigenic agents which 

must be determined for a complete assessment of inherent risk. 

solutions or elemental analysis are not recommended. Those 

methods are not sufficiently specific to be associated with the 

carcinogenic activity of TPM. 

content should be based on the FTC-method (22), described in 

detail in Chapter B. Reproducibility of the data will be 

confirmed by including a University of Kentucky standard 

reference cigarette during the analyses. 

Macro-methods of assessment, such as fluorescence of TPM 

The method for TPM determination, including its moisture 
D5 

2. pH of Mainstream Smoke 

Nicotine, the major habituating agent and an important toxic 

compound in tobacco smoke (19, 24), is protonated in the MS of 

U.S. blended cigarettes and of cigarettes that are made entirely 

with bright tobacco, because the smoke of these cigarettes rarely 

exceeds pH 6.2 (18). Higher smoke pH increases the toxicity. At 

pH above 8.0, which occurs in smoke from cigarettes made entirely 

with black or burley tobacco, more unprotonated nicotine is 

present in the vapor phase. This tends to raise blood pressure 

far more rapidly and to a greater extent than does the protonated 

(bound) nicotine in the smoke (24, 25). Thus, the pH of the 

mainstream smoke should be measured. 

Two methods are usually followed for determining smoke pH. 

The method developed by Sensabaugh and Cundiff (26) and applied 

by others (18) provides for measurement of the pH of individual 

puffs. In this case, single cigarettes are smoked by a 

piston-type machine. Each puff is led over a modified electrode; 

the latter is connected to a pH-meter (Beckman Model SS-2) and 

the signals are recorded by a strip-chart recorder (18). The 

test is run in triplicate to obtain representative values. The 

advantage of this method lies in its ability to record the pH of 

each puff from the first to the last puff. This method has been 

utilized primarily for research; it requires constant 

recalibration and maintenance. 

Grob describes another method in which three cigarettes are 

machine-smoked under standard conditions through a 40-ml glass 

tube that is loosely filled with cotton (27). After the smoking, 

the cotton is washed with 40 ml C0,-free distilled water and 

subsequently twice more with 10 ml water. The pH of the combined 

water extracts is measured with a pH meter resulting in an 

average value from all puffs of a cigarette. 

The average pH of a cigarette can also be crudely measured 

by smoking a cigarette puff by puff through 2 interconnected 

impingers filled with 10 ml C0,-free distilled water each (total 

free volume 40 ml). After 2 or 3 cigarettes have been smoked, a 

clearing puff is taken, the contents of the impingers are 

combined with 20 ml of water used for rinsing the impingers and, 

after filtration, the pH of the whole smoke is measured. To 

obtain representative pH values this test has to be done in 

triplicate. 

large-scale determinations of the smoke pH of cigarettes. The 

device is reported to deliver reproducible, average pH values 

within 20 minutes (28). 

A smoking machine-extractor device has been developed for 
D6 

3. Redox Potential of Cigarette Smoke 

It has been demonstrated that the reducing property of 

cigarette smoke adversely affects the respiration of the 

mammalian cell. Specifically, the redox potential of the smoke 

of a given tobacco product is correlated with the degree of 

cytochrome C reduction (29, 30). It is known that the redox 

potential in each puff of cigarette smoke gradually declines from 

the 230-240 mV initially measured for air with the reference 

calomel electrode to the 140-160 mV in the smoke of the last 

puff. Smoking a cigarette through a glass fiber filter 

demonstrates that the gaseous phase is practically free of 

reducing agents and that the latter reside primarily in the 

particulate phase. Ammonia and hydroquinone do not contribute to 

the redox potential of cigarette smoke, and nicotine does so only 

to a minor extent. 1,4-Benzoquinone, naphthoquinones and 

anthraquinones appear to have a stabilizing effect on the redox 

potential of cigarette smoke (31, 32), thus diminishing the 

reducing effect of cigarette smoke on subcellular components of 

the mammalian cell. 

To determine the redox potential of individual puffs, a 

piston-type smoking machine is connected with an apparatus 

containing a platinum (Pt) electrode which operates in 

conjunction with a reference calomel electrode (Figure 2; 32). 

The smoke is led over the Pt electrode only. Figure 3 shows the 

redox potential of 85-mm US blended non-filter cigarettes for 

puffs 1 through 8 (32). 

VaDor Phase 

4. Carbon Monoxide 

The toxic burden of environmental carbon monoxide exposure 

is of special significance to cardiovascular health. The 

methodology for the determination of carbon monoxide in cigarette 

smoke is described in Chapter B. 

5. Nitrogen Oxides (NO,) 

It is suspected that nitrogen oxides (NO,) in cigarette 

smoke contribute to the development of pulmonary emphysema (30, 

33) and the acceleration of platelet aggregation (34). They are 

potent inhibitors of thiol-dependent enzymes (35) and, at high 

concentration, they can induce bradycardia and arrhythmias (36). 

In addition, NO, in cigarette smoke may prevent the activation of 

scavenger cells such as macrophages in the respiratory system. 

NO, in cigarette smoke are also of concern because of their 

potential to nitrosate precursor amines and thus contribute to 

the formation of carcinogenic N-nitrosamines. 
D7 

Cigarette smoke contains 3 forms of NO,. These are nitrous 

oxide (N,O; (1 pgfcigarette), nitrogen dioxide (NO,; <10 

pgfcigarette) and nitric oxide (NO; 6-600 pgfcigarette). The 

fresh smoke of a US non-filter cigarette contains 200-300 pg NO, 

depending on the nitrate content of the tobacco, which represents 

the major precursor for the nitrogen oxides in smoke (37). It is 

important for the analytical chemist to realize that unaged MS 

contains hardly any NO,, but only NO, and that the latter 

oxidizes quickly to NO, (half-life in smoke, 6-10 minutes; 38, 

39). This is of major consequence since NO, is essential for the 

toxicity and the N-nitrosamine formation in cigarette smoke 

(section 111-9). 

Several methods have been employed for determining NO, in 

cigarette smoke. These include the widely used colorimetry 

(40-42), gas chromatography (43), nitrate ion electrode (44), 

infrared (39), and chemiluminescence (45, 46). 

The colorimetric method is based on the Saltzman procedure 

(41). It involves the Griess reagent which reacts only with NO,. 

For cigarette smoke analysis, NO must first undergo oxidation to 

NO,. Sloan and Morie have discussed the shortcomings of the 

Saltzman method for the analysis of NO, in cigarette smoke in 

great detail (44). 

The preferred method for the determination of NO, in 

cigarette smoke is by chemiluminescence. In this technique 

nitric oxide is measured by photoelectric amplification the 

chemiluminescent reaction of NO with ozone. NO, (NO+NO,) are 

measured by photoelectric amplification of the chemiluminescent 

reaction of NO, and atomic oxygen, which is derived from thermal 

decomposition of 0,. 

For chemilumniscent analysis, cigarettes are selected by 

weight and draw resistance and are smoked by a piston-type 

smoking machine through a Cambridge filter. The gas phase of 

each individual puff is directed through a gas sampling valve. 

An aliquot of the puff is injected into a GC column filled with a 

100f200 mesh porous polymer (Chromosorb 104). The column 

temperature is set at 45OC. Argon, the carrier gas, is adjusted 

to a flow rate of 15 mlfmin. The column exit is connected with a 

chemiluminescence detector (Figure 4). For each NO, analysis, 4 

cigarettes have to be smoked individually; this results in an 

experimental deviation for commercial US blended cigarettes of 

f6% (detection limit = 0.5-1.0 fig NO per puff). The British 

Tobacco Research Council, London, refined the chemiluminescence 

method for NO, in cigarette smoke and adopted it as a standard 

method (47). 
D8 

6. Hydrogen Cyanide 

Hydrogen cyanide (HCN) is an inhibitor of several 

respiratory enzymes; as such it can influence cellular metabolism 

in the myocardial and arterial wall. As a major ciliatoxic agent 

in cigarette smoke HCN greatly inhibits the clearance of tar 

components from the respiratory tract (19). Nitrate is a major 

precursor for HCN in the smoke (48), even though tobacco proteins 

are also precursors for HCN in smoke (49). 

include ion-selective electrode titration, gas chromatography and 

coulometric methods (50). With one exception all methods measure 

cyanogen [(CN),] as part of HCN. However, (CN), in the smoke of a 

cigarette amounts to less than 2.5% of the total HCN (51). To 

separate HCN from (CN),, the smoke of individual puffs of the 

cigarette is directed through a Cambridge filter and subsequently 

through gas wash bottles containing 100 ml 0.1N NaOH. When 

loaded with the smoke of 1 cigarette, the Cambridge filter is 

extracted with 100 ml 0.1N NaOH, washed, and combined with the 

solution from the gas wash bottles (total volume including 

washings 250 ml). A 1-ml aliquot is pipetted into a mixture of 2 

ml 1.0 M NaH,PO, with 1 ml chloramine-T solution and 20 ml 

n-hexane; it is then thorough1 mixed. One ~1 of the n-hexane 

layer is analyzed by GLC with 'Ni-EC detector (51). The cyanogen 

chloride, formed by the reaction of HCN with chloramine-T gives a 

distinct peak which is clearly separated in the GC. The relative 

standard deviation of this HCN method is less than 5&, the 

detection limit is 50 ng HCN per cigarette. (Modification of the 

method by smoking more than 1 cigarette can greatly increase the 

detection limit). 

Coulometric analysis for hydrogen cyanide by the method of 

Sloan (50) is recommended. It requires machine-smoking of 2 or 

more cigarettes through a scrubber containing 50 ml 0.1 N NaOH 

and through a Cambridge filter. Aliquots of the lVsmoked" 

scrubber solution plus washings are transferred to a titration 

cell which contains pH 11 buffer solution and 0.001 M lead 

acetate solution. The generator electrodes are connected to a 

coulometer, and the indicator electrodes are connected to the 

terminals of a potentiometer. The output of the potentiometer is 

connected to a strip-chart recorder. HCN that is not retained in 

the scrubber solution but trapped by the Cambridge filter is 

extracted with 0.1 NaOH and an aliquot is titrated 

coulometrically as discussed for the aqueous scrubber solution. 

The two sets of data are combined and compared with data obtained 

from the other more involved methods. The standard deviation for 

this simple and rapid method for HCN in cigarette smoke was less 

than 6% (50). 

The methods developed for HCN analysis in cigarette smoke 
D9 

7. Volatile Hydrocarbons 

The gaseous phase of cigarette smoke has been shown to 

contain about 20-25 alkanes, up to 20 alkenes, some alkynes such 

as acetylene, a number of dienes, especially 1,3-butadiene and 

isoprene, and in addition to benzene, up to 30 volatile aromatic 

hydrocarbons (2, 3, 59). Together these hydrocarbons constitute 

0.5-1.0% of the weight of the total mainstream smoke effluent of 

a cigarette. Despite this, few of them have been discussed as 

possible contributors to the toxicity of the smoke. 

The exceptions are 1,3-butadiene and benzene. In inhalation 

studies in mice and rats, 1,3-butadiene is carcinogenic; however, 

the International Agency for Research on Cancer considers "the 

evidence for carcinogenicity of 1,3-butadiene to humans as 

inadequate" (17). Benzene, on the other hand, is a recognized 

occupational carcinogen, which upon long-term exposure increases 

the risk of workers for various types of leukemia (17). Case 

control studies and large-scale prospective follow-up studies 

have shown an association between cigarette smoking and leukemia 

especially myeloid leukemia (52-54). There is a correlation in 

cigarette smokers between urinary cotinine, a major metabolite of 

nicotine, and urinary trans,trans-muconic acid, a metabolite of 

benzene (55). 

Rapid advances in chemical-analytical instrumentation have 

led to capillary GC-MS methods which enable the investigator to 

determine quantitatively within minutes dozens of volatile 

components in the vapor phase of only a fraction of individual 

puffs of a cigarette (56-58). These instruments are primarily 

helpful for research, they require great expertise; the methods 

are time consuming and generally not suitable for routine 

analyses of toxic hydrocarbons in the gaseous phase of cigarette 

smoke. 

The following GC-MSD method is suggested for routine 

analysis of 1,3-butadiene, isoprene, benzene, and toluene. The 

gas phase of individual puffs from freshly generated mainstream 

smoke of selected cigarettes (section 11) is led through a 1.0 ml 

sample loop. Upon reaching atmospheric conditions the sample 

loop is switched in line with gas chromatographic (GC) columns, 

first passing through a 5 m x 0.53 mm HP-1 precolumn and then 

onto a 30 m x 0.25 mm DB-5 analytical column. The effluent of 

the analytical column is diluted by the carrier gas, helium 1:25, 

and the emerging peaks representing individual volatile smoke 

components are determined by mass selective detection (GC-MSD). 

The advantage of GC-MSD is that volatiles that were masked 

by the GC peak of a hydrocarbon in a regular GC-MS trace can be 

eliminated by selective ion monitoring and thus allow accurate 

quantification. The disadvantage of this method lies in the 

fact, that the hydrocarbons to be assayed have to be determined 
D10 

for each individual puff separately in order to avoid losses 

during aging of the smoke. This is especially so for 

1,3-butadiene (conventional smoking of a cigarette requires in 

general 8-12 puffs or 7-11 minutes before an aliquot of all puffs 

can be analyzed). For routine analysis, especially when 

comparing different cigarettes, the seventh puff is chosen as a 

representative puff of the cigarette (59). 

8. Aldehydes 

Formaldehyde, acrolein, and acetaldehyde together with 

hydrogen cyanide are the major ciliatoxic agents in cigarette 

smoke and are known irritants to the mucous membranes of the 

upper respiratory system and to the eyes. Upon inhalation they 

clearly contribute to the inhibition of lung clearance 

mechanisms, thus allowing extrinsic particles, such as tobacco 

smoke particulates, to settle (21, 60). Formaldehyde and 

acetaldehyde are known animal carcinogens. The International 

Agency for Research on Cancer regards formaldehyde as "probably 

carcinogenic to humans", acetaldehyde as "possibly carcinogenic 

to humanst' and acrolein as "not classifiable as to its 

carcinogenicity to humansgt (17). 

individually through an 800-ml Xjeldahl flask containing a trap 

with 200 ml of saturated solution of 2,4-dinitrophenylhydrazine 

(DNPH) in 0.2 N HC1. After the smoking, the trapping solutions 

are extracted repeatedly with chloroform and triphenylene is 

added as an internal standard. The combined chloroform solutions 

are washed twice with 2 E HC1, then twice with water, and are 

then concentrated under a stream of nitrogen. The dried residue 

is dissolved in 5 ml methylene chloride. Aliquots are injected 

into a HPLC system which is described in great detail (61). The 

recovery rate is reported as better than 90% and the 

reproducibility better than 5%. 

Formaldehyde is determined by smoking 2 cigarettes 

Acetaldehyde, acrolein, and propionaldehyde may be analyzed 

by the method of Manning (62) at Oak Ridge National Laboratory. 

The cigarettes are individually smoked and the volatile aldehydes 

are trapped by reacting them with DNPH in 2 HC1 solution. The 

concentrates of the 2,4-dinitrophenylhydrazones of the aldehydes 

are separated and analyzed by reverse phase HPLC with the 

absorbance detector at 365 nm. The detection limit is 10 pg 

aldehyde/cigarette, the relative standard deviation is about 12%. 

9. Volatile N-Nitrosamines (VNA) 

Volatile N-nitrosamines in cigarette smoke originate from 

the tobacco by transfer into the smoke, and from thermal 

degradation of nitrosamino acids, as well as from pyrosynthesis 

during smoking. For example, during tobacco processing proline 

is nitrosated to N-nitrosoproline (NPRO); its yield in the 
D11 

tobacco is greatly influenced by the processing of the tobacco 

and by its nitrate concentration. 

to some N-nitrosopyrrolidine (NPYR) in MS (0.1-1%) and to much 

higher yields of NPYR in SS (20). Model studies have shown that 

volatile secondary amines can be nitrosated to nitrosamines in 

the vapor phase, however, the yields are low because freshly 

generated smoke contains primarily NO and only traces of NO,. 

The latter is essential since the nitrosating agent for the 

formation of nitrosamines is N,O, (64). 

During smoking NPRO gives rise 

All 8 VNA identified in cigarette smoke (Figure 5) are 

organ-specific carcinogens in animals (65). For routine analyses 

usually only the 3 major VNA in cigarette smoke are quantitated. 

These are N-nitrosodimethylamine (NDMA; 0-75 nglcigarette), 

N-nitrosodiethylamine (NDEA; 0-5 nglcigarette) and 

N-nitrosopyrrolidine (5-40 nglcigarette; 20). 

filter tips or 3 times 20-40 filter cigarettes; these have to be 

smoked individually under standard laboratory conditions (section 

11). [Cellulose acetate filter tips selectively remove VNA by 

70% and more; thus, the VNA analysis in the smoke of filter 

cigarettes requires 20 cigarettes] (63, 66). The MS is led 

through a gas wash bottle containing 100 ml citrate-phosphate 

buffer, pH 4.5, with 20 mM ascorbic acid and an internal standard 

([14C]NDMA or N-nitrosodipropylamine). A Cambridge filter treated 

with a solution of ascorbic acid, is placed between gas wash 

bottle and smoking machine (after smoking 10 cigarettes the 

loaded Cambridge filter is replaced). The loaded Cambridge 

filter is thoroughly washed with dichloromethane (CH,Cl,) and the 

washings are filtered. The buffer solution in the gas wash 

bottle is extracted 4 times with 100 ml CH,C12, all 

organic-extracts are washed with 2 NaOH (to remove interfering 

nitroalkanes), dried (Na,SO,) and concentrated to 5 ml. The 

concentrate is chromatographed on 65 g basic alumina (Woelm, 

activity 11-111). The VNA are eluted from the column with 200 ml 

CH2C1,, vacuum concentrated to about 1-2 ml and measured by 

GC-thermal energy analysis (63). The recovery rate is better 

than 70%. The detection limit is 0.05 ng NDMA per injection; the 

deviation coefficient for NDMA and NPYR is f 5%; for NDEA it is 

The MS analysis requires 3 times 10-20 cigarettes without 

up to * 10% 

Particulate Matter 

10. Nicotine 

The standard FTC-method for nicotine, the main pharmacologic 

agent in cigarette smoke (22), is described in Chapter B. 
D12 

11. Phenols 

More than 40 semivolatile phenols have been identified in 

cigarette smoke (3). The major precursors for these phenols in 

tobacco smoke are glucose, polysaccharides, pectins, rutin and 

other polyphenols. Minor amounts of semivolatile phenols that 

were formed in the tobacco during processing transfer into the 

smoke (14). It has been reported that the nonfilter 85-mm 

cigarette, made entirely from bright tobacco, delivered in the 

mainstream smoke 95 pg phenol, one made from Turkish tobacco 

yielded 120 pg, and from Maryland tobacco 60 pg; a burley tobacco 

cigarette produced 43 pg, and a US blend delivered 100 pg phenol 

(14). 

major role in the yields of volatile phenols in cigarette smoke. 

These findings indicate that the type of tobacco plays a 

The volatile phenols contribute significantly to the tumor 

promoting activity of cigarette tar (14, 67, 68) and are active 

as ciliatoxic agents (21). The latter effect is one of 

inhibition or temporary paralysis of the mucus clearance that is 

normally provided by the ciliated epithelium of the respiratory 

tract. Ciliastasis allows foreign particles to remain in the 

respiratory tract where they can exert their particular activity 

or allow other agents to impair physiologic or biochemical 

functions. 

The preferred analytical method for volatile phenols is gas 

chromatography of the weakly acidic portion of cigarette smoke 

condensate (69-71). Twenty to 40 cigarettes are smoked 

individually through a gas wash flask containing 2N NaOH with an 

internal standard (e.g. 2-chlorophenol, ['4C]phen~l) and a 

Cambridge filter. The "loaded" Cambridge filter is extracted 

with 2N NaOH, filtered, combined with the NaOH solution from the 

gas wash bottle and the washings. This combined NaOH solution is 

extracted 3 times with ether to liberate the phenol concentrate 

obtained by solvent extractions without the need for 

concentrating by water steam distillation (72). 

Cellulose acetate filter types, and especially those with 

specific plasticizers and with perforated filter tips reduce 

volatile phenols highly selectively (up to 85%); therefore, in 

some cases more than 40 or even 60 cigarettes are required for 

each analysis. 

12. Catechols 

The most abundant phenolic component in cigarette smoke is 

catechol (1,2-dihydroxybenzene; 80-400 pg/cigarette). Although 1 

g of processed tobacco contains microgram levels of catechol, 

most of the catechol in the smoke is formed during the burning of 

tobacco from cellulose, monosaccharides, chlorogenic acid and 

pectins as precursors (73). Cigarette smoke also contains small 

. . -. . 
D13 

amounts of alkylated catechols including 3-methylcatechol (<20 

pgjcigarette), 4-methylcatechol (-20 pgjcigarette) and 

4-ethylcatechol (<25 pgjcigarette; 74). 

cocarcinogen in tobacco smoke. Upon co-application with 

benzo(a)pyrene or with other carcinogenic polynuclear aromatic 

hydrocarbons (PAH), it greatly enhances the carcinogenic activity 

of these agents (75, 76). 

Catechol is not a carcinogen but a very effective 

Two methods are primarily employed in the analysis of 

catechol, 1) enrichment of catechol from tar by distribution 

between solvent pairs, followed by a spectrophotometric method 

(77), and 2) enrichment of the catechols from the acidic fraction 

of cigarette tar by extraction with boric acid, followed by GLC 

(74). Both methods are simple, require only 20-40 cigarettes per 

analysis and are reproducible, when an internal standard is used 

(e.9. [“C]catechol), within f 6%. 

Schlotzhauer (72) enriched the dihydroxybenzenes, catechols, 

resorcinols and hydroquinones from the weakly acidic fraction by 

gel filtration chromatography and analyzed the catechol 

concentrate by GC-MS. This method found catechol and six 

alkylcatechols, as well as other dihydroxybenzenes. It is used 

for the profile analysis of these types of chemicals in cigarette 

smoke. 

13. Polynuclear Aromatic Hydrocarbons (PAH) 

Inhalation studies with laboratory animals have demonstrated 

that the particulate matter of tobacco smoke induces malignant 

tumors of the respiratory tract, most notably in the larynx of 

the Syrian golden hamster (16, 79, 80). The particulate phase is 

much more carcinogenic than the gas phase (79). Fractions and 

subfractions of the particulate matter have been extensively 

assayed for tumorigenicity on rabbit skin and on mouse skin. It 

has been clearly demonstrated that the most tumorigenic fractions 

in these assays are those with highly concentrated PAH (14, 68). 

However, the carcinogenic activity of cigarette smoke 

particulates cannot be explained by the presence of carcinogenic 

PAH alone. When PAH concentrates of the neutral fraction (<1% of 

whole tar) are combined with the tumor-promoting weakly acidic 

fraction, which by itself does not induce tumors, the tumor yield 

on mouse skin reaches 70-90% of the carcinogenic activity 

observed with the whole tar (68, 81). Thus, the PAH serve as 

tumor initiators. To date about 80-100 PAHs have been identified 

in cigarette smoke (most <10 ng cigarette). A PAH concentrate of 

the neutral fraction of cigarette smoke condensate was the only 

portion that induced squamous tumors in the lung of rats upon 

intratracheal instillation (82). Benzo(a)pyrene (BaP) , a major 
D14 

carcinogenic PAH in smoke (20-40 nglcigarette), induced tumors in 

the hamster lung upon inhalation (83). 

The International Agency for Research on Cancer regards 11 

PAH and 3 nitrogen-containing PAH (aza-arenes) as established 

animal carcinogens. BaP, benz(a)anthracene and 

dibenz(a,h)anthracene are rated as "probably carcinogenic to 

humans" (Fig. 6; 17). 

in cigarette smoke (14, 84). Often the PAH are enriched by 

distribution of cigarette tar between solvent pairs such as 

methanol-water (4:l) and cyclohexane followed by a second 

partition between cyclohexane and nitromethane which leads to a 

10-fold enrichment of the PAHs (81). The PAHs are further 

concentrated by column chromatography followed by paper 

chromatography, or TLC; the individual PAH are then identified 

and quantitated by W-spectrophotometry (85). 

A great many studies were concerned with the analysis of PAH 

During the last 2 decades the final step of PAH analysis 

relies on capillary GC (86). Using an internal standard (e.g. 

[I4C] BaP) the recovery is better than 70%. The reproducibility 

for the major PAH (>5 nglcigarette) is f 8% with at least 100 

cigarettes. Several PAH profile studies of the MS of nonfilter 

cigarettes showed the highest PAH yields for the smoke of 

ciqarettes made entirely with bright tobacco (BaP= 35-53 

nglcigarette) and lowest PAH yields for cigarettes made entirely 

with burley tobacco (BaP= 20-24 nglcigarette; 14, 18). 

Most of the carcinogenic PAH in cigarette smoke (>go%) are 

pyrosynthesized via highly reactive C,H-radicals (14, 84) which 

result from thermal degradation of nonvolatile organic tobacco 

components. Since BaP is one of the most abundant carcinogenic 

PAH in cigarette smoke and its mechanism of formation is similar 

to the pyrosynthesis of other PAH, BaP is often quantitated as a 

monitor for the entire class of compounds in the smoke of a given 

cigarette. For this purpose, several quick methods for the 

analysis of BaP were developed recently (87, 88). Five to 10 

cigarettes are smoked through a Cambridge filter assembly. One 

ml of cyclohexane for each 1 mg of TPM is used to agitate the 

mixture of the filter and tar for 1 hour. After filtration, the 

volume is reduced to 10 ml by rotary evaporation. Following 

refiltration through a 0.45 pm membrane filter, a 2 ml aliquot is 

chromatographed through a NH,-Sep-Pak column, pre-conditioned 

with 10 ml hexane. The BaP fraction is eluted with 8 ml of 

hexane, evaporated to dryness, redissolved in 1 ml of 

acetonitrile and analyzed by reverse-phase HPLC using an 

isocratic solvent system (65% acetonitrile in water) and 

fluorescence detector (excitation = 289 nm; emission = 412 nm); 

benzo(e)pyrene served as internal standard (88). 
D15 

14. Tobacco-Specific N-Nitrosamines (TSNA) 

The TSNA are exclusively formed from nicotine and the minor 

Nicotiana alkaloids during tobacco processing and during smoking. 

So far 7 TSNA have been identified (Figure 7). Four of these are 

usually determined in cigarette smoke. These include the 

powerful organ-specific carcinogens N'-nitrosonornicotine (NNN) 

and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK). In 

mice, rats and hamsters these TSNA induce benign and malignant 

tumors of the lung, upper aerodigestive tract, pancreas and/or 

liver. 

N'-nitrosoanabasine (NAB) and the non-carcinogenic 

Nr-nitrosoanatabine (NAT; 89). 

The other two major TSNA are the weakly carcinogenic 

To determine the four major TSNA in cigarette mainstream 

smoke, 3 x 20 weight-selected cigarettes are smoked individually 

under standard laboratory conditions (section 11; in the case of 

filter cigarettes selection must also be done according to 

average draw-resistance). The mainstream smoke is retained on a 

Cambridge filter (9.0 cm diameter) which is treated with a 

solution of ascorbic acid (90). The filter assembly is placed 

between the smoking machine and two gas wash bottles in line, 

each containing 60 ml distilled water to which 2 ml of 20% 

ammonium sulfamate solution in 3.6 sulfuric acid is added, and 

also containing 0.5 pg [14C]NNN as an internal standard (others 

have used N-nitrosodibenzylamine, N-nitrosopentylpicolylamine or 

2-(methylnitrosamino)-l-(2-pyridyl)ethane; 91, 92). The total 

particulate matter trapped on the Cambridge filters is extracted 

twice with 100 ml ethyl acetate and the combined buffer solutions 

of the wash bottles are extracted with 3 times 100 ml ethyl 

acetate. The ethyl acetate extracts are dried (Na,S04), 

concentrated to about 2 ml and chromatographed on 50 g basic 

alumina (Woelm, activity 11 to 111) on a 2 x 20 cm column with 

150 ml dichloromethane and a 4:l mixture of dichloromethane: 

acetone (200 ml). The latter solvent mixture eluates the TSNA 

which are concentrated to 1-2 ml (recovery rate of [I4C]NNN= 75- 

85%). 

The method of Adams (93) is recommended for the gas 

chromatography-thermal energy analysis (GC-TEA). The specific 

detector for NO-containing substances assures clear separation of 

NAB from NAT. The TSNA values are determined in a triplicate 

analysis within ? 7% (detection limit = 1 ng of a single TSNA per 

cigarette). Other analytical methods for TSNA were recently 

reported (20, 92). 

IV. Postscript 

It was the goal of this chapter to suggest analysis of those 

tobacco smoke parameters and smoke components that are considered 
D16 

major contributors to the toxicity of cigarette smoke and are 

likely to be quantitatively affected by modifications that might 

reduce the ignition propensity of cigarettes. Athough this 

proposal is based on longstanding experience in the tobacco 

sciences and on a thorough study of the literature, it is not 

comprehensive for all toxicants which may occur in cigarette 

smoke. It was deemed important to keep the number of 

measurements practical. 

For example, the determination of polonium-210 (2'%o) was 

not suggested, although the U.S. National Council on Radiation 

Protection and Measurement ascribed about 1% of the risk of lung 

cancer in long-term cigarette smokers to 210Po (94). Polonium-210 

in the smoke originates from the tobacco by transfer; a change in 

the make-up of a cigarette will not greatly alter the 'loPo 

concentration in the smoke (0.03-1.0 pCi "'Pofcigarette; 14). 

Similarly, analysis of nickel (0.1-0.6 pg/cigarette) or 

cadmium (<0.5 lgfcigarette) was not suggested even though the 

International Agency for Research on Cancer considers these 

elements as "carcinogenic to humans" and "probably carcinogenic 

to humans", respectively (17). As discussed earlier (section I), 

inorganic additives, including silicates, that are added to the 

tobacco or paper to reduce ignition propensity must be determined 

in the smoke in order to ascertain that the toxicity of the smoke 

is not increased. 

Cigarette smoke also contains traces of a few known human 

carcinogens (17), such as 4-aminobiphenyl (2.4 ngfcigarette; 95), 

2-naphthylamine (1.0 ngfcigarette; 95) and vinyl chloride (5-16 

nglcigarette; 96). Because these compounds are present in minute 

amounts, and analytical methods required for their determination 

are rather involved, we have not included the determination of 

these three chemicals in the overall analysis of toxic 

constituents. However, the analytical profiling of cigarette 

smoke can be extended to include these andfor any other agents, 

deemed to be of significance in respect to human health. 

Acknowledaments 

Our research program in tobacco carcinogenesis is supported by U.S. 

National Institutes of Health grant No. CA-29580. The editorial assistance by 

Ilse Hoffmann and Laura Disciorio is greatly appreciated. 
D17 

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D26 

Table 1 

smoke of nonfilter cigarettes 

Major constituents of the vapor phase of the mainstream 

Compound 

Nitrogen 

oxygen 

Carbon dioxide 

Carbon monoxide 

Water 

Argon 

Hydrogen 

Ammonia 

Nitrogen oxides (NOx) 

Hydrogen cyanide 

Hydrogen sulfide 

Methane 

Other volatile alkanes (20)' 

Volatile alkenes (16) 

Isoprene 

Butadiene 

Acetylene 

Benzene 

Toluene 

Styrene 

Other vol. aromatic hydrocarbons 

Formic acid 

Acetic acid 

Propionic acid 

Methyl formate 

Other volatile acids (6) 

Formaldehyde 

Acetaldehyde 

Acrolein 

Other volatile aldehydes (6) 

Acetone 

Other volatile ketones (3) 

Methanol 

Other volatile alcohols (7) 

Acetonitrile 

Other volatile nitriles (10) 

Furan 

Other volatile furans (4) 

Pyridine 

Picolines (3) 

3-Vinylpyridine 

Other volatile pyridines (25) 

Pyrrole 

Pyrrolidine 

N-Methylpyrrolidine 

concentrationjcigarette 

(% of total effluent) 

280 - 320 mg (56-64%) 

50 - 70 mg (11-14%) 

45 - 65 mg (9-13%) 

14 - 23 mg (2.8-4.6%) 

7 - 12 mg (1.4-2.4%) 

5 mg (1.0%) 

0.5 - 1.0 mg 

10 - 130 pg 

100 - 600 pg 

400 - 500 pg 

20 - 90 pg 

1.0 - 2.0 mg 

1.0 - 1.6 mgb 

0.4 - 0.5 mg 

0.2 - 0.4 mg 

25 - 40 fig 

20 - 35 fig 

12 - 50 pg 

20 - 60 fig 

10 I.L9 

(29) 15 - 30 pg 

200 - 600 pg 

300 - 1700 pg 

100 - 300 pg 

20 - 30 fig 

5 - 10 pgb 

20 - 100 pg 

400 -1400 pg 

60 - 140 pg 

80 - 140 pg 

100 - 650 pg 

50 - 100 pg 

80 - 180 pg 

10 - 30 pgb 

100 - 150 pg 

50 - 80 figb 

20 - 40 pg 

45 - 125 pgb 

20 - 200 pg 

15 - 80 pg 

10 - 30 pg 

20 - 50 pgb 

0.1 - 10 pg 

10 - 18 pg 

2.0 - 3.0 pg 

. 
D2 7 

Volatile pyrazines (18) 3.0 - 8.0 pg 

Methylamine 4 - 10 pg 

Other aliphatic amines (32) 3 - 10 jig 

a Numbers in parentheses represent the individual compounds 

identified in a given group. 

Estimate 
D28 

Table 2 

mainstream smoke of nonfilter cigarettes 

Compound pglcigarette 

Nornicotine 

Major constituents of the particulate matter of the 

Nicotine 1000 - 3000 

Anatabine 5 - 15 

50 - 150 

Anabasine 5 - 12 

n-Hentriacotane [ n-C,,H,] 100 

Napthalene 2 - 4 

Phenanthrenes (7) 0.2 - 0.4c 

Other tobacco alkaloids (17)' n.a. 

Bipyridyls (4) 

Total nonvolatile hydrocarbons (45)' 

10 - 30 

300 - 400' 

Napthalenes (23) 3 - 6' 

Anthracenes (5) 0.05 - 0.1' 

0.6 - 1.0' 

0.3 - 0.5' 

Fluorenes (7) 

Pyrenes (6) 

Carcinogenic polynuclear aromatic hydrocarbons (ll)b 0.1 - 0.25 

Phenol 80 - 160 

Other phenols (45)' 60 - 180' 

Catechol 200 - 400 

Other catechols (4) 100 - 200c 

Other dihydroxybenzenes (10) 200 - 400' 

Scopoletin 15 - 30 

Other polyphenols (8)' n.a. 

Cyclotenes (10) 40 - 70' 

Quinones (7) 0.5 

Solanesol 600 - 1000 

Neophytadines (4) 200 - 350 

Limonene 30 - 60 

Fluoranthenes (5) 0.3 - 0.45' 

Other terpenes (200-250)' n.a. 

Palmitic acid 

Oleic acid 

Linoleic acid 

Linolenic acid 

100 - 150 

40 - 110 

150 - 250 

150 - 250 

Stearic acid 50 - 75 

Lactic acid 60 - 80 

Indole 10 - 15 

Skatole 12 - 16 

Other indoles (13) n.a. 

Quinolines (7) 2 - 4 

Other aza-arenes (55) n.a. 

Benzofurans (4) 

Other 0-heterocyclic compounds (42) n.a. 

Stigmasterol 40 - 70 

Sitosterol 30 - 40 

Campesterol 20 - 30 

200 - 300 

Cholesterol 10 - 20 
D29 

Anline 

Toludines 

Other aromatic amines (12) 

Tobacco-specific N-nitrosamines (6)b 

Glycerol 

0.36 

0.23 

0.25 

0.34 - 2.7 

120 

Number in parentheses represent individual compounds 

identified. 

For details, see Figure 6. 

Estimate. 

n.a. Not available. 
D30 

Table 3. Compounds That Contribute To The Toxicity 

Of Cigarette Mainstream smoke of US Nonfilter Cigarettes 

Compound ~ 

Yield/CigaretteToxic Effects 

Range 

1. Total Particulate Matter 

(TPM, dry) 

2. pH 

3. Redox Potential 

Vavor Phase 

4. Carbon Monoxide 

5. Nitrogen Oxides (NO,) 

6. Hydrogen Cyanide 

7. Hydrocarbons 

benzene 

1,3-butadiene 

8. Aldehydes 

formaldehyde 

acrolein 

acetaldehyde 

Carcinogen' 

12-40 mg 

Influences nicotine toxicityb 

Influences toxicity of whole smokec 

14-23 mg 

100-600 pg 

400-500 pg 

12-50 pg 

25-40 pg 

20-100 pg 

400-1400 fig 

Reacts with hemoglobin, inhibits 0, 

transport' 

Nitrosating agentd, inhibitor of 

thiol dependent enzymes" 

Ciliatoxic"; inhibitor of 

respiratory enzymes' 

Suspected or known carcinogens' 

Ciliatoxic", animal carcinogens' 

9. Volatile N-Nitrosamines (VNA) Strong animal carcinogens" 

N-nitrosodimethylamine 13-65 ng 

N-Nitrosopyrrolidine 7-34 ng 

Particulate Matter 

10. Nicotine 

11. Phenols 

phenol 

other phenols 

1-3 mg Associated with cardiovascular 

disease' 

Tumor promoters 

80-160 pg 

60-180 pg 

12. Catechol 200-400 pg Major cocarcinogen 

13. Polynuclear Aromatic Hydrocarbons Major tumor initiators' 

benzo (a) pyrene 20-60 ng 
D3 1 

14. Tobacco-Specific N-Nitrosamines Strong organ-specific animal 

NNN' 80-90 ng carcinogens' 

"K' 60-470 ng 

"According to the International Agency for Cancer Research (17) TPM and 

benzene are human carcinogens, formaldehyde, benzo(a)pyrene and some other 

PAH as well as some volatile N-nitrosamines are probably carcinogenic to 

humans and acetaldehyde and the tobacco-specific N-nitrosamines are 

possibly carcinogenic to humans. 

Brunnemann and Hoffmann, 1974 (18). 

U.S. Surgeon General, 1983 (19). 

Brunnemann and Hoffmann, 1991 (20). 

Battista, 1976 (21). 

* NNN - "-Nitrosonornicotine 

* NNK - 4-(Methylnitrosamino)-l-(3-pyridyl)-l-butanone 
D3 2 

Table 4. Bome toxic and tumorigenic agents in undiluted cigarette 

sidestream smoke 

Compound Type of Amount in sidestream Sidestream: 

toxicity smoke per cigarette mainstream smoke 

ratio 

Vapor phase 

Ammonia 

Carbon monoxide 

Carbonyl sulfide 

Benzene 

Formaldehyde 

3-Vinylpyridine 

Hydrogen cyanide 

Hydrazine 

Nitrogen oxides 

N-Nitrosodimethy 

N-Nitrosopyrroli 

T 

T 

T 

C 

C 

sc 

T 

C 

NO,) T 

amine C 

ine C 

Particulate ,phase 

Tar C 

Nicotine T 

Phenol TP 

Catechol COC 

o-Toluidine C 

2-Napthylamine C 

4-Aminobiphenyl C 

Benz[a]anthracene C 

Benzo[a]pyrene C 

Quinoline C 

NNN C 

NNK C 

N-Nitrosodiethanolamine C 

Cadmium C 

Nickel C 

Zinc T 

~olonium-210 C 

50.0 - 130 pg 

26.8 - 61 mg 

2-3w 

240 - 290 pg 

330 - 450 pg 

14 - 110 pg 

500 - 2000 pg 

1500 pg 

90 ng 

200 - 1040 ng 

30 - 390 ng 

14 - 30 mg 

2.1 - 46 mg 

70 - 250 pg 

58 - 290 pg 

3 P9 

70 ng 

140 ng 

40 - 200 ng 

40 - 70 ng 

15 - 20 pg 

0.15 - 1.7 pg 

0.2 - 1.4 pg 

43 ng 

0.72 pg 

0.2 - 2.5 pg 

6.0 ng 

0.5 - 1.6 pCi 

40 - 170 

2-5 - 14.9 

0.03 - 0.13 

8 - 10 

50 

24 - 34 

0.06 - 0.4 

3 

3.7 - 12.8 

20 - 130 

6 - 120 

1.1 - 15.7 

1.3 - 21 

1.3 - 3.0 

0.67 - 12.8 

18.7 

39 

31 

2 - 4 

2.5 - 20 

8 - 11 

0.5 - 5.0 

1.0 - 22 

1.2 

7.2 

13 - 30 

6.7 

1.06 - 3.7 

C, Carcinogenic; CoC, cocarcinogenic; SC, suspected carcinogen; T, toxic; 

TP, tumor promoter. 

NNN - N'-Nitrosonornicotine 

NNK - 4-(Methylnitrosamino)-l-(3-pyridyl)-l-butanone 
D3 3 

Table 5. Some toxic and tumorigenic agents in indoor environments polluted 

by tobacco smoke" 

Pollutant 

Nitric oxide 

Nitrogen dioxide 

Hydrogen cyanide 

Benzene 

Formaldehyde 

Acrolein 

Acetone 

Phenols (volatile) 

N-Nitrosodimethylamine 

N-Nitrosodiethylamine 

Nicotine 

Benzo[a]pyrene 

NNN 

NNK 

Location 

Workrooms 

Restaurants 

Bar 

Cafeteria 

Workrooms 

Restaurants 

Bar 

Cafeteria 

Living room 

Public places 

Living room 

Public places 

Public places 

Coffee houses 

Restaurant, public place 

Restaurant, public place 

Public places 

Restaurants 

Workrooms 

Restaurant, public place 

Public places 

Public places 

~oncentrationjm~ 

50 - 440 pg 

17 - 270 pg 

80 - 520 pg 

2.5 - 48 pg 

68 - 410 pg 

40 - 190 pg 

2 - 116 pg 

67 - 200 fig 

8 - 122 pg 

20 - 317 pg 

23 - 50 pg 

30 - 120 pg 

360 - 5800 pg 

7.4 - 11.5 ng 

0 - 240 ng 

0 - 200 ng 

1- 6 Pg 

3 - 10 pg 

1- 13.8 pg 

1.8- 22.8 pg 

1.4- 29.3 pg 

3.3 - 23.4 ng 

'References: Klus and Kuhn (97); IARC (17); US National Research Council 

(98); Klus et al. (99); Brunnemann (100). 

NNN - N'-Nitrosonornicotine 

NNK - 4-(Methylnitrosamino)-l-(3-pyridyl)-l-butanone 
D3 4 

Lesends to Ficrures 

1. Total Cigarette Smoke Compostition [% w/w] (2) 

2. Approaches for Measuring Redox Potential of Cigarette Smoke 

(30) 

3. Redox Potential of Puffs 1 to 8 of an 85 mm Plain U.S. 

Cigarette (30) 

4. Diagram of Thermal Energy Analyzer (20) 

5. Structures of Volatile N-Nitrosamines in Cigarette Smoke 

6. Carcinogenic Polynuclear Aromatic Hydrocarbons in Cigarette 

7. Formation of Tobacco-Specific N-Nitrosamines (20) 

(20) 

Smoke (16, 17) 

Voltmeter 

-cTo Smoking 

Modine PC Electrode 

Aemsol 

Chamber 

Cigarette 

.Calomel 

Electrode 

Figure 2 Appamtua for Meaaaring Max Potmtial of CigarrttC Smoke (30) 
> 

E 

C0NTROf:COMMERClAL 85mm, 

NON-FILTER US. CIGARETTE- z 

0 WHOLE SMOKE. 

180 

W 

a 

140 

Figure 3 Redm Potential of Puffs 1 to 8 of M 85 mm Plain US. Cigarette (30) 
I 

03 NO - Nq-N%+hu 

Figure 6 Carcinogenic Polynuclear Aromatic Hydrocarbans and hArares in Qarette Smoke (16,17) 

@g 0 

@@@ 
1 

Reduction fY 

D3 5 

Glossary of Terms 

carcinogen 

ciliatoxic agent 

cocarcinogen 

complete carcinogen 

draw resistance 

flavor additives 

mainstream smoke 

Substance that results in the 

production of tumors 

Substance that inhibits the 

movement of cilia in the 

mucus-secreting respiratory 

epithelium. Total inhibition of 

such movement ciliastasis) 

prevents lung clearance and leads 

to accumulation of foreign 

substances in the respiratory 

airways. 

Not a carcinogen by itself but 

potentiates the activity of a 

carcinogen when co-administered. 

A carcinogen with both tumor 

initiating and tumor promoting 

activity. 

Pressure developed by the full 

length of a cigarette when air is 

drawn through at a rate of 17.5 

ml/sec (2OoC, 760 torr). 

Expressed as inches (or nun) of 

water column. 

Plant extracts or synthesized 

chemicals that are added to the 

tobacco to impart flavor to the 

smoke. 

For analytical purposes and for 

collecting the smoke in a closed 

system, mainstream is the smoke 

issuing from the mouth end of a 

cigarette. For the smoker, 

mainstream is the smoke that is 

drawn from the mouth end of a 

cigarette during puffing. 

nonvolatile smoke constituents 

All particulate phase 

constituents. 

Exerts carcinogenic activity in 

host tissues that have appropriate 

organ-specific carcinogen 
D3 6 

activating enzymes; acts on 

specific organs regardless of the 

route of application. 

Tipping paper, opaque-white paper, 

or cork wrapped around the filter 

and the cigarette rod to join 

both. 

Tiny holes in cigarette paper or 

filter wrapper increases air 

permeability. Can be made by 

mechanical or electrostatic 

methods or by laser beams. 

Potential for electrobiochemical 

reduction/oxidation. 

For analytical purposes, 

sidestream is that part of the 

smoke of a cigarette that emits 

from the burning end during puff 

intervals and that diffuses 

through the paper. In freesmoking situations, some 

sidestream smoke exits from the 

mouthpiece during puff intervals. 

overwrap 

perforation 

redox potential 

sidestream smoke 

total particulate matter (TPM) 

Fraction of smoke collected on a 

Cambridge (glass fiber) filter in 

machine smoking. 

TPM dry 

toxic agent 

tumorigenic agent 

tumor initiator 

tumor promoter 

Total particulate matter minus 

water and minus nicotine. 

Adversely pharmacoactive 

substance, for example, nicotine. 

Substance known to elicit 

neoplasms in animal assays. 

A substance or an active 

metabolite that forms chemical 

lesions with DNA having potential 

to develop into benign and 

malignant neoplasms. 

An agent that facilitates tumor 

development in an initiated cell 

when applied subsequent to 

initiation. 
D37 

vapor phase (gas phase) The fraction of tobacco smoke that 

passes through a Cambridge filter. 

Contains some condensable smoke 

particulates that have not been 

retained on the glass fiber 

filter . 

volatile smoke constituent Chemical having more than 50% in 

the vapor phase. 
D38 

Estimated Coats For the Analysis of 

Individual Cigarette Smoke 

Smoke Component Eatimated Coat 

Total Particulate Matter, dry 

PH 

Redox Potential 

Carbon Monoxide 

Nitrogen Oxides 

Hydrogen Cyanide 

Volatile Hydrocarbons 

(esp. 1,3-Butadiene, Isoprene, Benzene, Toluene) 

Volatile Aldehydes 

Volatile N-Nitrosamines 

Nicotine (by GC) 

Phenols 

Catechols 

Polynuclar Aromatic Hydrocarbons (GC) 

Benzo (a) pyrene only 

Tobacco-Specific N-Nitrosamines 

$350 

$250 

$500 

$250 

$400 

$350 

$600 

$700 

$800 

$250 

$500 

$350 

$1,500 

$500 

$800 

'These estimates pertain to the direct cost of each determination 

(duplicate analyses) for one brand or prototype of cigarette. 

They exclude overhead as approved for the individual institute by 

the U.S. Dept. of Health and Human Services. 

'For practical purposes, we recommend that analytical profiles of 

the smoke of cigarettes are done for at least two different 

brands or prototypes and one reference cigarette at any time. 
Chapter E 

SHORT-TERM TESTS FOR THE EVALUATION OF CIGARETTE 

SMOKE TOXICITY 

C. Gary Gairola, Ph.D. 

Division of Pharmacology and Experimental Therapeutics 

College of Pharmacy 

University of Kentucky 

Lexington, KY 40546-0236 

Prepared for the U.S. Consumer Product Safety Commission 

Under Contract #S-92-5474 

March 16, 1993 

Mailing Address: 

130 Tobacco and Health Research Institute 

University of Kentucky 

Lexington, KY 40546-0236 
. . . . 
Chapter Contents 

1. Introduction 

2. General Consideration 

3. Recommended Tests 

3.a. Tests for Evaluating Genotoxicity of Smoke Condensates 

i) Test 1: Ames’ Salmonella tvphimurium Mutagenicity 

Assay 

Culture Maintenance and Growing Conditions 

Preparation of Rat Liver S-9 

Preparation of S-9 Mix 

Assay Procedure 

Experimental Design 

Data Evaluation and Interpretation 

Limitations of the Assay 

ii) Test 2: Mammalian Cell Transformation Assay 

Cell Culture Methods 

Cytotoxicity Assay 

Cell Transformation Assay 

Data Evaluation and Interpretation 

Limitations of the Assay 

3.b. Tests for Evaluating the Genotoxicity of Fresh Whole 

Cigarette Smoke Genotoxicity 

3.c. Tests for Evaluating Pulmonary Inflammatory Response to 

Fresh Whole Cigarette Smoke in Animals 

i) 

ii) 

Rat ionale 

Bioassay procedure 

Animal Model 

Smoke Exposure Protocol 

Indicators of Smoke Exposure 

Bronchoalveolar Lavage 

Assessment of BAL Cells 

Data Evaluation and Interpretation 

Limitations of the Bioassay 

4. Estimates of Cost and Duration of Tests 

5. Other Toxicological Endpoints Associated with Smoke Exposure 

References 

Glossary 

El 

1. Introduction: 

Several epidemiological and experimental studies have 

implicated cigarette smoking with the increased incidence of a 

variety of human diseases. Major health consequences of smoking 

have been discussed in Chapter A. Also the physicochemical 

complexity of tobacco smoke and its major toxic constituents have 

been described in Chapter D. The main aim of this Chapter is to 

describe a testing strategy for comparing the toxicity of low 

ignition propensity cigarette prototypes using short-term tests. 

Cigarette smoke is a complex aerosol which is composed of 

gaseous and particulate phases and contains thousands of 

different types of compounds. Any alteration in cigarette design 

and/or composition is likely to alter the physical and chemical 

characteristics of smoke and thereby its potential to cause 

toxicity. Some examples of such alterations have been described 

in Chapter A. To provide a comparison of toxicity of different 

cigarette prototypes, it is important that the tests selected for 

their toxicological evaluation are quantitative and have 

relevance to smoking-related health consequences described in 

Chapter A. 

have been developed for routine assessment of the toxicity of 

chemicals. The main focus of these tests has been the 

identification of potential carcinogens in the environment. As a 

result of such testing, large databases of chemical genotoxicity 

and carcinogenicity have been developed by the National Toxicity 

Program, US Environmental Protection Agency, and IPCS (Parodi and 

Waters 1991). Analyses of these data have demonstrated a 

relationship between genotoxicity and carcinogenicity of 

chemicals (Huff and Haseman 1991, Tennant 1991, Parodi et al. 

During the past several years, a number of short-term tests 

1991). 

Currently, there are several genotoxicity tests that are 

used for identification of chemical carcinogens. These tests can 

be generally classified into four categ0ries;tests for detecting 

(i) gene mutations, (ii) chromosomal aberrations, (iii) primary 

DNA damage, and (iv) induction of mammalian cell transformation 

(Brusick 1987). Many of the tests from these four categories 

have been applied to genotoxic assessment of cigarette smoke and 

its condensates. These tests demonstrate that tobacco smoke 

possesses cytotoxic, mutagenic and carcinogenic activities 

(DeMarini 1983, Hoffmann et al. 1987). 

A plan for the evaluation of toxicity of low ignition 

potential cigarettes is presented. Two types of tests are 

proposed. The prokaryotic Ames' Salmonella mutagenicity assay 

and a eukaryotic mammalian cell transformation assay are included 

to assess the genotoxic potential of the test cigarette smoke 

condensates. An inflammatory lung cell response bioassay is 
E2 

proposed to assess the general pulmonary toxicity of the whole 

smoke from cigarette prototypes. These three tests should be 

included in a reasonable evaluation of test cigarette smoke 

toxicity. 

2. General Considerations: 

There are three types of smoke preparations that are used 

for vitro testing of cigarette smoke: i) condensate 

collected by freezing smoke in a cold trap, ii) smoke 

particulates collected at room temperature on Cambridge filters, 

and iii) freshly generated whole smoke or its gas phase. The 

first two preparations have been used quite often as a 

solution/suspension in DMSO or acetone for testing in various 

short-term tests (DeMarini 1983). The use of fresh whole smoke 

for vitro testing has been limited. 

Since most biological assays have baseline noise,it is 

important that the toxicity of test cigarettes be compared to 

known reference compounds and cigarettes that serve as positive 

control. Compounds like benzo(a)pyrene or 3-methylcholanthrene 

and the University of Kentucky reference research cigarettes are 

included in each experiment to serve as references for comparison 

of test cigarettes. In our hands, the condensates from the 

University of Kentucky reference cigarettes have given fairly 

consistent response in the Ames' Salmonella assay and has allowed 

identification of condensates of low and high mutagenic activity 

(Gairola 1979). 

nicotine delivery that are available from the University of 

Kentucky Tobacco and Health Research Institute (Davis et al. 

1984). These cigarettes were developed to minimize the 

experimental variability introduced by the use of commercial 

cigarettes in analytical and biological studies of smoke (Benner 

1970) and have been used worldwide for experimental studies of 

tobacco smoke. Two newer research cigarettes, designated 1R4F and 

1R5F, were manufactured in the 1980s and contain 0.8 and 0.16 mg 

nicotinelcigarette, respectively. These two cigarettes probably 

represent low and ultralow nicotine cigarettes currently sold on 

the market. 

Standard reference materials for complex environmental 

There are several research cigarettes of different tar and 

mixtures developed by the National Institute of Standards and 

Technology (May et al. 1992) may also be useful as reference 

materials, but have not been tested in conjunction with tobacco 

smoke studies. 

3. Recommended Tests: 
E3 

The Salmonella mutagenicity assay and a mammalian cell 

transformation assay (C3H/lOT1/2 or BALB/3T3) are proposed for 

routine testing of genotoxicity of smoke condensates. To 

evaluate general pulmonary toxicity of whole smoke, a lung 

inflammatory cell response assay in mice has been proposed. Gene 

mutation and cell transformation assays are selected because of 

the high relevance of these two genotoxic endpoints in the 

overall carcinogenic process and tobacco smoke carcinogenesis in 

particular, which is known to involve the action of both tumor 

initiators and tumor promoters (Hoffmann and Wynder 1971, 

Melikian et al. 1989). 

The Ames Salmonella assay is a gene mutation assay which 

quantitatively measures the ability of the test condensates to 

induce specific point mutations in a prokaryotic bacterial 

system. Since mutational events are generally believed to be 

essential for the initiation phase of carcinogenesis, this test 

may provide an estimation of the potential tumor initiators in 

tobacco smoke preparations. 

Mammalian cell transformation assays use eukaryotic 

mammalian cell culture systems to measure the potential of smoke 

to induce malignant cell transformation. Cell transformation is 

a multistep process and is considered a close approximation of 

events occurring during in vivo oncogenesis. This assay may, 

therefore, measure the activity of both tumor initiators and 

promoters. Past studies have shown that cigarette smoke 

condensates give a positive response in both of these tests (Kier 

et a1.1974, Benedict et al. 1975). A good dose response 

relationship between the amount of test preparation and genotoxic 

response is generally observed in at least the Salmonella assay. 

Selection of the bronchoalveolar lavage (BAL) cell response 

assay for pulmonary toxicity is based on the observation that 

chronic inflammatory conditions exist in the lungs of smokers and 

the BAL cells obtained from smokers exhibit altered 

characterstics which play an important role in the development 

of chronic obstructive pulmonary diseases associated with 

cigarette smoking (Hunninghake et al. 1979, Niewoehner 1988). 

Studies in the animal models have also demonstrated several 

alterations in the lungs of mice that resemble those reported in 

human smokers (Matulionis 1984, Gairola 1986). 

This test, however, has never been employed to differentiate the 

toxicity of different types of cigarettes and may, therefore, 

need developmental work before routine use in toxicity 

evaluation. 

3.a. Tests for Evaluatina Genotoxicitv of Smoke Condensates: 

The Ames Salmonella assay can be performed on 

dimethylsulfoxide solutions of freshly collected particulates 

from 1-5 test cigarettes. The particulates are collected on 0.3 
E4 

pm Cambridge filters (Wartman et al. 1959), as noted in Chapter 

B. Larger quantities of condensates, needed for transformation 

assays, are collected in cold traps. The condensates are 

suspended in acetone for testing in mammalian cell assays. 

Test 1: AMES' SALMONELLA TYPHIMURIUM MUTAGENICITY ASSAY: 

Ames' Salmonella assay is the most widely used mutagenicity 

assay which has been used to predict the carcinogenicity of pure 

chemicals and complex mixtures (Ashby and Tennant 1991, Claxton 

et al. 1992). This is a simple assay which is easy to perform and 

provides results within a relatively short period of time. The 

test employs genetically constructed strains of a bacterium, 

Salmonella twhimurium, to detect the ability of chemicals to 

induce gene mutations. The tester strains possess defined 

mutations which prevent them from growing in a medium that does 

not contain an essential aminoacid, histidine. Upon treatment 

with a mutagenic chemical, some of these mutants revert back to 

their wild type form and regain capacity to grow in the absence 

of histidine (Ames et al. 1975). This simple feature of the test 

allows detection of chemicals that interact with bacterial DNA to 

cause reverse mutations. Up to five strains of S.tvvhimurium have 

been used for routine testing of chemicals. 

The test is performed by mixing the low-histidine top agar, 

tester strain, test compound, and the S-9 mix in a sterile tube. 

The mixture is poured into petridishes containing bottom agar and 

the dishes are incubated at 37' C for 48-72 hrs. Revertant 

colonies are scored and the data are analysed. The assay 

procedure has been described in detail by Ames and coworkers 

(Ames, McCann and Yamasaki, 1975, Maron and Ames, 1983). 

and TA100, provide an adequate evaluation of condensate 

mutagenicity ( Mizusaki et al. 1977, Sat0 et al. 1977, Gairola 

1979, Yoshida and Matsumoto 1980). The most sensitive strains for 

condensate evaluation are strains TA1538 and TA98 (DeMarini 

1983), which detect those substances that induce frameshift types 

of mutations. Since genetic backgrounds of these two strains are 

basically the same and TA98 is somewhat more sensitive due to the 

presence of a plasmid, use of the latter strain is recommended. 

Strain TA1535 and TAlOO which detect chemicals inducing base 

substitution types of mutations also give a positive response for 

smoke condensates and are especially useful for assaying the 

levels of direct mutagens eg.,nitrate-rich tobacco smoke 

condensates (Kier et al. 1974, Sugimura et a1.1977). In view of 

increased concern for the role of oxy-radicals in tobacco 

carcinogenesis (Church and Pryor 1985), a newer tester strain TA 

102 may also be considered in the evaluation of condensate 

mutagenicity. This strain has been successfully used to detect 

the mutagenicity of oxidizing agents (Levin et al. 1982) and some 

Past studies indicate that two strains of S.tvvhimurium, TA98 
E5 

metal species that act via oxidative mechanisms, e.g., selenite, 

(Kramer and Ames 1988). However, data on the mutagenic evaluation 

of smoke condensates using strain TA102 are presently not 

available. Also this strain has a high spontaneous reversion rate 

which may necessitate a feasibility study of this strain for 

evaluation of smoke condensate mutagenicity before its use for 

routine genotoxicity evalation. 

Aroclor-induced rat liver S-9 fraction to evaluate the presence 

of direct mutagens and those that require metabolic activation. 

In our experience, freshly prepared condensates give a near 

linear dose response curve when tested at concentrations of 

100-400 pglplate. If the condensates are stored for more than a 

week or two, a flattening effect is observed at higher 

concentrations, possibly due to cytotoxicity. 

A general description of the procedure is provided below: 

cultures are maintained as frozen stocks and are grown in Oxoid 

nutrient broth No. 2 to a density of 1-2 x lo9 cellslml. This is 

achieved by inoculating media flask with a tester strain and 

incubating at 37OC overnight as a stationary culture. Early next 

morning the cultures are placed in a gyrorotary incubator running 

at approximately 200 rpm at 37OC for 4-6 hrs. Once the cultures 

are ready they are stored on ice before use. 

b) Preparation of Rat Liver 8-9: Rat liver enzymes are 

induced with Aroclor 1254 suspension in corn oil (200 mglml). A 

single intraperitoneal injection of 500 mglkg body wt of rat is 

given 5 days prior to sacrifice. Rats are sacrificed by cervical 

dislocation and their livers are excised under sterile conditions 

for placing in ice cold sterile PBS. All the steps are performed 

at 0-4OC under sterile conditions. The liver is chopped into 

small pieces and homogenized in chilled 0.15 M KC1 using 3 ml of 

solution for each gm wet liver weight. The homogenate is 

centrifuged at 9,000G for 10 minutes and the supernatants are 

collected for storage in sterile plastic tubes at -8OOC. S-9 

preparations are standardized with respect to their cytochrome 

P-450 content and mixed function oxidase activity. 

Tests are performed in the absence and presence of 

a) Culture Maintenance and Growing Conditions: Bacterial 

c) Preparation of 8-9 mix: S-9 mix is composed of 100 mM 

phosphate buffer, pH 7.4, 8 mM magnesium chloride, 33 mM 

potassium chloride, 4 mM NADP and 0.05-0.1 ml of S-91ml of mix. 

The mix is made fresh for each experiment and can be stored on 

ice for the day without significant loss of activity. 

d) Assay Procedure: Bottom agar plates and top agar are 

prepared in advance as described by Moran and Ames (1983). On 

the day of the experiment, to the 100 ml of melted top agar, 10 

ml of a solution containing 0.5 mM histidine and 0.5 mM biotin 
E6 

are added and thoroughly mixed. This low histidine top agar is 

then dispensed at 2 mls per sterile 13 x 100 mm tube and 

maintained at 45OC in heating blocks. To each tube, the test 

sample and 0.1 ml of the tester strain culture with and without 

0.5 ml of S-9 mix are added and mixed before pouring onto plates 

containing 25 ml of bottom agar. The plates are incubated at 

37OC for 48 to 72 hours and the revertant colonies are counted to 

obtain the number of induced revertantslplate. 

e) Experimental Design: Each experiment should include 

negative (spontaneous revertants) and positive controls (one 

compound which is a direct mutagen and one which requires 

metabolic activation). A solvent control is also necessary. 

Four to 5 concentrations of each condensate sample with three 

replicates for each concentration are generally tested in each 

experiment. For reference cigarettes, 50 to 300 pg and 100 to 

500 pg condensate/plate are tested in the absence and presence of 

S-9 mix, respectively. Numbers of revertants/plate for each 

concentration are obtained by taking an average of three 

replicates for that concentration of the sample. The number of 

revertants vs. concentration of condensate/plate are plotted to 

obtain a dose-response curve. If necessary, log transformations 

of the response can be plotted against condensate concentration 

to obtain a dose-response curve. 

f) Data Evaluation and Interpretation: The data are 

generally presented as the number of induced revertants per 

plate, which are obtained by subtracting the number of 

spontaneous revertants from the total revertantslplate for each 

concentration of the test sample. When plots are developed for 

induced revertants vs. condensate concentration, a near linear 

dose-response curve is obtained; this is particularly common at 

lower concentrations. Specific activity of each condensate can 

be calculated from these plots. Specific activity is defined as 

the number of induced revertants/mg of condensate in the absence 

of metabolic activation, or the number of revertantslmg 

condensate, when metabolically activated with S-9 containing a 

given amount of cyt. P-450 or S-9 capable of metabolizing a given 

amount of benzo(a)pyrene/min/mg protein. Once the data have been 

generated for each of the test condensates, direct comparisons 

can be made to a reference or with each other to determine their 

mutagenic potential. Given the delivery of total particulate 

matter for each cigarette, the total number of 

revertantslcigarette can also be calculated to compare cigarette 

types. 

In the mutagenic evaluation of tobacco smoke condensates, it 

has been our experience and that of other's (Matsumoto et al. 

1977) that condensates generally do not show significant activity 

for direct mutagens, except for some experimental cigarettes, 

e.g., high nitrate tobacco cigarettes. Therefore, it is difficult 
E7 

to obtain a dose-response curve when condensates are tested in 

the absence of S-9s. 

g) Limitations of the Assay: Because of the high toxicity of 

fresh whole smoke to bacterial strains, the assay is limited to 

providing a genotoxic evaluation of only the particulate phase of 

cigarette smoke. Any changes in the constituents of gas phase 

resulting from low ignition modifications of cigarettes are, 

therefore, not evaluated in the plate incorporation version of 

this assay. 

organometallics, halogenated compounds, give negative results in 

the Ames' test (Ashby and Tennant 1988). If such chemicals are 

present in the low ignition potential cigarettes, their mutagenic 

activity will not be detected by the Ames' test. 

Test-2: EIAMMALIAN CELL TRANSFORMATION ASSAY: 

Certain mutagens and carcinogens, e.g., metallic salts, some 

A number of mammalian cell transformation assays have been 

used in the evaluation of genotoxicity of chemicals. C3H/lOT1/2 

mouse embryo, BALB/c 3T3 mouse fibroblast and Syrian hamster 

embryo (SHE) cell assay systems are the most commonly used assays 

for which reasonable baseline data exist (IARC/NCI/EPA 1985, IARC 

1985, Dunkel et a1.1991). Cell transformation has been defined 

as the induction of certain neoplasia-related phenotypic changes 

in the cultured cells. 

The most commonly examined endpoints in cell transformation 

assays have been the morphological alteration of cell colonies. 

Normally, cells in culture grow to form a confluent monolayer and 

then stop dividing when surrounded by the cells (contact 

inhibition). However, if the cells have been treated with a 

carcinogen, some cells continue to grow and form foci of 

transformed cells. These foci exhibit dense, haphazard 

overgrowth on the monolayer. When injected into appropriate host 

animals, the transformed cells form tumors, but the normal cells 

generally do not give rise to tumors. Because of their ability to 

form a tumor in the host, the cells from such foci are considered 

malignantly transformed (Landolph 1985). 

evaluation is based on a very low rate of spontaneous 

transformation of these cells in culture and positive results 

with smoke condensates (Benedict et al. 1975). Furthermore, 

C3H/10T1/2 and BALB/c 3T3 cells are continuous cell lines which 

give relatively more reproducible results than SHE cells, which 

are primary culture cells and are by nature variable. Since the 

main purpose of this testing program is to provide a quantitative 

comparison of the activity of different smoke condensates, it is 

desirable that the selected assays are reproducible. 

The selection of the C3H/lOT1/2 cell assay for condensate 
E8 

The theory that tumor initiator and tumor promoter 

activities of chemicals play important roles in the carcinogenic 

process reinforces the relevance of the cell transformation 

assay. Cigarette smoke condensates are known to contain both 

tumor initiators and promoters (Hoffman and Wynder 1971). 

Mammalian cell transformation assays can be performed with 

either C3H/lOT1/2 or BALB/c 3T3 cells. Detail test protocols for 

these assays have been described (IARC 1985). The basic features 

of the C3H/lOT1/2 assay procedure are described below which also 

apply to BALB/c 3T3 cell assay. 

Cell Transformation Assay: Transformation assays are 

performed essentially as described by Reznikoff et al., (1973) 

with some later modifications (Landolph 1985, Dunkel et al. 

1991). For each concentration of the test condensate, 30-40 60- 

mm petri dishes are seeded with 2 x lo3 cells. 

experiments a set of dishes for positive control 

(3-methylcholanthrene) and another set for solvent control are 

run in parallel. After 24 hr of incubation at 37OC in a 5% CO, 

atmosphere, appropriate concentrations of the test condensates in 

dimethylsulfoxide (DMSO) or acetone are added to the cultures. 

After another 24 hr incubation, the old medium is changed with 

fresh medium. Thereafter, the medium is replaced every fourth 

day until the cultures are confluent. Once the cultures have 

reached confluency, the medium is changed every seventh day with 

Eagle‘s basal medium (BME) containing 5% heat-inactivated fetal 

calf serum (FCS). The experiments are terminated after 6 weeks 

of incubation. The medium is removed and the cultures are washed 

with phosphate buffer solution (PBS), fixed with methanol, 

stained with Giemsa and scored for type 2 and type 3 foci of 

transformants (Reznikoff et al. 1973). 

In all 

Cell Culture Methods: Mouse embryo fibroblast cell line 

(C3H/lOT1/2), clone 8 is grown in BME supplemented with 10% heat 

inactivated FCS and 5 mg/ml gentamycin (Kennedy, 1985). It is 

important that several batches of sera are pretested for plating 

efficiency, cell attachment and transformation with 1 mg/ml 

3-methylcholanthrene prior to purchase to insure the consistency 

of the transformation assay. Only those lots that give a plating 

efficiency of over 20% are purchased for use in the assays. The 

C3H/lOT1/2 stock cultures are seeded at a density of 2 x lo4 

cells per 25 cm2 flask and grown in a humidified incubator in a 

5% CO,/air atmosphere at 37OC. Every 7 days, the stock cultures 

are passaged using trypsin (0.1% in PBS for 3 min) to detach the 

cells from the flask. After detachment, the cells are 

resuspended in complete BME, counted, and reseeded into 25 cm’ 

flasks. 
E9 

It is important that the cultures used for the assay are in 

early passage. For storage, the cells from the log phase of 

growth are harvested by trypsinization and suspended in 

antibiotic-free medium containing 10% FCS and 10% DMSO. The cell 

suspensions are frozen and stored in liquid nitrogen. 

advance and in parallel with the cell transformation assays. The 

information derived from the cytotoxicity experiments is 

necessary for running the transformation experiments and 

calculating the transformation frequencies. The toxicity of the 

test solution is assessed by determining the plating efficiency 

of untreated and treated cells in each experiment. Five 60-mm 

Petri dishes/group are seeded with 200 cells and treated with 

different concentrations of the test solution or the solvent in 

the same manner as described above for the cell transformation 

assay. After 10-12 days of culture, the cell monolayers are 

washed and stained. Colonies are counted and the plating 

efficiency, which is defined as the number of colonies formed as 

a percentage of the number of cells seeded per dish, is 

calculated. 

Cytotoxicity Assay: Cytotoxicity assays are performed in 

This assay is capable of detecting the cell transformation 

activity of 3-methylcholanthrene in the absence of a metabolic 

activation system (Reznikoff et al. 1973) and has also been 

reported positive for condensates in the absence of S-9 (Benedict 

et al. 1975). Presence of some metabolic activation capacity in 

C3H/lOT1/2 cells for polycyclic aromatic hydrocarbons has been 

demonstrated (Gehly and Heidelberger 1982). 

Data Evaluation and Interpretation: Data from these 

experiments are generally expressed as transformation 

frequencies, defined as the percentage of type 2 and type 3 foci, 

based on the number of survivors that form colonies in the 

dishes. The data generated can be plotted as % transformed 

colonies on a log scale vs the concentration of test condensate. 

Cytotoxicity data can also be plotted on the same graph to 

express percent cell survival in treated cultures. 

Transformation frequencies depend heavily on the number of 

survivors. The frequencies decrease if the surviving number of 

cells/dish is too high or too low. In view of this problem, 

tabulation is currently preferred of the number of type 2 and 

type 3 foci per total number of treated dishes and the total 

number of dishes containing type 2 and type 3 foci out of the 

total number of dishes treated. Both formats of data expression 

allow the development of a dose-response curve. 

When polycyclic aromatic hydrocarbons are tested in the 

assay, a dose response is usually obtained. Comparison of the 

transformation potential of smoke condensates from different test 

cigarettes will require tightly controlled conditions. In each 
E10 

experiment a reference compound or a reference condensate or both 

are run in parallel. The data from the test condensate can then 

be compared to the values for the reference substance in each 

experiment and a potency value assigned to each test condensate. 

These manipulations are necessary to minimize the impact of 

variability of response in the assay from experiment to 

experiment. 

fresh cigarette smoke to mammalian cells, C3H/lOT1/2 assay may 

also be limited to assessing the genotoxicity of cigarette smoke 

condensates. Any changes in gas phase constituents resulting from 

low ignition modifications of cigarettes will,therefore, not be 

evaluated in this assay. Very few studies of smoke condensates in 

cell transformation assays have been performed. It will be 

necessary to further develop assay conditions for routine 

evaluation of condensate activity. 

Limitations of the Assay: Because of the high toxicity of 

Other limitations of the cell transformation assay exist 

(Landolph 1985, IARC 1985, Dunkel et al. 1991). First, 

C3H/lOT1/2 cells have a maximum plating efficiency of about 30%. 

In the standard transformation assay when toxic concentrations of 

the smoke condensates are tested the plating efficiency will be 

further reduced and the number of survivors may be too few for a 

valid transformation assay. Second, too few or too many cells 

per dish can affect the number of transformed colonies thus 

making the test somewhat semiquantitative even under tightly 

controlled conditions. 

Third, the spontaneous transformation rate of C3H/lOT1/2 

cells is extremely low, which makes it difficult to obtain a 

baseline transformation frequency in each experiment. As many as 

500 control plates may be needed to obtain the true spontaneous 

transformation rate of C3H/lOT1/2 cells. However, this 

characteristic of C3H/lOT1/2 cells also makes the test 

attractive, because the induction of even a few transformed 

colonies on the plates can be attributed to the action of the 

test condensate with confidence. In this respect, it may be 

noted that the BALB/c 3T3 cell system, which has a low but still 

easily obtainable basal transformation rate, has been often 

utilized for routine testing of various chemicals but sufficient 

information about smoke condensate activity in this system is 

presently not available. In contrast, the C3H/lOT1/2 system has 

been successfully used to detect cell transformation activity of 

smoke condensate and its fraction (Benedict et al. 1975). 

3.b. Tests for Evaluatinq Genotoxicitv of Fresh Whole 

Cisarette Smoke: 

While microgram quantities (50 to 500 pg/plate) of smoke 

condensates can be tested in the bacterial and mammalian cell 

assays, direct exposure of tester cells to fresh whole cigarette 
Ell 

smoke can cause high cell mortality due to the presence of highly 

toxic constituents, such as carbon monoxide. Therefore, the above 

assays are generally difficult to use in their standard format 

for genotoxic evaluation of fresh cigarette smoke. A yeast cell 

system which tolerates cigarette smoke much better than do 

bacterial or mammalian cell systems may be used for fresh smoke 

evaluation, if necessary. 

In cases where chemical and physical analyses indicate a 

significant alteration in smoke composition, especially that of 

the gas phase constituents, the induction of mutations and other 

types of genetic damage by fresh whole smoke may be assessed by 

using the D-7 strain of Saccharromvces cerevisiae. This tester 

organism is a diploid strain of yeast which is used to identify 

chemicals that induce mitotic crossing-over, mitotic gene 

conversion, and gene mutations (Zimmermann et al. 1975). An 

exposure system has been developed to directly expose tester 

yeast cells to fresh smoke and has been found useful in 

quantitating various types of genetic damage by fresh smoke from 

different types of tobacco cigarettes (Gairola 1982). 

3.c. Tests for Evaluatinq Pulmonarv Inflammatorv Response in 

Animals 

i) Rationale: Cigarette smoking is a major risk factor in 

the development of chronic obstructive pulmonary disease, as 

discussed in Chapter A. Considerable evidence exists to suggest 

that chronic pulmonary inflammation plays an important role in 

the development of various pulmonary diseases, including those 

associated with cigarette smoking (Hunninghake et al. 1979, 

Neiwoehner 1988, Costabel and Guzman 1992). A test that 

evaluates the ability of test smokes to induce pulmonary 

inflammation will therefore be a useful indicator of the in vivo 

biological activity of cigarette smoke. The assay described below 

utilizes an analysis of the BAL cells from smoke-exposed mice for 

toxicological evaluation of fresh cigarette smoke. 

ii) Bioassay procedure 

Animal model: Rodents (mice, rats, guinea pigs and 

hamsters) have been generally used as animal models for studying 

the inhalation toxicity of cigarette smoke. Past studies have 

shown that exposure of rodents to cigarette smoke induces an 

inflammatory cell response in their lungs (Rylander 1974, Hoidal 

and Niewoehner 1982, Matulionis 1984, Gairola 1986). The studies 

have further shown that the pulmonary response of mice to 

cigarette smoke inhalation, as monitored by bronchoalveolar 

lavage (BAL), is significantly more pronounced than that of rats 

(Gairola 1986). Furthermore, it has been found that, as in human 

smokers, the BAL cells recovered from smoke-exposed mice show an 

infiltration of inflammatory cells including polymorphonuclear 

neutrophils (PMN) into the lungs, while those recovered from 
E12 

smoke-exposed rats hardly show any PMNs. Among other changes 

induced in BAL cells of smoke-exposed mice are increased oxidant 

production and lysosomal enzyme content of pulmonary alveolar 

macrophage (Gairola 1986) which resemble those reported in human 

smokers (Hunninghake et al. 1979, Finch et al. 1982, Fisher et 

al. 1982). 

proposed as an animal model for this assay. An additional 

advantage of using mouse is its smaller size which allows for 

increasing the number of animals in treatment and control groups 

at relatively lower costs. 

exposed to smoke in a nose-only exposure system (Griffith and 

Standafer 1985) to fresh smoke from one cigarette in the morning 

and one in the afternoon. Since nose-only exposures require the 

use of restrainers for animals during exposures, it is important 

that a group of animals be given the same treatment as 

smoke-exposed animals but in the absence of smoke (sham control) 

to simulate stress conditions similar to those of the 

smoke-exposed group. The first week of the experiment is a 

"break in" period during which the animals are gradually 

acclimatized to treatments by exposing them to 3, 6, and 9 puffs 

of smoke each session for two days at a time. 

animals receive exposure to 10 puffs of smoke per session, twice 

a day, seven days a week. Groups of animals (6-8) are sacrificed 

at different exposure points and the free lung cells are obtained 

by bronchoalveolar lavage (BAL) for further study. 

monitored to ascertain the inhalation of smoke by the animals. 

Total particulate matter (TPM) intake, blood carboxyhemoglobin, 

and urinary cotinine excretion are generally measured, (Griffith 

and Standafer 1985, Gairola 1986, 1987, Stanley et al. 1991). 

Animal TPM intake values are determined by measuring the removal 

of smoke particulates by the animals from the exposure chamber 

during each exposure session (Griffith and Hancock 1985). Some of 

the exposure markers, e.g., urinary cotinine and blood 

carboxyhemoglobin levels, used in animal studies are the same as 

those described for human studies in Chapter C. 

In view of the observations described above, mouse is 

Smoke exposure protocol: Male or female C57B1 mice are 

Thereafter, the 

Markers of smoke exposure: A number of markers are 

Bronchoalveolar lavage: The morning after the last 

treatment, animals are anesthetized by an ip injection of 

pentobarbital and are exsanguinated by severing the abdominal 

aorta. The lungs are lavaged and the BAL cells are obtained for 

analyses (Gairola 1986). 

Assessment of BAL cells: Total cell counts and viable 

cell counts by trypan blue exclusion are made with a 

hemocytometer. Small aliquots of BAL cell suspension are used to 

prepare Diff-Quik stained slides for differential leukocyte 
E13 

counts. These data allow an assessment of the degree of 

macrophage, lymphocyte and polymorphonuclear neutrophil (PMN) 

infiltration into the lungs. 

Differential counts for each batch of lavage cells at 

different exposure points indicate the time course of PMN 

infiltration into the lungs and can be plotted against exposure 

duration to determine the rate and extent of their influx. Since 

neutrophilic alveolitis has been implicated in the development of 

cigarette smoke-induced pulmonary diseases, such plots prepared 

for animals exposed to various test cigarette smokes are likely 

to provide a reasonable indication of their toxicity and the data 

can be used to differentiate the biological activity of test 

smokes. 

The cells remaining after total, viable, and differential 

counts are cultured for one hour at 37OC to isolate macrophages 

as monolayer. 5'-nucleotidase activity of the cell lysates is 

measured by a radiometric procedure, which is a good indicator of 

smoke-induced macrophage activation in mice (Gairola 1986). 

Data Evaluation and Interpretation: Graphs of the BAL 

cell data obtained from the study can be used to determine the 

time course of inflammatory cell influx into the bronchoalveolar 

lumen, which will indicate the toxicity of smoke. A useful 

endpoint is the time of PMN infiltration which may reflect the 

potency of the test smoke toxicity. Macrophage enzyme activities 

can also be used to assess the potential biological activity of 

test cigarette smoke. 

iii) Limitation of the Bioassay: Even though markers of 

smoke exposure are used, it is difficult to ascertain the dose of 

smoke inhaled by the animals. Also, interanimal variation may 

complicate interpretation of data, but this problem can be 

controlled by increasing the number of animals per group. The use 

of this assay has been limited for comparative assessment of 

smoke from different cigarette types and therefore may require 

further development of standardized assay conditions before use 

in any routine evaluation program. Also the expense of performing 

this assay may be a deterent for its routine use. 

The presence of particulates in the ambient atmosphere, 

respiratory infections,etc., can also induce an inflammatory 

response in the lungs of animals, thus complicating an assessment 

of smoke effects. Therefore, it is very important to house the 

animals in Bioclean rooms equipped with HEPA filters and 

maintained at higher (30 to 40) air changes/hour to minimize the 

exposure of animals to particulates andfor any infectious agents. 

4. Estimates of Cost and Duration of Tests: 

Ames' Test: The length of time for testing five concentrations 

of one reference and four unknown condensate samples, in the 
E14 

absence and presence of S-gs, is estimated at about 4-6 weeks. 

The current cost of testing five concentrations of one compound 

in the absence and presence of S-9, in two to five strains of the 

test bacteria, varies between $1,500-2,200/condensate sample. 

Cell Transformation Assay: The length of time for testing 5 

concentrations of one condensate in the absence of S-9 in cell 

transformation assay is about 6 months and costs range from 

$7,000-8,0OO/condensate sample. 

Inflammatory Cell Response Bioassay: The length of time for 

testing smoke from one type of cigarette in this bioassay will 

take 8-10 months. The cost of testing one cigarette type in this 

bioassay is estimated at about $50,000. If more than one 

cigarette type is tested at the same time, the cost will be 

reduced. 

5. Other Toxicoloqical EndDoints Associated with Smoke Exvosurerelated Health Problems: 

In addition to genotoxicity and pulmonary toxicity, 

cigarette smoke has been implicated in other health effects 

discussed in Chapter A. Smoking-induced immunosuppression in 

human smokers may be one of the mechanisms contributing to the 

development of smoking-associated health effects. Although the 

incidence of immunosuppression in smokers is well documented 

(Holt 1987, Johnson et al. 1990), the exact nature of 

immunotoxicants in smoke is not known. It may be useful to 

include some tests for evaluating the immunotoxic activity of 

low-ignition potential cigarette smoke. Unfortunately very few 

studies addressing immunotoxicity of smoke have been performed in 

animal models. Only recently some experimental evidence 

suggesting an impairment of thymic-dependent and independent 

functions of lymphocytes from long-term smoke-exposed mice and 

rats has been reported (Sopori et al. 1989, Chang et al. 1990, 

Goud et al. 1992). 

Reproductive toxicity and atherogenesis are two other health 

problems associated with cigarette smoking. A number of 

epidemiological studies have suggested that chronic smoking is 

deleterious to female reproductive health and fetal development 

(Abel 1980, Mattison et al. 1989, Werler 1986). Relatively fewer 

data regarding the effects of smoke on reproductive system are 

available in animal models (Mattison 1982). Preliminary studies 

in mice have shown that long-term exposure to cigarette smoke 

lengthens the murine estrous cycle and causes accelerated loss of 

oocytes from the ovaries (Gulati et al. 1989, Gairola and Gulati 

1991). 

Still fewer studies have examined the formation of 

atherosclerotic plaques following exposure to cigarette smoke in 

animal models. One study reported the formation of aortic 
E15 

lesions in Syrian hamsters following a 12-15 month exposure to 

cigarette smoke (Haley and Axelrad 1982). Recently, enhanced 

development of atherosclerosis has also been reported in rabbits 

which had been fed high cholesterol diets and were exposed to 

environmental tobacco smoke (Zhu et a1.1993). 

The long duration of exposures required to induce many of 

the above described immunological, reproductive, and atherogenic 

toxicities in animals would make bioassays based on these 

endpoints prohibitively expensive for routine use in the 

evaluation of low-ignition cigarette prototypes. 
E16 

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activity of cigarette smoke condensate. Mutation Res. 48:319-326, 

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Niewoehner, D.E.; Cigarette smoking, lung inflammation and the 

development of emphysema. Review. J. Lab. Clin. Med. 111:15-27, 

1988. 

Parodi, S., Malacarne, D. and Taningher, M.; Examples of uses of 

databases for quantitative and qualitative correlation studies 

between genotoxicity and carcinogenicity. Environ.Hlth.Perspect. 

96:61-66, 1991. 
E2 2 

GLOSSARY 

carcinogenesis: or Oncogenesis: Processes through which tumors 

are produced. 

Genotoxicity: 

Initiators: 

Any adverse effect on 

structure, function, or 

expression of genetic material 

in a living cell. 

Agents that begin but do not 

necessarily promote the 

carcinogenic process. 

Malignant Cell Transformation: Change in the phenotypic 

characterstics of cells in 

culture that indicates their 

capability to induce tumors 

when injected into animals. 

Mutagenesis: 

Mutations: 

Promoters: 

Processes by which heritable 

alterations in genetic 

material or functions are 

produced. 

Any heritable alterations in 

the expression of genetic 

material or functions. 

Agents that encourage but do 

not necessarily initiate 

cancer development. 
Chapter F 

In Vivo Bioassays for Carcinogenicity 

Dietrich Hoffmann, Ph.D. 

American Health Foundation 

Valhalla, New York 10595-1599 

Prepared for the U.S. Consumer Product Safety Commission under contract 

CPSC-s-92-5473 

March 14, 1993 

Chavter Contents 

Introduction 

I. Inhalation Bioassays 

11. Bioassays with Cigarette Smoke Particulate Matter 

111. Bioassays on Mouse Skin 

IV. Inhalation Bioassay with Syrian Golden Hamsters 

Critique 

Acknowledgements 

References 

Tables 

Legends to Figures 

Figures 

Glossary 

Cost Estimates 

F1 

Introduction 

In the 1989 Report on the Health Consequences of Smoking, 

the Surgeon General of the U.S. Public Health Service, in 

evaluating the health effects of smoking, concluded that "smoking 

is responsible for more than one of every six deaths in the 

United States" (1). Cigarette smoking alone increases the risk 

for coronary heart disease from 23/1000 to 54/1000, together with 

hypercholesterolemia cigarette smoking raises the risk to 

103/1000, and together with high blood pressure, to 92/1000. All 

three risk factors, smoking, hypercholesterolemia, and high blood 

pressure, synergistically increase the risk for coronary heart 

disease to 189/1000 (1). 

Cigarette smoking is also a major risk factor for chronic 

obstructive pulmonary disease, and here primarily for emphysema 

and chronic bronchitis. Eighty to 90% of the morbidity from 

chronic obstructive lung disease in the United States has been 

attributed to cigarette smoking (2). 

Smoking of cigarettes is causally associated with cancer of 

the lung, larynx, oral cavity, esophagus, pancreas, renal pelvis 

and urinary bladder and is also linked with an increased risk for 

cancer of the nasal cavity, liver and the uterine cervix and 

possibly, related to cancer of the stomach (1). In 1992, the 

National Cancer Institute published a population-based 

case-control study that provided evidence for the association of 

cigarette smoking with several types of leukemia and thereby 

confirmed earlier prospective and case control studies (3). The 

National Cancer Institute estimated that in 1991 of the 514,000 

cancer deaths at the seven sites causally associated with 

cigarette smoking, 30.6% are due to smoking (4). 

Chemical analyses for the major known carcinogens offer a 

meaningful indication of the carcinogenic potential of cigarette 

smoke, especially in conjunction with chemical analytical data 

for the smoke of cigarettes already bioassayed for carcinogenic 

activity. In vitro assays for genotoxicity such as the Ames test 

with various bacterial strains, the DNA repair assay with primary 

rat liver cells, and the sister chromatid exchange assay have 

remained inconclusive in regard to the quantitative aspects of 

the genotoxic potencies of cigarette smokes (5). At present, 

conclusive data on the carcinogenicity of the smoke of new 

cigarettes can only be ascertained with long-term bioassays with 

laboratory animals (5-7). 

Three animal species are primarily utilized for bioassays of 

whole cigarette smoke in inhalation experiments. These are mice, 

rats, and Syrian golden hamsters. All of the inhalation studies 

have the inherent shortcoming that the animals are obligated to 

breathe through the nose and that their inhalation of tobacco 
F2 

smoke is shallow. They do not inhale smoke through the mouth as 

human smokers do. Nevertheless, cigarette smoke inhalation 

studies with mice, rats and Syrian golden hamsters have led to 

the induction of significant numbers of benign and malignant 

tumors in the respiratory tract of these animals. 

have not been fully accepted since only lung adenoma and lung 

adenocarcinoma have been elicited and not squamous cell tumors in 

the lung. Furthermore, most strains of mice have a fairly high 

rate of spontaneous lung adenoma. The rates of both 

adenocarcinoma and squamous cell carcinoma of the lung have 

increased in cigarette smokers and the current ratio of lung 

adenocarcinoma to squamous cell carcinoma in male smokers is 

1:2-3 (8). 

The data from cigarette smoke inhalation studies with mice 

As will be discussed under "Inhalation Bioassays", the 

critique on the data from smoke inhalation studies with mice is 

no longer fully justified. A large-scale inhalation study with 

rats using highly advanced methodology presents encouraging data 

(9). However, until additional long-term inhalation bioassays 

have been completed with this exposure system, the database is 

too limited to recommend this rat bioassay for routine studies. 

The largest database from cigarette smoke inhalation studies 

stems from assays with Syrian golden hamsters. As will be 

discussed, these long-term inhalation studies have only in a few 

cases led to lung tumors; however, they have induced highly 

significant incidences of benign and malignant tumors in the 

upper respiratory tract of hamsters. The tumors occurred 

primarily in the larynx. 

achieved in respiratory carcinogenesis. We have become well 

aware of the existence of carcinogens with organ-specificity for 

the respiratory tract of laboratory animals, and bioassays of 

aerosols and volatilized chemicals have also provided 

considerable evidence for their potential to induce tumors in the 

respiratory tract of mice, rats and hamsters (10). 

Since the early 1960's, remarkable progress has been 

I. Inhalation Bioassays 

Three decades ago, the Leuchtenbergers (11) reported the 

first extensive inhalation experiments in which mice were exposed 

daily to air-diluted cigarette smoke in specially designed 

chambers. This smoke exposure led to early histological, 

cytological, and cytochemical changes in the major bronchi of the 

mice. The smoke exposure also caused various degrees of 

bronchitis associated with atypical proliferation of the 

bronchial epithelium. The investigators observed extracellular 

deposition of a brown pigment in the lungs of all the mice that 
F3 

underwent long-term exposure to cigarette smoke aerosols. After 

about 12-15 months, the smoke-exposed mice began to develop lung 

adenoma and lung adenocarcinoma in significantly higher numbers 

than did the control mice. 

phase of cigarette smoke, lung adenomas have also been observed, 

though to a significantly lesser extent than with the whole smoke 

(12). The findings of the Leuchtenbergers (11, 12) were 

confirmed by Otto (13) who exposed inbred albino mice to 

cigarette smoke daily. After at least 12 months of smoke 

exposure, 23 of 60 mice developed lung adenomas, while only 3 of 

60 control mice were found with such tumors. One mouse in the 

exposed group developed a squamous cell carcinoma of the lung 

after 16 months (13). 

Several criticisms have been voiced in regard to the 

In inhalation studies with the gas 

induction of lung adenoma and lung adenocarcinoma in mice by 

exposure to cigarette smoke. Concerns include the fact that such 

exposures caused tumors in the peripheral lung, and not in the 

bronchi, and that some of the tested strains of mice had a 

relatively high rate of spontaneous lung adenomas. It has been 

observed that not only the carcinoma in the bronchi, but also the 

incidence of lung adenocarcinoma, has significantly increased in 

cigarette smokers and that such tumors are now even seen in 

nonsmokers who have been exposed to environmental tobacco smoke, 

to carcinogenic chemicals, or to radiation (1). 

In the past, it was not understood how the topical applica- tion of tobacco lltarlt to the skin of mice could lead to the 

development of lung adenoma and adenocarcinoma. Today, we are 

aware that tobacco smoke contains also organ-specific carcinogens 

such as the tobacco-specific N-nitrosamines, which can induce 

adenoma and adenocarcinoma in the lung upon application to the 

skin (14) and other sites in mice. 

A major breakthrough in inhalation assays came with the 

development of new smoke-inhalation devices that facilitate the 

exposure to diluted tobacco smoke aerosols (15-17). When 80 rats 

were exposed seven times daily for intermittent periods (8.4 x 30 

seconds) to 10% cigarette smoke aerosol for up to 2.5 years, most 

animals developed hyperplastic and metaplastic changes in the 

nasal turbinals, larynges and tracheas. Seven of the 80 

smoke-exposed F344 female rats developed tumors in the 

respiratory tract, including 1 adenocarcinoma and 1 squamous cell 

carcinoma in the lung, compared to 1 alveologenic carcinoma only 

in the 93 control rats (9). 

In another study, rats were exposed to diluted cigarette 

smoke twice a day for 10 minutes, 5 days a week for up to 40 

weeks (18). Subsequently, DNA from nasal, lung and liver tissues 

was extracted and analyzed by the 32P-postlabeling procedure. 

the nasal mucosa at least four new DNA-adducts were seen; the 

In 
F4 

amount of these adducts increased with the duration of smoke 

exposure. In the lung, one new DNA-adduct was detected; it also 

accumulated as smoke exposure progressed. It appears that the 

DNA adducts were aromatic and/or hydrophobic in nature (18). In 

a similar assay, rats were exposed for 22 days to diluted 

cigarette smoke. 

nose-only continuous exposure 14 f 0.9 and 9.9 + 0.7 DNA adducts 

per lo9 bases were determined in the lung (19). These studies 

demonstrate that minute amounts of genotoxic smoke components 

reach the lungs of rats in inhalation assays. 

In the nose-only intermittent exposure and 

Dontenwill and associates developed the "Hamburg 11" smoke 

inhalation device in which small animals can be exposed to 

air-diluted smoke (Figure 1). Eighteen groups, each consisting 

of 80 female and 80 male random-bred Syrian golden hamsters, 

comprised this cigarette smoke inhalation lifetime assay. 

Animals in group 1 were exposed once daily for about 10 minutes, 

seven times each week to air-diluted smoke (7:l); those in group 

2 had twice daily exposures to diluted smoke, hamsters in group 3 

had 3 exposures to diluted smoke; and those in group 4 were 

exposed twice daily to the gas phase of diluted smoke, while 

group 5 consisted of sham-treated controls. In group 1, 38 

animals developed papilloma and one animal had a carcinoma of the 

larynx (total 24%), hamsters in group 2 developed 69 papilloma 

and 17 carcinoma of the larynx (total 54%), corresponding tumor 

yields in group 3 were 77 papilloma and 11 carcinoma of the 

larynx (total 55%). Laryngeal tumors were not observed in group 

4 (gas phase only) nor in group 5 (controls). Three hamsters in 

group 2 developed papilloma of the pharynx; tumors of the lung 

were not seen in any of the hamsters in this study (20). 

In another assay, male Syrian golden hamsters from 2 inbred 

lines were exposed five times a week for up to 100 weeks to 

air-diluted smoke (21). In one inbred strain, 7 of 84 hamsters 

developed papilloma in the larynx, 9 had microinvasive cancer; in 

the second inbred strain, 11 of 87 animals had papilloma and 2 

microinvasive cancers occurred in the larynx; none of the control 

hamsters developed laryngeal tumors (21). 

In a dose-response lifetime study with a hamster strain 

susceptible to the induction of laryngeal tumors, twice daily 

exposures to 22% cigarette smoke resulted in 70% with papilloma 

and 47% with carcinoma of the larynx (22). The corresponding 

incidences in the hamsters exposed twice daily to 11% cigarette 

smoke were 27% and 7%. Those in the control group were 6% and 

0%, respectively. In the high-dose group, 3 of 62 hamsters also 

developed tracheal papilloma (22). 

These studies demonstrated the dose-repsonse carcinogenic 

effect of cigarettes. The Syrian golden hamsters are less 

susceptible than other laboratory animals to the toxicity of 
F5 

nicotine and of carbon monoxide and are therefore preferred for 

inhalation studies with tobacco smoke. 

11. Bioassays with Cigarette Smoke Particulate Matter 

Inhalation assays with Syrian golden hamsters have 

demonstrated that only whole smoke induces benign and malignant 

tumors of the respiratory tract in a dose-dependent fashion. 

However, inhalation of smoke which is free of particulate matter 

(lltarll) does not lead to tumors. This indicates that the dose of 

carcinogens in the gas phase by itself is not sufficient to 

induce tumors and that the majority of the carcinogens reside in 

the particulate matter of tobacco smoke. This consideration has 

led to in-depth fractionation studies and bioassays with tobacco 

smoke condensate in mice, rats and rabbits (6, 23, 24). The 

neutral subfractions B and BI that contain a concentrate of the 

polynuclear aromatic hydrocarbons (PAH), harbor the major tumor 

initiators (Figure 2). The PAH subfraction is also the only 

portion of the tar that, upon repeated intratracheal instillation, elicits tumors in the respiratory tract of rats (25). 

Assays of the PAH concentrate explain only a small fraction 

of the total carcinogenicity of the tar. Results from bioassays 

of the PAH-subfraction in combination with the weakly acidic, 

non-carcinogenic fraction explain 70-90% of the carcinogenicity 

of the whole tar (23, 24). The weakly acidic fraction contains 

the major tumor promoters, volatile phenols, and the major cocarcinogens, catechols. In addition to tumor initiators, tumor 

promoters and cocarcinogens, tobacco smoke also contains 

carcinogens with organ-specificity. These act independently of 

the mode of exposure or site of application, by inducing benign 

and malignant tumors in specific organs. Table 1 presents a list 

of the known tumorigenic agents in tobacco smoke, their concentrations in the smoke of one cigarette, and the evaluation of 

evidence of their carcinogenicity by the International Agency for 

Research on Cancer (26 27). Table 2 is a listing of the likely 

causative agents for tobacco smoke-related cancers on the basis 

of organ-specificity of carcinogens and their various biological 

activities and concentrations in cigarette smoke. 

of cancer of the respiratory tract are PAH, the tobacco-specific 

N-nitrosamine 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone 

(NNK), the volatile aldehydes, acetylaldehyde and formaldehyde 

and, to a minor extent, polonium-210 (from agricultural and 

environmental sources). 

The agents in tobacco smoke most likely to cause induction 
F6 

111. Bioassay on Mouse Skin 

demonstrated that the major carcinogenic activity of whole 

cigarette smoke resides in its particulate matter (tar), as 

discussed earlier. This has led to extensive bioassays of 

cigarette tar in both the connective tissue of rats and the skin 

of mice (6). Since the induction of sarcoma in the connective 

tissue of rats can be influenced by the physical form of the tar, 

by the presence of insoluble particles (Oppenheimer-Nothdurft 

effect; 28, 29) the mouse skin bioassay is now the preferred 

method for estimating the tumor potency of smoke condensates 

especially when comparing tars of experimental cigarettes that 

vary from the control cigarette only in a few aspects. 

The tars obtained from a smoking machine (Chapter B), such 

as a Borgwaldt-30 cigarette smoker (30) or other devices (6), are 

stored in the dark at refrigerator temperature until needed for 

biological testing, but should not be older than 3 weeks when 

applied. Before use, the suspensions in acetone are thoroughly 

mixed in a mechanical shaker for at least 3 hours, a sample is 

poured into a 60-ml glass-stoppered reagent bottle. Since tars 

are not always fully dissolved in the acetone (l:l), vigorous 

shaking of the bottle is essential before each use. 

Anywhere from 30 to 100, usually 50 but preferably 100, 

Ha/ICR/Mil (Swiss albino) female mice are used for each tar to be 

tested. The random bred Ha/ICR/Mil (Swiss albino) mice are 

sturdy animals, and they are quite resistant to nicotine 

toxicity. Compared to two inbred strains of mice often used in 

skin carcinogenesis (CAF,, C57BL), they are more susceptible to 

the carcinogenic activity of tobacco tars (31). Female mice are 

used for the bioassay since they do not fight as do the males, 

which results in skin scratches. Thus, females can be housed 5 

to a cage, while males require one cage for each mouse. The 

maintenance of female mice is therefore significantly more cost 

effective (6). At the onset of bioassays the mice are 5 to 7 

weeks of age and weigh 22 to 25 g. They receive feed and water 

ad libitum. Their cages are cleaned twice weekly. 

Inhalation studies with Syrian golden hamsters have clearly 

Before each tar application, the dorsal hair of the mice is 

shaved with a Model A2 (size 40) Oster animal clipper. The tar 

is then applied in 0.1 ml of an acetone suspension containing 50 

mg tar with a full No. 5 camel hair brush, or by pipette. The 

treatment is repeated three times weekly, allowing at least one 

day between applications for absorption of the tar before the 

next application. It is sometimes necessary, especially at the 

onset of the experiment, to skip a painting if the mice exhibit 

poor absorption or low tolerance of the tar. 
F7 

Mice that survive the first month usually tolerate the toxic 

effects of the tar solutions (LD,). All mice lost during the 

first month in an experiment are replaced by mice of the same 

age. Therefore, the initial number of animals to be scheduled 

for each assay must exceed the requirements for the control and 

experimental groups by about 10%. If the toxicity persists, even 

though the number of applications is cut down, the tar must be 

applied at a lower concentration with the necessary revision of 

the protocol. In recent years, however, such modifications have 

usually not been necessary, owing to the generally lower levels 

of nicotine in tobaccos. The bioassay is not terminated until 

90% of the mice in the tar group with the longest survival rate 

have died or were moribund and had to be killed; this takes 

usually 18-20 months of tar application. 

Average weights of the mice are recorded at the onset of the 

experiment and again at 2 weeks, 4 weeks, and thereafter at 

monthly intervals as an indicator of the general health of the 

mice. A reduction in weight could be associated with a reduced 

tumor yield, particularly if the weight loss takes place in the 

tumor promotion phase. It is essential that weight records be 

kept in experiments with tobacco carcinogenesis. 

Constant observation of the animals is also essential. Any 

change in appearance, habit, or reaction is noted; any lesion on 

the back is described as to [l] type, i.e. ulcer, infection, or 

tumor; [2] date of appearance or change in appearance; and [3] 

exact location. When such a notation is first made, the animal, 

is marked on the head with a yellow dye (picric acid). 

Diagrammatic representations of the animal's back are used to 

facilitate the recording. 

The application of the test material by painting or 

pipetting may lead to benign skin tumors which are recorded when 

they have attained a diameter of 1 mm. They enlarge by nodular 

growth (papilloma) or by lateral invasion (carcinoma); some may 

not enlarge, but regress. Those tumors that remain 1 mm or grow 

larger for 21 consecutive calendar days are counted and become 

the raw tumor yield data. Continued growth of such lesions, 

however, is required before they can be recorded as 

macroscopically observed carcinomas (raw tumor yield data). A 

revised count is reported after histopathological confirmation of 

the macroscopically observed lesions. Lateral invasion of the 

tumor into adjacent skin is considered as transformation into a 

carcinoma. 

Mice with carcinomas are killed by cervical dislocation and 

tumors are excised for subsequent histopathological analysis. 

All suspicious lesions are likewise examined. The animals are 

autopsied for distant metastases and the occurrence of other 

tumors, especially pulmonary adenomas and lymphomas. 
F8 

The most careful recording of experimental observations 

demands equally careful statistical evaluation of the final data. 

Therefore, sone pertinent statistical considerations will be 

discussed. 

More than 90 mice, rats, or hamsters per group should be 

used in carcinogenicity bioassays with tobacco smoke condensates. 

We calculated that to find a difference with 80% statistical 

power between the carcinogenicity of condensates from an 

experimental cigarette [with reduced ignition propensity] vs. a 

control would require at least 90 animals in each group. 

additional 30 are also needed for the negative control group. 

bioassays be carried out with sufficient animals to achieve 

statistical soundness. 

experiment are replaced. 

control group has a tumor and 6 or more animals in the 

experimental group have tumors, one may utilize the table 

prepared by Vos based on chi-square analysis with "Yates 

correction". 

is significant at P <0.05 when the number of animals in each 

group varies from 10 to 50 or more. 

tumorigenic agent and does not allow a quantitative assay of 

tumor-producing agents of varying potency. If, €or example, 

there are as many as 4 tumor-bearing animals in a given group 

treated with a weak carcinogen, the number of animals in each 

group becomes important. If there were 10 animals in each group, 

all of them must bear tumors to indicate a difference at the P 

<0.05 level of significance. In a group of 50 animals treated 

with a stronger tumorigenic substance, only 12 need to show 

tumors. Information about differences in tumor yield necessary 

for significance at P <0.05 between two groups for groups of 10 

to 50 animals may be readily extracted from tables by the Food 

Protection Committee of the U.S. Academy of Science (30). 

An 

The lethal toxicity of some smoke condensates require that 

Mice lost during the first month of an 

Assuming, as an example, that none of the animals in a 

This shows that the difference between the groups 

This situation merely identifies that one is dealing with a 

IV. Inhalation Bioassay with Syrian Golden Hamsters 

Inhalation bioassays with whole smoke are also strongly 

indicated to confirm the relative carcinogenic potencies of 

respective condensates in mouse skin painting bioassays. As 

discussed earlier, the Syrian golden hamster (SGH) is presently 

the animal of choice for long-term inhalation assays with whole 

cigarette smoke (6, 20-22). It should be noted that inhalation 

bioassays with whole cigarette smoke will rarely lead to lung 

tumors in SGH or rats (9), but it will lead to papilloma and 

carcinoma in the larynges of the animals. Since the larynges of 

. . ... 
F9 

inbred strains of male SGH are apparently most susceptible to the 

carcinogenic effects of cigarette smoke (21, 22) one is inclined 

to prefer this animal model. 

Inbred strains may be difficult to obtain are not as 

resistant to the acute toxicity of the smoke as are random-bred 

SGH. Thus, the latter are generally used for inhalation studies 

(7, 31). 

Three inhalation devices have been developed for exposure of 

SGH and rats to cigarette smoke. These are the Wamburg 11" 

device (20, 34), the "Oak Ridge" smoke inhalation exposure device 

(9, 17) and the "Walton-reverse smoker" (21). All 3 machines are 

well developed and the "Oak Ridge" device has especially 

favorable features in respect to forced smoke inhalation by 

laboratory animals. However, the "Hamburg 11" device with SGH is 

recommended for comparing the tumorigenicity of whole smoke from 

various cigarettes. The device has been widely used and most 

data on the tumorigenicity of whole cigarette smoking, including 

a dose-response study, were generated with it (15, 20, 34). In 

general this bioassay requires 24-26 months. 

daily exposures to air-diluted smoke (7:l) of one cigarette each, 

seven times weekly, for the entire lifespan of the animals. 

Because of the high CO concentration in undiluted smoke (2.8 - 

4.6 ~01%) the maximum tolerated dose is typically a 10 minute 

exposure twice daily of cigarette smoke diluted by air 1:7. Ten 

SGH at at time can be exposed concurrently to diluted cigarette 

smoke from one Hamburg I1 device. Since 80 male hamsters are 

needed for each test cigarette, the bioassay is very labor 

intensive and is recommended only as a last step in the cascade 

of assays. Details for the inhalation assay with SGH are 

presented in Dontenwill (15, 20). These exposure protocols are 

not intended to mimic human smoking behavior (see Topography 

chapter), but are intended to produce tumorigenic effects that 

can be statistically evaluated. 

Inhalation assays using F344 rats and employing the "Oak 

Ridge" inhalation device for exposure to cigarette smoke (9, 17) 

appear promising. Although it can not yet be recommended for 

toxicity testing, it is hoped that the methodology will be 

confirmed by additional studies. 

The inhalation studies with SGH generally consists of twice 

critiaue 

Ideally one establishes the carcinogenicity of an inhalable 

substance by bioassays leading to the induction of benign and 

malignant tumors in the respiratory tract of laboratory animals. 

In the case of cigarette smoke this goal has been only partially 

reached. In the Syrian golden hamster, papilloma and carcinoma 
F10 

have been induced in the larynx with cigarette smoke in a 

dose-related fashion. However, with a few exceptions, squamous 

cell tumors of the lung, which are associated with cigarette 

smoking in humans, are not produced by this model (15, 20-22). 

Even with an advanced smoke inhalation device, only a few lung 

carcinomas were produced in rats (9, 17). 

Epidemiologists identify several hundred prospective and 

case-control studies demonstrating that cigarette smokers face an 

increased risk for lung cancer. Therefore, confirmation by 

inhalation bioassays is not necessary. 

Simulation of human smoking behavior in terms of deep 

inhalation of cigarette smoke into the lungs has not been 

successful in laboratory rodents. However, in comparisons of the 

relative tumorigenicity of the whole smoke of cigarettes with 

reduced ignition propensity to that of a control, the inhalation 

bioassay with hamsters should clearly reflect possible changes in 

the carcinogenic potential in the number of tumors observed in 

the larynges. 

Acknowledaements 

Our research program in tobacco carcinogenesis is supported by U.S. 

National Institutes of Health grant No. CA-29580. The editorial assistance of 

Ilse Hoffmann and Laura DiSciorio is greatly appreciated. 
F11 

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Natl. Cancer Inst. 1973; 51: 1781-1832. 

Bernfeld, P., Homburger, F., and Russfield, A.B. Strain 

differences in the response of inbred Syrian hamsters to 

cigarette smoke inhalation. J. Natl. Cancer Inst. 1974; 53: 

1141-1157. 

Bernfeld, P., Homburger, F., Soto, E., and Pai, K.J. Cigarette 

smoke inhalation studies in inbred Syrian golden hamsters. J. 

Natl. Cancer Inst. 1979; 63: 675-689. 

173-189. 

64-71. 

12. 

13. 

14. 

15. 

16. 

17. 

18. 

19. 

20. 

21. 

22. 
F13 

Hoffmann, D. and Wynder, E.L. A study of tobacco carcinogenesis 

XI. Tumor initiators, tumor accelerators, and tumor promoting 

activity of condensate fractions. Cancer 1971; 27: 848-864. 

Dontenwill, W.! Chevalier, H.-J., Harke, H.-P., Klimisch, H.-J., 

Brune, €I., Fleischmann, B., and Keller, W. Experimentelle 

Untersuchungen ueber die tumorerzeugende Wirkung von 

Zigarettenrauch-Kondensaten an der Maeusehaut. VI Untersuchungen 

zur Fraktionierung von Zigarettenrauch-Kondensat. Z. 

Krebsforsch. 1976; 85: 155-167. 

Davis, B.R., Whitehead, J.K., Gill, M.E., Lee, P.N., Butterworth, 

A.D., and Roe, F.J.C. Response of rat lung to tobacco smoke 

condensate or fractions derived of it administered repeatedly by 

intratracheal instillation. Brit. J. Cancer 1975; 31: 453-461. 

International Agency for Research on Cancer. Overall Evaluations 

of Carcinogenicity: An Updating of IARC Monographs 1 to 42. 

IARC Monographs on the Evaluation of the Carcinogenic Risk of 

Chemicals to Humans 1987; Suppl. z: 440, p. 

International Agency for Research Cancer. Radon. IARC 

Monographs on the Evaluation of the Carcinogenic Risks of 

Chemicals to Humans, 1988; 43: 173-259. 

Oppenheimer, B.S., Oppenheimer, E.T., Stout, A.P. and 

Danishefsky, I. Malignant tumors resulting from embedding 

plastics in rodents. Science, 1955; 118: 305-306. 

Nothdurft, H. Uber die Sarkomausloesung durch 

FremdkTrperimplantationen bei Ratten. 

der Implantate. Naturwissenschaften, 1955; 42: 106. 

Elmenhorst, H. Eine vollautomatische Rauchmaschine fiir die 

Herstellung grosser Mengen von Cigarettenrauchkondensaten. 

Beitr. Tabakforsch., 1966; 3: 545-553. 

Wynder, E.L., Graham, E.A., and Croninger, A.B. Experimental 

production of carcinoma with cigarette tar. 11. Tests with 

different mouse strains. Cancer Res. 1955; 15: 445-448. 

Food Protection Committee, Food Nutrition Board. Natl. Acad. 

Sci-Natl. Res. Council Publ. 1959; 749 pp. 

Karbe, E. and Park, J.F. Experimental Luna Cancer. 

Abhangigkeit von der Form 

23. 

24. 

25. 

26. 

27. 

28. 

29. 

30. 

31. 

32. 

33. 

34. 

Carcinogenesis and- Bioassays-. Springer Publ. New York, 1974, 

611 pp. 

Kobayashi, N:, Hoffmann, D., and Wynder, E.L. 

carcinogenesis. XII. Epithelial changes induced in the upper 

respiratory tracts of Syrian golden hamsters by cigarette smoke. 

J. Natl. Cancer Inst., 1974; 53: 1085-1089. 

A study of tobacco 

Benz[a]onthrocene 

BenzODyluwanthene 

Benzomf luoronthene 

Benzo[k]fluoconthene 

-0I~lWr- 0.1 - Pong 

chrvsene 

Dibenz[a,h]anthracene 

Dibenzo[a,~pylene 

Dibenzo[a,llp/rene 

Meno[l,2,3cd]pwene 

5btWhrYsene 

Am-asnaF 

Quinoline 1 -2PQ 

Dibenzo[a,h]acridine 

Dibenzo[aJocridine - 7H-Dibenzo[c,g]ca~ 

N-N~rosodimthylamine ND - 21 5 

N-Ntrosoethylrne~omim 

N-Nitrosodit hytrnine 

4-(Methylnitrosornino)-lN-Nitrosonornicotine 0.3 ~ 89pg 

(3-pyridyi)-l -butonone 0.2-7p~ 

N-Ntroswnobasine 0.01 - 1.9pQ 

N-NitrosMocphdine ND-690w 

AJufnmscmhar 

2-Toluidine 

2-Napthylamine - 4-AmhObiphenyI 

Formaldehyde 1.6-7.4pQ 

Acetaldehyde 1.4 - 7.4pQ 

Crotmldehyde 0.2 - 2.4~9 

-apcrJcca?porndr 

Benzene 

Aclylonitrile 

1.1 -Dirnethylhydrozine 60- 147pQ 

2-Nitroptopone 

Ethylcarbornate 310 - 375w 

vnyi ChlOdde 

-=fw=- 

Hvdrazine 14-51 ng 

AneniC 500-900ng 

Nickel 2000-M3oong 

Chromium 1000-2000ng 

LWd 8 - 10pg 

Polonium-21 0 0.2- 1.2pcI 

Cadmium 1300- 1M)ong 

20-70W 

4-zng 

6-21 ng 

6-12W 

20-40ng 

40-6ong 

4ng 

1.7 - 3.2 t-Q 

present 

4-2OnQ 

0.6 ng 

0.1 ng 

3 - long 

0.7 ng 

0.1 -18Ong 

3-13w 

ND-25m 

0.1 2 - 3.7 /JQ 

0.08 - 0.77 pg 

0.14 - 4.6pg 

30-2oong 

1 -22ng 

2-5m 

70- 1 OOp@ 

10 - 2OpQ 

12 - 48pQ 

18 - 14Wpgb 

3.2 - 15pg 

0.73 - 1.21 M 

20-38m 

1 - 16W 

24-43t-Q 

40- 120ng 

0-M)ong 

4-70W 

41 -62m 

35-85ng 

0.03 - 1 .o pcl 

suttkient 

sutticient 

sufficient 

suffkient 

sufficient 

sufficient 

sufficient 

sufficient 

sufficient 

sufficient 

sufficient 

suffic.ent 

sufficient 

sufficient 

sufficient 

sufficient 

sufficient 

sufficient 

sufficient 

limited 

sufficient 

sufficient 

sufficient 

suficient 

sufficient 

sufficient 

sufficm 

sufficient 

sufficient 

sufficient 

suffkient 

sufficient 

sufficient 

irodeqwte 

suffiicm 

sufficient 

sufficient 

sufficient 

sufficient 

aNodesignohon ' Micales that on evalwtm by l4RC has not been carried obi. blhe Om report of the hdependent 

SCiintliC Commiitee on smoking and Health (1 988) published values for the 14 

them0rket)of 2~105pg/cigorette[meon59p~)forf~~ond~1150p~cigorette(meon910p~)for 

WetylOdehyde. PAH, polynuclear orornotic hydrocabws; ND. not detected. 

Biitii ciQafettes h 1986 (51.4% of 
F14 

Table 2 

Likely Causative Agents for 

Tobacco-Related Cancers 

orcran 

lung, larynx 

esophagus 

pancreas 

bladder 

oral cavity 

Carcinoaen 

NNK 

acetaldehyde 

formaldeh de 

poloniumJ (minor) 

PAH 

N" 

NAB 

NNK 

NNAL 

4-aminobiphenyl 

2-naohthvlamine 

Enhancina Aaents 

acrolein, crotonaldehyde(?) 

catechol (cocarcinogen) 

weakly acidic tumor promoters 

ethanol, catechol 

othe; aromatic amines 

nutrition 

infectious agents(?) 

PAH 

NNN 

"K 

ethanol 

herpes simplex 

NAB - N'-nitrosoanabasine 

NNAL - 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanol 

NNK - 4-(methylnitrosamino)-1-(3-pyridyl)-l-butanone 

NNN - N'-nitrosonornicotine 

PAH - polyaromatic hydrocarbons 
Fig.1 Hamburg I1 Smoke-Inhalation Device for 10 Hamsters (16). 
amohm condmnutm 

Ellher Z+i$O4 

Phemlk 

+ Acldlc c: - c: + 

c: - 

I 

18. Melhyl alcohol 

2.0% 5.3% 5.1% 1.5% "W 

C. - Goti : ++ 

Fig.2 Fractionation of Cigarette Smoke Condensate (23). 

. 
F16 

Glossary of Terms 

adenocarcinoma 

adenoma 

carcinoma 

DNA 

genotoxicity 

in vitro 

in vivo 

papi 1 loma 

malignant tumor of a 

glandular structure, such 

as in the peripheral lung 

benign tumor of a 

glandular structure, such 

as in the peripheral lung 

malignant tumor of 

epithelial orgin 

deoxyribonucleic acid. 

DNA is localized in the 

cell nucleus and is the 

molecular basis of 

heredity in many 

organisms 

damage to the DNA 

structure 

experimentation with 

microorganisms, isolated 

cells, tissues, or 

isolated organs in 

biological media 

experimentation with live 

animals, such as mice, 

rats and hamsters 

benign tumors (warts) due 

to a proliferation of 

epithelial tissue 
F17 

Cost Estimates for Carcinogenicity Bioassays 

Bioassay Animal Groups Number of 

~n ima 1s' 

Inhalation SG hamster exptl. cigarette I 60 

(random bred) exptl. cigarette 11 60 

sham control 60 

Skin mouse exptl. cigarette 1 90 

(Ha/ICR/Mil) exptl. cigarette 11 90 

sham control 60 

cost' 

$185 , 000- 

$250,0003 

$39,000 

'Estimates pertain only to direct cost. 

approved for individual institutes by the U.S. Department of Health and 

Human services. Direct total costs include animal purchase, health 

screening of the animals, maintenance, treatment (smoking of hamsters or 

tar application to mouse skin), weighing (first 8 weeks weekly, 

subsequently monthly), recording, autopsy and histology. 

requires about 280,000 cigarettes/group; mouse skin bioassay 1.5 kg/group 

requires about 75,000 cigarettes, assuming one cigarette yields 20 mg tar), 

or the smoking of cigarettes for the preparation of the tar for the mouse 

skin bioassays. 

2The number of animals per group is calculated for a difference between 

two groups with 80% statistical power either for the tumorigenic activity 

in the larynx of hamsters of whole cigarette smoke or for the tumorigenic 

activity on mouse skin of a tar. 

3This includes the overtime for twice daily exposure on Saturday and 

Sunday. 

The costs exclude the overhead as 

Estimates do not include purchase of cigarettes (inhalation study 
Part I1 

VOLUME 5 

PART I1 

LIMITED TIER I AND I1 DEMONSTRATION 

OF THE TOXICITY TESTING PLAN FOR 

LOW IGNITION-POTENTIAL CIGARETTES 

U.S. Consumer Product Safety Commission 

in consultation with the 

U.S. Department of Health and Human Services 

Tobacco and Health Research Institute 

University of Kentucky at Lexington 

(C. Gary Gairola, PhD, Associate Professor 

of Pharmacology and Experimental Therapeutics) 

CPSC contract #S-93-5440 

American Health Foundation 

(Klaus Brunnemann, MS, Section Head, Analytical Chemistry 

Dietrich Hoffmann, PhD, Associate Director and Chief, 

Environmental Carcinogenesis) 

CPSC contract #S-93-5441 

July 27, 1993 
.. . 
I. Introduction 

The Toxicity Testing Plan for Low Ignition-Potential Cigarettes 

was written to fulfill part of the responsibilities given to the 

Consumer Product Safety Commission (CPSC) by the Fire-Safe 

Cigarette Act of 1990. The Plan identified various toxicological 

tests and associated direct cost estimates. Limited testing from 

Tiers I and I1 was recommended as a first step toward the 

implementation of the Plan. 

CPSC staff, in consultation with the Department of Health 

and Human Services (HHS), directed a demonstration of this 

limited testing to verify its feasibility and costs. This study 

was designed demonstrate the ability of the testing to 

distinguish among and between cigarette brandsftypes. It was not 

designed to directly compare brands/types against each other 

since the prototypes were not chosen as intended replacements for 

the commercial cigarettes selected. All testing recommended in 

the first step was conducted, except for the pH test. 

11. Experimental 

Five cigarette brands/types were tested in this 

demonstration. Two commercial brands, "K" and "L", were selected 

from among those with the highest current sales in the U.S. The 

two prototypes selected, #530 and 8531, showed low ignition- potential in preliminary data from the National Institute of 

Standards and Technology (NIST). A University of Kentucky 

standard reference medium tar f medium nicotine cigarette, #1R3F, 

was included in the testing. All cigarettes contained filters. 

Except for the reference type, the cigarettes tested were 

randomly selected from among 300 (or 1.5 cartons) of each 

brand/type sent to each laboratory by NIST and CPSC staff. The 

average values were derived from machine smoking 100 cigarettes 

of each brand/type according to the Federal Trade Commission 

(FTC) protocol (Chapter B in the Toxicity Testing Plan). 

Two contract laboratories performed the testing. The 

Tobacco Health and Research Institute at the University of 

Kentucky measured the standard FTC parameters of tar, nicotine, 

total particulate matter, and water. The number of puffs per 

cigarette was recorded. The weight of tobacco burned was 

estimated by weighing the amount of tobacco that would normally 

be burned during machine smoking in 50 cigarettes of each type. 

This provided sufficient data to compare results on per 

cigarette, per tobacco weight burned, and per tar weight bases. 

Per puff data are not shown, but can be calculated from the data 

in Tables 1-6. 

The American Health Foundation conducted analyses of 

benzo(a)pyrene and four tobacco-specific nitrosamines, 

N'-nitrosonornicotine (NNN), 
4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK), 

N'-nitrosoanabasine (NAB), and N'-nitrosoanatabine (NAT), as 

described in Chapter D of the Plan. The Foundation also ran the 

Salmonella mutagenicity (Ames) assay, as described in Chapter E. 

Data from the Tobacco Health and Research Institute were used to 

express the results on per tobacco weight burned and per tar 

weight bases. 

111. Results 

Tables 1 and 2 summarize the results of testing the standard 

FTC parameters for cigarettes #530, #531, K, L, and the #lR3F 

reference. The words "soft packed cigarette", It total 

particulate matter" and "carbon monoxide" are abbreviated as 

"SPF", I'TPM", and "COtl respectively, in these tables. The 

averages are shown on a per cigarette basis in Table 1 and on a 

per tobacco weight burned in Table 2. The range of data for 

nicotine was 29-58 mg/g, "tar" 24-47 mg/g, and carbon monoxide 

20-39 mg/g (Table 2). Tables 3 through 7 show the data according 

to run and port number, and also the estimated weight of tobacco 

burned for each brandltype tested. 

Table 8 contains data for the levels of four tobaccospecific nitrosamines in the cigarettes. The range of data for 

NNN was 181-458 ng/g, NAT 210-421 ng/g, NAB 42-72 ng/g, and NNK 

148-319. The range of the total tobacco-specific nitrosamine 

data was 582-1140 ng/g. 

for publication of this document due to laboratory technical 

difficulties. An addendum will contain this data. 

Results for benzopyrene levels could not generated in time 

Tables 9 through 12 summarize the mutagenicity as indicated 

by the number of revertant colonies in the Ames assay. A 

positive mutagenic effect is indicated when the response is 

greater than twice the spontaneous revertant rate and is denoted 

by an asterisk. Table 9 indicates the three tester strains are 

operating properly in the Ames test system. 

fraction was added (Tables 10-12). About 1.5 times more 

revertants occurred with the more mutagenic tars, in the presence 

of S-9, compared to the other tars. 

All cigarette "tars" were mutagenic when the microsomal S-9 

IV. Discussion and Conclusions 

The limited testing conducted in this demonstration can 

distinguish differences between and among cigarette brandsltypes. 

This indicates that the tests recommended in the Toxicity Testing 

Plan, at least in Tiers I and 11, can generate data that will be 
3 

useful in evaluating the potential changes in toxicity of low 

ignition-potential cigarettes. 

Minor changes in the specifications of the Ames assay are 

recommended. The TA1535 strain of Salmonella gave negative 

results for all condensates tested in the demonstration study. 

However, the positive control for that strain, sodium azide, 

indicated that the TA1535 strain was functioning properly. Only 

a small response was obtained with TA1538. This suggests that 

these two strains may not provide useful data about differences 

in toxicity for the condensates. After consultation with the 

testing laboratories, it is recommended that the Toxicity Testing 

Plan use TA98 and TA1537 strains, unless it is suspected that 

non-frameshift type mutations might occur. 

A dose-related response is typically seen in the Ames assay 

when testing tobacco smoke condensates. However, the lack of a 

dose-related response in Tables 10-12 suggests that the 

concentration range tested might be too high or that other 

toxicities, such as cell death, might be occurring. Data from 

the dose-related response range is more useful for comparing 

toxicity. Therefore, the initial test concentration should be 

adjusted to define the beginning and slope of this range. 

The testing costs for this demonstration were within the 

$6,900 in direct costs per brand/type estimated in the Plan for 

the first step of implementation. CPSC staff contracted the 

testing for under $3,000 per brand/type, but the commercial rate 

could be somewhat higher. On the other hand, the economies of 

large volume testing that could be conducted by manufacturers can 

be expected to reduce the cost per brand/type. The estimates 

stated in the Plan appear to be reasonable, at least for Tiers I 

and 11. 
4 

TABLE 1 

#531 

sd 

K 

sd 

L 

sd 

#lR3F 

sd 

16.97 1.27 1.84 13.86 12.1 6.60 

1.06 0.06 0.45 0.65 1.4 0.29 

17.33 1.11 1.81 14.42 12.6 7.74 

0.98 0.04 0.37 0.70 0.7 0.21 

19.80 1.50 1.68 16.61 12.7 8.97 

0.63 0.05 0.27 0.45 0.6 0.33 

21.20 1.31 3.31 16.57 19.0 8.09 

0.95 0.07 0.42 0.63 0.9 0.28 
5 

TABLE 2 

#531 

sd 

K 

sd 

L 

sd 

81R3F 

sd 

35.35 2.64 3.84 28.86 25.2 13.75 

2.21 0.13 0.94 1.35 2.9 0.60 

29.18 1.86 3.04 24.28 21.2 13.04 

1.65 0.07 0.62 1.18 1.2 0.35 

31.19 2.37 2.65 26.16 20.0 14.13 

0.99 0.08 0.43 0.71 0.9 0.52 

30.50 1.88 4.76 23.98 27.34 11.64 

1.37 0.10 0.60 0.91 1.29 0.40 
6 

TABLE 3 

Measurement of FTC Parameters 

Cigarette #530 

EXPERIMENTAL 92-30 FK 100's 

TIPPING LENGTH 32 MM 

BUTT LENGTH 35 MM 

....................................................................... 

RUN # DATE PORT TPM NICOTINE WATER TAR co PUFFS 

mg/cig mg/cig mg/cig mg/cig mg/cig /cig ....................................................................... 

C06 19APR93 2 27.22 1.94 3.39 21.89 18.2 7.90 

C06 19APR93 12 28.56 1.94 3.73 22.89 20.0 8.98 

C06 19APR93 17 27.34 1.88 3.33 22.13 19.6 8.08 

C07 19APR93 3 27.84 1.97 3.83 22.04 19.2 8.24 

C07 19APR93 13 26.08 1.82 3.25 21.00 17.7 8.40 

C07 19APR93 18 26.48 2.03 3.05 21.40 17.6 9.02 

C08 20APR93 4 27.42 1.90 3.66 21.86 18.7 7.98 

C08 20APR93 9 28.42 1.99 3.69 22.73 18.8 8.04 

C08 20APR93 14 27.10 1.86 3.12 22.12 18.9 8.62 

C08 20APR93 19 28.78 2.02 3.60 23.16 19.6 8.24 

cog 20APR93 5 28.40 1.93 4.35 22.12 19.6 8.40 

cog 20APR93 10 27.32 1.95 3.90 21.48 18.1 8.24 

cog 20APR93 15 26.42 1.87 3.47 21.08 16.4 8.56 

cog 20APR93 20 28.56 1.98 3.94 22.63 19.4 8.32 

c10 21APR93 1 27.58 1.88 4.04 21.66 18.9 7.92 

c10 21APR93 6 28.16 1.97 3.59 22.60 19.3 8.12 

c10 21APR93 11 30.46 1.96 4.87 23.64 19.7 8.14 

c10 21APR93 16 32.80 1.91 2.95 27.94 18.4 8.00 

c11 21APR93 4 29.50 2.01 4.70 22.79 18.5 8.06 

c11 21APR93 7 25.60 1.81 2.92 20.86 17.6 8.00 

AVERAGE 28.00 1.93 3.67 22.40 18.7 8.26 

STD DEV 1.62 0.06 0.54 1.50 0.9 0.32 

AVG/GM TOB BURNED 58.22 4.01 7.63 46.57 38.9 17.18 

0.481 

.. . 
7 

TABLE 4 

Measurements of FTC Parameters 

Cigarette #531 

EXPERIMENTAL 92-31 FK 100's 

TIPPING LENGTH 32 MM 

BUTT LENGTH 35 MM 

C06 

C06 

C06 

C06 

C07 

C07 

C07 

C07 

C08 

coa 

coa 

coa 

cog 

cog 

cog 

cog 

c10 

c10 

c10 

c10 

c11 

c11 

19APR93 3 

19APR93 8 

19APR93 13 

19APR93 18 

19APR93 4 

19APR93 9 

19APR93 14 

19APR93 19 

20APR93 5 

20APR93 10 

20APR93 15 

20APR93 20 

20APR93 1 

20APR93 6 

20APR93 11 

20APR93 16 

21APR93 2 

21APR93 7 

21APR93 12 

21APR93 17 

21APR93 3 

21APR93 12 

AVERAGE 

STD DEV 

AVGIGM TOB BURNED 

0.480 

16.24 

17.44 

16.04 

15.46 

17.56 

16.12 

15.64 

15.66 

15.92 

17.94 

16.50 

17.22 

17.20 

16.46 

18. 08 

17. 08 

18-72 

18.30 

19.38 

16.70 

16.40 

17.20 

16.97 

1.06 

35.35 

1.21 

1.25 

1.25 

1.26 

1.33 

1.22 

1.20 

1.23 

1.32 

1.32 

1.21 

1.24 

1.28 

1.19 

1.26 

1.30 

1.32 

1.41 

1.26 

1.27 

1.34 

1.27 

0.06 

2.64 

1.18 

1.72 

2.01 

1.45 

1.16 

2.01 

1.43 

1.41 

1.60 

1.50 

2.20 

2.14 

1.42 

2.07 

1.69 

2.06 

3.12 

2.19 

2.52 

1.52 

1.72 

1.80 

1.83 

1-84 

3.84 

0.45 

13.31 

13.34 

13.03 

14.21 

13.47 

13.03 

13.24 

14.56 

14. 18 

12. 83 

14.48 

13. 88 

13.91 

14.23 

13.47 

13.76 

14.30 

14.79 

15.45 

13.92 

13.40 

14.02 

13.86 

28.86 

0.65 

11.0 

12.3 

10.5 

16.6 

12.2 

10.7 

11.0 

10.0 

11.3 

12.4 

12.0 

12.2 

12.3 

12.1 

11.3 

11.3 

12.0 

13.4 

13.2 

12.3 

14.4 

11.9 

12.1 

1.4 

25.2 

6.22 

6.52 

6.26 

6.96 

7.02 

6.62 

6.46 

6.54 

6.92 

6.46 

6.30 

6.96 

6.46 

6.34 

7.00 

7.02 

6.50 

6.20 

6.26 

6.60 

0.29 

13.75 

6.88 

6.38 

6.88 
8 

TABLE 5 

Measurement of FTC Parameters 

Cigarette K 

C06 

C06 

C06 

C06 

C07 

C07 

C07 

C07 

C08 

C08 

C08 

C08 

cog 

cog 

cog 

cog 

c10 

c10 

c10 

c10 

c11 

19APR93 4 

19APR93 9 

19APR93 14 

19APR93 19 

19APR93 5 

19APR93 10 

19APR93 15 

19APR93 20 

20APR93 1 

20APR93 6 

20APR93 11 

20APR93 16 

20APR93 2 

20APR93 7 

20APR93 12 

20APR93 17 

21APR93 3 

21APR93 8 

21APR93 13 

21APR93 18 

21APR93 2 

AVERAGE 

STD DEV 

AVG/GM TOB BURNED 

0.594 

18.00 

17.36 

17.48 

17.96 

17.60 

17.40 

15.66 

15.48 

17.02 

16.66 

17.20 

17.58 

17.90 

16.30 

18.28 

17.36 

17.96 

18.38 

19.32 

17.68 

15.42 

17.33 

0.98 

29.18 

1.24 

1.15 

1.11 

1.15 

1.14 

1.14 

1.06 

1.06 

1.07 

1.06 

1.09 

1.06 

1.08 

1.04 

1.12 

1.09 

1.13 

1.17 

1.12 

1.12 

1.04 

1.11 

0.04 

1.86 

1.75 

1.55 

1.62 

1.85 

1.99 

1.77 

1.65 

1.42 

1.60 

1.52 

1.64 

1.87 

2.47 

1.54 

2.25 

1.89 

2.12 

1.97 

2.75 

1.52 

1.19 

1.81 

0.37 

3.04 

15.01 

14.66 

14.75 

14.96 

14.47 

14.49 

12.95 

13.00 

14.35 

14.08 

14.46 

14.65 

14.35 

13.72 

14.91 

14.38 

14.71 

15.24 

15.45 

15.04 

13.19 

14.42 

0.70 

24.28 

13.0 

12.2 

13.4 

12.5 

12.6 

11.9 

10.8 

12.2 

12.7 

12.4 

12.2 

12.6 

13.3 

12.6 

13.9 

13.3 

12.8 

13.3 

12.9 

12.1 

11.9 

12.6 

0.7 

21.2 

8.32 

7.94 

7.82 

7.92 

7.74 

7.72 

8.28 

7.68 

7.60 

7.64 

7.50 

7.72 

7.52 

7.32 

7.88 

7.72 

7.64 

7.74 

7.50 

7.92 

7.50 

7.74 

0.21 

13.04 
9 

TABLE 6 

Measurement of FTC Parameters 

Cigarette L 

85MM SPF 

TIPPING LENGTH 25 MM 

BUTT LENGTH 28 MM 

C06 

C06 

C06 

C06 

C07 

C07 

C07 

C07 

C08 

C08 

C08 

C08 

cog 

cog 

cog 

cog 

c10 

c10 

c10 

c10 

,211 

19APR93 5 

19APR93 10 

19APR93 15 

19APR93 20 

19APR93 1 

19APR93 6 

19APR93 11 

19APR93 16 

20APR93 2 

20APR93 7 

20APR93 12 

20APR93 17 

20APR93 3 

20APR93 8 

20APR93 13 

20APR93 18 

21APR93 4 

21APR93 9 

21APR93 14 

21APR93 19 

21APR93 8 

AVERAGE 

STD DEV 

AVGIGM TOB BURNED 

0.635 

19.10 

20.32 

19.02 

19.12 

19.68 

20.34 

20.96 

20.52 

18.76 

19.52 

20.26 

20.32 

19.74 

20.10 

20.60 

20.58 

19.36 

19.28 

19.20 

19.40 

19.68 

19.80 

0.63 

31.19 

1.42 

1.59 

1.55 

1.53 

1.55 

1.46 

1.56 

1.55 

1.42 

1.45 

1.55 

1.50 

1.45 

1.48 

1.54 

1.57 

1.43 

1.47 

1.50 

1.45 

1.52 

1.50 

0.05 

2.37 

1.57 

1.83 

1.39 

1.42 

1.76 

1.97 

2.11 

2.14 

1.67 

1.68 

1.46 

1.62 

1.80 

1.75 

2.08 

1.73 

1.60 

1.63 

1.20 

1.18 

1.78 

1.68 

0.27 

2.65 

16.11 

16.90 

16.08 

16.16 

16.37 

16.91 

17.29 

16.82 

15.67 

16.39 

17.25 

17.21 

16.49 

16.86 

16.97 

17.28 

16.33 

16.18 

16.49 

16.77 

16.38 

16.61 

0.45 

26.16 

12.3 

12.4 

12.1 

13.6 

12.2 

13.6 

12.9 

12.9 

11.6 

12.6 

13.0 

13.4 

12.7 

13.4 

13.1 

12.4 

12.9 

11.5 

12.5 

13.2 

12.9 

12.7 

0.6 

20.0 

9.28 

9.32 

9.46 

9.00 

8.80 

8.74 

9.42 

9.08 

8.68 

9.18 

9.16 

8.86 

8.62 

9.12 

8.96 

9.36 

8.18 

8.92 

8.76 

8.46 

9.06 

8.97 

0.33 

14.13 
10 

TABLE 7 

Measurement of FTC Parameters 

Cigarette #1R3F 

CODE X 1R3F Reference Cigarette 85MM SPF 

TIPPING LENGTH 25 

BUTT LENGTH 28 

C06 

C06 

C06 

C06 

C07 

C07 

C07 

C07 

C08 

C08 

C08 

C08 

cog 

cog 

cog 

cog 

c10 

c10 

c10 

c10 

c11 

c11 

c11 

19APR93 1 

19APR93 6 

19APR93 11 

19APR93 16 

19APR93 2 

19APR93 7 

19APR93 12 

19APR93 17 

20APR93 3 

20APR93 8 

20APR93 13 

20APR93 18 

20APR93 4 

20APR93 9 

20APR93 14 

20APR93 19 

21APR93 5 

21APR93 10 

21APR93 15 

21APR93 20 

21APR93 1 

21APR93 6 

21APR93 11 

AVERAGE 

ST DEV 

AVG/GM TOB BURNED 

0.695 

21.26 

22.02 

22.86 

22.70 

20.46 

20.72 

21.34 

21.68 

20.14 

20.00 

21.38 

19.78 

21.78 

20.70 

21.20 

22.02 

22.74 

22.14 

21.06 

21.04 

20.00 

19.68 

21.20 

0.95 

30.50 

20.84 

1.27 

1.31 

1.41 

1.47 

1.40 

1.34 

1.35 

1.39 

1.25 

1.25 

1.34 

1.17 

1.33 

1.25 

1.22 

1.29 

1.32 

1.32 

1.20 

1.33 

1.30 

1.30 

1.41 

1.31 

0.07 

1.88 

3.51 

3.30 

3.77 

3.50 

3.15 

3.08 

3.51 

3.71 

2.75 

3.12 

3.25 

2.78 

3.81 

3.35 

3.35 

3.47 

4.43 

3.46 

3.45 

2.73 

3.27 

2.53 

2.89 

3.31 

0.42 

4.76 

16.48 

17.41 

17.68 

17.73 

15.92 

16.29 

16.48 

16.58 

16.13 

15.63 

16.79 

15.83 

16.64 

16.10 

16.63 

17.26 

16.99 

17.36 

16.42 

16.78 

16.47 

16.18 

15.38 

16.57 

0.63 

23.98 

19.5 

19.1 

19.9 

19.2 

16.7 

18.7 

18.3 

18.7 

18.2 

17.2 

19.8 

17.7 

19.2 

18.8 

20.2 

20.5 

19.8 

19.6 

19.5 

20.0 

19.3 

18.6 

18.7 

19.0 

0.9 

27.34 

7.86 

8.16 

8.58 

8.04 

8.08 

7.82 

8.32 

8.56 

7.62 

8.34 

8.20 

7.88 

7.86 

8.20 

7.98 

8.18 

7.74 

8.56 

8.10 

7.92 

8.28 

7.64 

8.09 

0.28 

11.64 

8.10 
11 

brand 

TABLE 8 

Tobacco-specific N-Nitrosamines in Test Cigarettes 

KY 1R3F, 85 mm 193 210 29.3 222 654 

L, 85 mm 287 216 26.5 194 724 

K, 80 mm 272 250 25.9 173 721 

#530, 100 mm 134 165 34.7 102 436 

#531, 100 mm 86.7 101 20.5 70.9 279 

KY 1R3F 

L 

K 

#530 

#531 

KY 1R3F 

L 

K 

#530 

#531 

(ng/g tobacco) 

278 302 42.2 319 941 

452 340 41.7 306 1140 

458 421 43.6 184 1107 

279 343 72.1 212 841 

181 210 42.7 148 582 

(ng/mg tar) 

8.05 8.76 1.22 9.26 27.3 

17.3 13.0 1.60 11.7 43.6 

18.9 17.3 1.80 12.0 50.0 

5.98 7.36 1.55 4.55 19.4 

6.26 7.29 1.48 5.12 20.2 
12 

AmeS Salmonella/Microsome Mutagenicity Test 

Mean Summary Data 

Sponsor: Brunnemann Date Initiated: 

6/4/93 

Study number: LRD57 Date Scored: 

6/7/93 

Test Articles: KY 1R3F, K, L, 92-30 (#530), 92-31 (#531) 

Description: 5 tobacco smoke condensates; 4 doses each 

Considerations: TA98, TA1535, TA1538; +/- Rat S9 

TABLE 9 

Controls 

Spontaneous Revertant 

Colonies/Plate (avg) 

Solvent Controls 

DMSO 

DMSO 

s-9 TA98 TA1535 TA1538 

(-1 22 21 15 

i+j 34 14 23 

Positive Controls (ucrfv 1) 

sodium azide 5 (-1 

2-Nitrofluorene 5 (-1 382 

2-Anthramine 5 (+) 1964 114 1874 

- 1000 - 

- 115 

TABLE 10 

Test Compound: XY 1R3F 

Total Revertant Colonies/Plate 

(avg) 

Dose Level (uqfv 1) 

50 

100 

200 

300 

100 

200 

300 

400 

TA98 

25 

41 

52* 

57* 

169* 

195* 

157* 

172* 

TA1535 

19 

15 

21 

18 

11 

11 

12 

13 

TA1538 

18 

15 

20 

22 

73* 

69* 

73* 

56* 

* Positive response (threshold=2.000 x corresponding solvent). 
13 

TABLE 11 

Test Compound: K 

Dose Level (uq/D 11 

50 

100 

200 

300 

100 

200 

300 

400 

Dose Level (uq/D 11 

50 

100 

200 

300 

100 

200 

300 

400 

Total Revertant Colonies/Plate 

(avg) 

TA98 TA1535 TA1538 

29 16 9 

37 14 14 

67* 20 26 

ai* 15 26 

194* 14 76* 

162* 12 72* 

172* 14 77* 

167* 15 71* 

Test Compound: L 

Total Revertant Colonies/Plate 

(avg) 

s-9 TA98 TA1535 TA1538 

(-) 36 19 17 

(-) 40 ia ia 

(-) 47* 20 22 

(-) 42 16 23 

(+) 150* 16 71* 

(+) 156* 14 61* 

(+I 142* ia 5a* 

(+) 143* ia 59* 

* Positive response (threshold=2.000 x corresponding solvent). 
14 

TABLE 12 

Test Compound: 92-30 (#530) 

Dose Level (uqlv 1) 

50 

100 

200 

300 

100 

200 

300 

400 

Dose Level (uqlv 11 

50 

100 

200 

300 

100 

200 

300 

400 

Total Revertant ColoniesfPlate 

( avg ) 

34 

32 

37 

46* 

83* 

102* 

95* 

94* 

TA1535 

22 

24 

24 

17 

13 

20 

14 

11 

TA1538 

13 

16 

15 

16 

53* 

46 

54* 

53* 

Test Compound: 92-31 (#531) 

Total Revertant ColoniesfPlate 

(avg) 

TA98 

27 

32 

36 

44 

112* 

129* 

104* 

85* 

TA1535 

15 

19 

20 

19 

13 

27 

15 

18 

TA1538 

16 

14 

18 

22 

43 

53 * 

51* 

48* 

* Positive response (threshold=2.000 x corresponding solvent).